Dec 25, 2017 - The enzyme is inhibited by pepstatin, diazoacetyl-DL- norleucine methyl ester and by 1,2-epoxy-3-(4-nitro- phenoxy) propane. Proteinase A ...
‘rHE JOURNALOF BIOLOGICAL CHEMISTRY
Val. 255. N o 24. lmue of December 25, pp. 12087-12093. 19M Prtnted m U S A .
Purification and Propertiesof Proteinase A from Yeast* (Received for publication, June 2, 1980)
Franz Meussdoerffer, Paolo Tortora, and Helmut Holzer From the Biochemisches Institut der Uniuersitat. D-7800Freiburg andGSF-Abteilung fur Enzymchemie, 0-8042 Neuherberg bei Munchen, West Germany
Proteinase A (EC 3.4.23.6) was purified from commercial bakers’yeast in five steps, including hydrophobic chromatography and affinity chromatography. After the last step the enzyme appeared homogeneous on polyacrylamide gel electrophoresis and in the analytical ultracentrifuge. A molecular weight of 41,500 was determined for proteinaseA. The amino acidcomposition includes 43% polar residues and 12% aromatic amino acids. ProteinaseA is a glycoprotein containing 7.5% mannose and 1% of glucosamine and galactosamine. The temperature and the pH stability of the enzyme have been determined. AtpH 6, the proteinase exhibits a remarkable stability even in 6 M urea. Proteinase A splits hemoglobin withan optimum atpH 3.0 and casein and azocasein with an optimum at pH 6.0. The enzyme is inhibited by pepstatin, diazoacetyl-DLnorleucinemethyl ester andby 1,2-epoxy-3-(4-nitrophenoxy) propane.
Yeast Commercial bakers’ yeast from BAKO Gesellschaft eGmbH, Freiburg, wasused for the purification of proteinase A and for the preparation of the proteinase A inhibitor, IA3. Enzyme Assay Proteinase A activity was determined with acid-denatured hemoglobin as described by Saheki et al. (9). One unit is defined as the amount of enzyme which gives an absorbance at 660 nm equivalent to 1 pg of tyrosine in one min at 25’C. The degradation of casein and azocasein by proteinase A was measured in a total volume of 0.6 ml with a final substrate concentration of 2 mg/ml in citrate/phosphate buffer (McIlvaine buffer) of constant ionic strength and the appropriate pH. A blank containing the same reagents as the incubation mixture, but without proteinase A was tested for each sample. The reaction mixtures were incubated for 40 min a t 25°C. The reaction was stopped by the addition of 0.5 mlof trichloroacetic acid (10%) and the suspension was allowed to settle for another 15 min. After centrifugation, the absorbance in the supernatant was measured at 280 nm in the case of casein and at 350 nm in the case of azocasein. ProteinaseB activity was measured as described by Saheki and Holzer (10) with azocoll as substrate. Activity was expressed as described earlier (11).The assay of carboxypeptidase Y and definition of units are the same as described by Matern et al. (12). Aminopeptidase I activity was determined according to Metz and Rohm (13) with leucine-p-nitroanilide. The activity is expressed as described by these authors.
