Int. J. Peptidehotein Res. 1 3 , 1 9 7 9 , 253-259 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)
P R O T E A S E B FROM S A C C H A R O M Y C E S C E R E V I S I A E Purification and Characterization Y. LOOZE, L. GILLET, M. DECONINCK, B. COUTEAUX, E. POLASTRO and J . LEONIS
Laboratoire de Chimie Genkrale I, Faculte des Sciences, Universite libre de Bruxelles, Belgium
Received 1 5 May, accepted for publication 10 July 1978 Protease B has been isolated f r o m Saccharomyces cerevisiae and purified in six steps as follows: autolysis o f the yeast cells, ammonium sulfate fractionation, activation o f the proteolytic enzymes, chromatography on DEAE-cellulose, chromatography on CM-cellulose and finally, a second chromatography on DEAE-cellulose. The preparation was shown t o be homogeneous on polyacrylamide gels in the absence as well as in the presence o f sodium dodecylsulfate. Furthermore, t h e molecular weight (43 000 daltons) and the isoelectric point (S.45) were in good agreement with earlier published values. The amino acid composition is reported. The absence o f disulfide bonds in protease B has to be outlined. The amino acid residues o f the protein have been found t o be folded nearly quantitatively (at least 80%) in a 0-conformation as deduced f r o m a circular dichroism study. Finally, the tryptophan residues (S molfmol protein) are largely buried in the hydrophobic core o f the enzyme. Key words: circular dichroism ;fluorescence; protease B; Saccharomyces cerevisiae.
Several intracellular proteolytic enzymes from to function quite well in denaturing media. prokaryotes and ascomycetes have been des- Furthermore, as a result of its amidase action, cribed and their possible role investigated this enzyme could be applied to the sequence analysis of peptides having amidated COOH(Holzer et al., 1975). From Saccharomyces cerevisiae, two en- terminal groups (Hayashi et al., 1973). Proteases A and B, on the other hand, dopeptidases, namely proteinases A and B, as well as two exopeptidases: a carboxypeptidase seem to be involved in the regulation of several (carboxypeptidase Y, earlier known as protease enzyme activities including tryptophan synC) and an aminopeptidase have been purified thetase, fructose 1,6-bisphosphatase and chitin (Lenney & Dalbec, 1967; Katsunuma el al., synthetase (Tsai et al., 1973; Schott & Holzer, 1972; Hayashi et al., 1973; Metz & Rhom, 1974; Saheki & Holzer, 1974; Manney, 1968; Molano & Gancedo, 1974; Cabib & Ulane, 1976). In sequence studies carboxypeptidase Y 1 9 7 3 ~ ) . had greater advantages compared with the The proteases have been located in the pancreatic-type carboxypeptidases. This is due yeast vacuoles (Lenney, 1973). Thus, an to its broader specificity and also to its ability important question which is not yet clearly 0367-8377/79/030253-07
$02.00/0 0 1979 Munksgaard, Copenhagen
253
Y. LOOZE ET AL.
understood, is whether the control of the proteolytic attack in vivo, the spacial separation of proteases and their potential substrates is indeed the rule. Also, up to now, little attention has been given to the molecular structure of the enzymes. It is the purpose of this communication to approach this question by means of circular dichroism and fluorescence studies. Protease B was chosen. Also, the enzyme being very labile, we felt it would be useful to reinvestigate its purification.
I
ELUTION VOLUME l m l l ~
FIGURE 1 Chromatogaphy of the preparation of protease B Bovine serum albumin (lot 46C-0253), soybean from step 4 on a CM-cellulose column (2.5 X 40cm) trypsin inhibitor (lot 94C-8 190), bovine pan- equilibrated with 0.1 M KH,PO, at pH 4.5. After fixing of the proteins, the column was washed with creatic ribonuclease (lot 56C-8020), dith- 1 litre 0.1 M KH,PO, at pH 4.5. A linear gradient from iothreitol (lot 45C-0277), Tris (lot 104C-5000) pH 4.5 (300ml of 0.1 M KH,PO,) to pH 7.0 (300ml and L-tryptophan (lot 83B-0960) were pur- of 0.1 M phosphate buffer) is then applied. Elution chased from Sigma. Serva provided guani- was carried out at 4" with a flow rate of 60ml/h. dimium hydrochloride, a 7 M aqueous solution Fractions of 1 5 ml were collected.
MATERIAL AND METHODS
which had an absorbance of less than 0.03 at 260nm; only freshly prepared solutions of this compound were used. Azocoll (lot 5 10.1 19), 5.5'-dithiobis (2-nitrobenzoic acid) and iodoacetamide were provided by Calbiochem, and Fluka & Schuchardt, Munich, respectively. Ampholytes were purchased from LKB (Stockholm, Sweden). All other chemicals used were of the best grade available with the exception of ammonium sulfate (biochemical grade).
cm) equilibrated with 0.01 M phosphate buffer, pH 7.0, containing 0.1 M NaC1. The column was then washed with the equilibrating buffer until the effluent was devoid of proteolytic activity against Azocoll. Elution was carried out at 4", with a flow rate of 60ml/h. Fractions of 15 ml were collected.
