T H E J O U R N A L OF BIOLOGICAL (>HEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol . 264, NO. 29, Issue of October 15, pp. 17583-17588,1989 Printed in U.S.A.

Purification and Characterization of Neutral Trehalase from the Yeast ABY S 1 Mutant*

(Received for publication, April 26, 1989)

Harald App and Helmut Holzer From the Biochemisches Institut, Universitat Freiburg, Hermann-Herder Strasse 7, Freiburg 0-7800, Federal Republic of Germany

Neutral trehalase was purified from stationary yeast ABYSl mutant cells deficient in the vacuolar protein- ases A and B and the carboxypeptidases Y and s. The purified electrophoretically homogeneous preparation of phosphorylated neutral trehalase exhibited a molec- ular mass of 160,000 Da on nondenaturing gel electro- phoresis and of 80,000 Da on sodi.um dodecyl sulfate- gel electrophoresis. Maximal activity (1 14 rcmol of tre- halose min-l X mg” at 37 “C) was observed at pH 6.8- 7.0. The apparent K,,, for trehalose was 34.5 RIM. Among seven oligosaccharides studied, the enzyme formed glucose only from trehalose. Neutral trehalase is located in the cytosol. A polyclonal rabbit antiserum raised against neutral trehalase precipitates the en- zyme in the presence of protein A. The antiserum does not react with acid trehalase. Dephosphorylation by alkaline phosphatase from Escherichia coli of the ac- tive phosphorylated enzyme is accompanied by 290% inactivation. Rephosphorylation by incubation with the catalytic subunit of beef heart protein kinase is accompanied by reactivation and incorporation of 0.85 mol of phosphate/mol subunit (80,000 Da). The phos- phorylated amino a’cid residue was identified as phos- phoserine.

In yeast, the trehalose-hydrolyzing enzyme trehalase was first described by Ernil Fischer (1). An inactive (zymogen) form of trehalase, which is activated by cyclic AMP-depend- ent phosphorylation, was reported by van Solingen and van der Plaat (2). In 1982, Wiemken and co-workers (3, 4) dem- onstrated that the phosphorylatable trehalase was localized in the cytosol, whereas a second, permanently active, trehalase was found in the vacuoles. Londesborough and Varimo ( 5 ) separated these two a.ctivities and determined different pH optima of the two enzymes. The permanently active vacuolar trehalase, which has its maximal activity at pH 4.5 and which was therefore designated “acid trehalase,” was purified and characterized in a previous paper from this laboratory (6). Partial purification of the cytosolic “neutral trehalase,” which, when phosphorylated, has its maximal activity at pH 7.0, has been worked out by several groups ( 5 , 7, 8). In this paper, purification to homogeneity of the phosphorylated neutral trehalase and characterization of the purified enzyme are presented. In accordance with the recommendations of the

*This experimental work was supported by the Deutsche For- schungsgemeinschaft, So~nderforschungsbereich 206, the Verband der Chemischen Industrie e.‘V. (Fonds der Chemischen Industrie), and the Gesellschaft fur Strahlen- und Umweltforschung (GSF), Neuher- berg bei Munchen. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Nomenclature Committee of the International Union of Bio- chemistry (1984), the phosphorylated active form of neutral trehalase is designed a, and the inactive nonphosphorylated form is named b.

MATERIALS AND METHODS

Reagents Auxiliary enzymes and biochemicals were purchased from Boehrin-

ger Mannheim, Merck, and Sigma. [y3’P]ATP (3 Ci/mmol) was a product of Amersham Buchler (Braunschweig, Federal Republic of Germany). Orcinol was from Sigma; concanavalin A, poly(A) Sepha- rose CL-4B, protein A-Sepharose CL-4B, PD-10 columns, TSK- DEAE column, and Pharmalyte, pH 2.5-5.0, were from Pharmacia LKB Biotechnology Inc. The Spherogel TSK-phenyl 5PW column for high performance hydrophobic interaction chromatography was from Beckman.

