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Purification of a carboxypeptidase B-like enzyme from the starfish Dermasterias imbricata
Biochimica et Biophysica Acta, 386 (1975) 260-269
© Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands BBA 36948 P U R I F I C A T I O N OF A C A R B O X Y P E P T I D A S E B-LIKE E N Z Y M E F R O M T H E STARFISH DERMASTERIAS IMBRICATA
ROBERT
E. F E R R E L L * ,
ZENAIDO CAMACHO** and G. BARRIE KITTO***
Clayton Foundation Biochemical Institute, Department of Chemistry, University of Texas, Austin, Texas 78712 (U.S.A.)
(Received August 9th, 1974)
SUMMARY A carboxypeptidase B-like enzyme which catalyses the hydrolysis of synthetic esters oflysine and arginine has been isolated from the starfish Dermasterias imbricata. This carboxypeptidase B-like enzyme has a molecular weight of approximately 34 000 and shares this and other properties with bovine pancreatic carboxypeptidase B. The existence of zymogen for this activity in the pyloric caeca of the starfish is demonstrated. This zymogen has a molecular weight near 40 000 and appears to be analogous to other monomeric procarboxypeptidases B. The zymogen possesses an intrinsic low-level activity toward synthetic substrates of carboxypeptidase B and is activated by trypsin.
INTRODUCTION The presence of an enzyme in the euglobulin fraction of autolyzed beef pancreas, which rapidly and specifically liberated the basic amino acids lysine and arginine from the carboxyl-terminal end of synthetic peptide substrates, was first recognized by Folk [1]. A later paper [2] extended the substrate specificity studies with this basic carboxypeptidase to include a wide range of synthetic and naturally occurring substrates and gave it the trivial name carboxypeptidase B. These workers also reported that the enzyme existed in the pancreas as a zymogen which could be activated by the action of the enzyme trypsin and purified the procarboxypeptidase B from acetone extracts of beef pancreas. As part of an investigation of echinoderm proteases, we have isolated a carboxypeptidase B-like enzyme from the pyloric caeca of the starfish Dermasterias imbricata which has several properties similar to those of bovine carboxypeptidase B as well as the carboxypeptidase B isolated from the spiny Pacific dogfish by Prahl and Neurath [3] and from the white shrimp by Gates * Present address: Department of Human Genetics, University of Michigan Medical School, 1137 East Catherine Street, Ann Arbor, Mich. 48104, U.S.A. ** Present address : Department of Veterinary Pathology, Washington State University, Pullman, Wash., U.S.A. *** To whom correspondence should be addressed.
261 and Travis [4]. Evidence will also be presented for the existence of a zymogen of the starfish enzyme in the pyloric caeca. MATERIALS AND METHODS The substrates hippuryl-L-arginine, hippuryl-L-phenylalanine, hippuryl-Llysine, and hippuryl-L-glycine were from Sigma Chemical Company, benzoyl-oLarginine-p-nitroanilide from Nutritional Biochemical, and N-carbobenzoxy-L-serine and hippuryl-L-alanine were a gift from Dr D. J. Cox, Department of Chemistry, University of Texas. Diisopropylfluorophosphate (Dip-F3) was obtained from CalBiochem. The starfish, D. imbricata, was supplied by Pacific Biomarine Supply, Venice, Calif. Protein standards used in the gel-filtration studies were obtained as a kit from Pharmacia Fine Chemicals. Reagents and apparatus for isoelectric focusing were supplied by LKB, Stockholm. Purified bovine carboxypeptidases A and B were obtained from Worthington.
Enzyme assays Carboxypeptidase B activity was determined using hippuryl-L-arginine as substrate according to the method of Folk et al. [5]. Cleavage of the substrate was measured at 25 °C using a Zeiss PMQ II Spectrophotometer at 254 nm. One unit of carboxypeptidase B activity was defined as the amount of enzyme which produced an absorbance change of 1 per min at 254 nm. Carboxypeptidase A activity was determined using hippuryl-L-phenylalanine as substrate essentially as described by Folk and Schirmer [6]. Cleavage of substrate was followed as described for carboxypeptidase B. Substrate specificity studies were carried out using the substrates hippurylL-arginine, hippuryl-L-phenylalanine, hippuryl-L-glycine, and hippuryl-L-lysine. These substrates were used in the standard carboxypeptidase B assay, with the value for cleavage of hippuryl-L-arginine being designated as 100 ~ activity. The enzyme was tested for trypsin-like activity using benzoyl-DL-arginine-pnitroanilide as substrate according to the method of Erlanger et al. [7]. Cleavage Gf substrate was followed at 25 °C using a Zeiss PMQ II Spectrophotometer at 410 nm. Carboxypeptidase activity against benzoyl-DL-arginine-p-nitroanilide and N-carbobenzoxy-L-serine was determined according to Wintersberger et al. [8]. Activity toward hippuryl-L-arginine, as determined by the same assay, was used to relate these activities to all others.
