electrophoretic heterogeneity of human α-mannosidase

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Biochimica et Biophysica Acta, 303 (1973) 171-174 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 36348 ELECTROPHORETIC HETEROGENEITY OF HUMAN a-MANNOSIDASE L. POENARU AND J.-C. DREYFUS Institut de Pathologie Mol~culaire ~*, 24, rue du Faubourg Saint-Jacques, 75or 4 Paris (France) (Received October i3th, 1972) SUMMARY Electrophoresis on cellulose acetate of human tissue extracts demonstrated the presence of three bands of a-mannosidase (a-D-mannoside mannohydrolase, EC 3.2.1.24) activity. One band stained at a neutral pH, the two others at an acid pH. The pattern of acid a-mannosidase was different in various tissues. INTRODUCTION The enzyme a-D-mannoside mannohydrolase (EC 3.2.I.24) or a-mannosidase, is responsible for the hydrolysis of a-mannoside bonds in mucopolysaccharides and glycoproteins. Its deficiency has been described as mannosidosis by ~)ckermann 1 and results in an accumulation of mannose-rich oligosaccharides2, a. This enzyme has been studied by a variety of techniques, including deter- minations in organs 4-6 in blood platelets 7 plasma and serumS, 9, fibroblasts 1° and finally gel fltration and electrofocusing n. None of these techniques resulted in the finding of definite molecular forms. Electrophoresis on cellulose acetate followed by specific staining allowed us to find at least three different bands of a-mannosidase activity. METHODS AND RESULTS Our material was human tissue. Kidneys were obtained at autopsy. Liver and brain came from biopsies. The brain samples were obtained after neurosurgery for functional brains disorders. Placenta was received I h after delivery. Fibroblasts were grown from skin biopsies. Tissues were extracted in a glass homogenizer with 3 vol. of water containing o.1% Triton Xloo in order to extract lysosomal enzymes. The homogenates were centrifuged at 30 ooo × g for 15 min and the supernatants were utilized immediately. Determinations of activity according to pH showed two maxima, one at pH 3.5, ~* Universit6 de Paris V, Groupe U 15 de l'Institut National de la Santd et de la Recherche Mddicale, Laboratoire associd au Centre National de la Recherche Scientifique.

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Biochimica et Biophysica Acta, 303 (1973) 171-174 © Elsevier Scientific Pub l i sh ing C ompany , A m s t e r d a m - P r in t ed in The N e t h e r l a n d s

BBA 36348

E L E C T R O P H O R E T I C H E T E R O G E N E I T Y OF HUMAN a-MANNOSIDASE

L. P O E N A R U AND J.-C. D R E Y F U S

Institut de Pathologie Mol~culaire ~*, 24, rue du Faubourg Saint-Jacques, 75or 4 Paris (France)

(Received October i3 th , 1972)

SUMMARY

Electrophoresis on cellulose acetate of human tissue extracts demonstrated the presence of three bands of a-mannosidase (a-D-mannoside mannohydrolase, EC 3 . 2 . 1 . 2 4 ) activity. One band stained at a neutral pH, the two others at an acid pH. The pattern of acid a-mannosidase was different in various tissues.

INTRODUCTION

The enzyme a-D-mannoside mannohydrolase (EC 3.2.I.24) or a-mannosidase, is responsible for the hydrolysis of a-mannoside bonds in mucopolysaccharides and glycoproteins. I ts deficiency has been described as mannosidosis by ~)ckermann 1 and results in an accumulation of mannose-rich oligosaccharides2, a.

This enzyme has been studied by a variety of techniques, including deter- minations in organs 4-6 in blood platelets 7 plasma and serumS, 9, fibroblasts 1° and finally gel f l t ra t ion and electrofocusing n. None of these techniques resulted in the finding of definite molecular forms. Electrophoresis on cellulose acetate followed by specific staining allowed us to find at least three different bands of a-mannosidase activity.

METHODS AND RESULTS

Our material was human tissue. Kidneys were obtained at autopsy. Liver and brain came from biopsies. The brain samples were obtained after neurosurgery for functional brains disorders. Placenta was received I h after delivery. Fibroblasts were grown from skin biopsies. Tissues were extracted in a glass homogenizer with 3 vol. of water containing o.1% Triton Xloo in order to extract lysosomal enzymes. The homogenates were centrifuged at 30 ooo × g for 15 min and the supernatants were utilized immediately.

Determinations of activity according to pH showed two maxima, one at pH 3.5,

~* Univers i t6 de Par i s V, Groupe U 15 de l ' I n s t i t u t Na t iona l de la Santd et de la Recherche Mddicale, Labora to i re associd au Centre Na t iona l de la Recherche Scientifique.

172 L. POENARU, J.-C. DREYFUS

i l 315 415 6,5 pH

Fig. I. a -Mannos idase ac t iv i ty of t i ssue ex t r ac t s as a func t ion of pH. l iver; , - - , , U1--E], pla- cen ta ; 0 - - 0 , k idney ; © - - O , f ibroblas ts ; A - - A , brain. I n c u b a t i o n s were pe r fo rmed for I h in a c i t r a t e - p h o s p h a t e buffer 0.04 M a t p H va lues 3.0 to 7.0. The scale is g iven as pe rcen tage of the m a x i m a l ac t iv i ty observed wi th t he m o s t act ive ex t rac t . This cor responds to an ac t iv i ty of 80 n m o l e s / m i n per g t issue, in general a g r e e m e n t wi th o ther authorsS, 14,

the other at pH 6.5, except for fibroblast extracts in which the neutral peak of activity was not found.