Organism The diploid yeast mutant ABYSl (MATa pral-1 prbl-1 prcl-1

cpsl-3 ade) (9) lacking the vacuolar proteinases A and B and carbox- ypeptidases Y and S was a gift from Prof. Dr. Dieter H. Wolf, Biochemisches Institut, Universitiit Freiburg, FRG. ABYSl cells were grown for 24 h at 30 “C in YEPD medium (1% yeast extract, 2% Bacto-peptone, 2% glucose).

Enzyme Assay Neutral trehalase activity was measured according to Uno et al. (7)

by incubating 20 p1 of enzyme solution with 180 p1 of 0.5 M trehalose in 50 mM imidazole-HC1, pH 7.0, for 10 min at 37 “C. The reaction was stopped by adding 0.5 ml of a solution containing 10 g/liter dinitrosalicylic acid, 16.8 g/liter NaOH, and 300 g/liter sodium/ potassium tartrate. The controls were stopped immediately. After addition of 0.3 ml of H20, the samples were boiled for 5 min at 95 “C. After centrifugation for 5 min at 4 “C, the supernatants were equili- brated at room temperature, and the absorbance was recorded at 530 nm with a X 7 Perkin-Elmer spectrophotometer.

Definition of Units One unit of neutral trehalase is defined as the amount of enzyme

which catalyzes the hydrolysis of 1 pmol of trehalose/min at 37 “C and pH 7.0.

Carbohydrate Analysis The sugar content was determined with the orcinol method as

described by Winzler (10) using mannose as the standard. The absorption was measured a t 505 nm instead of 540 nm (11). Assays of protein-bound carbohydrate were also done with the glycan detec- tion kit from Boehringer Mannheim after periodate oxidation.

Protein Determination Protein was determined according to Lowry et al. (12) or by the

method of Bradford (13) using bovine serum albumin as the standard.

Electrophoresis, Isoelectric Focusing, and Activity Staining Preparative electrophoresis on nondenaturing 5% polyacrylamide

gels were carried out as described by Maurer (14). For activity staining, gel slices were incubated in 0.2 M imidazole-HC1 containing

17583

17584 Neutral Trehalase from the Yeast AB YS1 Mutant 0.1 M trehalose, pH 6.2, for 10 min at 37 "C and washed three times with distilled water. The glucose liberated in the gel was assayed according to Gabriel and Wang (15). Polyacrylamide gel electropho- resis in the presence of SDS' was carried out according to King and Laemmli (16) on 15% gels in the presence of SDS. Isoelectric focusing and the determination of the molecular mass of the native neutral trehalase were done with the Pharmacia PhastSystem according to the recommendations of the company. Gels were stained for protein with Coomassie Brilliant Blue R-250 and destained by diffusion.

Antiserum Preparation

A rabbit was immunized against neutral trehalase (150 pg) and boosted with 100 pg after 4 weeks. Antiserum was collected 4 weeks thereafter.

Purification of Neutral Trehalase from the Yeast Mutant A 3 Y S l All operations were performed at 4 "C. Step I. Crude Extract-Yeast cells were harvested by centrifugation

at 2,000 X g for 5 min and washed twice with cold water and once with 50 mM imidazole-HC1, pH 7.0. A 50% cell suspension (wet weight/volume) in 50 mM imidazole-HC1, pH 7.0, was passed through a French pressure cell (1,250 Kp/cm2). The supernatant obtained by centrifugation of the cell homogenate at 27,000 X g for 2 min is referred to as crude extract.

Step 2. Polyethyleneimine Fractionation-2% (v/v) polyethylene- imine solution in 50 mM imidazole-HC1, pH 7.0, was added to the crude extract, which was constantly stirred and kept in an ice bath. The resulting precipitate was removed by centrifugation for 30 min at 27,000 X g at 4 "C.

Step 3. Phosphorylation of Neutral Trehalase in the Supernatant after Polyethyleneimine Treatment-The phosphorylation of neutral trehalase by endogenous protein kinase of the extract was carried out according to Londesborough and Varimo (5). The phosphorylation reaction was stopped by transferring the enzyme solution to an ice bath. A protease inhibitor mixture containing antipain, bestatin, chymostatin, €364, leupeptin (5 mg/ml each), benzamidinium hydro- chloride, Trasylol (10 mg/ml each), pepstatin (20 mg/ml), phenyl- methylsulfonyl fluoride (80 mg/ml) was added to a final concentration of 2% (v/v). All subsequent chromatographic procedures, except the hydrophobic interaction chromatography, were done in the presence of 2% (v/v) of this protease inhibitor mixture. The extract was dialyzed at 4 "C overnight against 50 mM imidazole-HC1, pH 6.8.

