Intra- and extracellular forms of α-Glucosidase from Aspergillus niger

ARCHIVES OF RIOCHEMTSTRY AND BIOPHYSICS Vol. 193, No. 2, April 1, pp. 509-520, 1979

Intra- and Extracellular Forms of a-Glucosidase from Aspergillus niger’

MICHAEL J. RUDICK,” ZOE E. FITZGERALD,* AND VICTORIA L. RUDICK’f

*Department of Biology, Texas Woman’s University, Denton, Texas 76204, and tDepartm.ent of An,atom.y, North Texas State Universityl’l’exas College of‘ Osteopathic Medicine, Fort Worth, Texas 76107

Received August 25, 1978; revised October 30, 1978

An intracellular a-glucosidase (ru-glu,) of Aspergillus niger was purified and its properties were compared to those of a secreted ol-glucosidase (cu-glu,). The estimated molecular weight of a-glu, was 95,000 by gel filtration (a-glu E = 63,000); it is a glycoprotein possessing 29 mol of mannose, 6 mol of glucosamine, and 14 mol of glucose (cx-gluE has 5-6 and 2 mol of mannose and glucosamine, respectively). The K,‘s of u-glu, for p-nitrophenyl-cu- D-glucopyranoside and maltose were 1.49 and 1.04, respectively, slightly lower than those of cu-glu,. In addition, at 65°C cy-glu, enzymatic activity decayed fivefold faster than that of ry-glu,, and anti-ol-glu, antibody did not recognize a-glu,. While some of these distinctions between the enzymes could be ascribed to conformational differences, the great dissimilarity in molecular weight (approximately 32,000) and lack of reactivity with anti-a-glu, argue against a-glul being related to a-glu,. The antibody covalently coupled to horseradish peroxidase (Ab-Px) was used as a probe to determine the cellular location of cu-glu, by electron microscopic immunocytology. It was found on both sides of the plasma membrane (pm) and in the outer of the two layers of the cell wall. This may mean that oI-gluE is synthesized at the inner surface of the pm, is extruded through the pm, becomes associated with the outer layer of the cell wall (perhaps as enzyme-substrate complex), and is eventually released into the growth medium.

Cellular secretion mechanisms all involve the passage of nascent polypeptide chains through a cellular membrane as the initial step in the process. These nascent chains are synthesized by polysomes which are bound to the membranes (l-3). In eucaryotes this takes place at the endoplasmic reticulum (e# and is followed by move- ment of the intracisternal protein through a well-defined series of organelles such as smooth er and Golgi vesicles, and finally into storage granules whose membranes fuse

’ Supported by Grant GM22707 from NIH and by research funds from Texas Woman’s University.

* Abbreviations used: er, Endoplasmic reticulum; pm, plasma membrane; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; Ab-Px, antibody covalently coupled to horseradish peroxidase; a-glu,, intracellular ol-glucosidase; a-glu,, secreted form of (Y- glucosidase; oc, outer layer of cell wall; rer, endoplasmic reticulum with associated polysomes.

with those of the plasma membrane (pm), resulting in the emptying of their contents of concentrated secretory proteins to the outside (1). In this scheme the cell contents include a fairly high level of secretory protein at various stages of production se- questered in membranous organelles, although the intracellular protein usually differs from the mature extracellular one in not being fully posttranslationally modified (4). Such modifications usually consist of proteolytic cleavage and glycosylation at specific amino acid residues, and the intracellular protein may or may not be bio- logically active (4 - 6).

On the other hand, the process of pro- caryotic protein secretion is more direct in that the nascent polypeptide is extruded through the pm to the outside (3). Several reports indicate that the protein is tran- siently membrane bound and in the case of a penicillinase secreted by Bacillus licheni-

509 0003-9861/‘79/040509-12$02.00/O Copyright 0 1979by Academic Press, Inc. All rights of reproductmn m any form reserved.

RUDICK, FITZGERALD, AND RUDICK

formis this is accomplished by covalent attachment of a phospholipid to the amino terminus which, along with a few acidic amino acid residues, is subsequently cleaved off causing release of the enzyme (7, 8). Thus, procaryotes have no internal pools of secretory protein and the protein is not glycosylated (3).