Electrophoresis was run on cellulose acetate (cellogel), according to Fluharty et al. 1~ with modifications, for 2 h at 200 V and 3 mA per band. The buffer was 0.04 M potassium phosphate (pH 6.5). The staining substrate was 4-methylumbelliferyl-a- mannopyranoside (Koch Light) as a 5oo-#M solution in a o.I M citrate-phosphate buffer. Incubation was performed for each sample at three different pH values: 3.5, 4.5 and 6.5. Bands of Whatman 3 MM paper, saturated with the substrate, were pressed against the electrophoresis bands on a glass sheet. After lO-3O rain incubation at room temperature (according to enzymatic activity) a band saturated with I M glycine-NaOH buffer (pH IO) was substituted for the substrate-saturated paper. The alkaline reaction stops the enzyme activity and transforms the product, 4- methylumbelliferone, into a strongly fluorescent anion. The fluorescent spots were visualized in a Chromatovue cabinet and photographed with a Polaroid ultraviolet camera. Photographs must be taken immediately since the fluorescence is diffusing rapidly. Results are shown in Fig. 2.

At pH 6.5 (Fig. 2a) all extracts show one sharp band migrating at the same position. This band is absent or very faint in extracts of fibroblasts (Channel 4). At pH 3.5 there is one major fluorescent band. This band migrates more slowly towards the anode than the "neutral" band. It is more diffuse, and in some samples there appears a second minor spot. Two types of major band can be distinguished (Fig. 2b): a fast type, observed in extracts from liver and placenta (Channels I and 2), a slow

ELECTROPHORETIC HETEROGENEITY OF C~-MANNOSIDASE

a + . . . .

z73

O--~ <---O

1 2 3 4 ,'5 1 2 3 4 ,5

c +

0" - -~

1 2 3 4 5

Fig. 2. Electrophoresis and staining of a-mannosidase at three different p H values, a, p H 6.5; b, p H 3.5; c, p H 4.5. Channels: i, liver; 2, placenta; 3, kidney; 4, fibroblasts and 5, brain.

type, in extracts from kidney, fibroblasts and brain. The latter organ has a low activity and its fluorescent spot is seen faintly or not at all.

At an intermediate pH (4.5) (Fig. 2c) both "acid" and "neutral" bands of ~-mannosidase are simultaneously visible.

DISCUSSION

As shown previously in our laboratory for ~-glucosidase 13, electrophoresis on cellulose acetate, followed by incubation with 4-methylumbelliferyl derivatives at different pH values, allows one to separate and characterize glucosidases which act at neutral or acid pH values. After incubation at pH 4.5 only, followed by staining for ~-mannosidase, we observed a complex picture which can be analyzed by incu- bations at a lower and higher pH. The "neutral" mannosidase is the same in all tissues and seems to be lacking in fibroblasts, as confirmed by the pH activity curve. The "acid" mannosidases seem to occur as at least two forms, one of which pre- dominates in each tissue. I t is not possible yet to tell from our electrophoretic experi- ments whether the two slow isozymes are simultaneously present in the tissues, although in different proportions.

Our results are in general agreement with the recent work of Carrol et al. ~4

who separated three different forms of mmannosidase from liver extracts on DEAE- cellulose, one of which is active at a neutral pH, and two at an acid pH.

x74 L. POENARU, J.-C. DREYFUS

R E F E R E N C E S

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366-372 3 ()ckerman, P. A. (1967) Lancet II, 239-24i 4 Van Hoof , H. and Hers, H. G. (1968) Eur. J. Biochem. 7, 34-44 5 Hultberg, B. and 13ckerman, P. A. (1972) Clin. Chim. Acta 39, 49 58 6 Vidershain, G. Ya., Rozenfeld, E. L., Brusilovskii, A. I. and Kolibaba, L. G. (1971) Bioche-

mistry 36 , 859-864 7 Bosman, B. N. (1972) Biochim. Biophys. Acta 258, 265 273 8 ~)ckerman, P. A. (1969) Clin. Chim. Acta 23, 479 482 9 Courtois, J. E. and Mangeot, M. (1972 ) Ann. Biol. Clin. 3 ° , 49 58

io Den Tandt , W. R. (1972 ) Clin. Chim. Acta 4 ° , 199 2o 4 i i Hultberg, B. (197o) Scan& J. Clin. Lab. lnvest. 26, 155-159 12 Fluhar ty , A. L., Lassila, F. L., Porter , M. T. and Kihara, H. (i97 I) Biochem. Med. 5, 158-164 13 Dreyfus, J.-C. and Alexandre, Y. (1972) Biochem. Biophys. Res. Commun. 48, 914 920 14 Carroll, M., Dance, N., Masson, P. K., Robinson, D. and Winchester , B. G. (1972) Biochem.

Biophys. Res. Commun. 49, 579-583