Step 4. Ion Exchange Chromatography-The dialysate was applied to a TSK-DEAE column equilibrated with 50 mM imidazole-HC1, pH 6.8. The column was washed with 54 mM KC1 in 50 mM imidazole- HCl, pH 6.8, and the enzyme activity was eluted using a 54-300 mM KC1 gradient in equilibration buffer.

Step 5. Hydrophobic Interaction Chromatography Using the High Performance Liquid Chromatography Column Spherogel TSK-phenyl SPW-To the peak fractions of the previous step (volume, 120 ml), solid ammonium sulfate was added to a final concentration of 140 g/ liter. Precipitated material was removed by 15-min centrifugation at 27,000 X g, 4 "C. The soluble proteins were then bound to a Spherogel TSK-phenyl 5PW column (2.15 X 15 cm) equilibrated with 140 g/ liter ammonium sulfate in 50 mM imidazole-HC1, pH 7.0. The column was washed with 105 g/liter ammonium sulfate in 50 mM imidazole- HC1, pH 7.0. The enzyme activity was eluted by a decreasing ammo- nium sulfate (105 g/liter prepared in 50 mM imidazole-HC1, pH 7.0) gradient against distilled water. Active fractions were pooled and immediately buffer exchanged on Sephadex G-25 (PD-10 columns) against 0.5 mM imidazole-HC1, pH 7.0, containing the protease inhib- itors (2%, v/v).

Step 6. Pdy(A)-Sepharose CL-43-The desalted eluate was loaded on a poly(A)-Sepharose CL-4B column (25 X 41 mm) equilibrated with 0.5 mM imidazole-HC1, pH 7.0. Neutral trehalase could be desorbed by applying an increasing gradient of imidazole-HC1, pH 7.0 (0.5-50 mM) to the column. The poly(A) chromatography was repeated twice. Each time, eluted fractions could be rebound to poly(A)-Sepharose by diluting with ice-cold distilled water. Enzymat- ically active fractions were pooled and concentrated by ultrafiltration using Centricon 30 (Amicon, Witten, FRG). Glycerol was added to a final concentration of 20% (v/v).

' The abbreviations used are: SDS, sodium dodecyl sulfate; MES, 4-morpholineethanesulfonic acid HEPES, 4-(2-hydroxyethyl)-l-pi- perazineethanesulfonic acid EGTA, [ethylenebis(oxyethylene- nitri1o)ltetraacetic acid.

Step 7. Preparative Electrophoresis-After preparative electropho- resis on a nondenaturing polyacrylamide gel, neutral trehalase was localized in the gel by activity staining as described above. The activity-containing gel piece was excised and gently homogenized in 0.2 M imidazole-HC1, pH 6.2, while cooled in an ice bath. The gel suspension was centrifuged at 17,000 X g for 15 min. The supernatant was concentrated by Ultrafiltration and made 2% in the protease inhibitor mixture.

RESULTS

During the initial course of this work, we observed a pro- found proteinase sensitivity of neutral trehalase when par- tially purified from commercial bakers' yeast and Saccharo- myces cereuisiae M1 (17) or X2180B. To avoid this problem, the yeast strain ABYS1, which is devoid of the four major proteinases (proteinases A and B, carboxypeptidases Y and S ) and which was constructed and characterized by Achstetter et al. (9), was used for the purification of neutral trehalase. In addition, the enzyme was phosphorylated during an early stage of the purification since the phosphorylated form of the enzyme turned out to be more stable than the nonphosphor- ylated one. Purification was started from a stationary cell culture (18-24 h), at which growth phase the specific activity of neutral trehalase was found to be highest irrespective of whether the enzyme was phosphorylated or not. The final purification procedure is summarized in Table I. Details on the purification steps are given under "Materials and Meth- ods" and in (18).