Determination of molecular weight The molecular weight of each protease was estimated by the method of gel filtration [9] using a calibrated Sephadex G-100 column of inner dimensions 2 cm × 50 cm. All experiments were conducted at 4 °C. Each of the standard proteins was dissolved in 1 ml of buffer for application to the column. Sucrose was added to each sample and the higher density protein charge carefully layered above the gel. Collection of the column effluent in 1.5-ml fractions was begun with the addition of the sample to the top of the gel bed. The flow rate of the column was maintained at a constant rate (45 ml/h) by variation in hydrostatic pressure obtained by adjustment of the position of the buffer reservoir. Fractions were collected in a LKB fraction
262 collector and the absorbance at 280 nm was monitored with the use of a LKB Uvicord II and recorder. Each protein sample was run individually. The exact height of the gel bed in the column was maintained constant throughout the experiment. Values used for the molecular weight of reference proteins were as follows: cytochrome e, 12 400; ovalbumin, 45 000; bovine carboxypeptidase B, 34 300; and bovine serum albumin, 66 500. The Dermasterias carboxypeptidase B was also applied to the column separately. The protein concentration and carboxypeptidase activity of the fractions were determined spectrophotometrically.
Temperature stability and pH studies These were carried out as described previously [10]. Parallel studies using commercial bovine carboxypeptidase B were carried out for comparative purposes. Temperature stability was determined in 0.02 M potassium phosphate, pH 7.5. To study the stabilities of the two enzyme with respect to pH, equivalent amounts of enzyme were incubated in buffers of varying pH for 1 h. Aliquots were then removed and placed in the standard assay mixture using 1 mM hippuryl-L-arginine as substrate. Inhibition studies Inhibition of the enzyme by the chelating agents 1,10-phenanthroline and EDTA was measured by preincubation of the enzyme in 10 -3 M inhibitor for 1 h at 0 °C and measuring its activity in substrate solutions containing the same inhibitor concentration. Isoelectric jbcusing Isoelectric focusing was carried out with a linear pH gradient between pH 3 and 10 in a total volume of 110 ml. Carrier ampholytes, the column and the linear density gradient maker were purchased from LKB Produkter, Stockholm, Sweden, and all procedures were performed according to the manufacturer's instructions. The temperature was maintained at 4 °C by a criculating water bath and focusing was carried out for 36 h with a final potential of 700 V. After this time, 1-ml fractions were collected from the column and monitored for protein at 280 nm and assayed for carboxypeptidase B activity as described above. RESULTS
Purification of the enzyme The carboxypeptidase B was purified from the pyloric caeca of the starfish, D. imbrieata. Initial stages of the purification were carried out in parallel with the isolation of tryptic-like proteases from this organism [11 ]. Pyloric caeca were removed from live starfish and homogenized in cold 0.05 M Tris.HC1 buffer, pH 8.2, at a concentration of 200 mg of tissue per ml of buffer. The homogenate was centrifuged at 3000 × g for 10 rain, polyoxyethylene lauryl ether was added to the supernatant fraction to give a final concentration of 0.2 ~ , and the solution was allowed to incubate at 4 °C for 24 h. The solution was then centrifuged at 16 000 × g for 30 min and solid (NH4)2SO4 was added to give 40 ~ saturation. The precipitate was collected by centrifugation as above and the (NH4)zSO4 concentration of the supernatant fraction was raised to 60 ~ of saturation. The precipitate was collected by centrifugation in
263 the same manner, dissolved in cold 0.05 M T r i s . H C l buffer, pH 8.2, and dialyzed against frequent changes of the same buffer for 12 h. An acetone powder was then prepared from this dialyzed solution by the method of Morton [12]. The acetone powder was stirred with cold 0.05 M Tris.HCl, pH 7.5, for 30 min and centrifuged to remove insoluble material. The supernatant fraction was applied to a 10 cm x 100 cm column packed with Sephadex G-100, and the column was eluted with an 0.1 M potassium phosphate buffer, p H 7.5. The fractions containing both tryptic and carboxypeptidase B activity were pooled, concentrated by pressure ultrafiltration and applied to a 5.5 cm × 65 cm column of DEAE-cellulose equilibrated with 0.1 M potassium phosphate buffer, pH 7.5. The column was eluted with a linear gradient of 0.1-1.0 M phosphate buffer, pH 7.5, the elution profile being shown in Fig. 1. In this O.B
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--
120
160
