the spatial arrangement of esterases in the microsomal ... · sicko-goad, l. m., r e. . crang, th....

CYTOBIOLOGIE Zeitschrift für experimentelle Zellforschung

Organ der Deutschen Gesellschaft für Elektronenmikroskopie e .V.

Band/Vol. 11 • 1975

VÜÜCA

W I S S E N S C H A F T L I C H E V E R L A G S G E S E L L S C H A F T M B H S T U T T G A R T

I N H A L T B A N D 11 • C O N T E N T S V O L U M E 11 III

A R N O L D , J . M .

A n effect of calcium in cytokinesis as demonstrated w i t h ionophore A 23187 1 C a l c i u m e f f e k t i n der C y t o k i n e s e d a r g e s t e l l t m i t l o n o p h o r A 2 3 1 8 7

B E H N K E , O .

Studies on isolated microtubules. Evidence for a clear space component 366 U n t e r s u c h u n g e n a n i s o l i e r t e n M i k r o t u b u l i . Nachweise für eine äußere K o m p o n e n t e

B L A K E R S , M . , siehe M E N Z E L , R. 279

B L A N D , C H . E . , C . Z . L U N N E Y

M i t o t i c apparatus of Pilobolus crystallinus 382 M i t o s e a p p a r a t bei P i l o b o l u s c r y s t a l l i n u s

B L E C H E R , S T . R. (Short Communicat ion)

A c t i n - l i k e filaments associated w i t h spread chromosomes 190 Actin-ähnliche F i l a m e n t e v e r b u n d e n m i t gespreiteten Chromosomen

B O U B L I K , M . , F. J E N K I N S , H . R. K A B A C K

Use of critical point drying in the preparation of Escherichia coli membrane vesicles for electron microscopy 304 A n w e n d u n g des c r i t i c a l p o i n t - T r o c k n e n s bei der e l e k t r o n e n m i k r o s k o p i s c h e n Präparation v o n E s c h e r i c h i a c o l i M e m b r a n v e s i k e l n

B R A A T Z - S C H A D E , K . , M . H A B E R E Y

Bioelectrical potentials and motile activity in A m o e b a proteus 87 B i o e l e k t r i s c h e P o t e n t i a l e u n d Beioegungsaktivität bei Amoeba p r o t e u s

C A M P B E L L , G . R., siehe C H A M L E Y , J . H . 358

C A S S E N S , R. G . , siehe Y A M A G U C H I , M . 335

C H A M L E Y , J . H . , G . R. C A M P B E L L

Isolated ureteral smooth muscle cells in cultnre. Including their interaction w i t h intrinsic and extrinsic nerves 358 G l a t t e M u s k e l z e l l e n des U r e t h e r s i n der G e w e b e k u l t u r u n d i h r e I n n e r v i e r u n g

C O O K E , P.

Filamentous aggregates of purif ied myosin f rom smooth muscle 346 Filamentöse A g g r e g a t e aus g e r e i n i g t e m M y o s i n des g l a t t e n M u s k e l s

C R A N G , R . E . , siehe S I C K O - G O A D , L . M . 430

C S A B A , G . , T . L A N T O S

Specificity of hormone receptors in Tetrahymena. Experiments w i t h Serotonin and histamine antagonists 44 Spezifität der H o r m o n r e z e p t o r e n v o n T e t r a h y m e n a . V e r s u c h e m i t A n t a g o n i s t e n v o n S e r o t o n i n u n d H i s t a m i n

D A H L , D . , siehe Y A M A G U C H I , M . 335

I V I N H A L T B A N D 11 • C O N T E N T S V O L U M E 11

D E N K E R , H . - W . , E . S . E . H A F E Z

Proteases and implantation i n the rabbit : role of Trophoblast vs. uterine secretion 101 Z u r B e d e u t u n g v o n Proteasen für die I m p l a n t a t i o n beim K a n i n c h e n : Abhängigkeit v o m T r o p h o b l a s t e n oder v o m U t e r u s s e k r e t ?

D E X H E I M E R , J .

Etüde ultrastructurale de quelques etapes de la differenciation des cellules ä reserves d u gametophyte femelle du G i n k g o bi loba 264 U l t r a s t r u c t u r a l s t u d y o f some d i f f e r e n t i a t i o n stages o f reserve cells i n t h e female g a m e t o p h y t e o f G i n k g o b i l o b a

D U S T M A N N , J . H .

D i e Pigmentgranula i m Komplexauge der Honigbiene Apis mell i f ica bei W i l d t y p u n d verschiedenen Augenfarbmutanten 133 The p i g m e n t g r a n u l e s i n t h e C o m p o u n d eye o f t h e honeybee A p i s m e l l i f i c a i n w i l d t y p e and d i f f e r e n t eye-color m u t a n t s

E C K E R T , W . A . , W . W . F R A N K E

Changes in fine structure and composit ion of macronuclei of Tetrahymena pyr i formis induced by drugs interfering w i t h R N A synthesis and processing 392 F e i n s t r u k t u r u n d chemische Z u s a m m e n s e t z u n g v o n M a k r o n u k l e i des C i l i a t e n T e t r a h y m e n a p y r i f o r m i s n a c h B e h a n d l u n g m i t I n h i b i t o r e n der R N A - S y n t h e s e

E G G E R , G .

T h e electron microscopic localisation of succinic dehydrogenase w i t h i n the membranes of the mitochondrial cristae 110 D i e e l e k t r o n e n m i k r o s k o p i s c h e L o k a l i s i e r u n g der S u c c i n a t - D e h y d r o g e n a s e i n n e r h a l b der M e m b r a n e n der C r i s t a e m i t o c h o n d r i a l e s

E L B E R S , P . F . , siehe S P I E S , F . 50

F A L K , H . , siehe Z E N T G R A F , H . 10

F R A N K E , W . W . , siehe E C K E R T , W . A . 392

F R A N K E , W . W . , siehe Z E N T G R A F , H . 10

G E N B A Ö E V , O . , V . SULOVIC*

Local iza t ion of human chorionic gonadotropin ( H C G ) by immunohistochemical method i n normal and pathological human placenta 95 I m m u n h i s t o c h e m i s c h e L o k a l i s a t i o n v o n m e n s c h l i c h e m C h o r i o n g o n a d o t r o p i n ( H C G ) i n der P l a z e n t a bei n o r m a l e r u n d pathologischer S c h w a n g e r s c h a f t

G O O D E , D .