On SDS-polyacrylamide gel electrophoresis, the purified enzyme exhibited one band corresponding to a molecular mass of approximately 80,000 Da (Fig. 1). From the electrophoretic mobility of the enzyme on polyacrylamide gels under nonde- naturing conditions, a molecular mass of native neutral tre- halase a of 160,000 Da was calculated. We conclude that the native enzyme with a molecular mass of 160,000 Da consists of two subunits of a molecular mass of 80,000 Da each. Comparison of the migration of purified native neutral tre- halase a with reference proteins on an Amphoiine-containing isoelectric focusing PhastGel revealed an isoelectric point of 4.7 (Fig. 2). Dependence of the activity of the purified enzyme on the pH is shown in Fig. 3. Maximal activity was observed at pH 6.8 when assayed in buffers containing 50 mM each of acetate, MES, and HEPES adjusted to various pH values with NaOH. When assayed in 50 mM imidazole-HC1 (the buffer used for the routine assays at pH 7.0), maximal activity was obtained at pH 7.0. The pH optimum at 6.8-7.0 therefore justifies the designation of the enzyme as neutral trehalase.

From a Lineweaver-Burk plot, 34.5 mM trehalose was cal- culated for the apparent Michaelis constant, i.e. the substrate concentration required for half-maximal activity. The maxi- mal rate of activity observed was 111 unitsjmg of protein. Glucose formation by purified neutral trehalase from cello- biose, lactose, maltose, mellibiose, sucrose, and raffinose as- sayed at a 100 mM concentration each at pH 7.0 was less than 1 unit/mg of protein.

A polyclonal antiserum from rabbits prepared against the purified neutral trehalase a does not neutralize the enzyme activity. The antibodies did, however, react with the enzyme as indicated by a complete disappearance of trehalase activity in the presence of protein A-Sepharose (Fig. 4). Purified acid trehalase (6) does not cross-react with the antiserum because acid trehalase activity was unaffected by the addition of anti- neutral trehalase antiserum and protein A-Sepharose (Fig. 4). Western blot analysis of a total cytosolic yeast lysate also showed specificity of the antiserum raised against neutral trehalase (lower part of Fig. 5). With the antiserum raised against purified neutral trehalase, the intracellular compart-

Neutral Trehalase from the Yeast AB YSl Mutant TABLE I

Purification of neutral trehalase from the veast ABYSI mutant

17585

Step Volume Total activity 2:::; Purification Yield

Crude extract rnl units unitslmg -fold %

220 49.5 0.0058 13.5

Supernatant after polyethyleneimine treatment 205 78.6 0.0161 21.5

I n vitro phosphorylation of polyethyleneimine supernatant 215 366 0.0756 1 100

Dialysate 220 403 0.1188 1.6 110

Ultropac T TSK-DEAE chromatography 120 294 0.56 7.4 73

TSK-phenyl5PW chromatography 30 146 3.0 40 40

Sephadex G-25 chromatography 60 219 7.6 101 60

Poly(A)-Sepharose CL-4B chromatography I I1 I11

48 145 20.3 269 40 38.4 123 30.2 400 34 43 111 44.5 584 30

Preparative electrophoresis 0.71 6.7 114 1500 2

lane 1 lane 2 - 180 kDa

c. 116 kDa - 84 kDa - 5 8 kDa c- 48.5 kDa

i I c- 36.5 kDa .a - 26.6 kDa

FIG. 1. SDS-polyacrylamide gel electrophoresis of neutral trehalase. 5 pg of purified neutral trehalase from preparative elec- trophoresis is shown as in Table I. Marker proteins (in kDa): a-2- macroglobulin (180), @-galactosidase (116), fructose-6-phosphate ki- nase (84), pyruvate kinase (58), fumarase (48.5), lactate dehydrogen- ase (36.5), triosephosphate isomerase (26.6).

mentation of neutral trehalase was also examined. As shown in the lower part of Fig, 5, an immunoresponse to the anti- serum is only found with the pure enzyme (lune 2) and with the cytosolic fraction of a spheroplast lysate ( l a n e 3) but not with the vacuolar lysate (lane 4 ) .