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Fig. 1. First DEAE-cellulose chromatography of D. imbricata carboxypeptidase B. The column (5.5 cm × 60 cm) was equilibrated with 0.1 M potassium phosphate buffer, pH 7.5, and eluted with a linear gradient from 0.1 to 1.0 M potassium phosphate, pH 7.5; 8-ml fractions were collected; carboxypeptidase B activity A; absorbance at 280 nm, ©. step, the enzyme was only partially resolved from one of the two trypsins found in this organism [11]. The two activities were separated by a second DEAE-cellulose step using a 1.5 cm x 30 cm column equilibrated with 0.01 M potassium phosphate buffer and eluted with a shallow gradient from 0.01 to 0.1 M potassium phosphate, p H 7.5, as shown in Fig. 2. Fractions containing carboxypeptidase B activity were pooled and concentrated by pressure ultrafiltration. Final purification of the enzyme involved preparative isoelectric focusing in a pH gradient from pH 3 to 12, as described in Materials and Methods. The carboxypeptidase activity focused as a discrete protein peak and these fractions were pooled, passed through a Sephadex G-25 column to remove the ampholytes and then concentrated by pressure ultrafiltration. The carboxypeptidase B obtained in this manner was judged to be homogeneous, as it gave a single protein band when examined by analytical polyacrylamidegel electrophoresis [13].
Isolation of procarboxypeptidase B The presence of zymogen forms of carboxypeptidase B in other organisms [3,
264 0.60
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Fig. 2. Separation of carboxypeptidase B and protease B (trypsin) activity by DEAE-cellulosechromatography. The column (1.5 cm x 30 cm) was equilibrated with 0.01 M potassium phosphate buffer, pH 7.5, and eluted with a linear gradient from 0.01 to 0.10 M sodium phosphate, pH 7.5; 2ml fractions were collected. Protease B (trypsin) activity, Q; carboxypeptidase B activity, Z~; absorbance at 280 nm, O. 8, 14, 15] prompted us to examine whether a procarboxypeptidase B could also be demonstrated in extracts of the starfish D. imbricata. The isolation procedures employed were modified from the method described by Prahl and Neurath [3]. A live starfish was dissected and the pyloric caeca removed and placed in cold 0.005 M potassium phosphate buffer, pH 6.0, containing 0.005 NaC1 and 0.01 M Dip-F3. The Dip-F3 was included to prevent the activation of any zymogen that might be present in the tissue by the trypsin-like enzymes known tO be present in the pyloric caeca. These trypsin-like enzymes are known to be inhibited by the Dip-Fa [11]. The Dip-F3-inhibited tissue was immediately homogenized in a Waring Blender for 2 min and the homogenate centrifuged for 10 min at 16 000 x g. The supernatant was removed and adjusted to 0.1 M in Dip-F3 and loaded on a 3.5 cm × 36 cm column of DEAE-cellulose equilibrated with homogenizing buffer. The column was developed /
'
,
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14o
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NUMBER
on DEAE-cellulose
o f a n e x t r a c t o f D . imbrieata p y l o r i c caeca. G a r b o × y -
peptidase A activity before, [], and after, I , activation; carboxypeptidase B activity before, ©~ and after, O, activation with. trypsin; absorbance at 280 nm, A .
265 at 4 °C with starting buffer followed by a linear gradient of 0.005 to 0.2 M NaCI in 0.0050 M potassium phosphate buffer, pH 6.0, followed by a final wash of 2.0 M NaC1 in the same phosphate buffer. The column fractions were assayed for carboxypeptidases A and B as described above. An aliquot (0.5 ml) was removed from each fraction and activated with 20 #g of bovine pancreatic trypsin for 1 h at room temperature and again assayed for the above mentioned enzymes. When extraction of the pyloric caeca was carried out in the presence of Dip-F3, very low levels of both carboxypeptidase A and carboxypeptidase B activities were observed in the homogenate. The results of DEAE-cellulose chromatography on the distribution of these activities is shown in Fig. 3. It is clear from the elution pattern that both carboxypeptidases A and B are present in extracts of pyloric caeca of this starfish and that both enzymes occur in zymogen form. Although the carboxypeptidase A and carboxypeptidase B activities were not completely resolved by this DEAE-cellulose chromatography, the two activities were completely separated in the succeeding step. No further attempt was made to purify the starfish carboxypeptidase A. The procarboxypeptidase B activity was further purified by isoelectric focusing
1.80
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Fig. 4. Isoelectric focusing of procarboxypeptidase B after DEAE-cellulose chromatography (see text). Linear pH gradient from pH 3 to 12. Absorbance at 280 nm, @; carboxypeptidase B activity (units/ml) before activation with trypsin, A, and carboxypeptidase B activity after activation with. trypsin, O.