M i t o s i s of embryonic heart muscle cells in vi tro . A n immunofluorescence and ultrastructural study 203 M i t o s e e m b r y o n a l e r H e r z m u s k e l z e l l e n i n G e w e b e k u l t u r : I m m u n f l u o r e s z e n z - u n d F e i n s t r u k t u r - U n t e r s u c h u n g e n

G R A T Z L , M . , W . N A S T A I N C Z Y K , D . S C H W A B

T h e spatial arrangement of esterases in the microsomal membrane 123 D i e A n o r d n u n g der Esterasen i n der m i k r o s o m a l e n M e m b r a n

I N H A L T B A N D 1 1 • C O N T E N T S V O L U M E 1 1 V

H A B E R E Y , M . , siehe B R A A T Z - S C H A D E , K . 8 7

H A F E Z , E . S. E . , siehe D E N K E R , H . - W . 1 0 1

H A X , W . M . A . , siehe S P I E S , F. 65

H E I N R I C H , G .

Über die L o k a l i s a t i o n verschiedener Phosphatasen i m N e k t a r i u m von A l o e 2 4 7 O n t h e l o c a l i z a t i o n o f d i f f e r e n t phosphatases i n t h e n e c t a r y o f A l o e

J E N K I N S , F., siehe B O U B L I K , M . 3 0 4

J E N S E N , T H . E . , siehe S I C K O - G O A D , L . M . 4 3 0

J E S E R I C H , G . , H . R A H M A N N (Kurzmitteilung)

Elektronenmikroskopisch-histochemischer Nachweis saurer Polysaccharid-verbindungen i m Z N S v o n Teleosteern 4 8 3 E l e c t r o n m i c r o s c o p i c - h i s t o c b e m i c a l d e m o n s t r a t i o n o f acid Polysaccharides

i n t h e C N S o f teleosts

J O B S T , K . , siehe K E L L E R M A Y E R , M . 2 4 0

K A B A C K , H . R . , siehe B O U B L I K , M . 3 0 4

K A L I N I N A , L . , siehe S I K O R A , J . 4 8 0

K E L L E R M A Y E R , M . , M . S O M F A I , K . J O B S T

Determinat ion of saline extractable material of H e L a cell nuclei 2 4 0 B e s t i m m u n g des i n Salzlösungen e x t r a h i e r b a r e n M a t e r i a l s v o n H e L a Z e l l k e r n e n

Kiss , A . , siehe KovÄcs, J . 3 0 9

K I T A J I M A , E . W .

A peculiar type of glycocalyx on the microvi l l i of the midgut epithelial cells of the thrips Frankl in ie l la sp. (Thysanoptera, Thripidae) 2 9 9 E i n besonderer G l y c o c a l y x - T y p a u f den M i k r o v i l l i der M i t t e l d a r m e p i t h e l z e l l e n v o n F r a n k l i n i e l l a spec. ( T h y s a n o p t e r a , T h r i p i d a e )

K O M N I C K , H . , M . S C H M I T Z , W . W I C H A R D

Cytologische, elektrolyt-histochemische und funktionelle Untersuchungen der analen Chlor idepithel ien aquatischer Brachycerenlarven (Insecta, Diptera) 4 4 8 C y t o l o g i c a l , e l e c t r o l y t e - h i s t o c h e m i c a l and f u n c t i o n a l s t u d i e s o n t h e anal c h l o r i d e

e p i t h e l i a o f a q u a t i c B r a c h y c e r a l a r v a e ( I n s e c t a , D i p t e r a )

K O S H I N O , Y . , siehe Y A S U Z U M I , G . 3 0

KovÄcs, J . , G . R E Z , A . K I S S (Short Communicat ion) Vinblast ine- induced autophagocytosis and its prevention by cycloheximide and emetine i n mouse pancreatic acinar cells in vivo 3 0 9 V i n b l a s t i n - i n d u z i e r t e A u t o p h a g o c y t o s e u n d seine Verhütung d u r c h C y c l o h e x i m i d u n d E m e t i n i n der A c i n u s z e l l e des Mäusepankreas i n v i v o

V I I N H A L T B A N D 11 • C O N T E N T S V O L U M E 11

K R I S T E N , U .

Feinstrukturveränderungen in den submersen Laubblattdrüsen von N o m a p h i l a stricta Nees während der Sekretion 438 A l t e r a t i o n s i n f i n e s t r u c t u r e o f t h e submerged l e a f g l a n d s o f N o m a p h i l a s t r i c t a Nees d u r i n g secretion

L A N T O S , T . , siehe C S A B A , G . 44

L E U N I S S E N , J . L . M . , siehe S P I E S , F. 50

L I N N E M A N S , W . A . M . , siehe S P I E S , F. 50

L I N N E M A N S , W . A . M . , siehe S P I E S , F. 65

L U N N E Y , C . Z . , siehe B L A N D , C H . E . 382

M E N Z E L , R . , M . B L A K E R S

Funct ional Organization of an insect ommat id ium w i t h fused rhabdom 279 D i e f u n k t i o n e l l e O r g a n i s a t i o n eines I n s e k t e n o m m a t i d i u m s m i t f u s i o n i e r t e m Rhabdom

N A K A I , Y . , siehe Y A S U Z U M I , G . 30

N A S T A I N C Z Y K , W . , siehe G R A T Z L , M . 123

N A T A R A J A N , A . T . , siehe R A P O S A , T . 230

N I L S H A M M A R - H O L M V A L L , M .