M$+, Mn2+, and Ca2+ at a 1.5 mM concentration have no effect on the activity of purified neutral trehalase. However, EDTA or EGTA at 0.1 mM decreases the activity of the enzyme by 50%. This is in accordance with Londesborough and Varimo (5), who demonstrated that 1 mM EDTA inhibited neutral trehalase completely, allowing the measurement of the activity of acid trehalase that is not affected by EDTA. Neutral trehalase a is sensitive to sulfhydryl reagents (50% inhibition by 0.01 mM HgC12 or 0.02 mM p-hydroxymercuri- benzoate) but not to heavy metal complexing reagents (dipyr- idyl, cupferron, o-phenanthroline).

We have shown in a previous paper (21) that polycations such as polyethyleneimine, poly-L-lysine-L-phenylalanine, or histones activate neutral trehalase in crude extracts from yeast. The activation by polycations was found to be the result

7.0

neutral I a 5.0

4.0 - 3.0 -

I I I I

0 1 2 3 4

distance from anode (cm)

FIG. 2. Isoelectric focusing of neutral trehalase. For details, see “Materials and Methods.” Calculated isoelectric point, 4.7. Iso- electric points for reference proteins are: a, amyloglucosidase (3.55); b, trypsin inhibitor (4.55); c, @-lactoglobulin A (5.13); d, carbonic anhydrase B from bovine erythrocytes (5.85); e, carbonic anhydrase B from human erythrocytes (6.57).

of precipitation of RNA and polyphosphates, which both inhibit neutral trehalase. Addition of polycations (polyethyl- eneimine) in excess to what is needed for a complete precipi- tation of RNA and polyphosphates, however, inhibits neutral trehalase (21). Accordingly, when polycations are added to the purified neutral trehalase a, no activation but only inhi- bition of the enzyme is observed. For example, 0.025 mM polyethyleneimine causes a 2.5-4-fold activation of neutral trehalase in a crude extract (21), whereas the same concen- tration of polyethyleneimine inhibits the purified neutral trehalase more than 99%. Not only polycations but also polyanions inhibit neutral trehalase. This inhibition of puri- fied neutral trehalase by polyanions is very similar to the inhibition of the enzyme in a crude extract (21). Half of the activity was inhibited by 1 mM ATP, which corresponds to the intracellular concentration of ATP and might therefore be of regulatory significance (21, 22).

Inactivation of purified phosphorylated neutral trehalase

17586 Neutral Trehalase from the Yeast AB YSl Mutant

4 5 6 7 8 9

PH

FIG. 3. Dependence of activity of purified neutral trehalase on pH. A, buffer mixture containing acetate, MES, and HEPES, 50 mM each; A, 50 mM imidazole-HC1 buffer (for details see “Materials and Methods”).

4 c - 0.4

- 0.3 - 0.2

- 0.1

0 -1 (1 0 100 200 300 400

serum ( p )

FIG. 4. Immunoprecipitation of acid trehalase and neutral trehalase from crude extract of ABYSl yeast. 100 pl of crude extract (see “Purification of Neutral Trehalase” under “Materials and Methods”) was incubated for 2 h at 25 “C with antiserum as indicated below. After centrifugation (10 min, 10,000 X g, 4 “C), supernatants were assayed for neutral trehalase at pH 7.0 (see “Ma- terials and Methods”) and for acid trehalase at pH 4.5 (6). Neutral trehalase was incubated without (A) and with (A) 10 mg of swollen protein A-Sepharose. Acid trehalase was incubated without (0) and with (4) 10 mg of protein A-Sepharose.

upon incubation with alkaline phosphatase from Escherichia coli is shown in the left panel of Fig. 6. Reactivation of the phosphatase-treated trehalase by incubation with protein ki- nase from beef heart in the presence of ATP and cyclic AMP is shown in the right panel of Fig. 6. The maximal activity obtained after reactivation is about 35 units/ml, whereas prior to dephosphorylation (see left panel of Fig. 6), the enzyme exhibited only 27 units/ml. The higher specific catalytic ac- tivity after extensive phosphorylation of the enzyme prepa- ration indicates that the purified enzyme used for the exper- iment shown in Fig. 6 had not been phosphorylated com-

lane1 lane 2 lane 3 lane 4

100 kDa -c

116 kDa -W

84 kDa + 5 0 kDa -

40.5 kDa -c

36.5 kDa - 26.6 kDa -I

”. ”