on a linear pH gradient from pH 3 to 12 (Fig. 4). This gave a product free of carboxypeptidase A, chymotrypsin and trypsin activities. The molecular weight of both the purified active enzyme and the purified zymogen from isoelectric focusing were determined by gel filtration on a calibrated
266 I
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Fig. 5. Estimation of molecular weight of D. imbricata carboxypeptidase B and procarboxypeptidase B by gel filtration on Sephadex G-100. Bovine serum albumin, [] ; procarboxypeptidase B from D. imbricata, A; ovalbumin, I ; carboxypeptidase B from D. imbricata, ~ ; bovine pancreas carboxypeptidase B, O; cytochrome c, 0 .
I00
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Fig. 6. Effect of pH on the activity of bovine pancreas carboxypeptidase B, ©, and carboxypeptidase B from D. imbricata, 0. Each enzyme was incubated for 1 h at room temperature at the appropriate p H and the enzymatic activity remaining was determined in the standard assay.
267 column of Sephadex G-100 according to the method of Andrews [9] as detailed under Experimental Procedure. The results are presented in Fig. 5.
p H optima and enzyme stability The pH versus activity profile for the enzyme as well as that for bovine carboxypeptidase B (Fig. 6) shows that both have an activity maximum in the region pH 7-7.5. Both enzymes also show a second peak in activity at the high pH end of the profile. However, this may be an artifact due to the spontaneous hydrolysis of substrate at high pH values. The enzymes seem to be quite stable, as little loss of activity was observed on incubation for 15 min at temperatures up to 50 °C. Incubation at 60 and 70 °C for this period of time resulted in losses o f 56 and 85 ~o respectively. Enzyme specificity The enzyme was tested for enzymatic activity against a variety of synthetic substrates. As shown in Table I, the greatest activity was shown toward the substrates hippuryl-L-lysine and hippuryl-L-arginine. As reported for bovine carboxypeptidase B [8], incubation of the D. imbricata enzyme with 10 -3 M 1,10-phenanthroline for one hour at 0 °C destroys all of its activity toward the substrate hippuryl-L-arginine. This loss of activity is not restored with removal of the chelating agens by dialysis. The enzyme was found to retain 100 ~o of its activity when incubated with 10 -3 M EDTA under the same conditions. DISCUSSION The present experiments have demonstrated the existence, in the pyloric caeca of the starfish D. imbricata, of a carboxypeptidase enzyme which has many properties in common with the carboxypeptidase B of bovine pancreas. On the basis of its substrate specificity, particularly its high activity toward synthetic substrates having either lysine or arginine as the carboxy-terminal residue, it has been called a carboxypeptidase B-like enzyme. The enzyme co-chromatographs with bovine carboxypeptidase B on gel filtration and the apparent molecular weight of 34 000 is similar to the molecular weights reported for carboxypeptidase B from pancreatic tissues of the lungfish [15], the dogfish [3], pig [6], cow [8], and the white shrimp [4]. As shown in Table I, the carboxypeptidase B of D. imbricata is not absolutely specific for arginine or lysine at the carboxy-terminus, but shows a low level of activity toward the synthetic substrate hippuryl-L-phenylalanine. This low level of activity toward a substrate for carboxypeptidase A has also been reported for the dogfish carboxypeptidase B [3]. The presence of a zymogen which gave this carboxFpeptidase B-like activity when activated with trypsin was detected when the original homogenate was treated with Dip-Fa to prevent activation by trypsin during ion-exchange chromatography. The zymogen of D. imbricata carboxypeptidase B is activated by bovine pancreatic trypsin. Pyloric caeca extracts, not treated with Dip-F3, show a very marked increase in carboxypeptidase B activity upon incubation and in this case the zymogen is presumably activated by the trypsin-like enzymes known to be present in the pyloric caeca of this starfish [l 1].
