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A M A C R O M O L E C U L A R R E P E A T I N G U N I T O FM I T O C H O N D R I A L S T R U C T U R E A N D F U N C T I O NC or r e l a te d E l e c t r on M i c r os c op ic an d B i oc h e m i c a lS t u d i e s o f I s o l a t e d M i t oc h on d r i a an d S u b mi t oc h on d r i a lP ar t i c l e s o f B e e f H e ar t M u s c l e

H . F E R N A N D E Z - M O R A N , M .D ., T . O D A , M .D .,P . V . B L A I R , P h .D . , a n d D . E . G R E E N , P h .D .From the M ixter Laborato ries for Electron Microscopy, Neurosurgical Service, Massachu settsGeneral Hospital , Boston, and T he D epar tm ent of Biophysics, University of Chicago; and th eInst i tute for Enzym e Research, Un iversity of Wisconsin, Madison. Dr. Fem~indez-Mor~n's pres entaddress is De partm ent of Biophysics, Un iversity of Chicago. Dr. O da's present address is De part me nto f Pa tho logy , Okayam a U nivers i ty Medica l Schoo l , Olmyama, Japa n

A B S T R A C TA r e p e a t i n g p a r t i c l e as s o c ia t e d w i t h t h e c r i s t a e a n d t h e i n n e r m e m b r a n e o f t h e e x t e r n a le n v e l o p e h a s b e e n r e c o g n iz e d a n d c h a r a c t e r i z e d i n b e e f h e a r t m i t o c h o n d r i a b y c o r r e l a t e de l e c t r o n m i c r o s co p i c a n d b i o c h e m i c a l s tu d i es . M a n y t h o u s a n d s (ca. 104 to 105) of the sep a r t i c l e s , d i s p o s e d i n r e g u l a r a r r a y s , a r e p r e s e n t i n a s i n g l e m i t o c h o n d r i o n . T h e r e p e a t i n gp a r t i c l e , c a l l e d t h e e l e m e n t a r y p a r t i c l e ( E P ) , c o n s i st s o f t h r e e p a r t s : ( 1 ) a s p h e r i c a l o rp o l y h e d r a l h e a d p i e c e ( 8 0 t o 1 0 0 A i n d i a m e t e r ) ; ( 2 ) a c y l i n d r i c a l s t a l k ( a b o u t 5 0 A l o n ga n d 3 0 to 4 0 A w i d e ) ; a n d ( 3 ) a b a s e p i e c e (4 0 X 1 10 A ) . T h e b a s e p i e c e s o f t h e e l e m e n t a r yp a r t i c l e s f o r m a n i n t e g r a l p a r t o f t h e o u t e r d e n s e l a y e r s o f t h e c r i s t a e . T h e e l e m e n t a r y p a r -t i c l es c a n b e s e e n in e l e c t r o n m i c r o g r a p h s o f m i t o c h o n d r i a i n s i t u , o f i s o l a te d m i t o c h o n d r i a ,a n d o f s u b m i t o c h o n d r i a l p a r t ic l e s w i t h a c o m p l e t e e l e c t r o n t r an s f e r c h a in . N e g a t i v e s t a i n -i n g w i t h p h o s p h o t u n g s t a t e i s o n l y o n e o f s e v e r a l t e c h n i q u e s t h a t c a n b e u s e d f o r r e p r o d u c i b l ed e m o n s t r a t i o n o f t h e r e p e a t i n g p a r t i c l e s a n d u n d e r l y i n g s u b u n i t o r g a n i z a t i o n o f m i t o c h o n -d r i a l m e m b r a n e s . A p a r t i c u l a t e u n i t c o n t a i n i n g a c o m p l e t e e l e c tr o n t r an s f e r c h a i n c a n b ei s o l a te d f r om b e e f h e a r t m i t o c h o n d r i a . T h e i s o la t e d u n i t a p p r o x i m a t e s i n s iz e t h a t o f th ee l e m e n t a r y p a r t i c l e i n s i t u . T h e m o l e c u l a r w e i g h t o f th e p a r t i c l e i n s i t u i s c a l c u l a t e d t o b e1 .3 X 1 0 ~. E v i d e n c e i s p r e s e n t e d f o r i d e n t i f y i n g t h e i s o l a t e d u n i t w i t h t h e e l e m e n t a r y p a r -t i c l e v i s u a l i z e d i n s i t u . T h e e l e m e n t a r y p a r t i c l e o f t h e m i t o c h o n d r i o n i s b e li e v e d t o b e ap r o t o t y p e o f a c l a s s o f f u n c t i o n a l p a r t i c l e s o r m a c r o m o l e c u l a r a s s e m b l i e s o f s i m i l a r s i z ef o u n d i n a s s o c i a ti o n w i t h m e m b r a n e s g e n e r a ll y .

I N T R O D U C T I O NT h e p i o n e e r i n g e l e c t r o n m i c r o s c o p e s t u d i e s o fP a l a d e ( 5 3 , 5 4 ) a n d S j 6 s t r a n d ( 6 7 , 6 8 ) e s t a b l i s h e dm o r e t h a n a d e c a d e a g o t h e d i s t i n c t i v e f i n e s t r u c -

t u r e o f t h e m i t o c h o n d r i o n a s e s s e n t i a ll y t h a t o f a ne l o n g a t e d s u b c e ll u l a r b o d y b o u n d e d b y a m e m -b r a n e c o n s i s t i n g o f t w o l a y e r s . T h e e x t e r n a l l a y e r

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is considered to be the limiting membrane, whileregular infoldings of the inner membrane formnumerous internal ridges termed "cristae" byPalade. During the same period the mitochondrionwas identified as the locale of citric cycle oxida-tions, electron transfer, and oxidative phosphoryl-ation (49, 65). The concept of the mitochondrionas an organized system in which there was a pre-cise arrang ement of the many enzymes involved inthe sequential reactions underlying mitochondrialfunction (32, 36, 50) became firmly established inbiochemical thinking.

Large scale isolation of stable mitochondria(33, 51) unde r conditions which do not impa irtheir main enzymic activities has made thesehighly organized, membranous organelles avail-able for chemical analysis. Equally important,there are multiple, exact criteria by which thefunctional integrity of the mitochondrion can beevaluated . Of all the specialized lamell ar systems,mitochondria appeared, therefore, to be particu-larly well suited for a study of correlations betweenbiochemical function and ultrastructure (23-25, 37).

Respiratory energy transformations have beenshown to take place in the organized membranestructures of the mitochondrion. The mitochon-drial membranes contain the different multi-enzyme complexes involved in these transforma-tions; these complexes are arranged in highlyordered arrays (33, 37, 40, 51). Although the firstphase of the fine structural analysis of the mito-ehondrion (53, 54, 67) established the base linefor a correlation of structure and fun ction (4, 14,65, 79, 80), the shortcomings of the preparativetechniques and of the resolution of the electronmicroscope limited the extent of meaningful corre-lation. With mitochondria, as with other lamellarsystems (18, 68, 78), it is now apparent that thesubmicroscopic patterns of sectioned specimensfixed with osmium tetroxide reveal merely ageneral structural framework consisting essen-tially of lipoprotein. Thus, in the mitochondrialmembranes only certain stereotyped, uniformfeatures of the "osmium-stained" smooth mem-brane s are seen, with no indica tions of the specificenzymic complexes and other constituents. Themitochondrion is a lamellar system in which thebiochemical and enzymic properties are bettercharacterized than is the ultrastructure.

Recent improvements in preparative tech-niques, particularly that of negative staining

applied to the study of virus ultrastructure (7, 8),have made possible the examination of biologicalsystems in far greater detail than could heretoforebe achieved in sectioned material. Fortunately,mitochondria (both those seen in situ and afterisolation), by virtue of their ul trathi n membranes,are particularly suitable for application of manyof these new techniques which do not necessarilyrequire sectioning (16, 23).

Three years ago, we applied these techniques,including negative staining and improved lowtemperature methods which yield better morpho-logical and histochemical preservation in sec-tioned material (20-23), to the study of mi-tochondria. These techniques, combined withimproved high-resolution electron microscopy, re-vealed for the first time the presence of a char-acteristic polyhedral or round structural unit, 80to 100 A in diamete r, as a basic compon ent ofmitochondr ial memb ran es (23, 25, 26). This find-ing casts a new light on our views of the organiza-tion of the mitochondrion. Recognition of thisrepeating particulate subunit, which we desig-nated "elementary particle" (EP), was the start-ing point for a program of biochemical isolation.

In essence, the experimental approach of ourstudies correlating structure w ith function involvedfragmentation of mitochondria into subunits. Themitoc hondr ion was, in effect, treat ed as a chemi-cal entity capable of stepwise disassembly into itscomponent parts (and reassembly into the originalunit) (40). This approach, from a biochemicalpoint of view, has already led to the discovery ofnew electron transfer components such as co-enzyme Q, and of proteins containing non-hemeiron and copper. The study of electron transferin intact cells and in particles containing therespiratory chain (as exemplified by the studiesof Chance and his collaborators (9, 10) has beenof great value in promoting understanding of theover-all kinetics and the sequence of oxidoreduc-tion of the spectroscopically visible componen ts ofthe respiratory chain. A third approach involvedreconstitution of a fully functional electron trans-fer particle from its purified segments and fromthe highly purified and independent componentparts (37, 44, 45).

Studies on the stepwise degradation of themitochondrion into particles of decreasing com-plexity have led to the recognition of three basiccomponents of mitochondrial organization: (1)the particulate electron transfer chain; (2) the

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Insoluble network composed of structural proteinand lipid; and (3) the "solubiliza ble" dehydroge-nase'complexes. This was the biochemical pictureat the time when the collaborative effort of ourtwo laboratories began.

Sonic irradia tion dissociates mito chon dria intoa particulate structured fraction and a solublefraction; this resolution makes it clear that therecannot be a single mitochondrial subunit whichis all-embracing, but rather several subunits de-pending upon the function under consideration.We are particularly concerned here with the sub-unit that contains the complete electron transferchain--the apparatus for the transfer of electronsfrom succinate and DPNH to molecular oxygen.

We have worked mainly with mitochondriaprepared from beef heart muscle, because theyare relatively stable and can be isolated on a largescale; an additional advantage of heart mito-chondria over liver mitochondria is their higherdensity of cristae per unit volume with a corre-spondingly greater oxidative rate. By carefulcomminution of beef heart mitochondria in asucrose medium, well defined fractions were pre-pared, one of which (ETP) contains particleswith an essentially intact electron transfer chain,divested of the primary dehydrogenating enzymecomplexes. The capacity for both electron transferand oxidative phosphorylation was retained in ananalogous particle (ETPI~). Electron microscopicstudied-of these submitochondrial fractions bystandard osmium fixation and thin sectioningtechniques (14, 33, 79, 80) indicated that thedouble-membraned structure was preserved inspecimens of ETPm whereas only a single-mem-braned structure was observed in ETP. More ex-tensive correlation of fine structure with function,however, could not be achieved in these earlyinvestigations.

Concentrating on the electron transfer particle(ETP) as the primary unit of mitochondrialfunction, Green and his colleagues (33, 37, 40)succeeded in isolating and characterizing elevenoxidation-reduction components of the electrontransfer chain. At least nine proteins with oxida-tion-reduction groups participate in the termi nalelectron transfer process. Approximately 30 percent of the total dry weight of the mitochondrionand of the ETP is accounted for by lipids, thebulk of which (95 per cent) is phospholipid(29 b). Green and O da (38) tenta tively assumed,from preliminar y data, that the struc tural unit of

ETP was a cylindrical particle about 445 A longand 100 A in diameter, wit h a particle weight ofthe order of 4.3 million.