T h e effects of calcium deficiency on cell wal l format ion and autospore release of the green alga Scenedesmus 419 W i r k u n g e n des C a l c i u m - M a n g e l s a u f Z e l l w a n d b i l d u n g u n d E n t l e e r u n g der A u t o s p o r e n der Grünalge Scenedesmus

P E T Z O L D T , U . (Short Communicat ion)

A m i n o acid incorporat ion into embryonic proteins fiuring rabbit preimplantation development 490 Aminosäure-Einbau i n e m b r y o n a l e n P r o t e i n f r a k t i o n e n während der Frühentwicklung des K a n i n c h e n s

R A H M A N N , H . , siehe J E S E R I C H , G . 483

R A P O S A , T . , A . T . N A T A R A J A N

T h e use of the f luorochrome bis-benzimidazol derivative (Hoechst 33258) in the study of spontaneous and induced chromosome aberrations 230 D i e A n w e n d u n g des F l u o r o c h r o m e s B i s - B e n z i m i d a z o l d e r i v a t ( H o e c h s t 3 3 2 5 8 )

bei der U n t e r s u c h u n g s p o n t a n e r u n d i n d u z i e r t e r C h r o m o s o m e n a b e r r a t i o n e n

R E Z , G . , siehe KovÄcs, J . 309

R U B I O - H U E R T O S , M . , siehe V E G A , J . 186

R U Y T E R D E W I L D T , T H . M . D E , siehe SPIES , F. 65

I N H A L T B A N D 1 1 • C O N T E N T S V O L U M E 1 1 VII

S C H M I D T , K .

D a s Johnstonsche Organ der primär flügellosen Ectognatha (Lepisma, Zygentoma; M a c h i i i s , Archaeognatha) 1 5 3 Johnston's o r g a n o f t h e p r i m a r y apterous E c t o g n a t h a ( L e p i s m a , Z y g e n t o m a ; M a c h i i i s > A r c h a e o g n a t h a )

S C H M I T Z , M . , siehe K O M N I C K , H . 4 4 8

S C H W A B , D . , siehe G R A T Z L , M . 1 2 3

S H I R A I , T . , siehe Y A S U Z U M I , G . 3 0

S I C K O - G O A D , L . M . , R . E . C R A N G , T H . E. J E N S E N

Phosphate metabolism i n blue-green algae. I V . In situ analysis of polyphosphate bodies by x-ray energy dispersive analysis 4 3 0 Phosphatstoffwechsel bei B l a u a l g e n . I V . U n t e r s u c h u n g der P o l y p h o s p h a t - G r a n u l a m i t H i l f e der e n e r g i e d i s p e r s i v e n Röntgenanalyse

S I K O R A , J . , L . K A L I N I N A (Short Communicat ion)

Substrate detachment as a test of antigenic diversity of A m o e b a proteus strains 4 8 0 Substrat-Ablösung als Test der a n t i g e n e n V e r s c h i e d e n h e i t v o n Stämmen v o n Amoeba p r o t e u s

S O M F A I , M . , siehe K E L L E R M A Y E R , M . 2 4 0

S P I E S , F . , W . A . M . L I N N E M A N S , P. H . J . T H . V E R V E R G A E R T , J . L . M . L E U N I S S E N , P. F . E L B E R S

Encystment of Acanthamoeba castellanii (Neff) . A combined freeze etch -thin sectioning analysis of the cell surface 5 0 E n c y s t i e r u n g v o n Acanthamoeba c a s t e l l a n i i ( N e f f ) . K o m b i n i e r t e Gefrierätz-u n d Dünnschnittanalyse der Zelloberfläche

S P I E S , F . , W . A . M . L I N N E M A N S , T H . M . D E R U Y T E R D E W I L D T , W . M . A . H A X

G r o w t h phase dependent Concanaval in A agglutinability of Acanthamoeba castellanii (Neff strain) 65 D i e Abhängigkeit der A g g l u t i n i e r b a r k e i t v o n Acanthamoeba c a s t e l l a n i i d u r c h C o n c a n a v a l i n A v o n der Wachstumsphase

S U L O V I C , V . , siehe G E N B A C E V , O . 9 5

T R A U T , W .

Die Transkriptionsaktivität der Chromosomen i n den Oocyten von Ephestia (Lepidoptera) 1 7 2 T r a n s c r i p t i o n a l a c t i v i t y o f t h e chromosomes i n oocytes o f E p h e s t i a ( L e p i d o p t e r a )

V E G A , J. , M . R U B I O - H U E R T O S (Short Communicat ion)

Atypica l cristae i n wheat coleoptile mi tochondria 1 8 6 Ungewöhnliche C r i s t a e - S t r u k t u r e n i n M i t o c h o n d r i e n v o n W e i z e n k o l e o p t i l e n

V E R V E R G A E R T , P. H . J . T H . , siehe S P I E S , F . 5 0

W A L Z , B. (Short Communicat ion)

M o d i f i e d ci l iary structures i n receptor cells of M a c r o b i o t u s hufelandi (Tardigrada) 1 8 1 M o d i f i z i e r t e C i l i e n s t r u k t u r e n i n Rezeptorzellen v o n M a c r o b i o t u s h u f e l a n d i ( T a r d i g r a d a )

VIII I N H A L T B A N D 11 • C O N T E N T S V O L U M E 11

W E R Z , C , siehe Z E R B A N , H . 314

W E Y G O L D T , P., siehe Z I S S L E R , D . 466

W I C H A R D , W . , siehe K O M N I C K , H . 448

Y A M A G U C H I , M . , R . G . C A S S E N S , D . D A H L

Congenital rod disease: some biochemical aspects of nemaline rods 335 S t r u k t u r e l l e u n d biochemische U n t e r s u c h u n g e n der Kristallstäbchen bei einer F o r m angeborener M y o p a t h i e