268 TABLE I RELATIVE ACTIVITY OF CARBOXYPEPTIDASE B-LIKE ENZYME D. 1MBRICATA AGAINST SYNTHETIC SUBSTRATES Substrate
7oo relative activity
Hippuryl-L-arginine Hippuryl-L-lysine Hippuryl-L-glycine Hippuryl-L-alanine Hippuryl-L-phenylalanine Benzoyl-oL-arginine-p-nitroanilide N-Carbobenzoxy-L-serine
100 129 10 0 18 0 0
The molecular weight of the procarboxypeptidase of D. imbricata, as determined by gel filtration, is approximately 40 (3(30. This is similar to the reported molecular weights for the bovine, lungfish [15] and dogfish [3] zymogens. These reported values are 42 000-47 000, 45 000 and 45 000, respectively. The similarities between the procarboxypeptidase B and carboxypeptidase B of the starfish D. imbricata and the reported properties of these enzymes from both higher organisms and from the white shrimp strongly suggest that they are of common evolutionary origin. While Gates and Travis [4] did not specifically investigate the existence of zymogen forms of the carboxypeptidases in the white shrimp Penaeus setiferus, the increase in total activity they observed, following chromatography, over that of the applied sample is suggestive of the presence of pro-enzymes in that species also. An earlier report [16] indicated that a single carboxypeptidase possessing both A and B activities was present in the crayfish. Gates and Travis [4] have suggested that the inability to separate these two activities in the crayfish might have been due to the similar ionic properties of the two enzymes since these latter workers were able to clearly demonstrate separate proteins with A and B activities in the related crustacean, P. setiferus. They propose that the separation of the two carboxypeptidases from a single precursor may have occurred far back in evolutionary history, possibly long before the arthropods. Our finding of separable carboxypeptidases A and B in another invertebrate further supports that view. Histological and physiological studies by Anderson [17, 18] and Mauzey [19] suggested that the pyloric caeca in starfish serve many of the functions of an exocrine pancreas. Biochemical evidence is accumulating that this is indeed the case. The isolation of trypsin-like enzymes has now been reported from pyloric caeca extracts of several starfish [11, 20, 21]. Chymotryptic-like activity has been demonstrated in extracts of D. imbricata [10], and we now report the presence of carboxypeptidase-like enzymes in this species. ACKNOWLEDGEMENTS
This work was supported in part by grants from the Clayton Foundation for Research in Biochemistry and from the National Institutes of Health (HD02889).
269 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Folk, J. E. (1956) J. Am. Chem. Soc. 78, 3541-3542 Folk, J. E. and Gladner, J. A. (1958) J. Biol. Chem. 231, 379-391 Prahl, J. W. and Neurath, H. (1966) Biochemistry 5, 4137-4145 Gates, B. J. and Travis, J. (1973) Biochemistry 12, 1867-1874 Folk, J. E., Piez, K. A., Carroll, W. R. and Gladner, J. (1960) J. Biol. Chem. 235, 2272-2277 Folk, J. E. and Schirmer, E. W. (1963) J. Biol. Chem. 238, 3884-3894 Erlanger, B. F., Kokowsky, N. and Cohen, W. (1961) Arch. Biochem. Biophys. 95, 271-278 Wintersberger, E., Cox, D. J. and Neurath, H. (1962) Biochemistry 1, 1069-1078 Andrews, P. (1964) Biochem. J. 91,222-233 Camacho, Z. (1970) Ph.D. Dissertation, University of Texas Camacho, Z., Brown, J. R. and Kitto, G. B. (1970) J. Biol. Chem. 245, 3964-3972 Morton, R. K. (1955) in Methods in Enzymology (Colowick, S. P. and Kaplan, N. O., eds), Vol. I, pp. 25-51, Academic Press, New York Ornstein, L. (1963) Ann. N.Y. Acad. Sci. 121, 321-349 Sanders, T. G. (1970) Ph.D. Dissertation, University of Illinois Reeck, G. R. and Neurath, H. (1972) Biochemistry 11, 3947-3955 Kleine, R. (1969) Z. Vgl. Physiol. 56, 142-153 Anderson, J. M. (1953) Biol. Bull. 105, 47-61 Anderson, J. M. (1960) Biol. Bull. 119, 371-398 Mauzey, K. P. (1966) Biol. Bull. 131, 127-144 Bunday, H. F. and Gustavson, J. (1973) Comp. Biochem. Physiol. 44B, 241-251 Winter, W. P. and Neurath, H. (1970) Biochemistry 9, 4673-4679
© Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands BBA 36948 P U R I F I C A T I O N OF A C A R B O X Y P E P T I D A S E B-LIKE E N Z Y M E F R O M T H E STARFISH DERMASTERIAS IMBRICATA
ROBERT
E. F E R R E L L * ,
ZENAIDO CAMACHO** and G. BARRIE KITTO***
Clayton Foundation Biochemical Institute, Department of Chemistry, University of Texas, Austin, Texas 78712 (U.S.A.)