Three developments have pointed to a down-ward revision of the estimated value proposed(38) as the molecular weight of the repeating unitof the electron transfer particle: (1) the demon-stratio n by Fernfindez-Morfin (23, 25) that themembrane structure of isolated mitochondriaand subfractions appeared to be built up of pairedarray s of the 80 to 100 A particles, no ted above,separated by a "middle " or mesolayer of variablewidth; (2) the isolation by Green et a l . (12, 13, 39)of a structural protein, devoid of oxidation-reduc-tion groups, accoun ting for 60 to 70 per cent of thetotal mitochondrial protein; (3) the resolution ofthe electron transfer chain into four complexesfunctionally indepe ndent (44, 45), each of highpurity, and the reconstruction of the originalparticle with essentially undiminished activity byrecombination of the four complexes carried outby Hatefi and his colleagues (30, 44, 45). Thepostulated mo lecula r weight of 4.3 106 for theelectron transfer chain was hardly compatiblewith the dimensions of the 80 to It0 A repeatinguni t seen by Fernfindez-Morfin. ~I he observat ionthat a large proportion of the total pro~ein of themitochondrion was not concerned with the elec-tron transfer process p e r se suggested the possibilitythat the electron transfer particle as isolated couldbe resolved into a fraction containing structuralprotein and a fraction containin g the electrontransfer chain. The latter unit would have a cal-culated m olecula r weight of 1 to 2 106; thisvalue would bring the size of the unit better intoline wit h~h e dimensions of the repeating particleseen by the electron microscope. The particleweight of the reconstructed electron transfercha in (from the four purif ied complexes) was esti-mat ed to be 1.4 X 106 on a prote in basis, calcu-lated from the known particle weights of each ofthe four constituent complexes (established fromcomposition and ultracentrifuge data). These de-velopments made the conclusion inescapable thatthe n~olecnlar weight of the uni t of electron trans--fer had to be no more t han 1 to 2 X 106 and led usto a pr ogram directed toward isolation of a unit ofelectron transfer of molecular weight less thanthat of ETP. Indeed, such a particle has beenisolated (5). It con tains the complete electrontransfer chain with all the components in the samemolecular ratios as those found in the mitochon-

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drial electron transfer chain. Increase in activityand increase in concentrati on of the oxidation-reduction proteins per unit weight run parallelthroughout all but the last stage in the purifica-tion. The molecular weight of the particle at thehighe st puri ty level att ain ed is 1.4 X l06 on aprotein basis (this would correspond to a molec-ular weight of 2 X l0 s for the lipoprotein com-plex). The particle reconstructed from the fourisolated complexes has a molecular weight thatcorresponds closely to that of the integrated par-ticle isolated directly from the mitochondrion.

In consequence of these developments, we haveelected to designate the particle obtained both byisolation and by reconstruction as the elementaryparticle, and we have postulated that the isolatedor reconstructed elementary particle correspondsto the repeating particle with a head piece of 80to 100 A, a stalk, and a base piece, seen in electronmicrographs.

After the initial phase was completed, resultingin a definition of the basic structural parametersof the new repeating particle and the establish-ment of its relationship to the mitochondrial mem-branes, it remained to be established that thevisualized particle is a b o n a f i d e constituent of themembrane system. Mitochondria from a widevariety of sources were examined and the prepara-tive proce dures for the elecl-mn micro scopic ex-amination were carefully checked for the possi-bility of artifact formation. In addition, a comple teset of particles was examined ranging in size fromthe mitochondrion to the individual complexes;an internal control of artifact formation thus be-came available since only particles with a com-plete electron transfer chain could be expected toconstitute the repeating unit.

The present communication will deal specifi-cally with: (1) the electron microscopic evidenceof the existence of a repeating particle in wholemitochondria and in those submitochondrialparticles that have intact electron transfer chains;(2) the isolation and properties of the particles;(3) the experimental basis for identifying the iso-lated or reconstituted particle with the repeatingparticle visualized by the electron microscope.Preli minary accounts of facets of the pr esent studyhave been reported elsewhere (23-27, 34).MATER IAL~ AND METHODS

MATERIALS : i n the course of extensive correla-tive studies, mltochondria from many sources were

examined either i n s i t u or after isolation from heartmuscle, retina, liver, pancreas, and brai n of the rat,mouse, beef, chicken, and guinea pig; but theprincipal effort was directed to the study of isolatedbeef heart rnitochondria.

IS O LA TIO N O F MITO C H O N D R IA : Large scaleisolation of mitochondria from bee f heart muscle wascarried out under conditions that minimized con-tamination by other cell particulates and by myosin(5, 11). Suspensions in 0.5 M sucrose (kept at 0 to 5 )conformed to the most rigorous standards for preser-vation of mitochondrial structure and activity.Two alternative preparative procedures were used.In t he first procedure ground beef heart muscle ishomoge nize d in 0.5 M sucrose at pH 7.0 and themitochondrial fraction is separated from otherstructures and compounds by differential centrifuga-tion. In turn, the rnitochondrial fraction is refrac-tionated; a heavy fraction containing only intactwhole mitochondria with well preserved cristae iscollected. For ease of presentation the details of thispreparative procedure are summarized in Table I.In the second procedure ground beef heart musclewas suspended in 0,66 M sucrose and treated withNagarse proteinase according to the method ofHagihara (43). In the klagihara metho d the use ofthe high speed blendor is eliminated. The proteolyticenzyme attacks the myofibrils and connective tissueand thus releases mitochondria from the muscle mass.

We have found no recognizable difference in thepurity or integrity of the mitochondria prepared bythe two procedures outlined above. Both types ofpreparations were used for electron microscopy ofintact mitochondria, Freezing of the suspensions wasavoided and generally no more tha n 8 to 24 hoursintervened between the preparation of the mito-chondria and examination by electron microscopy,

ELEMENTARY PARTICLES: The de taile d pro-cedure for the isolation of elementary particles hasbeen described in the compa nion article by Blair e t a l .(5). A summa ry of the preparati ve procedur e is givenin Table II. It should be pointed out that elementaryparticles isolated from the standard preparation ofmit ochon dria in 0.25 M sucrose are equal ly as satis-factory as those prepared from mitochondria thathad been purified more rigorously for good electronmicroscopy.

The mitochondrial suspensions which served as thestarting point for the isolation of EP had " theoret ical"P/ O ratios (the value was two for succinate and threefor pyruvate plus malate). Furthermor e, they showedall the characteristics of intact mitochondria: non-reduction of external cytochrome c or DPN +, non-oxidation of external DPNH, non-oxidation of citrateor isocitrate, low rate of oxidation for succinate andnegligible ATPase activity.

Essentially the isolation of the eleme ntary particle

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involves a quantitative separation of the mito-chondrial subunits; this separation is achieved byammonium sulfate fractionation of mitochondriathat have been frozen in presence of 0.3 per centKCI washe d with 0.9 per cen t KC1, and finally"solubilized" with a mixture of cholate and de-oxycholate. The insoluble structural protein fractionsediments at low concentrati ons of ammoni um sulfate;the floating fraction containing the elementaryparticle separates at about 0.5 saturation; the solublefraction (not precipitated by 0.5 saturation) contains

some of the primary dehydrogenase complexes andcytochrome c.

When the procedure outlined in Table II is re-peated, the elementary particle thus obtained isstripped further of contaminating structural proteinand is correspondingly enriched with respect to theoxidation-reduction components. Thus, the cy-tochrome a cont ent of the one-cycle material is about2.8 re#moles per mg of protein and of the two-cyclematerial as high as 4.2 re#moles per rag of protein.

For ultracentrifugal and x-ray scattering analyses,

T A B L E IMe tho d/o r Large Scale Preparation of Mitochondria in 0.5 M Sucrose

1. Suspen d 3200 gm of grou nd beef hear t muscle in 9.6 1. of 0.5 M sucrose con tai n-ing 0.02 M K2HPO , (final vo lum e ca. 13 1.).

2. Adjust pH to 7.0 with N KO H and filter the suspension through cheese cloth(double l ayer). The filte red residue is resu spen ded in 9.6 1. of 0.5 M sucrosecont aini ng 0.02 M phos phat e buffer of pH 7.0.

3. Homogenize in macro Waring blendor for 1 min. with the rheostat set ting at 60.Neutraliz e the suspension with N KO H to pH 7.0.4. Two fractions are removed after centrifugati on at 0-5 : Rl- -af te r centrifu gationfor 20 rain. at 1600 g (13-1iter refri gera ted cent rifug e) ; R2-- aft er cen tri fuga tio nat 50,000 g in the Sharpies continuous centrifuge.

5. R~ is resus pende d in 1 1. of a mixt ure 0.5 M in sucrose an d 0.01 g in ph osph ate(pH 7.0); t he ho moge niz ed suspension is cent rifug ed at 15,000 g for 20 rain.The residue consists of a well packed as well as a loosely packed layer. The twolayers are separated, suspended in about 500 ml of the sucrose -phosphate me-dium, and the homogeniz ed suspensions are recentri fuged at 15,000 g for 20min. The suspension prepared from the well packed residue layer on cen-trifugation yields a predominantly well packed layer topped by a small layerof loosely packed material. Only the well packed material is resuspended in0.5 ~ sucrose and this suspension constitutes the final mitoc hondrial pr epara -tion.

TAB LE I ISummary of the Procedure/or Isolation of Elementary Particles from Bee f HeartMitochondria

(Blair et al., 5)1. Standard large scale isolation of beef heart mitochondria by the method of

Crane et al. (9). Suspension med ium ~0. 25 ~ sucrose.2. Freezing of mitoch ondria in a m ixture containi ng 0.3% KC1 and 0.17 M sucrose

and the n washing the thawed rni tochondria in 0.9% KC1. Residue resuspendedin 0.66 M sucrose to a final suc rose conc ent rat ion of 0.3 M.

3. M itoc hondri a exposed to deoxycholate (0.3 nag per mg protein) and chol ate (0.3rag/rag prote in) at pH 8.0 (0.02 M Tris buffer) and fracti onated with sa turat edammonium sulfate. The first fraction at 33 per cent saturation was discarded;the second at 50 per cent saturation (floating pellet) was retained.

4. Fl oat ing pel let di lute d to 0,5 mg of prot ein pe r ml in 0.25 M sucrose and se di-men te d at 30,000 RPM (4 hours). Pelle t res uspe nded in buffere d 0.25 M sucrose;the suspension was clarified by sonic irradiation. Insoluble mater ial was re-moved by centrifugation. The clear supernatant solution contains a finesuspension of the elementary particles.

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the suspension of elementary particles (two cycles oftreatment; 20 mg of protein per ml) was "solu-bili zed" wit h 1.5 X 10 3 M sodium phosph otung sta tein presence of 0.2 per cent a-tocophe rol (to minimizelipid peroxidation). The particles were separated intoa tightly packed sediment which was discarded, anda translucent liquid precipitate which was used forphysical measurements as well as for electron micro-scopy.

COMPLEXES OF THE ELECTRON TRANSFERCHAIN: The four complexes that make up theelectron transfer chain were prepared by the follow-ing procedures : succinic-coenzyme Q reductase bythe method of Ziegler and Doeg (81), or of Tisdaleet al. (75 a); DPNH-coenzyme Q rcductase by themethod of Hatefi et al. (47); QH2--cytochromereductase by the method of Rieske, Zaugg, andWharton (61); and cytoehrome oxidase by themethod of Griffiths and Wharton (42) or that ofWharton and Tzagoloff (77). These four complexesinteract to form a reconstituted elementary particleunder the conditions specified by Hatcfi et al. (44, 45)and by Fowler and Richardson (30).ELECTRON TRANSFER PARTI CLE: The clec-tren transfer particle with phosphorylating properties(ETPn) was prepared from heavy beef heart mito-chondria by the method of Smith and Hansen (69).METHODS OF ELEC TR ONMIC R OSC OPYA variety of complementary methods was applied inthe course of the correlated studies of mitochondria.