Y A S U Z U M I , G . , T . S H I R A I , Y . N A K A I , Y . K O S H I N O

Fine structure of nuclei as revealed by electron microscopy. VIII . Possible o r i g i n and function of nuclear bodies appearing i n precancerous and degenerating cell nucle i 30 D i e F e i n s t r u k t u r des K e r n s i m e l e k t r o n e n m i k r o s k o p i s c h e n B i l d . V I I I . W a h r s c h e i n l i c h e H e r k u n f t u n d F u n k t i o n der i n präkanzerösen u n d degenerierenden Z e l l k e r n e n a u f t r e t e n d e n Nuklear-Körper

Z E N T G R A F , H . , H . F A L K , W . W . F R A N K E

Nuclear membranes and plasma membranes f rom hen erythrocytes. I V . Characterization of nuclear membrane attached D N A 10 K e r n m e m b r a n e n u n d P l a s m a m e m b r a n e n aus Hühnererythrocyten. I V . C h a r a k t e r i s i e r u n g der kernmembranständigen D N A

Z E R B A N , H . , G . W E R Z (Short Communicat ion)

Ultrastructure of flagellar microtubules in the green algae Acetabularia mediterranea and Dunal ie l la salina as revealed in freeze-etch preparations 314 F e i n s t r u k t u r v o n Geißel-Mikrotubuli der Grünalgen A c e t a b u l a r i a m e d i t e r r a n e a u n d D u n a l i e l l a s a l i n a n a c h Gefrierätzung

Z I S S L E R , D . , P. W E Y G O L D T

Feinstruktur der embryonalen Lateralorgane der Geißelspinne Tarantula marginemaculata C . L . K o c h (Amblypygi , Arachnida) 466 F i n e s t r u c t u r e o f t h e l a t e r a l organs o f t h e e m b r y o o f t h e W h i p Spider T a r a n t u l a m a r g i n e m a c u l a t a C. L . K o c h ( A m b l y p y g i , A r a c h n i d a )

Bericht Ultrastrukturelle Probleme normaler und pathologisch veränderter H a u t 321 U l t r a s t r u c t u r a l Problems o f n o r m a l and a b n o r m a l s k i n

BUCHBESPRECHUNGEN

A S H W O R T H , J . M . : Zelldifferenzierung 494

B A R R E L L , B. G . , B. F. C C L A R K E : H a n d b o o k of Nuc le i c A c i d Sequences 201

D A V I E S , M . : Funkt ionen biologischer Membranen 494

G A R R O D , D . R . : Ze l lentwicklung 494

G R E L L , K . G . : Protozoology 202

J A C O B I , G . (Hrsg.): Biochemische Cytologie der Pflanzenzelle 201

L A S H , J . , J . R . W H I T T A K E R (Hrsg.): Concepts of Development 333

W O O D S , R . A . : Biochemische Genetik 494

Symposium: Mechanoreception 496

C Y T O B I O L O G I E V O L U M E 11 • N O . 1 Pages 123-132 • 1975

The spatial arrangement of esterases in the microsomal membrane

D i e A n o r d n u n g der Es terasen i n der m i k r o s o m a l e n M e m b r a n

M A N F R E D G R A T Z L *), W O L F G A N G N A S T A I N C Z Y K , and D I E T E R S C H W A B

Fachbereich Theoretische M e d i z i n der Universität des Saarlandes,

Homburg/Saar, Germany

Received February 7, 1975

A b s t r a c t

Rat l i v e r - microsomal esterases - asymmetric d i s t r i b u t i o n

T h e location of esterases is studied w i t h microsomal vesicles f rom rat liver using membrane impermeable charged Substrates and inhibitors . By comparing esterase activity against charged and uncharged Substrates it is shown that microsomal esterases are not latent. Furthermore charged inhibitors were unable to block both esterase - and amidase - activity differently i n disrupted and in intact vesicles. F r o m these biochemical studies it was concluded that the major part of the microsomal esterases/amidases is attached to the cytoplasmic side of the microsomes, which was confirmed by electron microscopic studies w i t h enzyme specific staining showing the electron dense reaction products of the microsomal esterases at the cytoplasmic side exclusively.

Introduct ion

M i c r o s o m e s i so la ted by the u s u a l m e t h o d s f r o m rat l i v e r are c l o s e d vesicles, w h o s e i n n e r surface represents the i n t r a c i s t e r n a l s ide of the e n d o p l a s m i c r e t i c u l u m . T h e outer surface b e a r i n g r i b o s o m e s i n r o u g h m i c r o s o m e s [ 1 7 , 1 8 ] , is e x p o s e d to the c y t o p l a s m i n the intact c e l l . V e r y recent ly it w a s c o n c l u d e d f r o m i o d i n a t i o n studies w i t h l a c t o -p e r o x i d a s e that m o s t of the m i c r o s o m a l p r o t e i n s are faced to the c y t o p l a s m i c side o f the vesicles [15]. S o m e p a r t i c u l a r m i c r o s o m a l p r o t e i n s , h o w e v e r , seem to be l o c a t e d o n the inner surface o f the m i c r o s o m a l m e m b r a n e .

T h e r e is n o d o u b t a b o u t the o r i e n t a t i o n of m i c r o s o m a l n u c l e o s i d e d i p h o s p h a t a s e [16] : T h i s e n z y m e s h o w s a h i g h degree of la tency (that means the e n z y m a t i c ac t iv i ty b e i n g great ly s t i m u l a t e d by t reatments w h i c h affect the l i p o p r o t e i n s t ructure of the m i c r o s o m a l vesicles) . It is a l so p r o t e c t e d f r o m p r o t e o l y t i c at tack as w e l l as f r o m i n h i b i t i o n by a speci f ic a n t i b o d y i n in tac t m i c r o s o m e s b u t is suscept ib le to h y d r o l y t i c

L) D R . M . G R A T Z L , Fachbereich Theoretische M e d i z i n der Universität des Saarlandes, 665 Homburg/Saar, Germany.