(Received August 9th, 1974)
SUMMARY A carboxypeptidase B-like enzyme which catalyses the hydrolysis of synthetic esters oflysine and arginine has been isolated from the starfish Dermasterias imbricata. This carboxypeptidase B-like enzyme has a molecular weight of approximately 34 000 and shares this and other properties with bovine pancreatic carboxypeptidase B. The existence of zymogen for this activity in the pyloric caeca of the starfish is demonstrated. This zymogen has a molecular weight near 40 000 and appears to be analogous to other monomeric procarboxypeptidases B. The zymogen possesses an intrinsic low-level activity toward synthetic substrates of carboxypeptidase B and is activated by trypsin.
INTRODUCTION The presence of an enzyme in the euglobulin fraction of autolyzed beef pancreas, which rapidly and specifically liberated the basic amino acids lysine and arginine from the carboxyl-terminal end of synthetic peptide substrates, was first recognized by Folk [1]. A later paper [2] extended the substrate specificity studies with this basic carboxypeptidase to include a wide range of synthetic and naturally occurring substrates and gave it the trivial name carboxypeptidase B. These workers also reported that the enzyme existed in the pancreas as a zymogen which could be activated by the action of the enzyme trypsin and purified the procarboxypeptidase B from acetone extracts of beef pancreas. As part of an investigation of echinoderm proteases, we have isolated a carboxypeptidase B-like enzyme from the pyloric caeca of the starfish Dermasterias imbricata which has several properties similar to those of bovine carboxypeptidase B as well as the carboxypeptidase B isolated from the spiny Pacific dogfish by Prahl and Neurath [3] and from the white shrimp by Gates * Present address: Department of Human Genetics, University of Michigan Medical School, 1137 East Catherine Street, Ann Arbor, Mich. 48104, U.S.A. ** Present address : Department of Veterinary Pathology, Washington State University, Pullman, Wash., U.S.A. *** To whom correspondence should be addressed.
261 and Travis [4]. Evidence will also be presented for the existence of a zymogen of the starfish enzyme in the pyloric caeca. MATERIALS AND METHODS The substrates hippuryl-L-arginine, hippuryl-L-phenylalanine, hippuryl-Llysine, and hippuryl-L-glycine were from Sigma Chemical Company, benzoyl-oLarginine-p-nitroanilide from Nutritional Biochemical, and N-carbobenzoxy-L-serine and hippuryl-L-alanine were a gift from Dr D. J. Cox, Department of Chemistry, University of Texas. Diisopropylfluorophosphate (Dip-F3) was obtained from CalBiochem. The starfish, D. imbricata, was supplied by Pacific Biomarine Supply, Venice, Calif. Protein standards used in the gel-filtration studies were obtained as a kit from Pharmacia Fine Chemicals. Reagents and apparatus for isoelectric focusing were supplied by LKB, Stockholm. Purified bovine carboxypeptidases A and B were obtained from Worthington.
Enzyme assays Carboxypeptidase B activity was determined using hippuryl-L-arginine as substrate according to the method of Folk et al. [5]. Cleavage of the substrate was measured at 25 °C using a Zeiss PMQ II Spectrophotometer at 254 nm. One unit of carboxypeptidase B activity was defined as the amount of enzyme which produced an absorbance change of 1 per min at 254 nm. Carboxypeptidase A activity was determined using hippuryl-L-phenylalanine as substrate essentially as described by Folk and Schirmer [6]. Cleavage of substrate was followed as described for carboxypeptidase B. Substrate specificity studies were carried out using the substrates hippurylL-arginine, hippuryl-L-phenylalanine, hippuryl-L-glycine, and hippuryl-L-lysine. These substrates were used in the standard carboxypeptidase B assay, with the value for cleavage of hippuryl-L-arginine being designated as 100 ~ activity. The enzyme was tested for trypsin-like activity using benzoyl-DL-arginine-pnitroanilide as substrate according to the method of Erlanger et al. [7]. Cleavage Gf substrate was followed at 25 °C using a Zeiss PMQ II Spectrophotometer at 410 nm. Carboxypeptidase activity against benzoyl-DL-arginine-p-nitroanilide and N-carbobenzoxy-L-serine was determined according to Wintersberger et al. [8]. Activity toward hippuryl-L-arginine, as determined by the same assay, was used to relate these activities to all others.