Fungi are primitive eucaryotes whose secretory pathway is relatively unchar- tered, although it has been known for a long time that they secrete several proteins. Earlier reports suggested that secretion in fungi and higher eucaryotes might be similar, involving sequestration in vesicles (9). Aspergillus niger, a filamentous fungus, secretes several glycoprotein enzymes into its growth medium and they all possess a single oligosaccharide chain bound to an asparagine residue and consisting of mannose and glucosamine (10-14). To date no attempt has been made to determine the mode of secretion of these enzymes nor to look for the existence of intracellular enzyme activities which might be related to the secretory forms.

This report describes the purification of a cell bound cr-glucosidase whose activity is very similar to, but whose physical properties are quite distinct from, that of a secreted cu-glucosidase. Also, the cellular location of the extracellular enzyme is determined by immunocytological methods and found to be like that expected of a procaryote.

MATERIALS AND METHODS

Chemicals. p-nitrophenyl-a-D-glucopyranoside and DEAE-cellulose were purchased from Sigma Chemi- cal Company; hydroxyapatite was from Bio-Rad; Glucostat was from Worthington Biochemical Cor- poration.

Cell growth. Aspergillus niger was grown for 2 days at 30°C with constant shaking, as previously described (IO).

Enzymatic assays. Unless otherwise stated, a- glucosidase activity was measured in the following reaction mixture (0.5 ml): sodium acetate buffer, pH 5.0, 100 pmol; substrate, 10 pmol; a suitable dilu- tion of enzyme. After intubation at 37°C the reaction was terminated in one of two ways: With p-nitrophenyl-a-n-glucopyranoside as substrate, 0.5 ml of 0.4 M glycine buffer, pH 10.5, was added and released p-nitrophenol was determined spectropho-

tometrically at 405 nm; with maltose as substrate the reaction mixture was heated in a boiling water bath for 5 min and glucose was determined by Glucostat.

Sugar and protein assays. Neutral sugars were quantitated by anthrone and by gas-liquid chromatography (lo), and amino sugars by the Elson-Morgan assay (15) after hydrolysis with 4 N HCl at 100°C for 4 h. Protein was determined as described by Lowry et al. (16).

Polyacrylamide gel electrophoresis. Tris-glycine, pH 8.9, polyacrylamide gels (PAGE) were prepared and run according to Davis (1’7). Sodium dodecyl sulfate (SDS)-PAGE was done, as described by Weber and Davis (18). Staining for carbohydrate was carried out according to Zacharias et al. (19).

Immunological methods. Rabbit antibody to a secreted A. niger a-glucosidase (11) was obtained by using a previously described inoculation regime (12). Double immunodiffusion was carried out according to Ouchterlony (20).

Electron microscopy. Mycelia from &-day cultures were prefixed in 2% paraformaldehyde in phosphate buffer, pH 7.4, at room temperature for 1 h and at 4°C for 4-16 h. The prefixed material was washed three times with phosphate-buffered saline (PBS), pH 7.4, and then incubated with 10% dimethyl sulfoxide for 1 h, after which it was quick frozen in an acetone- dry ice bath. Following thawing, the mycelia were cut into about lo-mm pieces. The freeze-thawing makes the hyphae permeable to the conjugate (21). Rabbit immunoglobulin isolated from anti-a-glucosidase serum was conjugated to horseradish peroxidase (Sigme VI r.z. 3.0) by the method of Nakane (22). The mycelia were treated with this conjugate (Ab-Px) for 18 h at 4°C and then rinsed for 18 h with PBS also at 4°C. Fixation was performed in phosphate-buffered 5% glutaraldehyde for 4-16 h followed by an overnight rinse in PBS, after which the material was incubated with 0.05-0.10% diaminobenzidine and 0.01-0.108 hydrogen peroxide in 0.05 M Tris-HCl buffer, pH 7.6, for 30 min to 1 h. A 2-h wash in PBS preceded 4-12 h of osmication. Finally the mycelia were dehydrated in graded ethanol and embedded in Spurr. Thin sections were cut on a Sorvall MT-2B ultramicrotome, poststained with 1% aqueous uranyl acetate for 30 min and then with lead citrate for 10 min. Specimens were ex- amined in the Siemens 1OOA electron microscope at 80 kV.

RESULTS

Puri$cation of Intracellular cr-Glucosidase

Unless otherwise stated all operations were carried out at 4°C.