100 kDa + 116 kDa

8 4 kDa -W

5 0 kDa -+ 40 .5 kDa -I

36.5 kDa -I

26.6 kDa +

FIG. 5. Intracellular compartmentation of neutral treha- lase. Molecular mass markers (see legend to Fig. 2), lane 1 ; 1 pg of purified neutral trehalase, lane 2; 100 pg of protein of a cytosolic spheroplast lysate, lune 3; 100 pg of protein of vacuolar lysate, lane 4 were submitted to SDS-polyacrylamide gel electrophoresis, blotted on nitrocellulose, and reversibly stained with Ponceau S (upperpanel) and immunostained (lower panel) according to (19) with antiserum raised against purified neutral trehalase. Spheroplasts and vacuoles were prepared from ABYSl yeast according to Diirr et al. (20).

pletely, i.e. was not maximally active. Reactivation, i.e. phosphorylation of the dephosphorylated enzyme, was done with [y3’P]ATP. The incorporation of radioactivity into the enzyme protein extracted from SDS-polyacrylamide gel elec- trophoresis is shown in Fig. 6. A calculation of the amount of incorporated phosphate yielded 0.85 mol of phosphate/mol subunit (molecular mass, 80,000 Da). After phosphorylation of purified neutral trehalase with [y3*P]ATP and M g + in the presence of the catalytic subunit of protein kinase from beef heart and hydrolysis of the electrophoretically separated 80-kDa band, the phosphorylated amino acid residue was identified as phosphoserine using thin-layer chromatography as described by Hunter and Sefton (23).

DISCUSSION

In order to avoid proteolytic breakdown of neutral trehalase during the isolation procedure, the ABYSl mutant of S. cereuisiae X2180, deficient in the four major yeast proteases (9), was used for the purification of the enzyme. Furthermore, a mixture of protease inhibitors was added after the polyeth- yleneimine precipitation step. Finally, the phosphorylated form of the enzyme appeared to be more resistant to proteol- ysis during the purification procedure. Therefore, early during the purification protocol (after polyethyleneimine fractiona- tion), the crude enzyme was phosphorylated. At this step, enough endogenous cyclic AMP-dependent protein kinase was present to enable phosphorylation following addition of ATP, M P , and cyclic AMP. The phosphorylated enzyme can be dephosphorylated after its isolation by the use of commer-

FIG. 6. Dephosphorylation (left panel) and cyclic ANIP-dependent phosphorylation (rightpanel) of pu- rified neutral trehalase. Left, purified phosphorylated trehalase (27 units/ml) was incubated with 5 mg/ml alkaline phosphatase from E. coli (Sigma) at pH 7.0 and 30 "C (total volume, 40 rl). Right, purified dephosphorylated neutral tre- halase (activity after phosphorylation was 35 units/ml) was incubated (total volume, 40 p l ) with 62.5 units of catalytic subunit of beef heart protein kinase (Sigma) in the presence of 12.5 mM M e , 2.6 mM ATP, and 50 mg/ml dithi- othreitol at 0 "C. Samples were assayed for neutral trehalase catalytic activity (0) and for incorporated 32P radioactiv- ity after extraction of the 80-kDa band from SDS-polyacrylamide gels (0).

Neutral Trehalase from the Yeast ABYSl Mutant 17587

' O I 30

lncubatkm at J0"C (mln)

- 600

-w)o

- 400

k - 300

R - 200

1 loo cially available alkaline phosphatase from E. coli. Rephos- phorylation is then possible with the commercially available catalytic subunit of protein kinase from beef heart (cf. Fig. 6).

Polyethyleneimine treatment of the crude extract, which increases the specific activity of neutral trehalase about 3- fold (cf. Table I), was shown in a previous publication to precipitate RNA, inorganic polyphosphate, and protein, which inhibit neutral trehalase (18, 21). A biologically significant regulatory function of the inhibition of neutral trehalase by RNA and/or inorganic polyphosphate was discussed previ- ously (18, 21). Further characterization of the RNA or poly- phosphate species, which might control activity of neutral trehalase in uiuo, can now be performed with the trehalase available in a pure for:m.