Step 5. Chromatography on CM-cellulose. The pH of the protease B solution was adjusted to 4.5 with 2 N HCl and the solution applied to Purification of yeast protease B the CM-cellulose column. Elution was carried The purification of yeast proteinase B was out as described in the legend of Fig. 1. The carried out as follows, in six steps. In the course fractions containing the protease B activity of the purification, protease B fractions were were pooled and extensively dialyzed at 4" localized using the proteolytic activity of the against lOmM phosphate buffer at pH 7.0. enzyme. Azocoll was used as the substrate (Cabib & Ulane, 1973b). Step 6. Second chromatography on DEAE-celluThe first three steps: autolysis of the yeast lose. The dialyzed protease B solution was cells, ammonium sulfate fractionation and applied to the DEAEcellulose column and activation of the yeast proteinases have been eluted as described in the legend of Fig. 2. The performed as outlined by Hayashi and co- fractions containing protease B were pooled, workers for the purification of carboxypep- dialyzed at 4" against distilled water and tidase Y (Hayashi et al., 1973). finally lyophilized. Step 4. First chromatography on DEAE-cellu- Polyacrylamide gel electrophoresis lose. The dialyzed sample from step 3 was Polyacrylamide gel electrophoresis was carried applied to a DEAE-cellulose column (2.5 x 40 out by the standard method of Ornstein (1964) 254
CHARACTERIZATION OF YEAST PROTEASE B
I I IW IH ! I
ELUTION VOCUIE.lrnl1
_-
lysates were subjected to amino acid analysis in a Beckman Unichrom and in a Durum amino acid analyzer (Moore et d.,1958). The total half-cystine content was estimated after reduction and S-carboxymethylation of the protein (Rask et a/., 1971), the free cysteine content being deduced after reaction of the protein with 5,5'-dithiobis(2-nitrobenzoic acid) according to Habeeb (1972). The tryptophan content was estimated by the fluorimetric method of Pajot (1976).
I --I -
FIGURE 2 Chromatography o f the preparation of protease B from step 5 on a DEAEcellulose column (2.5 X 40cm) equilibrated with 0.01 M phosphate buffer at pH 7.0. Elution was carried out at 4" with a flow rate of 50ml/h with a linear NaCl gradient in the same phosphate buffer (0.0 to 0.4M NaCl, 800ml total volume). Fractions of 15 ml were collected.
and Davis (1964). Electrophoresis at 4mA per gel column was performed at 4' for 30 min. Gels were stained with 0.025% Coomassie blue in 10% trichloroacetic acid. Dodecylsulfatepolyacrylamide gel electrophoresis was performed according to Fairbanks et al. (1971). Isoelectric focusing
Absorbance measurements
Absorbances were measured with either a PMQ I1 (Zeiss) or a Cary 15 M spectrophotometer. The concentration of protease B was determined spectro hotometrically. The specific absorbance (Aim at 280nm) was found to be equal to 13.1. It was deduced from the knowledge of the absorbance of a solution whose protein content was determined by the method of Lowry et al. (1951). Circular dichroism measurements
The circular dichroism curves were obtained with a Cary 61 spectropolarimeter. Measurements were made at 20" in quartz cells with a pathlength of 1 cm in the region above 250nm and of 1 mm below this wavelength. Protein concentration was 0.8 mg/ml. The mean residue molecular ellipticity, (O), is given in degrees. cm2/dmol amino acid residue. The mean residue weight was calculated from the amino acid composition.
The focusing experiment was prepared with ampholytes covering the pH range from 3 to 10 according to the method of Vesterberg & Swenson (1966). Samples of about 10mg of protease B were applied to a l l O m l LKB column. The electrolysis was run for 24 h at 4'. Fluorescence measurements Fluorescence was measured at 25' with a Hitachi Perkin Elmer model MPF-2A spectroAnalytical gel chromatography The molecular weight of protease B was esti- fluorimeter equipped with an Osram XBO 150 mated by gel chromatography on a Sephadex watt Xenon lamp and a RCA 1-P28 photoG-75 column (1 x 140cm) equilibrated with a multiplier. A solution of Ltryptophan in water served lOmM phosphate buffer at pH 7.0. Elution was carried out at room temperature and with a as a standard. In order to correct for small flow rate of 4.5 ml/h. Fractions of 1.5 ml were instrumental fluctuations, this solution was collected. Bovine serum albumin, soybean recorded simultaneously with that of the protrypsin inhibitor and bovine pancreatic ribonuc- tein solution. Excitation and emission bandwidths were 5 nm each. The absorbance (at the lease served as standards. excitation wavelength) of the protein solution was always less than 0.20. Under these conAmino acid analysis Acid hydrolyses were performed at 105' for 24 ditions, fluorescence intensity could be linearly h under vacuum in azeotropic HCl. The hydro- related to the protein concentration. Quantum 255
Y. LOOZE ET AL.
yields were estimated by reference to trypto- to achieve the purification by means of a phan neutral aqueous solution, assuming its chromatography on DEAEcellulose in the yield to be equal to 0.20 (Brand & Witholt, conditions described under the legend of Fig. 2. 1967). In his laboratory, protease B had previously been shown to form complexes with proteins, RESULTS AND DISCUSSION in particular with protease A. Such complexes had been readily fractionated by chromatoPurification of yeast protease B Yeast protease B has been obtained in the pure graphy on DEAEcellulose (Hinze et d., 1975). state in two laboratories (Bunning & Holzer, The results of our chromatography are shown 1976a; Ulane & Cabib, 1976). This success was in Fig. 2. The preparation obtained at the end of the due in both cases to the introduction of affinity six purification steps was shown to be homochromatography. The latter was made possible geneous on disc gel electrophoresis in the after the discovery of two proteins, inhibiting absence as well as in the presence of SDS. The protease B, found in the cytosolic fraction of isoelectric point (5.45) and the molecular the yeast cells (Bunning & Holzer, 1977). The (43 000 daltons) values are now in good weight purification of the protease B inhibitors, howagreement with those values published earlier ever, is at least as laborious as purification of & Holzer, 1976; Ulane & Cabib, (Bunning the protease itself. Furthermore, protease B is 1976). eluted from the affinity column by means of urea (4 to 6M). A loss of enzymatic activity Amino acid composition at t h i s level may be surmised to occur. The amino acid composition of protease B is The main difficulty encountered in the shown in Table 1. The methionine content was purification of protease B lies in the instability of the enzyme. Recently, to overcome this TABLE 1 difficulty, Bunning and Holzer have suggested Amino acid composition of protease B inhibiting protease B, using p-chloromercuribenzoic acid (Bunning & Holzer, 19762~). Residues Residues/ Nearest Reactivation could be achieved when desired mola integer by reacting the inhibited protein with an excess of 2-mercaptoethanol. However, we Aspartic acid 38.79 39 18.29 19 found such a reactivation to be far from com- Threonine Serine 21.75 22 plete. 33.25 33 The scheme presented in this paper seems Glutamic acid Proline 13.35 13 well suited for preparing protease B since it 36.12 36 is rapid, and since the homogeneous prepar- Glycine Alanine 31.97 32 ation obtained at the end of the six steps Half-cystineb 0.92 1 could not be distinguished from that obtained Valine 30.09 30 with the use of affinity chromatography. Me thionine' traces Fig. 1 shows the result of the chromato- Isoleucine 14.84 15 graphy on CM-cellulose of the protease B Leucine 22 22.06 preparation from step 5. At this level, the Tyrosine 6.39 6 14 14.24 preparation- was shown to be homogeneous Phenylalanine 21 26.98 on disc gel electrophoresis in the absence of Lysine 10.38 10 sodium dodecylsulfate. However, the iso- Histidine 11 10.69 electric point (5.10) and the molecular weight Arginine 5 5.15 (80 000 daltons) values differed significantly Tryptophand from those values published earlier (Bunning aCalculations were based on a molecular weight of & Holzer, 1 9 7 6 ~Ulane ; & Cabib, 1976). Also 43 000. three bands could be evidenced on disc gel Determined as S-carboxymethylcysteine. electrophoresis in the presence of SDS. Not determined, It was the suggestion of Professor Holzer Determined by the fluonmetric method of Pajot.