The principal preparation procedures used can beclassified as follows:

1. Modified negative staining techniques re-quiring minimal preparative manipulations weremainly used. These techniques do not involve sec-tioning, but only extension or surface spreading ofwhole mitochondria and mitochondrial membranes.

2. Thin sectioning of mitochondria by standardand modified techniques for fixation and e mbedding.

3. Cryofixation and related low tem peratur epreparat ion techniques. Most of the results describedin this report were obtained with methods of group 1,which will be described in greater detail.1 . P r e p a r a t i o n o f M i t o c h o n d r i a b y M o d i f i ed

N e g a t i v e S t a i n i n g T e c h n i q u e s(a) Isolated mitochondria suspended in micro-

droplets of a sucrose solution containing 0.1 to 1per cent potassium phosphotungstatc (pH 7.2) aresandwiched between carbon-coated plastic films, orimpermea ble single-crystal graphite, or mica lamellaein vacuum-fight microchambers of special design(20-25). When used in combination with low-in-tensity microbeam illumination and controlledspecimen cooling (0 to --130C), this techniq uefor electron microscopy of wet or partly hydratedbiological systems yielded the first useful pictures ofthe regular mitochondrial membrane particles (23).Cytochrome c can he used instead of phosphotungstateto enhance contrast through "negative staining"by embedding flattened mitochondrial membrane

Index to E lec t ron MicrographsN O T E WO R T H Y I T E M *

Subunit structure in sections cristaeArrays of EP in i solated mitochondriaArrays of EP in ETP (Electron transfer particle)Structure of head piece EPStructure of stalk and base piece EPIsolated elementary particle (EP)Reconstituted elementary particleComplexes of electron transfer chain* See arrows.

FIGUR]~ NUMBER1, 2, 34, 5, 6, 7, 8, 9, 15, 271410, 11, 12, 135, 7, 8, 9, 10, 12, 1317, 18, 19, 20, ~821, 22, 2923, 24, 25, 26

FmvaE s 1 and 2 a Thin sections of isolated beef hcart mitochondria, "Nagarse" prepara-tions (0.66 sucrose); osmium fixation, low temperature embe dding in methacr ylate,stained wit h Pb(OH)2. Not e regularity of cristae and preservation of fine structure. Fig. 1,X 180,000; Fig. 2 a, X 180,000.FmUItE 2 b Enlargeme nt of Fig. ~ a demonstrat ing particulate subuni t structure in denseoute r layers of each crista. X 650,000.Fiorv-nE 3 Enlarged segment of loop of a crista in a specimen similar to th at shown in Fig.2 a. Clearly visible arra y of globular subuni ts abou t 70 to 80 A in diamet er. X 500,000.

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i n t h e u l t r a t h i n l i q u i d l a y e r s ( c a . 5 0 0 to 1 0 0 0 A)w h i c h a r e e n c l o s e d b e t w e e n t h e m i c r o c h a m b e r f i l m" w i n d o w s . " S i n c e t h is t e c h n i q u e e s s e n t i a l l y y i e ld sn e g a t i v e s t a i n in g o f w e t s p e c i m e n s w i t h o u t m o s t o ft h e u s u a l d r y i n g a r t i f a c t s , i t i s p r o v i n g t o b e i n -d i s p e n s a b l e f o r s t u d i e s o f l a b i l e m e m b r a n e s t r u c t u r e sa n d l i p o p r o t e i n sy s t em s u n d e r c o n d i t i o n s a p p r o a c h i n gt h e n a t i v e h y d r a t e d s t a t e .

( b ) S u r f a c e c e l l - f i l m m e t h o d w a s u s e d i n c o m b i n a -t i o n w i t h p o s i t i v e o r n e g a t i v e s t a i n i n g a n d r e l a t e dv a r i a n t s o f o u r o r i g i n a l t e c h n i q u e ( 1 6) f o r s p r e a d i n gm i t o c h o n d r i a ( e i t h e r a s i s o l a t e d o r i n s i t u ) o n al i q u i d s u r f a c e t o f o r m a n u l t r a t h i n l a y e r . T h i s c a nb e p i c k e d u p d i r e c t l y o n a s p e c i m e n g r i d a n d e x -a m i n e d d i r e c tl y o r a f t e r s u i t a b le s t a i n i n g a n d s h a d o w -c a s t i n g . T h e b e s t r e s u l t s w e r e o b t a i n e d b y u s i n g t h ev a l u a b l e m o d i f i c a t i o n in t r o d u c e d b y D . P a r s o n s( 5 5 , 5 6 ) w h i c h e m b o d i e s t h e a d v a n t a g e s o f n e g a t i v es t a i n i n g - e m b e d d i n g . T h i s s i m p l e m e t h o d w a s c a r r i e do u t b y i n s e r t i n g a c l e a n g l a s s n e e d l e o r a f r e s h l yc l e a v ed m i c a s u r fa c e i n t o t h e s p e c i m e n o f n a t i v et is s u e o r t h e m i t o c h o n d r i a l s u s p e n s io n w i t h o u t p r i o rf i x a t i o n . T h e n e e d l e o r m i c a s u r f a c e c o a t e d w i t h t h es p e c i m e n w a s t h e n d i p p e d s l o w l y i n t o a 1 t o 2 p e rc e n t s o d i u m - o r p o t a s s i u m p h o s p h o t n n g s t a t e s o l u t i o na t p H 7 .2 , p r e f e r a b l y c o o l e d t o 0 t o 4 , a n d u l t r a -f i l t e r e d ( u s in g 1 0 m /~ M i l l i p o r e f i l t e r s ). T h e m i t o -c h o n d r i a l m e m b r a n e s p e c i m e n s p r e a d s o u t i n t o a ne x q u i s i t e l y p r e s e r v e d u l t r a t h i n l a y e r f l o a t i n g o n t h el i q u i d s u r f a c e . T h i s c o h e r e n t s u r f a c e f i l m ( o n l y af e w h u n d r e d A t h i c k a n d e v e n l es s i n c e r t a i n a r e a s )i s p i c k e d u p d i r e c t l y o n a t h i n F o r m v a r s p e c i m e ns u p p o r t , c a r r y i n g w i t h i t a t e n u o u s p h o s p h o t u n g s t a t el a y er . U p o n d r y i n g ( p r e f e r a b ly i n a c o ld c h a m b e r a t0 4 ) , t h i s p h o s p h o t u n g s t a t e f il m f o rm s a n a m o r -p h o u s e m b e d d i n g m a t r i x o f h i g h e l e c t ro n o p a c it y ,a n d p r o v i d e s b o t h p r o t e c t i o n a g a i n s t s u r f a c e t e n s i o na r t i f a c t s a n d e x c e l l e n t n e g a t i v e c o n t r a s t . T h e s es p r e a d - c e l l p r e p a r a t i o n s a r e i d e a l l y s u i t e d f o r h i g hr e s o l u t i o n e l e c t r o n m i c r o s c o p y o f m i t o c h o n d r i a lm e m b r a n e s a n d r e l a t e d l a m e l l a r s y s t e m s , p r o v i d e dt h e s p e c i m e n s a r e e x a m i n e d i m m e d i a t e l y u n d e ra p p r o p r i a t e c o n d i t i o n s t o r e d u c e i r r a d i a t i o n d a m a g ea n d c o n t a m i n a t i o n . T h u s , r e m a r k a b l e i n t e g r i t y o ft h e f i n e s t r u c t u r e o f t h e m e m b r a n e s y s te m o f a ne n t i r e m i t o c h o n d r i o n i s o b t a i n e d w h i c h c o m p a r e sf a v o r a b l y w i t h t h a t o b s e r v e d i n t h e b e s t u l t r a t h i n

s e c t i o n s . T h i s s i m p l e t e c h n i q u e i s s u s c e p t i b l e o ff u r t h e r r e f i n e m e n t b y s p r e a d i n g t h e s p e c i m e n sa g a i n s t c o n s t a n t p r e ss u r e, s p r e a d i n g o n u n d e r c o o l e dl i q u i d s u r f a c e s , o r o n c l e a n m e r c u r y s u r f a c e s , e t c .A l so , b y s p r e a d i n g t h e n a t i v e m i t o c h o n d r i a o n s o lu -t i o n s o f b u f f e r e d f o r m a l i n , g l u t a r a l d e h y d e , o s m i u mt e t r o x i d e , o r p o t a s s i u m d i c h r o m a t e ( 0. 1 t o 2 p e rc e n t ) t h e e f f ec t s o f p r i o r f i x a t i o n c a n b e s t u d i e d i nd e t a i l .( c ) M i c r o d r o p l e t c r o s s - s p r a y i n g t e c h n i q u e s (23 , 24)w e r e u s e d r o u t i n e l y i n t h e s t u d y o f m i t o c h o n d r i a lm e m b r a n e s a n d i s o l a t e d p a r t i c l e s r e q u i r i n g m i n i m a le x p o s u r e t o r e a g e n t s , a n d a h i g h d e g r e e o f r e p r o -d u c i b i l i t y . W i t h t h e u s e o f a s p e c i a l m u l t i p l e - s p r a y i n gd e v i c e w i t h s u i t a b l y a r r a n g e d s e p a r a t e c a p i l l a r i e s f o rs p e c i m e n s a n d r e a g e n t s , i t i s p o s s i b l e t o o b t a i n c o n -t r o l l e d , b r i e f i n t e r a c t i o n o f m i c r o d r o p l e t s o f t h e s p e c i -m e n w i t h m i c r o d r o p l e t s o f 1 t o 2 p e r c e n t p o t a s s i u mp h o s p h o t u n g s t a t e a t p H 7 . 2 , u r a n y l a c e t a t e , o r o t h e rh e a v y m e t a l s o l u t i o n s . T h e c r o s s - s p r a y e d m i c r o -d r o p l e t s c o l l i d e a n d i n t e r a c t v e r y r a p i d l y s h o r t l y b e -f o r e i m p i n g i n g o n t h e s p e c i m e n g r i d . T h e t w o f o l da d v a n t a g e i s h e r e b y g a i n e d o f im p o s i n g p r e -d e t e r m i n e d s p a t i a l a n d t e m p o r a l c o n s t r a i n t s o n t h es p e c i m e n - r e a g e n t s y s te m d u r i n g a h i g h l y r e p r o -d u c i b l e p r e p a r a t i v e p r o c e s s . T h i s m e t h o d h a s t o b ec o n t r a s t e d w i t h t h e s t a n d a r d n e g a t i v e s t a i n i n g p r o -c e d u r e b y w h i c h t h e P T A r e a g e n t i s m i x e d w i t h t h es p e c i m e n , a n d c a n p r o d u c e v a r i a b l e d e g r e e s o fm o d i f i c a t i o n b e f o r e b e i n g s p r a y e d a n d d r i e d o n t h es u p p o r t . M o r e o v e r , a s a r e s u l t o f t h e s m a l l s iz e a n dr e l a t i v e l y h i g h s p e e d o f t h e c r o s s - s p r a y e d m i c r o -d r o p l e t s , t h e r e i s a fa v o r a b l e c o n c u r r e n c e o f r a p i ds p e c i m e n c o o l i n g a n d l i m i t e d d r y i n g t o a c h i e v e au n i q u e d e g r e e o f p r e s e rv a t i o n o f t h e m o s t l a b i l es t r u c t u r e s ( 2 4 ) . F i n a l l y , t h i s t e c h n i q u e i s e a s i l yc a r r i e d o u t a n d p e r m i t s r a p i d p r o c e s s i n g o f a l a r g en u m b e r o f s pe c i m e ns . I n t h e p r e s e n t s tu d i es i t w a su s e d f o r e x a m i n a t i o n o f a l l c r i t i c a l s p e c i m e n s , a n dt o i n v e s t i g a t e t h e e f f e c ts o f fi x a t i o n a g e n t s a n d h e a v ym e t a l s o l u t i o n s o n t h e f i n e s t r u c t u r e o f n a t i v e m i t o -c h o n d r i a l c o m p o n e n t s .