(Y-GLUCOSIDASES OF Aspergillus niger 511

TABLE I

PURIFICATION OF a-glu,

Purification step

Total protein

(mg)

Total activity (units)*

Specific activity

(units/mg protein)

(1) Homogenate (after centrifugation)

(2) Homogenate (after dialysis)

(3) Dialysate (affer centrifugation)

(4) Acetone precipitate (5) Hydroxyapatite

chromatography (6) DEAE-cellulose

chromatography

1160 108 0.093

598 145 0.24

145 137 0.94 32 131 4.09

3.6 123 34.2

1.4 118 84.3

D Approximately 30 g of mycelia (eight l-liter cultures). * One unit equals 1 gmol of p-nitrophenyl-ol-D-glucopyranoside hydrolyzed in 1 h.

FIG. 1. (a) Polyacrylamide gel electrophoresis of purified cr-glu,. The 7.5% gels were stained as follows: A, with Coomassie blue; B, with periodate-fuchsin. The anode is at the bottom. (b) Poly- acrylamide gel electrophoresis of cY-glucosidases. Gel A, a-glu, (arrow 1) and a secreted pglucosidaae [molecular weight = 40,000 (lo), arrow 21; Gel B, a-glu,. The 7.5% gels were stained with Coomassie blue.

512 RUDICK, FITZGERALD, AND RUDICK

Step 2. Mycelia from ten 2-day old 1 liter cultures of Aspergillus niger were bar- vested by filtration through four layers of cheesecloth and washed thoroughly with cold 0.05 M Tris-HCI buffer, pH 7.0. Mycelia were disrupted in the same buffer with an equal weight of glass beads using a blender for 10 min after which the homogenate was filtered through four layers of cheesecloth. The filtrate was centrifuged at 2000 g for 10 min and the resultant supernatant was centrifuged at 10,OOCQ for 15 min. This final supernatant was dialyzed overnight against two changes of 0.1 M sodium acetate buffer, pH 5.0, followed by removal of the precipitate by centrifugation at 50009 for 10 min. To the soluble material were slowly added 2 vol of cold acetone with constant stirring, and the precipitate containing all of the cr-glucosidase activity was collected by centrifugation at 500% for 10 min, dissolved in water, and lyophilized.

Step 2. The lyophilized powder was dissolved in 50 ml of 0.01 M sodium phosphate buffer, pH 7.2, and placed onto a 2 x 12-cm column of hydroxyapatite equilibrated in the same buffer. After washing the column until no further protein was detected in the effluent, successive elution was carried out with 0.02 and 0.04 M sodium phosphate buf- fers, pH 7.2. The 0.04 M eluate was dialyzed against water and lyophilized.

Step 3. Protein from step 2 was dissolved in 0.01 M sodium acetate buffer pH 5.0 and placed onto a 2 x lo-cm column of DEAE- cellulose equilibrated with that buffer. The column was washed until no more protein was detected, and cr-glucosidase activity was eluted with 0.05 M KC1 in the same buffer. Results of this purification scheme are presented in Table I.

Purity and Properties of the Enzyme The intracellular a-glucosidase (a-glu,)

was shown to be at least 95% pure by PAGE in Tris-glycine buffer, pH 8.9, and to be a glycoprotein by periodate-fuchsin staining (Fig. la). A comparison between the mobilities of o+glul and the secreted form of cY-glucosidase (a-g&) on the same kind of gel demonstrated that cr-glu, migrated sub- stantially faster than a-glu, (Fig. lb) which

A B C FIG. 2. SDS-polyacrylamide gel electrophoresis of

a-glucosidases. Gel A, cy-glu, (arrow 2) and a secreted p-glucosidase, as in the legend to Fig. lb. (arrow 1); Gel B, a-glu,; Gel C, a-glu, (arrow 3), a-glu, (arrow 2), and p-glucosidase (arrow 1). The 7% gels were stained with Coomassie Blue.

indicated a difference in charge and/or mass between them. Electrophoresis on SDS- PAGE after treatment with SDS and mer- captoethanol (Fig. 2) demonstrated a dif-

FRACTION NUMBER

FIG. 3. Molecular weight estimation of a-gluI. A 1.6 x 90 cm Sephadex G-200 column was calibrated with the following markers: immunoglobulin G (IgG); bovine serum albumin (BSA); secreted a-glucosidase (cr-glu,); ovalbumin (0~).

wGLUCOSIDASES OF Aspergillus niger 513

TABLE II

ENZYMEACTIVITIESOFINTRA-ANDEXTRACELLULAR FORMS OF WGLUCOSIDASE

K, (rnM)

Substrate

Maltose p-nitrophenyl-a-D-glucoside

a-glu, cy-glu,

1.04 1.85 1.49 2.22

ference in their molecular weights and an estimate of 95,000 was obtained for cy-glu, by chromatography through a calibrated Sephadex G-200 column (Fig. 3). The mo- bility on SDS-PAGE and apparent molecular weight indicate that the enzyme is probably a monomer. Since the molecular weight of a-glu, is 63,000 (10, the mass difference is approximately 32,000.