256
CHARACTERIZATION OF YEAST PROTEASE B
found to be very low. Only traces of this amino acid could be seen on the chromatograms. The complete absence of disulfide bonds must also be noted. After treatment of the protein in 8 M urea with dithiothreitol followed by alkylation with iodoacetamide, only 1 mol Scarboxymethylcysteine/mol protein was found. The presence of this free cysteine residue of protease B could be confirmed after reaction with Ellman's reagent. The tryptophan content, determined by the fluorimetric method of Pajot, was found to be equal to 5 mol/mol protein. Circular dichroism studies
The circular dichroism curves in the far U.V. region of protease B at pH 4.5 and 7.0 are shown in Fig. 3. r
~
I
W 0:
OK-
FIGURE 3 Circular dichroism curves of protease B in the aromatic region (A) and far U.V. region (B, -pH 7.0 in 0.01 M phosphate buffer; --- pH 4.5 in 0.01M KH,PO,). The mean residue ellipticity, expressed in degrees. cm'/dmoi amino acid residue, is plotted as a function o f wavelength.
In this wavelength range from 200 to 250 nm, one minimum at 215 nm can be observed. This characteristic had previously been obtained with poly-L-lysine in the 0-conformation (Greenfield & Fasman, 1969). It corresponds also quite well to those spectra calculated for the regions of proteins with a 0-structure (Chen et al., 1972). In these spectra, the absence of shoulders at 207 and 2 2 2 m suggests the absence of any measurable &-helix structure in this protein. Thus the protein seems to be exclusively composed of a mixture of regions in a pconformation and of unorganized regions. Usually, for the analysis of the circular dichroism spectra of proteins, the model of Chen and coworkers gives more satisfactory results than the model of Greenfield and Fasman. This is mainly due to the presence in poly-L-lysine of long stretches of amino acid residues in a given conformation (the length of the stretches is known to influence the circular dichroism parameters). Such a situation is rarely found in globular proteins. From the high molecular ellipticities at 2 15 nm, such as those observed in Fig. 3 for protease B, a very high content (and therefore long stretches) of 0-conformation could be surmised. As a consequence, the model of Greenfield and Fasman was chosen for the quantitative analysis of the spectra. For protease B at pH 7.0, the spectrum could be accounted by assuming 80% of the amino acid residues to be folded in a 0-conformation and 20% to be random coiled. For protease B at pH 4.5, 61% of the amino acid residues folded in the p-conformation. Thus at pH 4.5 protease B seems less structured than with the protein at pH 7.0. This conformational change is accompanied by modifications of the fluorescence parameters as illustrated in Fig. 4. We have found that the conformational transition as observed by fluorescence was at least partly reversible. In the purification scheme of protease B, a chromatography on CM-cellulose at pH 4.5 was included. These conditions can thus be considered o posteriori as leading to only a small fraction of irreversibly denatured protein. In the aromatic region (from 250 to 300nm) a positive peak, centered at 285nm with two 2.57
Y. LOOZE ET AL.
FIGURE 4 Effect of pH on the fluorescence of protease B. Fluorescence intensity at 330nm is plotted as a function of pH. Protease B was dissolved in a phosphate-citrate buffer. The pH values were subsequently adjusted by adding aliquots of 1 N HCl or 1 N NaOH.
shoulders respectively at 275 and 292.5 nm, can be observed from Fig. 3A. Fluorescence studies
The parameters characterizing the fluorescence of protease B are listed in Table 2. The bandwidth, the quantum yield and the wavelength where the emission is maximal are independent of the excitation wavelength between 275 and 300nm. This observation strongly suggests that only one species of aromatic amino acid residue, namely tryptophan, is responsible for the measured fluorescence. The above cited parameters, however, are a TABLE 2 Parameters characterizing the fluorescence of protease B from Saccharomyces cerevisiae Properties
,,A emission Bandwidth Quantum yield
KQ (1.)' KQ (NO;)' KQ (CS+)'
Tryptophan
Protease B
350-353 nma 59-61 nma 0.2Od
325 nmb 45 nmb 0.lOb
11.5 (M-') 34.0 (M-') 1.95 (M-')
1.OS (M-' ) 8.10 (M-' ) 0.16 (M-')
a The free amino acid dissolved in water.
The protein was dissolved in 0.01 M phosphate buffer at pH 7. The amino acid as well as the protein were dissolved in 2 mM HEPES buffer at pH 7.4. An excitation wavelength of 290 nm was used.