I n t e r e s t i n g r e s u lt s w e r e o b t a i n e d i n t h e s t u d y o fi s o l a t e d E P ( F i g . 2 0 ) b y c o m b i n i n g p o s i t i v e o r n e g a -t i v e s t a i n i n g o f c r o s s - s p r a y e d m i c r o d r o p l e t s w i t hs u b s e q u e n t s h a d o w - c a s t i n g a t a n e x t r e m e l y l o ws h a d o w i n g a n g l e ( u s i ng p l a ti n u m , o r p l a t i n u m - c a r b o n

] h O ~ t E 4 N e g a t i v e l y s t a i n e d be e f h e a r t m i t o c h o n d r i o n ; i s ol a t e d in 0 . 5 M s u c ro s e , a n dp r e p a r e d b y s u r f a c e s p r e a d i n g o n 1 p e r c e n t p o t a s s i u m p h o s p h o t u n g s t a t e a t p H 7 . 2 w i t h o u tp r i o r fi x a ti o n . P a r t l y i n t a c t w h o l e m o u n t o f f l a t t e n e d m i t o c h o n d r i a l m e m b r a n e s s h o w i n gr e g u l a r a r r a n g e m e n t o f r e p e a t i n g p a r t i c u l a t e c o m p o n e n t s . ) < 6 ~ , 0 0 0 .I ~ G~m E 5 P r o f i l e v i e w o f c r i s t a e w i th a r r a y s o f e l e m e n ta r y p a r t i c l e s ( E P ) i n e n l a r g e ds e g m e n t o f F i g . 4 . P o l y h e d r a l h e a d p i e c es o f t h e e l e m e n t a r y p a r t ic l e s a r e a t t a c h e d b y s t a l k sto t h e c o n t in u o u s d e n se o u t e r l a y e r s o f t h e c r i s t a e . X 4 ~ 0, 00 0 .

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evaporation in a vacuum system with liquid nitrogencold-trap). The resulting reproducible "surfacedecoration" patterns may provide information onthe underlying subunit structure of isolated EP.

For attainment of highest resolution the carbon-coated, fenestrated specimen films (29) were usedwithout the normal ult rathin film supports (cast from0.05 per cent Formvar solutions in ultrafilteredethylene chloride). Instead, extremely thin em-bedding supports bridging th e holes of the fenestrate dfihn (ca. 100 to 1000 A in diamete r) were ob tainedby the addit ion of very dilute (0.01 to 0.05 per cent)sodium silicate, gelatin, or gum arabic solutions tothe specimen, preferably applied through micro-droplet cross-spraying (24).2 . T h i n S e c t io n i n g T e c h n i q u e s

In addition to the standard osmium fixation orglutaraldehyde fixation procedure followed bymethacrylate or Araldite embedding and thin sec-tioning, the following two variants proved useful inthe study of mitochondrial fine structure:a. Negative staining- -embedding of ultrathinsections. Fresh tissues or mitochondrial suspensions(either unfixed and dried, or partially fixed byformalin vapor, or by ultraviolet irradiation) weresectioned without embedding by a diamond knifemounted on a Moran-Leitz ultramicrotome. Whenthese coherent ultrathin sections were collecteddirectly on a 1 to 2 per cent buffered phosphotung-state solution (pH 7) and picked up on filmed speci-men grids, the fine structure of mitochondria andother l amellar systems showed up in striking negativecontrast images. This favorable result is simply dueto permeation of the "unsupported" ultrathin sectionby the " phosphotung state glass" which acts as an"embedding" medium of high density and permitsenhanced contrast and resolution. Despite its in-herent limitations (mainly caused by drying artifacts),this interesting and simple approach has proved to beof value as a control procedure for the evaluation ofnegative staining in sectioned material. The resultswill be reported in a separate publication.

b. Ultrathin frozen sections of fresh tissues (24)prepared by cutting the frozen tissue with a diamondknife in a c ryostat (at -- 30 to -- 180C) can also beexamined directly without thawing by embeddingthe sections in vitrified heavy metal layers. A liquidnitrogen cooling device and appropri ate low tempera -ture methods of electron microscopy are required toimplement this technique. However, numeroustechnical problems still remain to be solved beforethis method can be used routinely.

For thin sectioning of suspensions and pellets ofisolated mitochondria and submitochondrial frac-tions (ETP), the technique described earlier (29 a)for examination of oriented Tobacco Mosaic Virusgels proved very useful. The pellets or suspensionswere sucked into thin plastic capillaries (0.1 to 0.2mm internal diameter). The specimen capillarieswere cut into short segments which could then bestained, dehydrated, and embedded. Extraction anddehydrat ion or embeddi ng artifacts were considerablyreduced, particularly when using low temperaturepreparation techniques.

c. Cryofixation and related low temper aturetechniques (19-23), including low-temperature de-hydration and embedding in methacrylate afterstandard osmium fixation, were used primarily forcontrol purposes in these studies.

ELECTRON MICI~OSCOPY : A Siemens E hni -skop I was used, opera ting mainl y at 80 kv (alsoat 40, 60, and 100 kv for selected specimens ).Improved pointed filaments (20) of single-crystaltungs ten with a tip r adius of 1 to 10 # wereused routinely with the double condenser systemof the Elmiskop to provide intense microbeamillumination of high coherence and low angular di-vergence. With this arrangement, the image bright-ness (with a 2 to 10 /~ spot size, beam current2 to 8 /za at 80 kv) was adequate for directobservation of image detail at the highest electronoptical magnifications. Using clean multiple objectiveapertures of copper or platinum (50 #), the astig-matism of the objective lens could be readily correct edunder direct observation and reduced to less than 0.2micron; for high resolution studies, the measured

I~GURE 6 Isolate d beef heart mitochondrion embedded in thin phosphotung state (PTA)layer. Prepar ed by m icrodroplet cross-spraying procedure involving only brief interact ionof mitochondrial suspension in 0.5 m sucrose with 1 per cent PTA. Notice characteristicspaired arrays of elementary particles in profiles of fragmented cristae, which are readilydistinguishable from the envelope of the mitochondrion. X 120,000.FtGURE 7 Enlarged segment of crista in a specimen similar to tha t shown in Fig. 6. Dem-onstrati on of three parts of the eleme ntary particle: head piece, stalk, base piece. Inva rian tassociation of the arra ys of head pieces EP wit h t he underlyi ng dense layer of crista. Seg-mentation and knob-like proturherance of dense layer at point of attachment of stalk.X 600,000.

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a s t i g m a t i s m o f t h e o b j e c t i v e l e n s w a s 0 . 1 # o r l e ss .T h e h i g h e r e f fi c ie n c y o f t h e p o i n t e d f i l a m e n t e n a b l e du s t o u s e s p e c i a l Il f o r d H i g h R e s o l u t i o n P l a t e s a s ar o u t i n e ( 8 t o 1 5 s e c o n d s e x p o s u r e a t 8 0 , 0 0 0 t o 1 0 0 ,0 0 0e l e c t r o n o p t i c a l m a g n i f i c a t i o n s ) f o r r e c o r d i n g s a t i s -f a c t o r y e l e c t r o n m i c r o g r a p h s w h i c h c a n b e s u b s e -q u e n t l y e n l a r g e d t o y i e l d h i g h e r u s e f u l m a g n i f i c a -t i o n s t h a n o r d i n a r y p l a t e s ( 2 2, 2 4 ) . C o n v e r s e l y , i tw a s a l s o p o s s i b le t o o b t a i n m i c r o b e a m s ( 0 .5 t o 2 /~i n d i a m e t e r ) o f e x t re m e l y l o w i n t e n s i t y b y u s i n g t h en e w p o i n t e d f i la m e n t s u n d e r a p p r o p r i a t e c o n d i t i o ns ,a n d b y p r o v i d i n g t h e d o u b l e c o n d e n s e r s y s te m ( C I I )w i t h a p e r t u r e s o f 5 0 t o 1 0 0 / ~ . T h i s a r r a n g e m e n tp r o v e d t o b e e s s e n t i a l i n e x a m i n i n g c e r t a i n h i g h l yl a b i l e m e m b r a n e c o n s t i t u e n t s . I n f a c t , u s e f u l m i c r o -g r a p h s f r o m t h e s e sp e c i m e n s c o u l d b e r e c o r d e d o n l yo n h i g h - s p e e d e m u l s io n s (e.g. 3 5 m m T r i X K o d a k ,s e n s i t i z e d w i t h g o l d t h i o c y a n a t e s o l u t i o n s ) b y f i r s tf o c u si n g o n a d j a c e n t s p e c i m e n a r e as , a n d t h e n r a p i d l ys h i f t i n g t h e l o w - i n t e n s i t y m i c r o b e a m t o t h e p r e s e -l e e r e d s i t e . I r r a d i a t i o n d a m a g e a n d s p e c i m e n c o n -t a m i n a t i o n c o u l d b e c o n s i d e r a b l y r e d u c e d b y u s i n gi m p r o v e d s p e c i m e n c o o l i n g d e v i c e s ( L e i s e g a n g l i q u i dn i t r o g e n s t a g e w i t h s p e c i a l s h i e l d i n g a p e r t u r e s ) i nc o m b i n a t i o n w i t h l o w - i n t e n s i t y e l e c t r o n o p t i c s( 1 9 - 2 4 ) . F o r a l l c ri t i c a l m e a s u r e m e n t s , c a l i b r a t i o no f t h e m i c r os c o p e w a s c a r r i e d o u t a t t h e t i m e o fr e c o r d i n g t h e e l e c t r o n m l c r o g r a p h s , u s i n g a d i f f r a c -t i o n g r a t i n g r e p l i c a c a r e f u l l y a d j u s t e d f o r t h e s a m es p e c i m e n p o s i t i o n . F e r r i t i n m o l e c u l e s o f u n i f o r m s i z e( 1 1 8 t o 1 2 0 A d i a m e t e r ) , p r e p a r e d b y d e n s i t y g r a -d i e n t u l t r a c e n t r i f u g a t io n a n d u s e d b y J . W . A n d e r e g ga n d F . F i s c h b a c k f o r x - r a y s c a t t e r i n g s t u d ie s , w e r ef r e q u e n t l y u s e d a s a n i n t e r n a l c a l i b r a t i o n r e f e r e n c ef o r c e r t a i n s p e c i m e n s . H o w e v e r , i n a l l o f t h e s e e x -p e r i m e n t s p a r t i c u l a r c a r e w a s t a k e n t o g u a r d a g a i n s tp o s s i b l e c o n t a m i n a t i o n w i t h a p o f e r r i t i n o r m o d i f i e df e r r i t i n p a r t i c l e s b y r u n n i n g p a r a l l e l c o n t r o l s w i t h o u ta d d i t i o n o f f e r r i t i n o r o t h e r c a l i b r a t i o n m a r k e r s .T h e o b s e r v a t i o n s d e s c r i b e d h e r e a r e b a s e d o n t h ee v a l u a t i o n o f m o r e t h a n 3 , 0 0 0 p l a te s a n d f il m s , i nw h i c h a n a v e r a g e r e s o l u t i o n o f 1 0 t o 2 0 A w a s c o n -s i s t e n t l y a c h i e v e d .