The pH optimum for the two enzymes is identical (pH 5.0) and Table II shows that their K,‘s for two substrates, p-nitrophenyl-a-D-glucopyranoside and maltose are very close, those of cr-glu, being slightly lower. When the enzymes were incubated at

60

TIME AT 65°C (minutes)

FIG. 4. Stabilities of cy-glucosidases at 65°C. The enzymes were individually incubated at 65°C; samples were taken with time and assayed for 30 min at 37°C usingp-nitrophenyl-a-D-glucopyranoside as substrate.

TABLE III

CARBOHYDRATE CONTENTOF INTRA- AND EXTRACELLULARFORMSOF

a-GLUCOSIDASE'

Moles of sugar/mole of enzyme

Sugar

Glucosamine Mannose Glucose

a-glu, cu-glue.

6.3 2 23.5 5-6 14.0 0

a The sugar determinations were carried out twice.

65°C for increasing times and samples taken to assay for activity, the a-glu, was found to decay fivefold more rapidly than cr-glu, (Fig. 4) and was completely inactivated after 45 min.

Sugar content of acid-precipitated a-glu, was determined and found to differ from a-glu, in having more mannose (29 mol) and glucosamine (6 mol) per mole of enzyme. Also present in a-glul was a significant quantity of glucose which is not found at all in o-glu, (Table III).

A final comparison between the two enzymes was made by double immunodiffusion using purified cr-glu, and a-glu, in alternating peripheral wells and monospecific anti-cu-glu, in the center well (Fig. 5). A

FIG. 5. Double-diffusion immunoprecipitation of cY-glucosidases. I, peripheral wells containing a-glu, (50 pg); E, peripheral wells containing a-glu, (10 pg). The center well contained undiluted anti-a-glu, serum.


single precipitin line is obtained with cr-gluE, but none with cr-glul which may be taken to indicate that there are no antigenic sites on cw-glu, that anti-a-glu, recognizes. A further check on the antigenicity of a-glu, was carried out by quantitative micro complement fixation (23) and, while a normal curve was obtained with cu-glu,, no complement was fixed in the presence of a-glu, over a 200- fold range. (Thompson and Rudick, unpublished observations). This rules out the pos- sibility that a-glu, possesses a single anti- genie determinant rendering it incapable of being precipitated by a-glu,.

Electron Microscopic Immunocytology

The absence of a reaction between a-glu, and anti-cr-glu, enabled the use of anti-cr- glu, coupled to peroxidase to be used as a probe to determine the cellular location of

cr-glu,. Figure 6 shows a typical cross section through a hypha which has been treated as for immunocytologlcal examination but with no Ab-Px administered. While some structural detail is lost during the pro- longed treatment times, enough integrity is maintained to discern major cellular organelles. A similar cross section for an Ab- Px-treated cell (Fig. 7) illustrates that the electron-dense peroxidase reaction product is found in the outer of the two layers of the cell wall (oc) and on both sides of the plasma membrane (pm), and that there appears to be none anywhere in the cytoplasm. The difference in appearances of reactive and unreactive membrane is obvious. In many cases it has been found that cells with a high level of pm labeling have little oc labeling (Figs. 7a and 9a) and vice versa (Figs. 7b and 9b). While the reason for this is unknown, it is possibly related to the dis-

FIG. 6. Electron micrograph of the hyphal cell. This cell was treated as for immunocytological examination, except that no Ab-Px was used. The following structures may be seen: outer cell wall (oc), inner cell wall (ic), endoplasmic reticulum (er), nucleus (nu), and vacuoles (v). The bar equals 0.5 pm.

a-GLUCOSIDASES OF Aspergillus niger 515

FIG. ‘7. Immunocytological localization of a-glucosidase in Aspergillus naer. Mycelia were stained by direct immunoeytochemical technique with anti-a-glucosidase horseradish conjugate, as described under Materials and Methods. Ultrastructural details are as in F’ig. 6. Note the electron- dense peroxidase reaction production: (a) in the plasma membrane (pm); (b) in the outer cell wall (oc), but not in the inner cell wall (ic), other cell organelles, or the cytoplasm. The stars and arrows indicate reaction product. (The bar equals 1.0 pm.)