'
258
function of pH as shown in Fig. 4. The fluorescence intensity is maximal and constant for pH between 5.0 and 8.5. On both sides of these pHs it decreases. Above pH 8.5 the decrease of the intensity is possibly due to a quenching by hydroxide ions. Below pH 5.0, a conformational transition leading to a disorganized protein occurs. As a consequence, all subsequent measurements were performed at pH values around 7. In proteins, as outlined by Bumstein and coworkers, three classes of tryptophan residues can be identified by means of their fluorescence parameters (Burnstein et d., 1973). Class I, composed of tryptophan residues located at the surface of proteins in an apolar (aqueous) environment, is characterized by a bandwidth of 59-61 nm, ,a, A emission around 350nm and a quantum yield of 0.20. Class 11, composed of tryptophan residues partly buried and partly exposed t o solvent, is characterized by a bandwidth of 54-58nm, ,a , A emission of 340-343nm and a quantum yield of 0.30. Finally, class 111 composed of residues completely buried in an apolar environment, is characterized by a bandwidth of 48-49 nm, a ,,X emission of 330-332nm and a quantum yield of 0.1 1. From the parameters listed in Table 2, the tryptophan residues of protease B would seem to belong to class 111. To check this possibility, the study of the effect on tryptophan fluorescence of ionic quenchers was undertaken (Burnstein et al.,
CHARACTERIZATION OF YEAST PROTEASE B
1973; Lehrer, 1971). Cesium cations and iodide and nitrate anions were choosen for that purpose. The values of the Stern-Volmer constants (KQ) obtained for tryptophan as the free amino acid and for protease B are listed in Table 2. From these values, accessibility percentages of 8% (Cs'), 9% (I-) and 24% (NO;) can be estimated. The tryptophan residues of protease B were thus confirmed to be largely buried in the hydrophobic core of the protein. ACKNOWLEDGMENTS The authors are grateful to Professor Holzer for many helpful suggestions. We thank also Professors Kanarek and Strosberg for the use of the Cary 61 spectropolarimeter and the Durum amino acid analyzer. This work was supported by grants from the Fonds National Belge de la Recherche Fondamentale et Collective and from the Fonds Emile Defay. Polastro Enrico is Aspirant de Recherches of the Fonds National Belge de la Recherche Scientifique.
REFERENCES Brand, L. & Witholt, B. (1967) in Methods in Enzymology (Hirs, C.H.W., ed), vol. 11, pp. 776-856, Academic Press, New York Bunning. P. & Holzer, H. (19760) Xth I.U.B. Congress held in Hamburg, Abstract No. 07-8-101 Bunning, P. & Holzer, H. (1 9766) Communicated at the Fahrestagung der Osterreich Biochem. Gesell. Innsbruck Bunning, P. & Holzer, H. (1977) J. Biol. Chem. 252, 5316-5323 Burnstein, E.A., Vedenkina, N.S. & Ivkova, M.N. (1973) Photochem. Photobiol. 18, 263-279 Cabib, E. & Ulane, R. ( 1 9 7 3 ~ )J. Biol. Chem 248, 1451-1458 Cabib, E. & Ulane, R. (19736) Biochem. Biophys. Res. Commun. 50,186-191 Chen, Y.C., Yang, J.T. & Martinez, H. (1972) Biochemistry 11,4 120-41 31 Davis, B.J. (1964) Ann. N.Y. Acod. Sci. 121, 404427 Fairbanks,G., Stech, T.L. & Wallach, D.F.H. (1971) Biochemistry 10,2611-2624 Greenfield, N. & Fasman, G.D. (1969) Biochemistry 8,4108-4116
Habeeb, A.F.S.A. (1972) in Methods in Enzymology (Hus, C.H.W. & Timasheff, S.N., eds.), vol. 25, pp. 457-464, Academic Press, New York Hayashi, R., Moore S. & Stein, W.H. (1973) J. Biol. Chem. 248,2296-2302 Hinze, H., Betz, H., Saheki, T. & Holzer, H. (1975) Hoppe-Seyler's Z. Physiol. Chem. 356, 12591264 Holzer, H., Betz, H. & Ebner, E. (1975) in Current Topics in Cellular Regulations, (Horecker, B.L. & Stadtman, B.R., eds.), pp. 103-156, Academic Press, New York Katsunuma, T., Schott, E., Elasser, S. & Holzer, H. (1972) Eur. J. Biochem. 27,520-526 Lehrer, S . S . (1971) Biochemistry 10, 3254-3263 Lenney, J.F. (1973) Fed. Proc. 32,659 Lenney, J.F. & Dalbec, J.M. (1967) Arch. Biochem. Biophys. 1 2 0 , 4 2 4 8 Lowry, O.H., Rosebrough, N.J., Fan, A.L. & Randall, R.J. (1951)J. Biol. Chem. 193,265-275 Manney, T. R. (1968) J. Bacteriol. %, 403-408 Metz, G. & Rhom, K.M. (1976) Biochim. Biophys. Acta 429,933-949 Molano, J. & Gancedo, C. (1974) Eur. J. Biochem. 44, 213-217 Moore, S., Spackman, D.H. & Stein, W.H. (1958) Anal. Chem. 30,1185-1190 Ornstein, L. (1964) Ann.N.Y. Acad. Sci. 121, 3213 29 Pajot,P. (1976) Eur. J. Biochem. 63, 263-269 Rask, L., Peterson, P.A. & Nilsson, S.F. (1971) J. Biol. Chem. 246,6087-6097 Saheki, T. & Holzer, H. (1974) Eur. J. Biochem. 42,621-626 Schott, E.H. & Holzer, H. (1974) Eur. J. Biochem. 42,61-66 Tsai, H., Tsai, J.H.J. & Yu, P. (1973) Eur. J. Biochem. 40,225-232 Ulane, R. & Cabib, E. (1976) J. Biol. Chem. 251, 3367-3374 Vesterberg, 0 . & Svensson, H. (1966) Acta Chem. Scand. 20,6 20 Address:
Yvan Looze Laboratoire de Chimie GCnCrale I FacultC des Sciences UniversitC libre de Bruxelles 50, Avenue F.D. Roosevelt 1050, Bruxelles Belgium
259
P R O T E A S E B FROM S A C C H A R O M Y C E S C E R E V I S I A E Purification and Characterization Y. LOOZE, L. GILLET, M. DECONINCK, B. COUTEAUX, E. POLASTRO and J . LEONIS
Laboratoire de Chimie Genkrale I, Faculte des Sciences, Universite libre de Bruxelles, Belgium
Received 1 5 May, accepted for publication 10 July 1978 Protease B has been isolated f r o m Saccharomyces cerevisiae and purified in six steps as follows: autolysis o f the yeast cells, ammonium sulfate fractionation, activation o f the proteolytic enzymes, chromatography on DEAE-cellulose, chromatography on CM-cellulose and finally, a second chromatography on DEAE-cellulose. The preparation was shown t o be homogeneous on polyacrylamide gels in the absence as well as in the presence o f sodium dodecylsulfate. Furthermore, t h e molecular weight (43 000 daltons) and the isoelectric point (S.45) were in good agreement with earlier published values. The amino acid composition is reported. The absence o f disulfide bonds in protease B has to be outlined. The amino acid residues o f the protein have been found t o be folded nearly quantitatively (at least 80%) in a 0-conformation as deduced f r o m a circular dichroism study. Finally, the tryptophan residues (S molfmol protein) are largely buried in the hydrophobic core o f the enzyme. Key words: circular dichroism ;fluorescence; protease B; Saccharomyces cerevisiae.