Proteinase A (EC 3.4.23.6)was among the first enzymes to be described in yeast (1-3). It was partially purified by Hata et al. (4), Lenneyand Dalbec (5), and Betz et al. (6). The introductionofaffinitychromatography yielded an almost pure enzyme (7). During the course of this work a preliminary report on the Protein Determination preparation of a similar enzyme has appeared (8). However, Protein was determined by the method of Lowry et al. (14) with little is known about the propertiesofpure proteinase A. Therefore we developed a simple procedure for the large scale crystalline bovine serum albumin as standard. preparation of homogeneous proteinase A. Characteristics of Electrophoretic Procedures the pure enzyme are reported. These features differ in some Disc electrophoresis was carried out in polyacrylamide gels with respect from properties attributed to proteinase A in earlier 10%and 15%acrylamide concentration according to Davis (15) at pH reports. 9, and at pH 7.5 using gel system 6 of Maurer (16). Gels were run at MATERIALS AND METHODS
Chemicals CH-Sepharose 4B, DEAE-Sephacel, phenyl-Sepharose and a low molecular weight standard protein kit were obtained from Deutsche Pharmacia. Casein, PhCH2So?F,’and hemoglobin were from Merck, Darmstadt; azocasein, EDC, N-hydroxysuccinimide, and DAN were from Sigma. Carrier ampholytes, pH 3.5 to 5.0, were from LKB. EPNP was from Calbiochem. Mercaptoethanesulfonic acid (Pierce) was from Gunther Karl OHG. Pepstatin was from Peptide Institute Inc., Osaka, Japan. Ethylene glyml was a gift from Farbwerke Hoechst. All other chemicals were of reagent grade obtained from Roth, Karlsruhe. ~~
* This work was supported by Sonderforschungsbereich 46 (Deutsche Forschungsgemeinschaft) and Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ’ The abbreviations used are: PhCHzSOzF, phenylmethylsulfonyl fluoride; DAN, diazoacetyl-DL-norleucinemethyl ester; EDC, l-ethyl3-(dimethyla~ninopropyl)carbodiimide; EPNP, 1,2-epoxy-3-(4-nitrophenoxy)propane; SDS, sodium dodecyl sulfate; IA3,specific proteinase A inhibitor from yeast (9).
4’C with 2.5 mA/gel. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was performed in slab gels composed of a linear gradient from 10 to 30% acrylamide. The SDS/Tris/HCI buffer system of King and Laemmli (17) was used. Gels were pre-equilibrated with the buffer by electrophoresis at 70 V for 1 h. After the samples were layered on the gel, a voltage of 300 V was applied for 10 min. When the proteins had entered the gel, the voltage was lowered to 120 V and theelectrophoresis was continued for 15 h at 12°C. The gels were stained with Coomassie Blue as described by Mason et al. (18). Carbohydrate staining was performed by the thymol-sulfuric acid method of Racusen (19). Analytical electrofocusing in polyacrylamide gels was carried out as described byWrigley (20) in 7.5% gels containing 1% carrier ampholyte a t 4°C for 2 h at 400 V. Gels were cut into 2-mm slices, which were disrupted mechanically and eluted with 750p1of water. The pH and proteinase A activity were measured in these extracts. Amino Acid Analysis Protein samples (200pg each) were hydrolyzed with 0.5 ml of constant boiling HCI in sealed, evacuated glass tubes or with 0.5 ml of 3N mercaptoethanesulfonic acid according to Penke et al. (21) at 105°C for different times. The hydrolysates were analyzed in a Biotronic amino acid analyzer LC 6OOO. Cysteic acidwas determined after performic acid oxidation as described by Moore (22).
12087
Purification of ProteinaseYeast A from
12088
Carbohydrate Analysis Total neutral sugar contentof proteinase A was determined by the orcinol-sulfuricacid method (23) withmannose as standard. The hexoses were identifiedby gas chromatography according to Lenhardt and Winzler (24). Total hexosamine content of proteinaseA was determined according to Gatt and Bermann(25) withglucosamine as standard. Amino sugars were identified after hydrolysis in 4 N HC1 for 4 h using an amino acid analyzer, Ultracentrifugation Analytical ultracentrifugation was performed a t 12,000 rpm at 4°C in a six-channel centerpiece accordingto Yphantis(26) in a Beckman Model E ultracentrifuge. The absorption was recorded with aphotoelectric scanner.