124 M A N F R E D G A R T Z L , W O L F G A N G N A S T A I N C Z Y K and D I E T E R S C H W A B

enzymes a n d a n t i s e r u m after v a r i o u s treatments w h i c h affect the i n t e g r i t y o f the membrane s t ruc ture . E s p e c i a l l y s o m e p h o s p h o h y d r o l a s e a n d p h o s p h o t r a n s f e r a s e reac t ions ca ta lysed b y m i c r o s o m a l g l u c o s e - 6 - p h o s p h a t a s e are i n a latent State [ 2 , 3 1 ] . E l e c t r o n -m i c r o s c o p e [17, 18] a n d c e n t r i f u g a t i o n studies [19] c o n c e r n i n g the t r a p p i n g of p h o s p h a t e b y P b 2 + a lso f a v o u r an i n t r a m i c r o s o m a l l o c a t i o n of this e n z y m e . E n z y m e spec i f i c s t a i n i n g of the acyl transferases i n v o l v e d i n the a c y l a t i o n of a - g l y c e r o p h o s p h a t e p r o v i d e s ev idence f o r the a t tachment of this e n z y m e at the i n n e r surface of the m i c r o s o m a l m e m b r a n e [13]. U D P - g l u c u r o n y l transferase s h o w s a lso la tency a n d m a y be a n o t h e r e n z y m e w h i c h is l o c a t e d at the i n t r a c i s t e r n a l s ide of the m i c r o s o m a l m e m b r a n e [8].

D i g e s t i o n of m i c r o s o m e s w i t h proteases nei ther s o l u b i l i z e s n o r inact ivates m i c r o s o m a l a m i d a s e a c t i v i t y [1] a n d an a n t i b o d y to the a c e t a n i l i d e - h y d r o l y z i n g esterase fa i l s to react w i t h the m e m b r a n e b o u n d e n z y m e . F r o m these o b s e r v a t i o n s it w a s c o n c l u d e d that m i c r o s o m a l esterase m a y be a n c h o r e d to the i n n e r surface of the v e s i c u l a r m e m b r a n e . If this assumption is correct this enzyme should s h o w latency at least w i t h charged Substrates a n d i n h i b i t o r s since m i c r o s o m a l vesicles are r e g a r d e d to be i m p e r m e a b l e to a n i o n i c Compounds [22].

In the present p a p e r w e invest igated therefore the h y d r o l y s i s of espec ia l ly des igned charged Substrates i n c o m p a r i s o n w i t h the uncharged parent Compounds i n in tac t m i c r o s o m e s a n d i n p r e p a r a t i o n s p r e v i o u s l y treated w i t h agents a f f e c t i n g the in tegr i ty of the m e m b r a n e s t ruc ture . T o reveal , w h e t h e r esterase a c t i v i t y is la tent o r not , a lso studies w i t h a charged i n h i b i t o r w e r e p e r f o r m e d . In a d d i t i o n e n z y m e spec i f i c s t a i n i n g m e t h o d s w e r e e m p l o y e d to detect, o n w h i c h side of the m i c r o s o m a l m e m b r a n e the esterase m a y be l o c a t e d .

M a t e r i a l s a n d methods

Microsomes were isolated from a 20°/o homogenate of rat liver in 0.25 M sucrose/0.01 M sodium phosphate buffer, p H 1 A by gel f i l tration [26]. M a l e Sprague-Dawley rats of body weight 150 to 200 g were used.

Protein was determined according to L O W R Y et a l . [21], using a cal ibrat ion curve wi th crystalline bovine serum albumin (Serva, Heidelberg, Germany) . Esterase activity was determined by the f o l l o w i n g methods: Spectrophotometric determination of liberated 4-nitrophenol or 4-carboxy-2-nitrophenol from 4-nitrophenyl acetate (= nitrophenyl acetate) and 4-carboxy-2-nitrophenyl acetate ( = carboxynitrophenyl acetate) at 400 n m . Carboxyni t rophenyl acetate was synthesized by acetylation of 4-hydroxy-3-nitrobenzoic acid [7]. The hydrolysis of two esters could be fo l lowed very sensitively by f luorometric estimation of 7 -hydroxycoumarin [28] and 4-carboxy-7-hydroxycoumarin formed from umbelliferone acetate and 4-carboxyumbel l i -ferone acetate (carboxyumbelliferone acetate), respectively. As the excitation and emission spectra of 7-hydroxycoumarin was only slightly modif ied by introduct ion of the carboxy group the same primary and secondary filters could be used for both assays [28]. C a r b o x y u m b e l l i ferone acetate was prepared by acetylation of 4-carboxyumbell i ferone, prepared from resorcin and diethyl oxaloacetate [30], acetylation of umbelliferone yielded umbelliferone acetate [27]. The amidase activity of the esterase was determined by measuring the formation of aniline as described [1].

A l l enzyme assays were performed at p H 7.4 in 0.01 M sodium phosphate buffer containing 0.25 M sucrose at a temperature of 20° C . T o avoid uncertainties f rom incomplete ionisation of phenols or quenching of fluorescence a k n o w n amount of product was added for calibration to the cuvettes after each assay. Initial reaction velocities were determined using an external recorder.

For electron microscopic investigations microsomes were prepared f rom fasted rats by gel f i l trat ion in 0.01 M cacodylate buffer, p H 7.0, containing 0.14 M N a C l . One m l of microsomal

Locat ion of microsomal esterases 125

Suspension (3 m g of protein/ml) was spun down several times in an Eppendorf centrifuge model 3200 to form a pellet. The supernatant was removed and the pellet was f ixed in 2,5 °/o glutaraldehyde in 0,03 M cacodylate buffer p H 7.0 for 60 m i n . at r o o m temperature. After postf ixation in 1 % osmium tetroxide in the same buffer the pellets were dehydrated with ethanol and embedded in E p o n 812. T h i n sections were cut w i t h a Reichert Ultramicrotome, stained with uranyl acetate and lead citrate in the conventional manner and examined w i t h a Siemens E lmiskop 101. T h i o p h e n o l acetate, the Substrate used for electron microscopic Observation of esterase activity, was prepared f r o m thiophenol and acetyl chloride [10]. Enzyme specific staining [29] was achieved by preincubation of the unfixed microsomal pellets in 5 m M gold sodium thiosulphate (Sanocrysin ®, a gift f rom Ferrosan, Copenhagen, Denmark) dissolved in 0.01 M cacodylate buffer p H 7/0.14 M N a C l for five minutes at the temperature of melt ing ice. After addit ion of 10 ul thiophenyl acetate (destilled just before use to remove traces of thiophenol f r o m this unstable ester and diluted 1/1000 by sonification i n incubation buffer), the pellets were shaked gently for ten minutes at 30° C , stopped w i t h 2,5 °/o glutaraldehyde and processed as described above. For controls microsomes were incubated 15 m i n . before centrifugation w i t h 1 m M bis(4-nitrophenyl)phosphate.