tance of the cell from the’ growing hyphal was used in place of the immunocytological tip, those closer (and, therefore, newer) reagents, no reaction product was seen, having more pm enzyme than those further and, when the osmication step was omitted, away. To ensure the specificity of the to illustrate that the electron-opaque reac- procedure used to localize the enzyme, sev- tion products were not due to osmophilic eral controls were prepared as follows. cellular particles, electron-opaque deposits Substitution of saline for Ab-Px but addi- were present. Antibody against partially tion of the diaminobenzidine-peroxide at the purified a-glu, (through the hydroxyapatite appropriate steps did not produce electron- step) was coupled to Px and substituted dense areas, indicating the absence of en- dogenous peroxidase activity. When saline

for the anti-a-glu, Ab-Px, yielding a completely different and intracellular labeling


FIG. 8. Immunocytological localization of intracellular antigens. IgG from rabbit antiserum against intracellular antigens (cy-glu, purified through the hydroxyapatite step) was coupled to horseradish peroxidase and used as described under Materials and Methods. Note the complete absence of peroxidase reaction product in the plasma membrane (pm) and inner (ic) or outer (oc) cell wall, both of which are very transparent in this preparation, and the dense pockets of reaction product in the cytoplasm. Some endoplasmic reticulum (er) is apparent, perhaps with associated intracellular antigens. Also present is a large vacuole (V) which is found in many of the cells. The bar = 0.5 pm.

pattern (Fig. 8) which showed that labeling was not due to nonspecific adsorption of Ab and that the entire cell was permeable to the Ab-Px. It might be pointed out that no detectable precipitin reaction was seen when this antiserum was tested with a-glu,.

Higher magnification examinations of cell walls and plasma membranes (Figs. 9 and 10) show the uniform appearance of reaction product in the cell wall, but the sometimes intermittent nature of reaction product in the pm. It is interesting to note that in the latter situation whenever enzyme is found on one side of the pm it is also found on the other. In addition, there is no evidence of enzyme in the periplasmic space, i.e., the space between pm and cell wall.

DISCUSSION

The a-glq, whose purification is described herein, is shown to have enzymatic properties very similar to those of a-glu,, but to differ significantly from the extracellular enzyme by being approximately 32,000 daltons heavier, by being fivefold more labile at 65”C, by possessing covalently bound glucose and more mannose, and by not binding to anti-cy-glu, antibody. It should be emphasized that, since cr-glu, is not detected in the A. niger growth medium, it is solely intracellular.

If a-glu, and cu-glu, are related as pre- cursor and product, respectively, the difference in stabilities at 65°C could be a re- sult of conformational difference, possibly brought about by the additional 32,000-

wGLUCOSIDASES OF Aspergillus niger 517

FIG. 9. Immunocytological localization of a-glucosidase, in plasma membrane and outer cell wall. (a) and (b) Ab-Px treated cells. Note the heavy and symmetrical deposition of reaction product in the plasma membrane (a), and the intermittent, but symmetrical deposition in plasma membrane and outer cell wall (b). (c) A control preparation showing the normal appearance of pm and cell wall in cells which have not been treated with Ab-Px. The stars indicate reaction product. (The bar equals 0.5 km.)

dalton peptide of Iy-glu,, the antigenic sites being masked by the conformational differences a&or the extra peptide. This ra- tionale has been invoked to explain the lo- to l&fold decreased antigenicity of proparathormone to antiparathormone serum compared to that of parathormone (24). How- ever, there is a precipitation reaction with proparathormone, which is not the case with a-glu, and anti-a-glu,. Other intracellular precursors which may differ by 2000-3000 daltons or more from their secreted counterparts (4-6) are capable of being bound by antisera to the secreted protein (6, 25-27). Also, the molecular weight difference between cu-glu, and a-glur would necessitate the removal of a 32,000-dalton peptide. Attempts have been made to determine the possible relationship between the two enzymes by tryptic peptide map- ping and by cyanogen bromide cleavage and analysis of the peptides by SDS-PAGE. It

appears that both enzymes are fairly resist- ant to trypsin. Cyanogen bromide con- sistently produced four discrete cr-glu, peptides, which agrees with the amino acid content (ll), but did not give consistent results with a-glu, for unknown reasons. Therefore, a definite statement about the relationship between the two enzymes cannot yet be made.