B I O C H E M I C A L M E T H O D ST h e c o m p a n i o n p a p e r b y B l a ir et al. (5 ) g ives a fu l ld e s c r i p t i o n o f t h e m e t h o d s o f a s s a y o f t h e e n z y m i ca c t i v i t y o f m i t o c h o n d r i a a n d s u b m i t o e h o n d r i a lp a r ti c l es a n d o f th e a n a l y t i c a l m e t h o d s u s e d i nq u a n t i f y i n g t h e c o m p o n e n t s i n t h e s e p a r t i c l e s .R E S U L T SE l e c tr o n M i c r o s c o p y o f I s o la t e d M i t o c h o n d r i a

I s o l a t e d b ee f h e a r t m i t o c h o n d r i a p r e p a r e d a sd e s c r i b e d i n t h e s e c t io n o n M e t h o d s s h o w e d g o o dp r e s e r v a t i o n o f f o r m a n d f in e s t ru c t u r e . R o u t i n ec h e c k s w e r e m a d e f o r c o n t a m i n a t i o n o f m i t o -c h o n d r i a l p r e p a r a t i o n s w i t h m y o f i l a m e n t s , r i b o -s o m e s, a n d o t h e r e x t r a n e o u s p a r t i c u l a t e m a t e r i a lb y s y s t e m a t i c e x a m i n a t i o n o f p e ll e t s o f i s o l a t e dm i t o c h o n d r i a . N o n e o f t h e s e c o n t a m i n a n t s w e r ef o u n d i n o u r s t a n d a r d p r e p a r a t i o n s .

S i n c e m i t o c h o n d r i a a r e e s s e n t i a l l y f l u i d - f i l l e dv e ss e ls w i t h a n i n v o l u t e d i n t e r n a l m e m b r a n e s ys -t e m o f a f e w m o l e c u l e s i n t h i c k n e s s , t h e y a r e , i np r i n c i p le , w e l l s u i t e d f o r d i re c t e x a m i n a t i o n w i t h -o u t s e c t i o n i n g . O n c e t h e i n t e r n a l f l u i d h a s b e e nr e m o v e d , t h e c o l l a p s e d a n d d i s t e n d e d m i t o c h o n -d r i a l m e m b r a n e s c a n b e p e r m e a t e d b y b u f fe r e ds o l u ti o n s o f p h o s p h o t u n g s t a t e a n d s u p p o r t e d b yn e g a ti v e s ta i n in g e m b e d d i n g . T h e e m b e d d e dl a y e r s a re t h i n e n o u g h f o r h i g h - r e s o l u t i o n e l e c t r o nm i c r o s c o p y .

D i s r u p t i o n , d i s s o c i a t i o n , a n d s p u r i o u s r e a r -r a n g e m e n t s o f t h e d e l ic a t e m e m b r a n e s t r u c t u r e sm u s t b e t a k e n i n t o a c c o u n t a s p o s s ib l e ca u s e s o fa r t if a c ts . V a r i o u s m e t h o d s o f n e g a ti v e s t a i n i n gw e r e u s e d t o c o n t r o l t h e p o s s i b i l it y o f a r t i f a c tf o r m a t i o n . W e h a v e h a d n o i n d i c a t i o n t h a t t h es t r u c t u re s w h i c h a r e t h e p r i n c i p a l s u b j e c t o f t h ep r e s e n t c o m m u n i c a t i o n a r e p r o d u c t s o f a p a r t i c u -l a r t y p e o f p r e p a r a t i v e p r o c e d u r e . O n t h e c o n -

F m v ~ v . 8 E n l a r g e d p o r t i o n o f c r i s t a f r o m s p e c im e n s h o w n i n F ig . 4 . N o t i c e c h a r a c t e r i s t i ck n o b - l i k e s tr u c t u r e o f t h e b a s e p i e c e a t t h e i n s e r t i o n p o i n t o f e a c h E P s t a l k . T h e b a s e p i e ces u b u n i t s a n d u n d e r l y i n g r e g u l ar s e g m e n t e d a r r a n g e m e n t o f d e n s e l a y e r d is c e r n ib l e i n b o t hprof i le and sur face v iew of cr is ta . )< 600 ,000 .F IG U R E 9 S e g m e n t o f n e g a t i v e l y s t a i n e d c r l s ta i n a P T A s p r e a d -c e l l p r e p a r a t i o n o f b e e fh e a r t m i t o e h o n d r i o n . T h e r e g u l a r k n o w s t r u c t u r e s d e m a r c a t i n g t h e b a s e p i e c e o f e a c he l e m e n t a r y p a r t i c l e f o r m a n i n t e g r a l p a r t o f t h e d e n s e l a y e r o f t h e c r i s t a . P e n e t r a t i o n o fP T A b e t w e e n t w o a p p o s e d d e n s e l a y e r s o f t h e c r i s t a m a y a c c o u n t f o r t h e c e n t r a l d e n s eb a n d o f t h e "m e so z o n e " r e g io n . X 6 5 0 ,0 0 0.

7 4 T H E J O U R N AL O F C E L L B I O L O G Y V O L U M E g ~ , 1 9 6 4



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t r a r y , a w i d e r a n g e o f p r e p a r a t i v e p r o c e d u r e s h a sb e e n u s e d s u c c e s sf u l ly f o r t h e v i s u a l i z a t i o n o f th e s es t r u c t u r e s .

I n v i e w o f t h e l a r g e n u m b e r o f e l e c t r o n m i c r o -g r a p h s p r e s e n t e d i n t h is c o m m u n i c a t i o n , w e h a v ep r e p a r e d a n I n d e x s u m m a r i z i n g a l l th e e s se n t ia ls t r u c t u r a l i t e m s t o b e d i s c u s s e d i n t h e s e c t i o n o nR e s u l t s a n d t h e c o r r e s p o n d i n g f i g u r e s t o b e e x -a m i n e d i n t h e s e c o n te x t s.

F I N E S T R U C T U R E O F I S O L A T E D M 1 T O -C H O N D R I A I N S E C T I O N S : P r e p a r a t i o n s f i x e dw i t h o s m i u m t e t r o x id e a n d d e h y d r a t e d a n d e m -b e d d e d a t l o w t e m p e r a t u r e s ( F i g s. 1 t o 3) r e v e a la d e g r e e o f p r e s e r v a t i o n o f f i n e s t r u c t u r e o f t h em e m b r a n e s w h i c h c o m p a r e s f a v o r a b ly w i t h t h a tf o u n d i n t h e b e s t p r e p a r a t io n s o f r e t i n a l m i t o -c h o n d r i a ( 2 2 - 2 5 ). I n b o t h t y p e s of p r e p a r a t io n s ,t h e e l e c t r o n o p a q u e l a y e r s o f t h e c r i s t a e s h o wc h a r a c t e r is t i c g r a n u l a r o r g l o b u l a r s u b u n i ts a b o u t7 0 t o 80 A i n d i a m e t e r . T h i s p a r t i c u l a t e s u b u n i ts t r u c tu r e o f t h e m i t o c h o n d r i a l m e m b r a n e s i s a l sor e g u l a r l y e n c o u n t e r e d i n s e c t i o n e d p r e p a r a t i o n sf i x ed w i t h g l u t a r a l d e h y d e o r f o r m a l i n a n d s t a in e dw i t h u r a n y l o r l e a d a c e ta t e . A p p o s i n g m e m b r a n e so f a d j a c e n t m i t o c h o n d r i a ( i n s e c ti o n s o f p e l l e ts ) e x -h i b i t e d a r e g u l a r p a t t e r n w i t h a p e r i o d o f 1 0 0 t o1 60 A . S u c h p a t t e r n s w e r e f i r s t d e s c r i b e d b y P e a s e( 5 7) . T h e t y p i c a l a n g u l a r c o n f i g u r a ti o n s o f t h ec r i s t a e , p r e v i o u s l y n o t e d b y R e v e l e t a l . (5 9 ) , a rep a r t i c u l a r l y n o t i c e a b l e i n p r e p a r a t i o n s t r e a t e d a tl o w t e m p e r a t u r e s . T h e r e i s a f o r m a l r e s e m b l a n c eb e t w e e n t h e c r i s t a e i n o s m i u m - f i x e d a n d n e g a -t i v e l y s t a i n e d p r e p a r a t i o n s i n r e s p e c t to t h e d e n s el a y e r s . I n b o t h c a s e s , t h e s t r u c t u r e d e l e m e n t s a r el o c a l i z e d i n t h e s e t w o l a y e r s a n d i t i s o n l y i n t h ee l e c t r o n o p a q u e l a y e rs t h a t a r e g u l a r i t y o f st r u c-t u r a l p a t t e r n i s r e c o g n i z a b l e . T h e p o s s i b i l i t y m u s tb e b o r n e i n m i n d t h a t t h e m a t e r i a l p r e s e n t i n t h ep a r t i c l e s a s s o c i a t e d w i t h t h e c r i s t a e i n t h e n e g a -t i v e l y s t a in e d p r e p a r a t i o n s m a y b e c o m e i n -c o r p o r a t e d i n t o t h e t h i c k e r e le c t r o n o p a q u e l a y er so f th e o s m i u m - f i x e d p r e p a r a t i o n s .

T H E E L E M E N T A R Y P A R T I C L E S : I n w h o l em e m b r a n e m o u n t s o f n e g a t i v e l y s t a in e d m i t o -

c h o n d r i a , a l l o f t h e v i s i b le m e m b r a n e s u r f a c e s a p -p e a r t o b e c o v e r e d w i t h u n i f o r m p a r t i c l e s h a v i n gd i a m e t e r s t h e o r d e r o f 8 0 t o 1 0 0 A ( c f . Figs. 4 to7 ). T h e v a s t n u m b e r s o f t h e s e p a r t i c l e s - - a s w e l l a st h e i r r e g u l a r i t y a n d p e r i o d i c i t y - - a r e t h e m o s t r em a r k a b l e f e a t u r e s . W h e n f i r s t o b s e r v e d ( 2 3, 2 5 ) ,t h i s r e p e a t i n g s t r u c t u r a l e n t i t y w a s d e s i g n a t e d t h e" e l e m e n t a r y p a r t i c l e " ( E P ) . I n t y p i c al n e g a t iv e l ys t a i n e d p r e p a r a t i o n s ( F i g s. 4 t o 1 0 ), t h e e l e m e n t a r yp a r t ic l e s a r e a r r a n g e d i n r e c u r r i n g a r r a y s a s so -c i a t e d w i t h t h e c r i s t a e a n d w i t h t h e i n n e r m e m -b r a n e o f t h e e x t e r n a l m i t o c h o n d r i a l e n v e l o p e .E x a m i n a t i o n o f w h o l e - m o u n t s p e c i m e n s r e v e a l sa p p r o x i m a t e l y 2 , 0 0 0 t o 4 , 0 0 0 p a r t i c l e s p e r s q u a r em i c r o n . I f a f a i r l y u n i f o r m d i s t r i b u t i o n i s a s s u m e d ,t h e t o t a l n u m b e r w o u l d b e o f t h e o r d e r o f 1 0 ,0 0 0t o 1 0 0 , 0 0 0 e l e m e n t a r y p a r t i c l e s p e r m i t o c h o n d r i o nd e p e n d i n g o n t h e s i z e a n d t y p e o f m i t o c h o n d r i o n .T h e a v e r a g e d i s t a n c e s e p a r a t i n g t w o a d j a c e n tp a r t i c l e s w a s f o u n d t o b e a b o u t 1 10 t o 1 1 5 A . I ft h e p a r t i c l e s a r e a s s u m e d t o b e s p h e r e s , 1 0 0 t o1 20 A i n d i a m e t e r , t h e y w o u l d a c c o u n t f o r 1 0 t o1 5 p e r c e n t o f t h e t o t a l m i t o c h o n d r i a l v o l u m e .S i m i l a r e s ti m a t e s h a v e b e e n m a d e b y S m i t h ( 7 0,7 1 ) f o r t h e v o l u m e o c c u p i e d b y t h e s e p a r t i c l e si n i n s e c t s a r c o s o m e s .