The pH optimum of' the purified enzyme (pH 6.8-7.0) is in good agreement with the value obtained previously (pH 6.7) of Londesborough and Varimo (5) determined with a partially purified enzyme. The pH optimum corresponds to the average pH value of the yeast cytosol, as determined with the 32P NMR method (24, 25).

The apparent K , value of the enzyme with trehalose as substrate was found to be 34.5 mM. van Asche and Carlier (26) observed a similar K , value of 55 mM. Other authors published K, values around 5-10 mM measured with partially purified and probably only partially phosphorylated enzyme preparations (5,27, 28). The high concentrations of trehalose observed in yeast under certain conditions (up to 23% of the dry weight or 0.7 mol/liter of the soluble space of the yeast cells (29, 30)) may explain why a trehalase with a high K , value may in fact be of physiological significance.

The purified neutnsl trehalase is not bound by Con A Sepharose and does n'ot positively react in the orcinol assay for carbohydrate after polyacrylamide gel electrophoresis (data not shown).

The molecular mass of the subunit was calculated from SDS-polyacrylamide gel electrophoresis to be 80,000 Da. This corresponds well with values found by Uno et al. (7) and Dellamora-Ortiz et al. (27). The molecular mass of the native enzyme was found by nondenaturing polyacrylamide gel elec- trophoresis to be 160,0100 Da. This is in contrast to a molecular mass of 320,000 Da calculated by Uno et al. (7) from gel filtration of a partially purified preparation.

The polyclonal rabbit antiserum prepared against the na- tive phosphorylated neutral trehalase a as antigen does not inhibit or precipitate the purified neutral trehalase. However, in the presence of protein A-Sepharose, neutral trehalase is precipitated (cf. Fig. 4). Purified acid trehalase is not precip- itated under these conditions. On the other hand, an anti- serum prepared against purified acid trehalase (6) does not affect neutral trehalase and does not cross-react on Western blots with neutral trehalase (18). In addition to the different intracellular compartmentations (cf. Fig. 5), the immunolog- ical unrelatedness underlies that the two trehalase isoenzymes are encoded by different genes. The antisera are now being used for selection of clones and to isolate and sequence the respective genes. Neutral trehalase is sensitive to sulfhydryl reagents, whereas acid trehalase is not (6). This is another characteristic difference between the activities of acid and neutral trehalases, in addition to the different sensitivity toward EDTA mentioned above.

Phosphorylation with endogenous cyclic AMP-dependent protein kinase or with the cyclic AMP-dependent protein kinase from beef heart activates neutral trehalase. The phos- phorylation of the purified enzyme takes place at serine residues to an extent of 0.85 ( i e . about 1) phosphate per subunit. Dephosphorylation of the phosphorylated neutral trehalase can be achieved with crude yeast extract (31) or with commercially available alkaline phosphatase from E. coli (Fig. 6) but not with the purified phosphoprotein phosphatase from yeast, which dephosphorylates phosphorylated fructose- 1,6-bisphosphatase (31,32). Specific activity of the phosphor- ylated neutral trehalase a is 114 units/mg of protein; the dephosphorylated trehalase b exhibits less than 9% of the specific activity of the fully phosphorylated enzyme. We do not know at present if the remaining small activity of the dephosphorylated enzyme represents indeed a property of neutral trehalase b or if it is the result of contamination of enzyme b with enzyme a.

Panek et al. (33) observed that trehalose-6-phosphatase synthase is inactivated by cyclic AMP-dependent phos- phorylation and reactivated by dephosphorylation. As shown by previous workers (2, 5, 8) and in this paper, the trehalose- degrading neutral trehalase is activated by cyclic AMP-de- pendent phosphorylation and inactivated by dephosphoryla-

17588 Neutral Trehalase from the Yeast ABYSl Mutant

tion. This represents another example for the reciprocal reg- ulation of degradation and synthesis of a polymer by phos- phorylation/dephosphorylation of the respective synthesizing and degrading enzymes (34).

Acknowledgments-We are grateful to Dr. Matthias Muller for a critical reading of the manuscript, to Hanne Muller for preparation of cytosolic and vacuolar lysates from ABYSl spheroplasts, and to Wolfgang Fritz and Ulrike Eitel-App for help with the figures and for help with the manuscript.

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