Several intracellular proteolytic enzymes from to function quite well in denaturing media. prokaryotes and ascomycetes have been des- Furthermore, as a result of its amidase action, cribed and their possible role investigated this enzyme could be applied to the sequence analysis of peptides having amidated COOH(Holzer et al., 1975). From Saccharomyces cerevisiae, two en- terminal groups (Hayashi et al., 1973). Proteases A and B, on the other hand, dopeptidases, namely proteinases A and B, as well as two exopeptidases: a carboxypeptidase seem to be involved in the regulation of several (carboxypeptidase Y, earlier known as protease enzyme activities including tryptophan synC) and an aminopeptidase have been purified thetase, fructose 1,6-bisphosphatase and chitin (Lenney & Dalbec, 1967; Katsunuma el al., synthetase (Tsai et al., 1973; Schott & Holzer, 1972; Hayashi et al., 1973; Metz & Rhom, 1974; Saheki & Holzer, 1974; Manney, 1968; Molano & Gancedo, 1974; Cabib & Ulane, 1976). In sequence studies carboxypeptidase Y 1 9 7 3 ~ ) . had greater advantages compared with the The proteases have been located in the pancreatic-type carboxypeptidases. This is due yeast vacuoles (Lenney, 1973). Thus, an to its broader specificity and also to its ability important question which is not yet clearly 0367-8377/79/030253-07
$02.00/0 0 1979 Munksgaard, Copenhagen
253
Y. LOOZE ET AL.
understood, is whether the control of the proteolytic attack in vivo, the spacial separation of proteases and their potential substrates is indeed the rule. Also, up to now, little attention has been given to the molecular structure of the enzymes. It is the purpose of this communication to approach this question by means of circular dichroism and fluorescence studies. Protease B was chosen. Also, the enzyme being very labile, we felt it would be useful to reinvestigate its purification.
I
ELUTION VOLUME l m l l ~
FIGURE 1 Chromatogaphy of the preparation of protease B Bovine serum albumin (lot 46C-0253), soybean from step 4 on a CM-cellulose column (2.5 X 40cm) trypsin inhibitor (lot 94C-8 190), bovine pan- equilibrated with 0.1 M KH,PO, at pH 4.5. After fixing of the proteins, the column was washed with creatic ribonuclease (lot 56C-8020), dith- 1 litre 0.1 M KH,PO, at pH 4.5. A linear gradient from iothreitol (lot 45C-0277), Tris (lot 104C-5000) pH 4.5 (300ml of 0.1 M KH,PO,) to pH 7.0 (300ml and L-tryptophan (lot 83B-0960) were pur- of 0.1 M phosphate buffer) is then applied. Elution chased from Sigma. Serva provided guani- was carried out at 4" with a flow rate of 60ml/h. dimium hydrochloride, a 7 M aqueous solution Fractions of 1 5 ml were collected.
MATERIAL AND METHODS
which had an absorbance of less than 0.03 at 260nm; only freshly prepared solutions of this compound were used. Azocoll (lot 5 10.1 19), 5.5'-dithiobis (2-nitrobenzoic acid) and iodoacetamide were provided by Calbiochem, and Fluka & Schuchardt, Munich, respectively. Ampholytes were purchased from LKB (Stockholm, Sweden). All other chemicals used were of the best grade available with the exception of ammonium sulfate (biochemical grade).
cm) equilibrated with 0.01 M phosphate buffer, pH 7.0, containing 0.1 M NaC1. The column was then washed with the equilibrating buffer until the effluent was devoid of proteolytic activity against Azocoll. Elution was carried out at 4", with a flow rate of 60ml/h. Fractions of 15 ml were collected.