A s s e e n b e s t i n p a i r e d a r r a y s a l o n g a c r i s t a ,e a c h e l e m e n t a r y p a r t i c l e c o n s is t s o f t h e f o l l o w i n gt h r e e r e c o g n i z a b l e c o m p o n e n t s : a h e a d p i e ce , as t e rn o r s t a lk , a n d a b a s e p i e c e w h i c h i s t h e r e g i o no f a t t a c h m e n t o f t h e s t a l k t o t h e c ri s t a. T h e h e a dp i e c e is t h e m o s t c o n s p i c u o u s p o r t i o n , b e i n g p o l y -h e d r a l a n d a s y m m e t r i c b y v i r t u e o f t h e s t e m r e g i o n( F i g s . 5 t o 1 2 , 1 3 a to c) . T h e m e a s u r e d d i a m e t e ri n m i c r o g r a p h s o f f r es h ly p r e p a r e d s p e c i m e n s isa b o u t 8 0 t o 1 0 0 A ( c f . Fi g s . 4 t o 1 2 ) . Th e ra t i o b e -t w e e n t h e l o n g a n d s h o r t a x es o f t h e a s y m m e t r i ch e a d p i e c e c o m e s t o v a l u e s o f 1 .2 t o 1 . 4. W h e nm i t o c h o n d r i a a r e f i xe d w i t h a l d e h yd e s ( f o r m a l d e -h y d e , g l u t a r a l d e h y d e ) , a n d t h e r e s ul t in g p r e p a r a -t i o n tr e a t e d w i t h i o d i n e o r d i c h r o m a t e , t h e h e a do f t e n a p p e a r s s u r r o u n d e d b y a " h a l o " o f g r a n u l a rc o m p o n e n t s ( 2 0 t o 3 0 A d i a m e t e r ) d e l i m i t i n gt h e p e r i p h e r y .

FIGVRE 1 0 E l e c t ro n mi c ro g ra p h o f n e g a t i v e l y s ta i n e d b e e f h e a r t mi t o c h o n d r i a l me m-b ra n e s re c o rd e d wi t h l o w - i n t e n si t y mi c ro b e a m i l l u mi n a ti o n , a n d sp e c i me n c o o li n g ( - 8 0 )t o re d u c e i r ra d i a t i o n d a ma g e . Un d e r t h e se fa v o ra b l e c o n d i t io n s , t h e d e n se su b s t ru c t u re o ft h e E P h e a d p i e c e s ca n b e m o re f re q u e n t l y o b se rv e d i n su i t a b ly o r i e n te d sp e ci men s . No t i c el a rg e r si ze o f d e t a c h e d EP . Sp e c i me n c o o le d a n d e x a mi n e d i m me d i a t e l y a f t e r p re p a ra t i o nby cross-spray ing technique. M 860 ,000 .

76 THE JOm~AL OF CELL BIOLOGY VOLUM~ ~ , 1964



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T h e h e a d p i e c e e x h i b i t s a c h a r a c t e r i s t i c , e l e c -t r o n o p a q u e c o r e w h i c h c a n b e d e m o n s t r a t e d c o n -s i s t e n t l y , p r o v i d i n g s p e c i a l p r e c a u t i o n s a r e t a k e n(F i g s . 1 0 t o 1 2 ,. 1 3 a t o d ) . Th e v i su a l i z a t i o n o f t h i se l e c t r o n - o p a q u e r e g i o n o f t h e h e a d i s i n f l u en c e db y e x p o s u r e o f t h e s p e c i m e n t o t h e i n t en s e e l e c t r o nb e a m . T h e a p p a r e n t f a d i n g a w a y o f t h e d e n sec e n t r a l r e g i o n w h i l e t h e h e a d p i e c e is b e in g o b -s e r v e d w i t h e l e c t r o n o p t i c s a t h i g h m a g n i f i c a t i o nh a s b e e n r e p e a t e d l y n o t e d . T h e h e a d c o r e is t h e r e -f o r e b es t r e c o r d e d w h e n l o w - in t e n s it y , m i c r o b e a mi l l u m i n a t i o n i s u s e d a t r e l a t i v e l y lo w e l e c t r o no p t i c a l m a g n i f i c a t i o n s ( 1 0 ,0 0 0 t o 2 0, 0 0 0 ). F o ro b s e r v a t i o n s a t h i g h e r e l e c t r o n o p t i c a l m a g n i f i -c a t i o n s ( X 4 0 , 0 0 0 t o 8 0 , 0 0 0 ) , t h e s ta g e c o n t a i n i n gt h e s p e c i m e n i s c o o l e d w i t h l i q u i d n i t r o g e n ( 1 9 ,2 3 - 2 6 ) t o a v o i d d e t e r i o r a t i o n a n d f a d i n g o f t h ei m a g e . A s d e m o n s t r a t e d i n t h r o u g h - f o c u s s er ie s(F i g s . 1 1 a, b a n d 1 2 a , b ) , t h e e l e c t ro n o p a q u ec o r e o f t h e h e a d p i e c e is o f v a r i a b l e w i d t h ( ca .2 0 t o 6 0 A ) . T h e c o r e s h o w s u p b e s t a g a i n s t a l o wc o n t r a s t b a c k g r o u n d s u c h a s i s a c h i e v e d b y e m -b e d d i n g t h e s p e c i m e n i n d i l u t e ( 0. 1 t o 0 .3 p e r c e n t )p h o s p h o t u n g s t a t e s o lu t io n s . I n m a n y w a y s t h ee l e c t r o n o p a q u e c o r e o f t h e h e a d p i e c e re s e m b l e st h e c o r r e s p o n d i n g e l e c t r o n o p a q u e c o r e s e en i ne l e c t r o n m i c r o g r a p h s o f c y t o c h r o m e o x i d a s e ( a n -n u l a r f o r m s o f t h e c o m p l e x ) . T h e e l e c t r o n o p a q u ec o r e h a s b e e n c o n s i s t e n t ly o b s e r v e d a l s o i n p r e p -a r a t i o n s t r e a t e d w i t h u r a n y l a c e t a t e o r d i c h r o m a t ea n d i n s p e c i m e n s f i x ed w i t h f o r m a l i n o r g l u t a r a l d e -h y d e (F i g . 1 3 d ) .

T h e s t e m o r s t a l k r e g i o n a p p e a r s t o h a v e v a r i -a b l e s i ze a n d c o n f i g u r a t i o n d e p e n d i n g o n t h et y p e o f n e g a t i v e s t a in i n g . W h e n t h e s p e c i m e n i sp r e p a r e d b y m i c r o d r o p l e t c r o ss - s p r a yi n g w i t h av e r y b r i e f p e r i o d o f i n t e r a c ti o n w i t h p h o s p h o -t u n g s t a t e , t h e s t a l k s e e m s to b e v e r y s h o r t ( F i g .1 0 ) a n d t h e h e a d p i e c e s e e m s t o b e w e d g e d i n d i -

r e c t l y w i t h i n t h e m e s o r e g i o n o f th e c r i s t a . I n t h e s ec a s e s , t h e e l e m e n t a r y p a r t i c l e s a s s u m e a t e a r -d r o p f o r m a t i o n (cf . F i g . 7 ) . I n s p r e a d c e l l p r e p -a r a t i o n s , t h e s t a l k i s s e e n t o b e w e l l d e v e l o p e d( F i g s. 5 , 8 ) a n d i s a p p r o x i m a t e l y 2 0 t o 4 0 A w i d ea n d 4 0 t o 5 0 A l o n g . A t t h e p o i n t o f a t t a c h m e n to f t h e s t a l k t o t h e c r i s t a a c h a r a c t e r i s t i c k n o b - l i k ef o r m a t i o n , a p p r o x i m a t e l y 4 0 to 5 0 A in d i a m e t e r ,i s r e g u l a r l y s e e n .

A n i n v a r i a n t f e a t u r e o f a ll t h e e l e c t r o n m i c r o -g r a p h s e x a m i n e d i s t h e c o r r e l a t i o n b e t w e e n t h ea r r a y s o f h e a d p i e c es o f t h e e l e m e n t a r y p a r t i c l ea n d t h e e l e c t r o n o p a q u e l a y e r o f t h e c r i s ta t ow h i c h t h e h e a d p i e c e s a r e a t t a c h e d b y s t a lk s ( cf .F i g s . 7 t o 9 , a n d 1 5) . W e c o n s i d e r t h e d e n s e l a y e rt o b e a n e x p r e ss i o n o f t h e e n d - t o - e n d a l i g n m e n to f t h e b a s e p i e c e s o f t h e e l e m e n t a r y p a r t i c le s . T h es e g m e n t a t i o n o f th i s d e n se l a y e r i n t h e f o r m o f re -c u r r i n g k n o b s p a r a l l e l s t h e p e r i o d i c i t y o f t h e h e a dp i e c e s a n d t h e s t a lk s . S i n c e t h e a v e r a g e d i s t a n c eb e t w e e n t h e h e a d p i e c e s is a b o u t 1 15 A , t h e s a m ed i s t a n c e m u s t b e a s s i g n e d t o t h e w i d t h o f a b a s ep i e c e i f t h e d e n s e l a y e r i s a s s u m e d t o b e m a d e u po f f u s e d b a s e p i e c e s . T h e t h i c k n e s s o f t h e d e n s el a y e r i s 4 0 t o 5 0 A . T h e t h i r d d i m e n s i o n o f th eb a s e p i e c e w o u l d h a v e t o b e 1 15 A t o b e c o n -s i s te n t w i t h t h e a s s u m p t i o n o f a c o n t i n u o u s l a y e r .