Determination of molecular weight The molecular weight of each protease was estimated by the method of gel filtration [9] using a calibrated Sephadex G-100 column of inner dimensions 2 cm × 50 cm. All experiments were conducted at 4 °C. Each of the standard proteins was dissolved in 1 ml of buffer for application to the column. Sucrose was added to each sample and the higher density protein charge carefully layered above the gel. Collection of the column effluent in 1.5-ml fractions was begun with the addition of the sample to the top of the gel bed. The flow rate of the column was maintained at a constant rate (45 ml/h) by variation in hydrostatic pressure obtained by adjustment of the position of the buffer reservoir. Fractions were collected in a LKB fraction
262 collector and the absorbance at 280 nm was monitored with the use of a LKB Uvicord II and recorder. Each protein sample was run individually. The exact height of the gel bed in the column was maintained constant throughout the experiment. Values used for the molecular weight of reference proteins were as follows: cytochrome e, 12 400; ovalbumin, 45 000; bovine carboxypeptidase B, 34 300; and bovine serum albumin, 66 500. The Dermasterias carboxypeptidase B was also applied to the column separately. The protein concentration and carboxypeptidase activity of the fractions were determined spectrophotometrically.
Temperature stability and pH studies These were carried out as described previously [10]. Parallel studies using commercial bovine carboxypeptidase B were carried out for comparative purposes. Temperature stability was determined in 0.02 M potassium phosphate, pH 7.5. To study the stabilities of the two enzyme with respect to pH, equivalent amounts of enzyme were incubated in buffers of varying pH for 1 h. Aliquots were then removed and placed in the standard assay mixture using 1 mM hippuryl-L-arginine as substrate. Inhibition studies Inhibition of the enzyme by the chelating agents 1,10-phenanthroline and EDTA was measured by preincubation of the enzyme in 10 -3 M inhibitor for 1 h at 0 °C and measuring its activity in substrate solutions containing the same inhibitor concentration. Isoelectric jbcusing Isoelectric focusing was carried out with a linear pH gradient between pH 3 and 10 in a total volume of 110 ml. Carrier ampholytes, the column and the linear density gradient maker were purchased from LKB Produkter, Stockholm, Sweden, and all procedures were performed according to the manufacturer's instructions. The temperature was maintained at 4 °C by a criculating water bath and focusing was carried out for 36 h with a final potential of 700 V. After this time, 1-ml fractions were collected from the column and monitored for protein at 280 nm and assayed for carboxypeptidase B activity as described above. RESULTS
Purification of the enzyme The carboxypeptidase B was purified from the pyloric caeca of the starfish, D. imbrieata. Initial stages of the purification were carried out in parallel with the isolation of tryptic-like proteases from this organism [11 ]. Pyloric caeca were removed from live starfish and homogenized in cold 0.05 M Tris.HC1 buffer, pH 8.2, at a concentration of 200 mg of tissue per ml of buffer. The homogenate was centrifuged at 3000 × g for 10 rain, polyoxyethylene lauryl ether was added to the supernatant fraction to give a final concentration of 0.2 ~ , and the solution was allowed to incubate at 4 °C for 24 h. The solution was then centrifuged at 16 000 × g for 30 min and solid (NH4)2SO4 was added to give 40 ~ saturation. The precipitate was collected by centrifugation as above and the (NH4)zSO4 concentration of the supernatant fraction was raised to 60 ~ of saturation. The precipitate was collected by centrifugation in
263 the same manner, dissolved in cold 0.05 M T r i s . H C l buffer, pH 8.2, and dialyzed against frequent changes of the same buffer for 12 h. An acetone powder was then prepared from this dialyzed solution by the method of Morton [12]. The acetone powder was stirred with cold 0.05 M Tris.HCl, pH 7.5, for 30 min and centrifuged to remove insoluble material. The supernatant fraction was applied to a 10 cm x 100 cm column packed with Sephadex G-100, and the column was eluted with an 0.1 M potassium phosphate buffer, p H 7.5. The fractions containing both tryptic and carboxypeptidase B activity were pooled, concentrated by pressure ultrafiltration and applied to a 5.5 cm × 65 cm column of DEAE-cellulose equilibrated with 0.1 M potassium phosphate buffer, pH 7.5. The column was eluted with a linear gradient of 0.1-1.0 M phosphate buffer, pH 7.5, the elution profile being shown in Fig. 1. In this O.B