Step 5. Chromatography on CM-cellulose. The pH of the protease B solution was adjusted to 4.5 with 2 N HCl and the solution applied to Purification of yeast protease B the CM-cellulose column. Elution was carried The purification of yeast proteinase B was out as described in the legend of Fig. 1. The carried out as follows, in six steps. In the course fractions containing the protease B activity of the purification, protease B fractions were were pooled and extensively dialyzed at 4" localized using the proteolytic activity of the against lOmM phosphate buffer at pH 7.0. enzyme. Azocoll was used as the substrate (Cabib & Ulane, 1973b). Step 6. Second chromatography on DEAE-celluThe first three steps: autolysis of the yeast lose. The dialyzed protease B solution was cells, ammonium sulfate fractionation and applied to the DEAEcellulose column and activation of the yeast proteinases have been eluted as described in the legend of Fig. 2. The performed as outlined by Hayashi and co- fractions containing protease B were pooled, workers for the purification of carboxypep- dialyzed at 4" against distilled water and tidase Y (Hayashi et al., 1973). finally lyophilized. Step 4. First chromatography on DEAE-cellu- Polyacrylamide gel electrophoresis lose. The dialyzed sample from step 3 was Polyacrylamide gel electrophoresis was carried applied to a DEAE-cellulose column (2.5 x 40 out by the standard method of Ornstein (1964) 254
CHARACTERIZATION OF YEAST PROTEASE B
I I IW IH ! I
ELUTION VOCUIE.lrnl1
_-
lysates were subjected to amino acid analysis in a Beckman Unichrom and in a Durum amino acid analyzer (Moore et d.,1958). The total half-cystine content was estimated after reduction and S-carboxymethylation of the protein (Rask et a/., 1971), the free cysteine content being deduced after reaction of the protein with 5,5'-dithiobis(2-nitrobenzoic acid) according to Habeeb (1972). The tryptophan content was estimated by the fluorimetric method of Pajot (1976).
I --I -
FIGURE 2 Chromatography o f the preparation of protease B from step 5 on a DEAEcellulose column (2.5 X 40cm) equilibrated with 0.01 M phosphate buffer at pH 7.0. Elution was carried out at 4" with a flow rate of 50ml/h with a linear NaCl gradient in the same phosphate buffer (0.0 to 0.4M NaCl, 800ml total volume). Fractions of 15 ml were collected.
and Davis (1964). Electrophoresis at 4mA per gel column was performed at 4' for 30 min. Gels were stained with 0.025% Coomassie blue in 10% trichloroacetic acid. Dodecylsulfatepolyacrylamide gel electrophoresis was performed according to Fairbanks et al. (1971). Isoelectric focusing
Absorbance measurements
Absorbances were measured with either a PMQ I1 (Zeiss) or a Cary 15 M spectrophotometer. The concentration of protease B was determined spectro hotometrically. The specific absorbance (Aim at 280nm) was found to be equal to 13.1. It was deduced from the knowledge of the absorbance of a solution whose protein content was determined by the method of Lowry et al. (1951). Circular dichroism measurements
The circular dichroism curves were obtained with a Cary 61 spectropolarimeter. Measurements were made at 20" in quartz cells with a pathlength of 1 cm in the region above 250nm and of 1 mm below this wavelength. Protein concentration was 0.8 mg/ml. The mean residue molecular ellipticity, (O), is given in degrees. cm2/dmol amino acid residue. The mean residue weight was calculated from the amino acid composition.
The focusing experiment was prepared with ampholytes covering the pH range from 3 to 10 according to the method of Vesterberg & Swenson (1966). Samples of about 10mg of protease B were applied to a l l O m l LKB column. The electrolysis was run for 24 h at 4'. Fluorescence measurements Fluorescence was measured at 25' with a Hitachi Perkin Elmer model MPF-2A spectroAnalytical gel chromatography The molecular weight of protease B was esti- fluorimeter equipped with an Osram XBO 150 mated by gel chromatography on a Sephadex watt Xenon lamp and a RCA 1-P28 photoG-75 column (1 x 140cm) equilibrated with a multiplier. A solution of Ltryptophan in water served lOmM phosphate buffer at pH 7.0. Elution was carried out at room temperature and with a as a standard. In order to correct for small flow rate of 4.5 ml/h. Fractions of 1.5 ml were instrumental fluctuations, this solution was collected. Bovine serum albumin, soybean recorded simultaneously with that of the protrypsin inhibitor and bovine pancreatic ribonuc- tein solution. Excitation and emission bandwidths were 5 nm each. The absorbance (at the lease served as standards. excitation wavelength) of the protein solution was always less than 0.20. Under these conAmino acid analysis Acid hydrolyses were performed at 105' for 24 ditions, fluorescence intensity could be linearly h under vacuum in azeotropic HCl. The hydro- related to the protein concentration. Quantum 255
Y. LOOZE ET AL.
yields were estimated by reference to trypto- to achieve the purification by means of a phan neutral aqueous solution, assuming its chromatography on DEAEcellulose in the yield to be equal to 0.20 (Brand & Witholt, conditions described under the legend of Fig. 2. 1967). In his laboratory, protease B had previously been shown to form complexes with proteins, RESULTS AND DISCUSSION in particular with protease A. Such complexes had been readily fractionated by chromatoPurification of yeast protease B Yeast protease B has been obtained in the pure graphy on DEAEcellulose (Hinze et d., 1975). state in two laboratories (Bunning & Holzer, The results of our chromatography are shown 1976a; Ulane & Cabib, 1976). This success was in Fig. 2. The preparation obtained at the end of the due in both cases to the introduction of affinity six purification steps was shown to be homochromatography. The latter was made possible geneous on disc gel electrophoresis in the after the discovery of two proteins, inhibiting absence as well as in the presence of SDS. The protease B, found in the cytosolic fraction of isoelectric point (5.45) and the molecular the yeast cells (Bunning & Holzer, 1977). The (43 000 daltons) values are now in good weight purification of the protease B inhibitors, howagreement with those values published earlier ever, is at least as laborious as purification of & Holzer, 1976; Ulane & Cabib, (Bunning the protease itself. Furthermore, protease B is 1976). eluted from the affinity column by means of urea (4 to 6M). A loss of enzymatic activity Amino acid composition at t h i s level may be surmised to occur. The amino acid composition of protease B is The main difficulty encountered in the shown in Table 1. The methionine content was purification of protease B lies in the instability of the enzyme. Recently, to overcome this TABLE 1 difficulty, Bunning and Holzer have suggested Amino acid composition of protease B inhibiting protease B, using p-chloromercuribenzoic acid (Bunning & Holzer, 19762~). Residues Residues/ Nearest Reactivation could be achieved when desired mola integer by reacting the inhibited protein with an excess of 2-mercaptoethanol. However, we Aspartic acid 38.79 39 18.29 19 found such a reactivation to be far from com- Threonine Serine 21.75 22 plete. 33.25 33 The scheme presented in this paper seems Glutamic acid Proline 13.35 13 well suited for preparing protease B since it 36.12 36 is rapid, and since the homogeneous prepar- Glycine Alanine 31.97 32 ation obtained at the end of the six steps Half-cystineb 0.92 1 could not be distinguished from that obtained Valine 30.09 30 with the use of affinity chromatography. Me thionine' traces Fig. 1 shows the result of the chromato- Isoleucine 14.84 15 graphy on CM-cellulose of the protease B Leucine 22 22.06 preparation from step 5. At this level, the Tyrosine 6.39 6 14 14.24 preparation- was shown to be homogeneous Phenylalanine 21 26.98 on disc gel electrophoresis in the absence of Lysine 10.38 10 sodium dodecylsulfate. However, the iso- Histidine 11 10.69 electric point (5.10) and the molecular weight Arginine 5 5.15 (80 000 daltons) values differed significantly Tryptophand from those values published earlier (Bunning aCalculations were based on a molecular weight of & Holzer, 1 9 7 6 ~Ulane ; & Cabib, 1976). Also 43 000. three bands could be evidenced on disc gel Determined as S-carboxymethylcysteine. electrophoresis in the presence of SDS. Not determined, It was the suggestion of Professor Holzer Determined by the fluonmetric method of Pajot.