E L E M E N T A R Y P A R T I C L E S I N S U B M I T O -C H O N D R I A L F R A G M E N T S : T h e s a m e t y p e o fr e p e a t i n g p a r t i c u l a t e s t r u c t u r e i s r e g u l a r l y o b -s e r v e d i n s u b m i t o c h o n d r i a l f r a g m e n t s ( E T P ) o b -t a i n e d b y s o n i c a t i o n ( @ Fi g s . 1 4 a n d 1 5 ). Th ep a r t i c l e s a r e a n i n t r i n s i c f e a t u r e o f t h e m i t o c h o n -d r i al m e m b r a n e s e v e n w h e n t h e m i t o c h o n d r io nh a s b e e n d i s r u p t e d . I n f a c t , t h e p r e s e n c e o f e l e -m e n t a r y p a r t i c l e s b o t h i n i n t a c t m i t o c h o n d r i a lm e m b r a n e s a n d i n m i t o c h o n d r i a l f r a c t io n s w a st h e s t a r t i n g p o i n t f o r t h e b i o c h e m i c a l i s o l a t i o n .T h e t h r e e p a r t s o f t h e e l e m e n t a r y p a r t i c le ( h e a d

Fra mE S l l a a n d b, a n d 1 2 a a n d b E l e c t ro n mi c ro g ra p h s se l e ct e d f ro m t h ro u g h - fo c u sse ri es o f n e g a t i v e l y s t a i n e d c r i s t ae p re p a re d a n d e x a mi n e d u n d e r sa me e x p e r i me n t a l c o n -d i t ions as described in legend for Fig . 11 . Even under such favorab le condi t ions, the e lec-t ro n o p a q u e c o re w i t h in d i c a t io n s o f su b s t ru c t u re c a n b e re p ro d u c i b l y d e m o n s t ra t e d o n l yin certain EP head pieces (arrows). Figs. 11 a, b, X 750,000; Figs. 12 a, b, X 450,000.FIGURES 13 a to d Inse t en largem ents showing e lec t ron opaque core in head p ieces ofse lec ted e lementary part ic les . Experimenta l condi t ions as described in legends for Figs . 10and 11 . Figs . 13 a to c , specimens s ta ined wi th 1 per cen t po tassium phosphotungsta tewi thout p rior f ixa t ion . Fig . 18 d , specimen f ixed wi th 2 per cen t g lu tara ldehyde a t pH 7 .2prior to s ta in ing wi th 1 per c~nt u rany laceta te . Ma gni fica t ions: Fig . 18 a , X 1 ,250 ,000Figs. 13 b to d, )< 950,000.

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p i e c e , s t a l k , a n d b a s e p i e c e ) h a v e t h e s a m e r e l a -t i v e d i m e n s i o n s i n t h e s u b m i t o c h o n d r i a l f ra g m e n t sa s t h e y d o i n t h e i n t a c t m i t o c h o n d r i o n .

W e h a v e f r e q u e n t l y u s e d f e r r i ti n a s a r e f e re n c ep a r t i c l e f o r e s t i m a t i o n o f s i z e. O n t h e b a s i s o fc a r e f u l m e a s u r e m e n t o f t h e d i a m e t e r s o f s p e c ia l lyg r a d e d f e r r i ti n p a r t ic l e s w h i c h s h o w u p a s s p h e r ic a lu n i t s w i t h a n e l e c t r o n - o p a q u e c o r e , w e h a v e a s -s i g n e d a v a l u e o f 1 1 8 A w h i c h i s i n g o o d a g r e e -m e n t w i t h t h e u n p u b l i s h e d x - ra y d a t a o f J . W .A n d e r e g g a n d F . F i s c h b a c k . T h e h e a d p i e c e o ft h e e l e m e n t a r y p a r t i c l e i s r e m a r k a b l y s i m i l a r t of e r r it i n , a l t h o u g h t h e a v e r a g e d i a m e t e r i s s li g h t lyl es s t h a n t h a t o f f e r r i ti n a n d t h e e l e c t r o n o p a q u ec o r e i s l e s s p r o n o u n c e d .

I n m a n y p r e p a r a t io n s o f m i t o c h o n d r i a a n ds u b m i t o c h o n d r i a l fr a c t io n s , c o m p l e t e l y d e t a c h e dp a r t ic l e s a r e o b s er v e d n e a r t h e m e m b r a n e s . I ft h e s e p a r t ic l e s a r e , i n fa c t , d e t a c h e d e l e m e n t a r yp a r t i c l e s , t h e i r s i z e w o u l d b e s i g n i f i c a n t l y l a r g e rt h a n t h a t o f t h e h e a d p i e c e ( a p p r o x i m a t e l y 1 20 to1 40 A i n d i a m e t e r c o m p a r e d t o 8 0 t o 1 0 0 A ). T h e s ed e t a c h e d p a r t i c le s a r e l a r g e r t h a n t h o s e o f f e r ri t in .

V I S U A L I Z A T I O N O F T H E E L E M E N T A R YP A R T I C L E B Y A L T E R N A T I V E T E C H N I Q U E S :M i t o c h o n d r i a ( in s i tu a n d a f te r i s o l a ti o n ) h a v e b e e nf i xe d a n d s t a i n e d w i t h a v a r i e t y o f r e a g e n t s ( i n -c l u d i n g b u f f e r e d s o l u t i o n s o f o s m i u m t e t r o x i d e ,f o r m a l d e h y de , g l u t a ra l d e h y d e , p o t a s s i u m p e r m a n -g a n a t e , u r a n y l a c e t a te , p o t a s s i u m d i c h r o m a t e ,s i l ic o t u n g s ta t e , p o t a s s i u m i o d i d e , s o d i u m b r o m i d e ,a n d c a d m i u m i o d i d e ). E x a m i n a t i o n o f s u c ht r e a t e d p r e p a r a t i o n s h a s c o n s i s t e n t l y r e v e a l e d e s -s e n t i a l l y t h e s a m e t y p e o f p a r t i c u l a t e s t r u c t u r e .T h e f o l lo w i n g ty p e s o f v a r i a t i o n i n t h e e x p e r i -m e n t a l c o n d i t i o n s w e r e a l s o e x p l o r e d : i n t e r a c t i o no f t h e m i t o c h o n d r i o n w i t h t h e f i x in g re a g e n t p r i o rt o s t a i n i n g f o r d i ff e r e n t t i m e s a n d a t v a r i o u s t e m -p e r a t u r e s ; c o o l i n g o f t h e s p e c i m e n d u r i n g o b s e r v a -t i o n ; e l e c t r o n m i c r o s c o p y o f p a r t i a l l y h y d r a t e d

m i t o c h o n d r i a l s p e c im e n s i n v a c u u m - t i g h t m i c r o -c h a m b e r s ( 2 0 , 2 2 -2 4 ) a n d r e l a t e d t e c h n i q u e s .V i s u a l i z a t i o n o f t h e s u b u n i t s t r u c t u r e w a s n o ts i g n i f i c a n t l y a f f e c t e d b y s u c h v a r i a t i o n s . P o s i t i v er e s u l t s w e r e a l s o o b t a i n e d i n t h e e x a m i n a t i o n o fs p e c i m e n s p r e p a r e d b y u l t r a t h i n s e c t i o n i n g a f t e rc r y o f i x a t i o n , b y n e g a t i v e s t a i n i n g c o m b i n e d w i t hu l t r a t h i n s e c t io n i ng , a n d b y e m b e d d i n g i n g e l a t ina n d o t h e r w a t e r - s o l u b l e m e d i a . T h e s e r e s u l ts w i llb e d e s c r ib e d i n d e ta i l i n a s e p a r a t e c o m m u n i c a -t i o n .B I O C H E M I C A L R E S U L T S

E L E M E N T A R Y P A R T I C L E A N D T H E U N I TO F E L E C T R O N T R A N S F E R : F u l l e v i d e n c e h a sa l r e a d y b e e n p r e s e n t e d ( 5 ) t h a t t h e c o m p o n e n t s o ft h e e l e c t r o n t r a n s f e r c h a i n a s w e l l a s e l e c t r o nt r a n s f e r a c t i v i t y ( a s m e a s u r e d b y s u c c i n o x i d a s ea n d D N P H o x i d a s e a c t iv i ti e s ) a r e c o n c e n t r a t e ds o m e 2 . 6 t i m e s d u r i n g i s o l a t io n o f t h e e l e m e n t a r yp a r t i c le f r o m m i t o c h o n d r i a . I n p r e s e n t i n g t h er e s u l t s t h a t f o l lo w , w e s h a l l a d o p t t h e c o n c l u s i o no f B l a i r et a l . ( 5 ) t h a t t h e e l e m e n t a r y p a r t i c l e i s ,i n f a c t, t h e i n t a c t e l e c t r o n t r a n s f e r c h a i n s t r i p p e dc o m p l e t e ly f r o m t h e p r i m a r y d e h y d r o g e n a s e c o m -p l e x es a n d l a r g e ly , i f n o t c o m p l e t e l y , f r o m t h es t r u c t u r a l p r o t e i n - l i p i d n e tw o r k . W e s h a l l n o wc o n s i d e r t h e e x p e r i m e n t a l e v i d e n c e t h a t b e a r s o nt h e m o l e c u l a r w e i g h t o f t h e i s o l a t e d e l e m e n t a r yp a r t i c le a n d o n i ts p r o b a b l e m o l e c u l a r d i m e n s i o n s .T h i s i n f o r m a t i o n is e s s en t i a l t o a n s w e r t h e q u e s t i o no f t h e i d e n t i t y o f th e i s o la t e d e l e m e n t a r y p a r t i c l ew i t h t h e c o u n t e r p a r t p a r t i c le s e e n i n t h e c r i st a e o fi n t a c t m i t o c h o n d r ia .

M O L E C U L A R W E I G H T O F T H E E L E M E N -T A R Y P A R T I C L E F R O M A N A L Y T I C A L D A T A :T h e c y t o c h r o m e a c o n t e n t o f t h e e l e m e n t a r yp a r t i c l e i s o la t e d b y p r o c e s s in g t h r o u g h t w o c y c l e so f e x p o s u r e t o t h e c h o l a t e - d e o x y c h o l a t e m i x t u r e ,f o l lo w e d b y f r a c t i o n a t i o n w i t h a m m o n i u m s u l fa t e ,

I ~GVRE 14 E lec t r on mic r ogr aph o f nega t ive ly s t a ined p r epar a t i on o f t he e l ec t ron t r ansf e rpa r t i c le ( E T P) ~a subm i tochondr i a l fr ac t i on . E l em en ta r y pa r t i c le a r r ays c lose ly r esemblethose i n i n t ac t c r i s tae ; ave r age d imens ions o f head p i ece and o the r com ponen t s o f E P sameas t hose f o r co r r espond ing s t r uc tu r es i n E P o f i n t ac t mi tocho ndr i a ; appos i t ion o f two denselayers to form a single fused centra l layer . Specim en prep arat io n as described in legend forFig. 6 . X 600,000.l~e u lm 15 Nega t ive ly s t a ined segmen t o f e r i s ta f r om i so l a t ed who le I n i t ochondr ion ( en -l a r gemen t o f F ig . 6 ) . Spec imen p r epar a t i on cond i t i ons a r e essen t i a l ly t he sam e as f o r spec i -men show n in F ig . 14 . No te i den t i t y o f fo r m an d a r r ang eme n t o f e l emen ta r y pa r t ic l es i ne r i s t a of i n t ac t mi toehon dr ion and in t he e l ec t r on t r ansf e r pa r t i c l e ( E T P) . X 660 ,000 .

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T A B L E I I IMi nim al Molecular Weigh ts of the Four Complexes of the Electron Transfer Chainat the Highest Purity Levels Achieved

Molcular weightLipoproteinComplex Designation Protein basis (3 ~0 lipid)

II II I II V

T o t a l

DP NH -co enz ym e Q r eductase* 550,000 786,000Succ ini c- coen zyme Q r eduet ase ~ 208,000 297,000QH~ -cyto ehro me c r eduetase 200,000 285,000Cy toe hro me oxidase][ 426,000 609,000

1,976,00(),384,000* U npub l i s hed da t a o f J . M er o l a .Dat a of Ziegler and D oeg (81). Data of Rieske , Zaugg, and W har to n (61).][ Data of Cr iddle , Bock, Green, and Ti sd ale (13); Ambe and V enk etara man ( l ) .