While it is known that a-glu, and all the other enzymes secreted by A. niger have a single asparagine-linked oligosaccharide chain (lo-14), nothing is yet known about the arrangement of sugar residues in a-glu,. Assuming asparagine linkage and chito- biose as the sugar chain linkage unit (28), there may be as many as three cr-glu, oligosaccharide chains. Interestingly, the presence of glucose in chains of this type, suggested originally by Leloir and co-work- ers (29), has recently been confirmed by Spiro (3) who demonstrated the transfer of

518 RUDICK. FITZGERALD, AND RUDICK

b.

FIG. 10. Immunocytological localization of cu-glucosidase, in plasma membrane. (a) and (b) (the bar equals 0.25 pm) Ab-Px-treated cells. As in Fig. 9, there is both continuous (a) and intermittent, but symmetrical (b) presence of peroxidase reaction product. (c) A control cell. Stars indicate reaction product. (The bar equals 0.5 pm.)

such a chain from a lipid carrier to protein acceptors.

Electron microscopy of A. niger hyphae has revealed the presence of most of the typical organelles, including in some cases a large central vacuole, but it has been difficult to find any but very low quantities of endoplasmic reticulum (er) which may or may not possess associated polysomes (rer). Recent studies of yeast when grown under aerobic or anaerobic conditions have shown that there is very little er (31) and this appears to be common among the fungi (32). On the other hand, higher eucaryotic cells, especially those involved in secretion, contain tightly stacked arrays of rer which are involved in the synthesis and sequestration of secretory proteins (1). Given this difference, the mode of secretion of proteins by A. niger and other fungi might be expected to be different from that of higher eucaryotic cells. Immunocytological examination of A. niger demonstrates that cw-glu, ap-

pears to be located only in the outer cell wall and on both sides of the pm, but nowhere in the cytoplasm or other organelles. Since the amount of er and rer is so small, it might be impossible to detect the presence of nascent or completed a-glu, polypeptides in that organelle by this method, and so its presence in er cannot be entirely ruled out. Those areas of the pm that have a patchy distribution of a-glu, (Figs. 9 and 10) always manifest it by a symmetrical arrangement, i.e., with patches opposite one another on the two sides of the pm. The specificity of the immunocytological method is demonstrated by using antibody against internal enzyme (Fig. 8) which appears to be entirely intracellular and not associated with cellular membranes.

Presence of the cr-glu, in the outer, and not the inner, part of the cell wall indicated an attachment of the enzyme to some com- ponent(s) of that part of the cell wall. Recent evidence shows that it is probably not a

c+GLUCOSIDASES OF Aspergillus niger 519

covalent attachment (Fitzgerald and Rudick, unpublished observation), so cu-glu, may be located there by substrate affinity having some function in cell wall degrada- tion associated with hyphal growth (32). A cy-(1,3)-glucanase has been found in Sclero- tium rolfsii cell walls by indirect immuno- fluorescence and a “masked,” enzymatically inactive, immunologically unreactive form of the enzyme has been suggested to exist associated with cellular membranes (33). The present report gives a more precise localization of a secreted fungal enzyme and also shows that the membrane-bound form is immunoreactive. Procaryotes are known to have membrane-bound forms of extracellular enzymes (‘7, 8, 34, 35) which, at least under some circumstances, serve as intermediates in the secretory process. The most well-characterized system of this type, Bacillus licheniformis, secretes a penicillinase whose membrane-bound form differs only by the addition of phosphatidyl serine and a few hydrophilic amino acids (7, 8).

The c”-glu, seems to be free in the cytoplasm, since no vesicles have been found which might contain it, but the enzyme must come into contact with a membrane at some stage of its synthesis to be glycosylated, no soluble glycosyl transferases having been detected (4). If a-glu, is converted to a-gIuE, then this must occur at the inner surface of the pm and lead to extrusion of the consequent a-glu, across the pm to the outside. However, if, as seems more likely, the two enzymes are completely unrelated, then a-glu, may be synthesized at the inner surface of the pm and extruded through it as synthesis proceeds. This would allow passage through the pm before attaining its native, active conformation and is really the same secretory scheme as that proposed for procaryotes (3). There remains the question of the function of a-glu,-an enzymatically active glycoprotein which is totally intracellular and not membrane bound. Its presence inside the cells apparently means, however, that glycosylation of proteins may be necessary but is not sufficient for secretion.

Work is now in progress to investigate the nature of the interaction of cy-glu, with the pm.

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Intra- and extracellular forms of α-Glucosidase from Aspergillus niger

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