A l l reagents not specified in detail were the best grade available and were used without further puri f icat ion.

Results

In o r d e r to f i n d a m e t h o d to d i s r u p t m i c r o s o m a l m e m b r a n e s a v o i d i n g a n i n a c t i v a t i o n o f m i c r o s o m a l esterases w e u n d e r t o o k p r e l i m i n a r y e x p e r i m e n t s . M a n y treatments c o m -m o n l y used f o r this p u r p o s e i n c l u d i n g t a u r o c h o l a t e , d e o x y c h o l a t e , acetone a n d a l k a l i i r e a t m e n t , o r s o n i c a t i o n w e r e a c c o m p a n i e d by c o n s i d e r a b l e i n a c t i v a t i o n o f the e n z y m e a c t i v i t y . A m o n g the C o m p o u n d s tested o n l y T r i t o n X 100 caused n o i n a c t i v a t i o n of the m i c r o s o m a l esterase. F i g u r e 1 s h o w s the i n s e n s i t i v i t y of the e n z y m e to v a r i o u s c o n c e n t r a t i o n s of T r i t o n X 100.

S ince the detergent d i d n o t inac t iva te the m i c r o s o m a l esterase, i t w a s expec ted , that the a c t i v i t y to the u n c h a r g e d Substrate ( n i t r o p h e n y l acetate) w a s u n a f f e c t e d . S u r p r i s i n g , h o w e v e r , w a s the f i n d i n g that even the h y d r o l y t i c ac t iv i ty against the charged Substrate c a r b o x y n i t r o p h e n y l acetate w a s u n c h a n g e d u p o n a d d i t i o n of d i f fe rent c o n c e n t r a t i o n s

2 t 0 ü 1 8"

Ol 02 03 CU 05

Percent Triton X 100

F i g . 1. Effect of T r i t o n X 100 on the esterase activity of microsomes. - O p e n c i r c l e s : H y d r o l y t i c activity against carboxynitrophenylacetate (10~4 M ) . - Closed c i r c l e s : H y d r o l y t i c activity against nitrophenylacetate (10~4 M ) .

120

80

o 40.

U 6 M A N F R E D G A R T Z L , W O L F G A N G N A S T A I N C Z Y K and D I E T E R S C H W A B

of the detergent. T o c o m p a r e the i n f l u e n c e o f detergent u p o n the h y d r o l y s i s o f u n c h a r g e d a n d charged Substrates b y m i c r o s o m a l esterases the i n i t i a l r e a c t i o n v e l o -cities w e r e d e t e r m i n e d at d i f f e rent Substrate c o n c e n t r a t i o n s . R e c i p r o c a l p l o t s of t h e d a t a o b t a i n e d are presented i n F i g u r e s 2 a n d 3 .

120

cn

£ 100-X c

£ 80-

X 1 60-

o

£ 40-

> 20-

I/(p -nitrophenylacetate) (mM )

10 20 30 40 50

I /(Carboxynitrophenylacetate) (mM"1)

Fig . 2. Plot of reciprocal ini t ia l reaction velocity against reciprocal m i l l i m o l a r concentration of nitrophenylacetate in the absence (o) and in the presence of 0 . 1 % (0) or 0 . 5 % (y) T r i t o n X 100.

Fig . 3. Plot of reciprocal ini t ia l reaction velocity against reciprocal m i l l i m o l a r concentration of carboxynitrophenylacetate in the absence (o) and in the presence {%) of 0.1 % T r i t o n X 100.

1.5

l/(Umbelliferone acetate) (mM ')

Fig . 4. Kinetics of umbelliferoneacetate hydrolysis in the presence (0) and in the absence (o) of 0.1 % T r i t o n X 100.

T h e k i n e t i c constants are u n c h a n g e d b y T r i t o n X 100 f o r charged a n d u n c h a r g e d Substrates. T h e a f f i n i t y o f the e n z y m e f o r a Substrate does n o t seem to be c h a n g e d great ly by the i n t r o d u c t i o n of a negat ive charge. In cont ras t the a p p a r e n t m a x i m a l v e l o c i t y is m o r e t h a n d o u b l e d w i t h n i t r o p h e n y l acetate c o m p a r e d t o c a r b o x y n i t r o p h e n y l acetate.


to m e a s u r e esterase a c t i v i t y against charged a n d u n c h a r g e d Substrates f l u o r o m e t r i c a l l y . P r e l i m i n a r y e x p e r i m e n t s a g a i n s h o w e d n o change i n esterase a c t i v i t y u s i n g c o n c e n t r a t ions of T r i t o n X 100 u p to 1 °/o. L i n e w e a v e r - B u r k p l o t s of the h y d r o l y s i s of u m b e l l i f e r o n e acetate a n d c a r b o x y u m b e l l i f e r o n e acetate b y m i c r o s o m a l esterases i n the presence a n d i n the absence of detergent are s h o w n i n F i g u r e s 4 a n d 5 .

T h e a f f i n i t y of the m i c r o s o m a l esterases f o r these t w o Substrates a l so w a s n e a r l y the same. T h e a p p a r e n t m a x i m a l v e l o c i t y w a s even 40 t imes h i g h e r w i t h the u n c h a r g e d Substrate c o m p a r e d w i t h the charged one . B u t a g a i n w i t h this c o u p l e of Substrates nei ther K m n o r V m a . \ w a s c h a n g e d b y detergents i n d i c a t i n g that the esterase ac t iv i ty of m i c r o s o m e s is n o t latent .