256
CHARACTERIZATION OF YEAST PROTEASE B
found to be very low. Only traces of this amino acid could be seen on the chromatograms. The complete absence of disulfide bonds must also be noted. After treatment of the protein in 8 M urea with dithiothreitol followed by alkylation with iodoacetamide, only 1 mol Scarboxymethylcysteine/mol protein was found. The presence of this free cysteine residue of protease B could be confirmed after reaction with Ellman's reagent. The tryptophan content, determined by the fluorimetric method of Pajot, was found to be equal to 5 mol/mol protein. Circular dichroism studies
The circular dichroism curves in the far U.V. region of protease B at pH 4.5 and 7.0 are shown in Fig. 3. r
~
I
W 0:
OK-
FIGURE 3 Circular dichroism curves of protease B in the aromatic region (A) and far U.V. region (B, -pH 7.0 in 0.01 M phosphate buffer; --- pH 4.5 in 0.01M KH,PO,). The mean residue ellipticity, expressed in degrees. cm'/dmoi amino acid residue, is plotted as a function o f wavelength.
In this wavelength range from 200 to 250 nm, one minimum at 215 nm can be observed. This characteristic had previously been obtained with poly-L-lysine in the 0-conformation (Greenfield & Fasman, 1969). It corresponds also quite well to those spectra calculated for the regions of proteins with a 0-structure (Chen et al., 1972). In these spectra, the absence of shoulders at 207 and 2 2 2 m suggests the absence of any measurable &-helix structure in this protein. Thus the protein seems to be exclusively composed of a mixture of regions in a pconformation and of unorganized regions. Usually, for the analysis of the circular dichroism spectra of proteins, the model of Chen and coworkers gives more satisfactory results than the model of Greenfield and Fasman. This is mainly due to the presence in poly-L-lysine of long stretches of amino acid residues in a given conformation (the length of the stretches is known to influence the circular dichroism parameters). Such a situation is rarely found in globular proteins. From the high molecular ellipticities at 2 15 nm, such as those observed in Fig. 3 for protease B, a very high content (and therefore long stretches) of 0-conformation could be surmised. As a consequence, the model of Greenfield and Fasman was chosen for the quantitative analysis of the spectra. For protease B at pH 7.0, the spectrum could be accounted by assuming 80% of the amino acid residues to be folded in a 0-conformation and 20% to be random coiled. For protease B at pH 4.5, 61% of the amino acid residues folded in the p-conformation. Thus at pH 4.5 protease B seems less structured than with the protein at pH 7.0. This conformational change is accompanied by modifications of the fluorescence parameters as illustrated in Fig. 4. We have found that the conformational transition as observed by fluorescence was at least partly reversible. In the purification scheme of protease B, a chromatography on CM-cellulose at pH 4.5 was included. These conditions can thus be considered o posteriori as leading to only a small fraction of irreversibly denatured protein. In the aromatic region (from 250 to 300nm) a positive peak, centered at 285nm with two 2.57
Y. LOOZE ET AL.
FIGURE 4 Effect of pH on the fluorescence of protease B. Fluorescence intensity at 330nm is plotted as a function of pH. Protease B was dissolved in a phosphate-citrate buffer. The pH values were subsequently adjusted by adding aliquots of 1 N HCl or 1 N NaOH.
shoulders respectively at 275 and 292.5 nm, can be observed from Fig. 3A. Fluorescence studies
The parameters characterizing the fluorescence of protease B are listed in Table 2. The bandwidth, the quantum yield and the wavelength where the emission is maximal are independent of the excitation wavelength between 275 and 300nm. This observation strongly suggests that only one species of aromatic amino acid residue, namely tryptophan, is responsible for the measured fluorescence. The above cited parameters, however, are a TABLE 2 Parameters characterizing the fluorescence of protease B from Saccharomyces cerevisiae Properties
,,A emission Bandwidth Quantum yield
KQ (1.)' KQ (NO;)' KQ (CS+)'
Tryptophan
Protease B
350-353 nma 59-61 nma 0.2Od
325 nmb 45 nmb 0.lOb
11.5 (M-') 34.0 (M-') 1.95 (M-')
1.OS (M-' ) 8.10 (M-' ) 0.16 (M-')
a The free amino acid dissolved in water.
The protein was dissolved in 0.01 M phosphate buffer at pH 7. The amino acid as well as the protein were dissolved in 2 mM HEPES buffer at pH 7.4. An excitation wavelength of 290 nm was used.