Step 5. Affinity Chromatography-The active fractions from Step 4 were pooled and diluted to three times their volume with 0.1 M potassium phosphate buffer, pH 6.0.HgCL was addedto a final concentration of 1 mM and portions containing approximately 30,000 units of proteinase A were applied to the affinity gel. The affinity column was washed with 200 ml of potassium phosphate buffer, pH 6.0, containing 2 M NaC1. Proteinase A was eluted from the gel with 6.5 M urea in 0.1 M potassium phosphate buffer, pH 6.0. Immediately afterelution,theeluate was diluted 3-fold with 0.1 M potassium phosphate buffer, pH 6.0, Then the eluate was dialyzed against 20 liters of water for several hours. The water was replaced by 50 mM ammonium acetate buffer, pH 5.5, and dialysis was continued overnight. The resulting solution of proteinase A was concentrated by ultrafiltration and stored in 500-pl portions a t -23°C. RESULTS
Purification of the InhibitorI A 3
Purification of Proteinase A-Proteinase A was purified The specific proteinase A inhibitor IA3was prepared as described from commercial bakers' yeast. The proteolytic activity in the by Nunez de Castro and Holzer (27) by sulfopropyl (SP)-Sephadex Cby incubation at acidic pH as 25 and QAE-Sephadex (quaternary aminoethyl Sephadex) A-25 ion homogenatewasactivated exchange chromatography, The resultinginhibitor had a specific introduced by Lenney andDalbec (5) andused by Hayashi et activity of2100 units/mg and was homogeneous as estimated by al. (28). After ammonium sulfate precipitation, the proteinase polyacrylamide gel electrophoresis. was chromatographed on phenyl-Sepharose and DEAE-Sephacel. The eluate from theDEAE-Sephacel column was Immobilization of the Proteinase A Inhibitor treated withHgC12to prevent degradationof the immobilized The lyophilized inhibitor was equilibrated with 0.2 M triethanola- inhibitor IA3 during affinity the chromatography by proteinase mine/HCl buffer, pH 8.2, by gel filtration. CH-Sepharose 4B (5 g) B and carboxypeptidase Y (9). Portions of the preparation was washed with 500 ml of IM NaCl and with 1 liter of water. N containing about 30,000 units of proteinase A were applied to Hydroxysuccinimide (0.5 g) and EDC (0.75 g) were dissolved in 100 the affinity column. This quantity of proteinase A was comml of water and the swollen gel was added to this solution. The suspension was adjusted to pH5.0 by adding IN HCI and kept at this pletely absorbed on the gel. The enzyme was eluted with 6.5 pH for 120 min by adding 1 N NaOH. After 2 h the gel was washed M urea and diluted 3-fold immediately after elution. It was to 20 ml of asolution with 1 liter of ice-cold waterandadded important to keep the pH at 6.0 during elution and dilution containing 0.5 mg of inhibitor/ml. The suspension was shaken over- until the urea was removed by dialysis. After concentration night a t 4°C and then washed successively with 1 liter of 0.5 M Tris/ by Ultrafiltration, the enzyme was stored at -23OC. At this HCI buffer, pH 8.0, 0.5 liter of water, and 1 liter of 0.05 M sodium acetate buffer, pH 4.5. The gel was stored in the acetate buffer after temperature, the enzyme was stable for several weeks, but repeated thawing and freezing, o r prolonged incubation at addition of 200 pg of sodium azide. Approximately 7 mg of inhibitor room temperature, led to inactivation. Proteinase A lostabout were bound to 5 g of gel (dry weight). 