N e g a t i v e l y charged i n h i b i t o r s a lso s h o u l d have a l i m i t e d access to the act ive site o f an e n z y m e l o c a t e d i n s i d e a m e m b r a n e b a r r i e r . W e expec ted , that after d i s r u p t i o n of the m e m b r a n e , b i s ( 4 - n i t r o p h e n y l ) p h o s p h a t e , a w e l l k n o w n esterase i n h i b i t o r , w o u l d be m o r e p o w e r f u l t h a n i n in tac t m i c r o s o m e s . H o w e v e r , as s h o w n i n F i g u r e 6 the t i m e course of i n h i b i t i o n of m i c r o s o m a l esterases w a s u n c h a n g e d i n T r i t o n X 100 c o m p a r e d to the assays i n absence of detergent .

It is k n o w n , that m i c r o s o m a l esterases f r o m rat l i v e r are heterogeneous . S o m e enzymes are ab le to s p l i t esters e x c l u s i v e l y a n d others c a n a lso cleave a m i d e s [ 3 , 4 , 5 ] . U n -f o r t u n a t e l y w e c o u l d n o t repeat the d e s c r i b e d e x p e r i m e n t s w i t h charged a n d u n c h a r g e d amides since w e d i d n o t measure a n y h y d r o l y t i c act ivety against negat ive ly charged a m i d e s . T h e e x p e r i m e n t c o n c e r n i n g the i n h i b i t i o n of a m i d a s e a c t i v i t y b y a charged i n h i b i t o r presented i n F i g u r e 7 l ed to the c o n c l u s i o n that the m a j o r p a r t of the acet-a n i l i d e h y d r o l y z i n g esterases is a lso accessible a n d therefore l o c a t e d o n the outer surface of the m i c r o s o m e s .

T h e e x p e r i m e n t s d e s c r i b e d w i t h charged Substrates a n d a charged i n h i b i t o r suggest that m i c r o s o m a l esterases are at tached to the c y t o p l a s m i c s ide of the m i c r o s o m a l vesicles a n d are n o t e x c l u d e d f r o m the s u r r o u n d i n g m e d i u m b y a m e m b r a n e w h i c h is i m p e r m e a b l e f o r charged substances . T h i s f i n d i n g c o u l d be c o n f i r m e d b y e n z y m e speci f ic s t a i n i n g of m i c r o s o m a l esterases u s i n g t h i o p h e n y l acetate as a Substrate. In p r e l i m i n a r y studies w e w e r e able to f o l l o w the Splitting of this ester by m i c r o s o m e s s p e c t r o p h o t o -

I / (Carboxyumbelliferone acetate) (mM"1)

Fig . 5. Plot of reciprocal ini t ia l reaction velocity against reciprocal mi l l imolar concentration of Substrate (carboxyumbelliferoneacetate). Closed c i r c l e s represent assays in 0.1 °/o T r i t o n X 100, open c i r c l e s control assays wi thout detergent.


m e t r i c a l l y based o n the p r o d u c t i o n of a y e l l o w c o l o r w i t h 5 , 5 ' - d i t h i o b i s - 2 - n i t r o b e n z o a t e [10]. T h i s e n z y m a t i c h y d r o l y s i s c o u l d be c o m p l e t e l y i n h i b i t e d b y 1 m M b i s ( 4 - n i t r o -p h e n y l ) p h o s p h a t e . A s s h o w n i n F i g u r e s 8 a a n d b the r e a c t i o n p r o d u c t ( A u + - t r a p p e d t h i o p h e n o l ) appears outs ide the m i c r o s o m a l vesicles e x c l u s i v e l y . In p r e p a r a t i o n s o f m i c r o s o m e s p r e v i o u s l y treated w i t h the i n h i b i t o r n e a r l y n o depos i t s c a n be seen i n the sections (F ig . 8 c).

10 20 30

Time of incubation (min)

10 20 30

Time of incubation (min )

Fig . 6. Inhibit ion of microsomal esterase (substrate 10~4 M p-nitrophenylacetate by bis(p-nitro-phenyl)phosphate. Circles represent control values wi thout detergent (o) and in the presence of 0.1 °/o T r i t o n X 100 Squares represent inhibi t ion of microsomal esterases (0.43 mg/ml) after supplementation of Suspension w i t h 1,31 u. mole inhibi tor per m l in the absence (•) and i n the presence (•) of 0.1 °/o T r i t o n X 100.

F i g . 7. Inhibit ion of microsomal acetanil ide-hydrolyzing esterases (substrate 25 m M acetanilide) by bis(p-nitrophenyl)phosphate. C i r c l e s represent control values wi thout detergent (o) and in the presence of 0.6 °/o T r i t o n X 100 (0), Squares show inhibi tor of microsomal esterases (0,36 mg/ml) after supplementation of the Suspension w i t h 0,3 u, mole inhibi tor per m l in the absence (•) and in the presence (<•) of 0,6 % T r i t o n X 100.

Discuss ion

T h e e x p e r i m e n t s d e s c r i b e d w e r e des igned to d e t e r m i n e the l o c a l i z a t i o n o f esterases i n the m i c r o s o m a l vesicles . T h e results o b t a i n e d d e m o n s t r a t e that esterase a c t i v i t y is n o t latent i n m i c r o s o m e s . H o w e v e r , la tency of m e m b r a n e b o u n d e n z y m e s i n m i c r o somes is c o n s i d e r e d to i n d i c a t e the presence of a p e r m e a b i l i t y b a r r i e r a n d a l o c a t i o n of the latent e n z y m e ins ide the vesic le [ 2 , 8 , 1 3 , 1 6 , 3 1 ] . In agreement w i t h o u r c y t o -c h e m i c a l d e m o n s t r a t i o ! ! o f es tero lyt ic enzymes i t m a y be c o n c l u d e d there fore that m i c r o s o m a l esterases are at tached to the c y t o p l a s m i c s ide of the m i c r o s o m a l vesic les .