'
258
function of pH as shown in Fig. 4. The fluorescence intensity is maximal and constant for pH between 5.0 and 8.5. On both sides of these pHs it decreases. Above pH 8.5 the decrease of the intensity is possibly due to a quenching by hydroxide ions. Below pH 5.0, a conformational transition leading to a disorganized protein occurs. As a consequence, all subsequent measurements were performed at pH values around 7. In proteins, as outlined by Bumstein and coworkers, three classes of tryptophan residues can be identified by means of their fluorescence parameters (Burnstein et d., 1973). Class I, composed of tryptophan residues located at the surface of proteins in an apolar (aqueous) environment, is characterized by a bandwidth of 59-61 nm, ,a, A emission around 350nm and a quantum yield of 0.20. Class 11, composed of tryptophan residues partly buried and partly exposed t o solvent, is characterized by a bandwidth of 54-58nm, ,a , A emission of 340-343nm and a quantum yield of 0.30. Finally, class 111 composed of residues completely buried in an apolar environment, is characterized by a bandwidth of 48-49 nm, a ,,X emission of 330-332nm and a quantum yield of 0.1 1. From the parameters listed in Table 2, the tryptophan residues of protease B would seem to belong to class 111. To check this possibility, the study of the effect on tryptophan fluorescence of ionic quenchers was undertaken (Burnstein et al.,
CHARACTERIZATION OF YEAST PROTEASE B
1973; Lehrer, 1971). Cesium cations and iodide and nitrate anions were choosen for that purpose. The values of the Stern-Volmer constants (KQ) obtained for tryptophan as the free amino acid and for protease B are listed in Table 2. From these values, accessibility percentages of 8% (Cs'), 9% (I-) and 24% (NO;) can be estimated. The tryptophan residues of protease B were thus confirmed to be largely buried in the hydrophobic core of the protein. ACKNOWLEDGMENTS The authors are grateful to Professor Holzer for many helpful suggestions. We thank also Professors Kanarek and Strosberg for the use of the Cary 61 spectropolarimeter and the Durum amino acid analyzer. This work was supported by grants from the Fonds National Belge de la Recherche Fondamentale et Collective and from the Fonds Emile Defay. Polastro Enrico is Aspirant de Recherches of the Fonds National Belge de la Recherche Scientifique.
REFERENCES Brand, L. & Witholt, B. (1967) in Methods in Enzymology (Hirs, C.H.W., ed), vol. 11, pp. 776-856, Academic Press, New York Bunning. P. & Holzer, H. (19760) Xth I.U.B. Congress held in Hamburg, Abstract No. 07-8-101 Bunning, P. & Holzer, H. (1 9766) Communicated at the Fahrestagung der Osterreich Biochem. Gesell. Innsbruck Bunning, P. & Holzer, H. (1977) J. Biol. Chem. 252, 5316-5323 Burnstein, E.A., Vedenkina, N.S. & Ivkova, M.N. (1973) Photochem. Photobiol. 18, 263-279 Cabib, E. & Ulane, R. ( 1 9 7 3 ~ )J. Biol. Chem 248, 1451-1458 Cabib, E. & Ulane, R. (19736) Biochem. Biophys. Res. Commun. 50,186-191 Chen, Y.C., Yang, J.T. & Martinez, H. (1972) Biochemistry 11,4 120-41 31 Davis, B.J. (1964) Ann. N.Y. Acod. Sci. 121, 404427 Fairbanks,G., Stech, T.L. & Wallach, D.F.H. (1971) Biochemistry 10,2611-2624 Greenfield, N. & Fasman, G.D. (1969) Biochemistry 8,4108-4116
Habeeb, A.F.S.A. (1972) in Methods in Enzymology (Hus, C.H.W. & Timasheff, S.N., eds.), vol. 25, pp. 457-464, Academic Press, New York Hayashi, R., Moore S. & Stein, W.H. (1973) J. Biol. Chem. 248,2296-2302 Hinze, H., Betz, H., Saheki, T. & Holzer, H. (1975) Hoppe-Seyler's Z. Physiol. Chem. 356, 12591264 Holzer, H., Betz, H. & Ebner, E. (1975) in Current Topics in Cellular Regulations, (Horecker, B.L. & Stadtman, B.R., eds.), pp. 103-156, Academic Press, New York Katsunuma, T., Schott, E., Elasser, S. & Holzer, H. (1972) Eur. J. Biochem. 27,520-526 Lehrer, S . S . (1971) Biochemistry 10, 3254-3263 Lenney, J.F. (1973) Fed. Proc. 32,659 Lenney, J.F. & Dalbec, J.M. (1967) Arch. Biochem. Biophys. 1 2 0 , 4 2 4 8 Lowry, O.H., Rosebrough, N.J., Fan, A.L. & Randall, R.J. (1951)J. Biol. Chem. 193,265-275 Manney, T. R. (1968) J. Bacteriol. %, 403-408 Metz, G. & Rhom, K.M. (1976) Biochim. Biophys. Acta 429,933-949 Molano, J. & Gancedo, C. (1974) Eur. J. Biochem. 44, 213-217 Moore, S., Spackman, D.H. & Stein, W.H. (1958) Anal. Chem. 30,1185-1190 Ornstein, L. (1964) Ann.N.Y. Acad. Sci. 121, 3213 29 Pajot,P. (1976) Eur. J. Biochem. 63, 263-269 Rask, L., Peterson, P.A. & Nilsson, S.F. (1971) J. Biol. Chem. 246,6087-6097 Saheki, T. & Holzer, H. (1974) Eur. J. Biochem. 42,621-626 Schott, E.H. & Holzer, H. (1974) Eur. J. Biochem. 42,61-66 Tsai, H., Tsai, J.H.J. & Yu, P. (1973) Eur. J. Biochem. 40,225-232 Ulane, R. & Cabib, E. (1976) J. Biol. Chem. 251, 3367-3374 Vesterberg, 0 . & Svensson, H. (1966) Acta Chem. Scand. 20,6 20 Address:
Yvan Looze Laboratoire de Chimie GCnCrale I FacultC des Sciences UniversitC libre de Bruxelles 50, Avenue F.D. Roosevelt 1050, Bruxelles Belgium
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