50% of its activity afterlyophilization. Purification of Proteinase A The proteinase A preparation obtained by the procedure All operations were carried out at 5°C except where stated other- described here had a specific activity of 3065 units/mg. The preparation containedless than 40 milliunits/mg of proteinase tothe wise. Steps 1 and 2correspondwithslightmodifications procedure described by Betz et al. (6). B, less than 17 milliunits/mg of carboxypeptidase Y and less Step 1. Acid Actiuation--1.2 kg of commercial bakers' yeast were than 13 milliunits/mg of aminopeptidase I. The results of a suspended in 1,200 ml of cold water and passed six times through a typical purification are summarized in Table I. Manton Gaulin homogenizer (Manton Gaulin Mfg., Everett, Mass.) Homogeneity-The proteinase A purified in the described a t 500 kg/cm'. After every run, the suspension was cooled again to manner yielded asingle band afterpolyacrylamide gel electro10°C. The homogenate was centrifuged a t 27,000 X g for 30 min and the pH of the supernatant was adjusted to pH 5.0 by adding glacial phoresis at pH 7.5 in gels containing 10% and 15%acrylamide, acetic acid. Streptomycin sulfate and penicillin (60 mg each) were respectively (Fig. 1, A and B).A single band was also found added and thesuspension was kept at 25°C for 20 h. after electrophoresisat pH 9 in gels containing 10%acrylamide Step 2. Ammonium Sulfate Precipitation-Solid ammonium sulwhen the enzymewas inhibited by pepstatin prior to electrofate was added to the activated crude extract 95% to saturation. The phoresis (Fig. 1D). A single band at the sameposition as the suspension was stirred for 2 h and then centrifuged a t 27,000 X g for band observed after staining with Coomassie Blue was also 30 min. The precipitate was dissolved in 400 ml of water and dialyzed found when a gel run under these conditions was stained for against 5 liters of water overnight. Step 3. Batchwise Chromatography onPhenyl-Sepharose-500 ml carbohydrate withthymol-sulfuric acid according to Racusen of phenyl-Sepharose were added to thedialysate and the suspension (19). However, when proteinase A was subjected to electrowas mechanically stirred for 30 min. Then the gel was transferred to a sintered glass filter and washed successively with 400 ml of 0.1 M potassium phosphate buffer, pH 6.0, containing 2 M NaCl and with 360 ml of thesame buffer containing 50% (v/v)ethylene glycol. Proteinase A was eluted from the gel by washing the phenyl-Sepharose on a sintered glass filter with a total volume of 500 ml of 0.1 M potassium phosphate buffer, pH 6.0, containing 85% (v/v) ethylene glycol. The eluate was dialyzed overnight against 15 liters of water. Step 4.DEAE-Sephacel Chromatography-120 ml of DEAE-Sephacel were equilibrated overnight with 1 M potassium phosphate buffer, pH 7.0, transferred into a chromatography column (5 X 6 cm) and washed extensively with 0.1 M potassium phosphate buffer, pH 7.0. The dialysate from step 3 was adjusted to pH 7.0 and applied to the ion exchange column. The column was washed with 300 ml of potassium phosphate buffer, pH 7.0, and eluted with 1.5 liters of a linear gradient from 0 to 0.8 M NaCl in 0.1 M potassium phosphate buffer, pH 7.0. A flow rate of 70 ml/h was maintained and fractions of 20 ml were collected.
TABLE I Purification ofproteinase A from bakers' yeast Step
Homogenization Activation Ammonium sulfate precipitation Phenyl-Sepharose chromatography DEAE-Sephacel chromatography Affinity chromatograPhY
tivity
Specific Purifiact.ivitv canon
units/mg
ml
unm
1,780 1,770 540
158,100 264,000 237,900
1,030
124,000 659
58
-fold
1 1.8 47 20.7 42 43.4
111,300 1,380 540 8
2 32
37,700 3,065
12 96.4
Yield '7,
100
90
Purification of Proteinase A from Yenst
12089
c
I p.
. .. I
A
B
C
d o
-
D
5
FIG.1. Disc electrophoresis of proteinase A. 50 pg o f protein were applied to each gel. A , gel svstem 6 (pH 7.5) of lief 16, 10'r acrylamide concentration. R , gel svstem 6 (pH 7.5) o f Kef. 16. 15? acrvlamide concentration. C , gel svstem of Kef. 15 (pH 9.0), IOc; acrylarnide concentration. ProteinaseA was applied t o the gel without further treatment. I). gel system as under C, proteinase A was inhibited bv pepstatin prior t o the electrophoresis. 1.