Fig . 8 a. T h i n section of a microsomal fraction previously treated w i t h thiophenyl acetate, showing besides microsomes (M) the reaction product of the microsomal esterases, the A u + -trapped thiophenol , as electron dense particles ( a r r o w s ) . - 12 000 X . - b. In higher magnifica-t ion the reaction product is clearly visible outside the microsomes ( M ) , partly contacting the microsomal membranes ( a r r o w s ) . - 50 000 X . - c. M i c r o s o m a l fraction after inhibi t ion of the enzymatic hydrolysis of thiophenyl acetate by bis-p-nitrophenyl-phosphate. Near ly no esterase reaction products are visible. - 12 000 X .


T h i s I n t e r p r e t a t i o n is at v a r i a n c e w i t h i m m u n o l o g i c a l e x p e r i m e n t s r e p o r t e d b y A K A O a n d O M U R A [1] s h o w i n g that a p r e c i p i t a t i n g a n t i b o d y p r e p a r e d to p u r i f i e d a c e t a n i l i d e -h y d r o l y z i n g esterase d i d n o t in terac t w i t h the e n z y m e assoc ia ted w i t h m i c r o s o m a l vesic les [1 ] . T h e resistance of m i c r o s o m a l a c e t a n i l i d e - h y d r o l y z i n g esterase to p r o t e o l y s i s a l so suggested that this e n z y m e is n o t e x p o s e d to the o u t s i d e m e d i u m [1] .

H o w e v e r , protease t rea tment m a y be n o i d e a l t o o l f o r s t u d y i n g the s ideness of esterases since s o l u b l e a n d m e m b r a n e b o u n d a c e t a n i l i d e - h y d r o l y z i n g esterase is i n s e n s i t i v e t o p r o t e o l y t i c attack [1] . F u r t h e r m o r e e n z y m a t i c a c t i v i t y against a r o m a t i c esters, w h i c h are sp l i t b y a l l species o f m i c r o s o m a l esterases [5] , is ne i ther i n a c t i v a t e d n o r s o l u b i l i z e d d u r i n g d i g e s t i o n of m i c r o s o m e s w i t h t r y p s i n i n c o n c e n t r a t i o n s u p t o 0 , 1 °/o 2 ) , a l t h o u g h i t is k n o w n f r o m e x p e r i m e n t s d e s c r i b e d i n th is p a p e r , that m a i n esterase a c t i v i t y is l o c a l i z e d o n the outer surface of the m i c r o s o m a l ves ic le .

T h e c o n f l i c t i n g results o b t a i n e d b y A K A O a n d O M U R A [1] a n d b y us c o u l d be d u e t o the heterogenei ty of m i c r o s o m a l esterases. F r o m rat l i v e r f ive esterases c o u l d be d i f f e r e n t i a t e d d u r i n g one p u r i f i c a t i o n p r o c e d u r e [ 3 , 4 , 5 ] . O t h e r p r e p a r a t i o n s o f m i c r o s o m a l esterases f r o m rat l i v e r are d e s c r i b e d i n the l i t e ra ture [ 1 , 1 1 , 1 2 , 2 0 ] b u t the r e l a t i o n s to the f ive m e n t i o n e d a b o v e are n o t c lear [5 ] .

T w o o f these e n z y m e s v a r i a n t s h y d r o l y z e c a r b o x y l esters o n l y (des ignated E i a n d E2),

w h e r e a s the r e m a i n i n g three e x h i b i t b o t h esterase a n d a m i d a s e ac t iv i t ies . T h e c o n t r o -verse c o n c l u s i o n s d r a w n i n this w o r k c o m p a r e d to i n t e r p r e t a t i o n s a r i s i n g f r o m i m m u n o l o g i c a l a n d d i g e s t i o n e x p e r i m e n t s [1] m a y be e x p l a i n e d b y d i f f e rent d i s t r i b u t i o n o f the esterase v a r i a n t s o n b o t h sides o f the m i c r o s o m a l m e m b r a n e . In this m o d e l , a c c o r d i n g t o the e x p e r i m e n t s r e p o r t e d i n this p u b l i c a t i o n , the t w o esterases e x h i b i t i n g h y d r o l y z i n g a c t i v i t y against c a r b o x y l esters ( E i , E2) are l o c a t e d o n the outer sur face o f the m i c r o s o m a l vesic les . O n e of the esterase var iants e x h i b i t i n g a m i d a s e a c t i v i t y seems t o b e l i n k e d t o the i n n e r surface of the v e s i c u l a r m e m b r a n e as s u p p o r t e d m a i n l y b y the f i n d i n g that a n a n t i b o d y to the esterase fa i l s to react w i t h the m i c r o s o m e - b o u n d e n z y m e [1] , w h i l e others , as c o n c l u d e d a b o v e f r o m s t u d i e d w i t h a m e m b r a n e i m p e r m e a b l e i n h i b i t o r o f a m i d a s e a c t i v i t y , together w i t h N A D H - c y t o c h r o m e bs reductase , N A D P H - c y t o c h r o m e c reductase , c y t o c h r o m e bs [14 , 2 3 , 2 4 , 2 5 ] a n d the esterases E i a n d E2 are f a c i n g the c y t o p l a s m i c s ide of the m i c r o s o m a l m e m b r a n e .

Acknowledgements. T h e authors thank D R . V . U L L R I C H for many helpful discussions and D R . T . O M U R A for his valuable advice during preparation of this manuscript. Parts of this paper have been presented at the Joint Meet ing 1974 of the Biochemical Societes of Belgium, the Federal Republ ic of Germany und the Netherlands. Düsseldorf 2nd to 5th O k t o b e r 1974 [9]. - T h i s w o r k was supported by the Sonderforschungsbereich 38, " M e m b r a n f o r s c h u n g " .

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