FIGURE 16 Sedim entation diagram of an elemen tary particle preparation. The particles were suspendedin 90 per ce nt D20 which was 0.0~ M in Tris chloride and 0.001 I~ in ED TA . P rotein concen tration, 11 m gper ml. R otor speed, 50,700 arM; rotor tempera ture, 6 . Tim e interva l between photographs, 4 minutes.The apparent sedimentat ion constant at 6 is 13.6 S~0.,~ = 5~. Th e p article s were solub ilized with phos-photungstate as described in the text . The s low minor peak in the diagram is that of phosphotungstate.The sedimentat ion diagrams read f rom r ight to lef t .

i s 4 . 2 m gm ol e s / m g o f p r o t e in . O n t he ba si s o f6 molecules of cytoc hrom e a per m olecule of e le-m en t a r y pa r t i c l e ( 41 ) , t he m i n i m a l m o l ecu l a rweig ht of t he par t i c l e would be 6 1064.2 1.4 X106 on a pro te in bas is . Cy toch rome a i s s e l ec t edfor t h i s computa t ion s ince i t s concent r a t ion i sm or e accu r a t e l y de t e r m i ned t han any o t he r com -ponen t o f t he cha i n .

T he m i n i m a l m o l ecu l a r w e i gh t i s no t nece s -sar i ly the ac tual molecular weight . I n a uni t ofmin ima l molec ular weight , 1 .4 X 106, i t is as -s um ed t ha t t he r e w ou l d be one m o l ecu l e e ach o fcy t och r om e q , s ucc i n ic dehyd r ogenas e ( fs ), andD P N H dehyd r ogenas e ( fD ). T he ac t ua l un i t o fe l ec t r on t r ans f e r cou l d con t a i n m or e t han onem ol ecu l e o f t he se com p onen t s .

MOLECULAR WEIGHT OF THE ELEM EN-TARY PARTICLE FROM THE DATA OF RE-CONSTITUTION EXPERIMENTS: The e l e men-t a r y pa r t i c le ha s been r econs t i tu t ed by r ecom b i na -

t i on o f t he f ou r com po nen t com pl exes unde r s pe -c i f i ed condi t ions (30, 44 , 45) . I n the c omp anio npape r by B l a i r et al. (5) , we have di scus sed theev i dence t ha t t he f ou r com pl exes com bi ne i n1 : 1 : 1 : 1 mo lecular s to i chiomet ry . Tab le I I I con-t a i n s t he m i n i m a l m o l ecu l a r w e i gh t s o f e ach o f thef ou r com pl exes a s de t e r m i ned by chem i ca l ana l y -s i s . The four complexes used in the r econs t i t u t ions tudies were a t t he s t age of pur i ty indica t ed inTab le I I I . A s we shal l d i scus s l a t er , comp lex Ia t t he pu r i ty l evel used in these exper imen t s i ss t i l l h ighly impure , but t he o ther t hr ee complexesare pro bably c lose to the l imi t of pur i f i ca t ion . Th es um o f t he m i n i m a l m o l ecu l a r w e i gh t s o f t he f ou rcomplex es come s to 1 .38 X 106 on a prote inbas i s - - a va l ue w h i ch i s i n good ag r eem en t w i t ht he m i n i m a l m o l ecu l a r w e i gh t c a l cu l a t ed on t hebas is of t he compos i t ion of t he i so l a t ed e l e men tarypar t icle.

S i nce t he f ou r com pl exes m ay com bi ne i n1 : 1 : 1 : 1 mo lecula r s to i chomet ry , i t i s neces sary to

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establish for only one of them t hat the minim al andactual molecular weights are the same in order toextrapolate to the actual molecular weight of thereconstituted unit. The molecular weight of theQH2-cytochrome c reductase (complex III) wasdete rmine d by sedimentation analysis (J. Rieskeand P. Yang, unpublished data) and found toconform to the minimal molecular weight listedin Table III . Takemori et al. (73) estimated themolecular weight of cytochrome oxidase (corn-

plex IV) to be about 500,000--a value in goodagreement with that for the minimal molecularweight of the complex (615,000 for the li poproteincomplex). Singer, Kearney, and Massey (66) haveisolated a form of succinic dehydrogenase almostidentical in composition with that of complex II .The molecular weight which they determined bysedimentation analysis of their preparation cameto about 250,000--in good agreement with thevalue for the minimal molecular weight of com-

TAB LE IVInhibition of Electron Transfer Activities by Sodium Phosphotungstate

Concentration ofsodium phospho*Activity measured Particle type tungstate Inhibition

1. Oxidation ofDPNH by 02

2. Oxi dation of suc-cinate by 02

per centMit oeh ondr ia (heavy) 5 X 10 7 I04 X 10 .-6 50

8 X 10_6 75Mitoc hondri a (washed 4 X 10_5 10

wit h 0.9% KC1) 5.5 X 10 5 506.5 X 10 4 75ETP ~ 2.0 X 10 5 10

6.0 X 10 5 501 . 2 X 1 0 4 75

EP 5.0 X 10 6 103.0 X 10 5 505.5 X 10 5 75

Mito chondr ia (heavy) 5.0 X 10 7 I04.0 X 10 6 507.0 X 10 -6 75

Mito chondr ia (washed 2.0 X 10- 6 10with 0.9% KCI) 5.0 X 10- 6 508.5 X 10- 6 75

ETPH 5.0 >( 10 7 102.0 X 10- 6 504.0 X 10 6 75

EP 1.0 X 10- 5 103.5 X 10 5 505.5 X 10 5 75

The above experiment s were all carried out at a particle prot ein concent rati onofS0 #g per assay. When the prot ein conc entra tion is increased to 5000 #g per assay,the concent ration of sodium phosphotungstate required to achieve the same degreeof inhibition as at the 50 #g protein level is 3 to 5 times higher.

When specimens are prepared for negative staining, the usual concentrationrange of sodium phosphotun gstate concent rations is 0.1 to 1%, which corresponds inmolar terms to 3.3 )< 10 5 ~ -- 3.3 )< 10 4 M.

Mitochondria or submitochondrial particles which have been exposed to phos-photungstate at inhibitory concentrations can be washed free of the reagent; theelectron transfer activity after washing is the same as the original, uninhibited ac-tivity.

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plex II. The available evidence supports the viewthat the minimal molecular weight calculatedfrom analytical data is, in fact, the actual molec-ular weight of the e lementary particle.

SEDIMENTATION ANALYSIS O F THE ELE-MENTARY PARTICLE : The isolated elemen-tary particle shows a strong tendency to aggregatedespite the presence of residual deoxycholate whichis not removed by exhaustive washing in 0.25sucrose. Prolonged son ication partia lly dispersesthe suspension, but neither per manentl y nor com-pletely, to the stage of the m onomeric species. Thistendency of the particles to aggregate has effec-tively blocked our attempts to establish the homo-geneity of the preparation and to determine itsmolecular weight by sedimentation analysis.

The ultracentrifugal analysis of suspensions ofthe elementary particle following dispersion bysonic irradia tion shows a broad distribution ofparticle sizes. We have sorted out the particlesfrom the most aggregated to the least aggregatedin successive fractions collected in the trunnionhead of the Spinco ultracentrifuge and have de-termined the composition of each of these frac-tions No measurable differences were found inthe chemical composition of the different frac-tions. Thus, it is clear that the heterogeneity ofparticles is an expression of differences in degree ofphysical aggregation, but not in chemical compo-sition,

The elementary particles can be brought intotrue solution by interaction with phosphotung-state. The optimal level for "solubilization" is al: l ratio by weight of elementary particle (asprotein) an d phosphotungstate, The solution ofthe elementary particle-phosphotungstate complexis centrifuged at 50,000 RPM for 15 minutes. Th eprecipitate consists of two layers--a hard packedsediment which is discarded and a fluffy layerwhich is mixed with 0.01 ~a Tris buf fer of pH 7.5.Sedimentation analysis, by Dr. Pauline Yang, of

this phosphotungstate complex of the elementaryparticle showed a majo r compon ent with a weightaverage sediment ation coefficient o f 52 X 10 ~ see.after extrapolation to zero concentration ( c f . Fig.16). Chemi cal analysis of the com plex showed tha t1 gm of elementary particle protein was combinedwith 0,9 gm of lipid and 1 gm of phosphotungstate.The minimal molecular weight of this complexshould, therefore, be 2.9 X 1.6 X 106 = 4.6 X106 (the mini mal molecul ar weight of this particu -lar pr epar ation of EP was 1.6 X l0 p on a prot einbasis). The observed molecular weight deter-mined by the modified Archibald approach tosedimentation equilibrium was 5.8 X 106, in fairagreement with the minimal molecular weightcalculated from analytical data. The differencecould be accounted for by water of hydration.

From the experimentally determined values forthe partial specific volume (0.727 cm~/gm) andthe sed imenta tion coefficient (52 X 10 13 sec.),the fric tional coefficient of the el emen tary particl e-phosphotungstate complex can be calculated bythe Svedberg equation:

M ( l - - ~ 0 )/- NS

where f is the frictiona l coefficient of the elemen-tary particle complex; M , the molecular weight ofthe complex (5.8 X 106 gin /mo le) ; 5, the par tialspecific volum e; p, the density of the solvent (1:0 0gm/cm3); S, the sedimentation coefficient; and N,Avogadro's number. Wh en the values indicatedare substituted in the equation, the value for fcomes to 5.06 X 10 7 (C.G.S. units). The fric-tional coefficient of a spherical molecule of thesame molecular weight was calculated by thefollowing equation :

( 3 M g , ~ l /a: o = 6 ~ , @ I ~ : /

FIGURE 17 Electron micrograph of isolated elementary particles negatively stained with1 per cent potassium phosphotungstate by microdroplet cross-spray technique. Isolationof elementary particles as described in text; electron transfer activity of particles fullypreserved. In this typical field, discrete and conglomerated particles are seen in differentorientations; ferritin molecules (dense core, diameter 1~0 A) added as a marker. X 4~0,000.FIGURE 18 Preparation of isolated elementary particles embedded in thin film of phos-photungstate, selected from another specimen series. Note: asymmetry of particles; rela-tive uniformity of particle size (1~0)< 160 A); minimal aggregation in this par ticularspecimen. )< 330,000.

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FEnN~,NDEz-MoR.~N E'r .eL. Macromolecular Repeating Unit of Mitochondrial Structure 85



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Fm ua~ 19 Isolated elementary part icles examined by the shadow-cast ing technique. D etai ls of prepar-ation as in legend for Fig. 17. Individual particles of variable size and polyhedral shape clearly visible.X 300,000.Fto ua E ~0 Microd roplet of isolated eleme ntary particles, prepa ration positively stained with uranyl ace-tare and shadowed with plat inum-carbon at very low grazing angle; the resul ting "surface decorat ion" pat-terns indicate possible underlying subunit structure of individual elementary particles. X 600,000.

86 T ~ JOURNAL OF CELL BIOLOGY ' VOLUME g~, 1964



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where y is the viscosity of the solvent (1.47 X 10 ~poise) and the other symbols have been definedabove. The value for fo comes to 3.28 X 10 7when the indic ated substitutions are made. There-fore,

f 5.06 X 10 -7 - 1.54fo 3.28 X 10 -7

The value for the ratio of f to fo clearly indicatesthat the elementary particle-phosphotungstatecomplex is asymmetric, but the degree of asym-metry cannot be evaluated in terms of axial ratiountil the hydration of the complex can be speci-fied with precision.

E F F E C T O F P H O S P H O T U N G S T A T E O NE N Z Y M I C A C T I V I T Y : Phosphotungstate, at thelevels used for the preparation of samples to beexamined in the electron microscope, is a rela-tively mild reagent in its effects on enzymaticactivity of mitochondria and submit

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