phoresis at pH9 in the absence of pepstatin, a broad range of stained products was observed (Fig. IC). The proteinase A preparation also appeared homogeneous after electrophoresis in the presence of sodium dodecyl sulfate when the enzyme was boiled in the presence of SDS prior to the electrophoresis (Fig. 2A).However, multiple bands were observed when proteinase A was incubated in a solution containing 16 SDS for 30 min at room temperature and was then applied to the gel without boiling (Fig. 2R). The homogeneity of the proteinase A preparation was further shown by analytical ultracentrifugation in sedimentation-diffusionequilibrium.A linear dependence of the logarithmof the absorbance measured at 280 nm and the square of the distancefrom the rotation center at different protein concentrations indicated that only one species was present (Fig. 3). Molecular Weight of Proteinase A-The molecular weight of proteinase Awas determined froma comparison of its electrophoretic mobility in the SDS-gradient gel (10 to 30B acrylamide) with the mobilities of marker proteins (Fig. 2 , A and C ) . A value of 41,700 for the molecular weight was calculated. The molecular weight of proteinase A was also determined by sedimentation-diffusion equilibrium ultracentrifugation. Assuming a partial specific volume of proteinase A of 0.718 cm:'/g as calculated from the aminoacid and sugar composition of proteinase A according to Schachman(29) and Gibbons (30), an average molecular weight of 41,400 k 800 was calculated from measurements at4 different protein concentrations. AminoAcidAnalysis,IsoelectricPoint, a n d UV-spectrum-The amino acid composition of proteinase A was determined after hydrolysis with constant boiling HCI or with 3 N mercaptoethanesulfonic acid at 105OC. The results shown in Table I1 are average values obtained from three different preparations. As can be seen in Table 11, no significant differences in the amino acid composition of proteinase A determined after either hydrolysis method are found except in the case of t-yrosine. Therefore the content of tyrosine and tryptophan was determined spectrophotometrically by the
A
B
FIG.2, SDS electrophoresis of proteinase A in a gradient gel containing 10 to 30% acrylamide. A , proteinase A. T h e enzyme was hoiled in the SIX solution prior t o the electrophoresis. W. proteinase A. The enzvme was kept in 1'; SI>Ssolution for 30 rnin at 25°C and then applied to thegel without further treatment. C.marker proteins: 1. phosphorvlase h ( M , = 94,"): 2, albumin (M,= R7,~H)O);
3. ovalbumin ( M , = 43.000); 4. carbonic anhydrase ( M , = 3O.(XM)); soybean trypsin inhibitor ( M , = 20,IOO): 6. lactalhumin
5,
( M , = 14,400).
t" 20
0
00 N
a C
12
,
,
42
43
\\
1;:
44
r2, cm2 FIG.3. Ultracentrifugation of proteinase A. Conditions are descrihed under "Materials and Methods." A:,, was recorded with a photoelectric scanner. r = distance from the center of rotation.
method of Edelhoch (31).Proteinase A contains 12%aromatic amino acids and4 3 8 polar residues. The isoelectric point of proteinase A was measured after isoelectric focusing in polyacrylamide gels over a range from pH 3.5 to 5.0. An isoelectric point of 4.4 was determined from independent isoelectric focusing experiments with three different preparations of proteinase A.
Purification of Proteinase A from Yeast
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TABLEI1 Amino acid composition ofproteinase A Mercaptoethane-
HCI
sulfonic acid
Amino acid 24"
Aspartic acid asparagine Threonine Serine Glutamic acid glutamine Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Tryptophan 1/2Cystine Total: I'
72"
22"
70"
Average or extrapolated inteeer
l
residues/rnoleculeb
+
39.75
39.0
39.0
39.0
39
+
20.67 19.65 23.75
18.0 13.25 24.0
20.33 21.33 25.0
20.67 20.67 25.33
21" 21" 25
14.75 31.25 20.0 9.50 1.0 17.0 24.75 12.75 15.33 14.30 4.0 4.33 4' 5'
15.50 35.0 21.67 10.67 1.50 19.0 25.0 12.50 15.50 16.0 4.0 4.0
15.50 33.13 23.0 11.30 2.0 17.0 26.0 17.67 16.33 15.0 4.0 4.0 3.0
15.33 35.0 23.30 12.0 1.70 18.33 27.0 17.67 16.33 16.33 4.0 4.0 2.5
15 34 22 11 2 19" 26" 16' 16 15 4 4 4