0.2
~O.B o oJ 15
0.4
O.I
z er
.~®~°'--~ z 0
A x 40
--
120
160
--r
200
FRACTION NUMBER
Fig. 1. First DEAE-cellulose chromatography of D. imbricata carboxypeptidase B. The column (5.5 cm × 60 cm) was equilibrated with 0.1 M potassium phosphate buffer, pH 7.5, and eluted with a linear gradient from 0.1 to 1.0 M potassium phosphate, pH 7.5; 8-ml fractions were collected; carboxypeptidase B activity A; absorbance at 280 nm, ©. step, the enzyme was only partially resolved from one of the two trypsins found in this organism [11]. The two activities were separated by a second DEAE-cellulose step using a 1.5 cm x 30 cm column equilibrated with 0.01 M potassium phosphate buffer and eluted with a shallow gradient from 0.01 to 0.1 M potassium phosphate, p H 7.5, as shown in Fig. 2. Fractions containing carboxypeptidase B activity were pooled and concentrated by pressure ultrafiltration. Final purification of the enzyme involved preparative isoelectric focusing in a pH gradient from pH 3 to 12, as described in Materials and Methods. The carboxypeptidase activity focused as a discrete protein peak and these fractions were pooled, passed through a Sephadex G-25 column to remove the ampholytes and then concentrated by pressure ultrafiltration. The carboxypeptidase B obtained in this manner was judged to be homogeneous, as it gave a single protein band when examined by analytical polyacrylamidegel electrophoresis [13].
Isolation of procarboxypeptidase B The presence of zymogen forms of carboxypeptidase B in other organisms [3,
264 0.60
-- 0.4
I
I
I
0.2
I
E 014~ - 10.3
c 0 go N
0.30
-
oi
~0.2
I'-,Z
u Z m
W 0.15 - ~E 0.1 ),.. O (/) N m Z I.&l _
~' 0
40
80
120
FRACTION
160
200
NUMBER
Fig. 2. Separation of carboxypeptidase B and protease B (trypsin) activity by DEAE-cellulosechromatography. The column (1.5 cm x 30 cm) was equilibrated with 0.01 M potassium phosphate buffer, pH 7.5, and eluted with a linear gradient from 0.01 to 0.10 M sodium phosphate, pH 7.5; 2ml fractions were collected. Protease B (trypsin) activity, Q; carboxypeptidase B activity, Z~; absorbance at 280 nm, O. 8, 14, 15] prompted us to examine whether a procarboxypeptidase B could also be demonstrated in extracts of the starfish D. imbricata. The isolation procedures employed were modified from the method described by Prahl and Neurath [3]. A live starfish was dissected and the pyloric caeca removed and placed in cold 0.005 M potassium phosphate buffer, pH 6.0, containing 0.005 NaC1 and 0.01 M Dip-F3. The Dip-F3 was included to prevent the activation of any zymogen that might be present in the tissue by the trypsin-like enzymes known tO be present in the pyloric caeca. These trypsin-like enzymes are known to be inhibited by the Dip-Fa [11]. The Dip-F3-inhibited tissue was immediately homogenized in a Waring Blender for 2 min and the homogenate centrifuged for 10 min at 16 000 x g. The supernatant was removed and adjusted to 0.1 M in Dip-F3 and loaded on a 3.5 cm × 36 cm column of DEAE-cellulose equilibrated with homogenizing buffer. The column was developed /
'
,
2o
40
,
,
,
ao
ioo
,
/
! ~0.6
so
FRACTION F i g . 3. C h r o m a t o g r a p h y
12o
14o
leo
NUMBER
on DEAE-cellulose
o f a n e x t r a c t o f D . imbrieata p y l o r i c caeca. G a r b o × y -
peptidase A activity before, [], and after, I , activation; carboxypeptidase B activity before, ©~ and after, O, activation with. trypsin; absorbance at 280 nm, A .
265 at 4 °C with starting buffer followed by a linear gradient of 0.005 to 0.2 M NaCI in 0.0050 M potassium phosphate buffer, pH 6.0, followed by a final wash of 2.0 M NaC1 in the same phosphate buffer. The column fractions were assayed for carboxypeptidases A and B as described above. An aliquot (0.5 ml) was removed from each fraction and activated with 20 #g of bovine pancreatic trypsin for 1 h at room temperature and again assayed for the above mentioned enzymes. When extraction of the pyloric caeca was carried out in the presence of Dip-F3, very low levels of both carboxypeptidase A and carboxypeptidase B activities were observed in the homogenate. The results of DEAE-cellulose chromatography on the distribution of these activities is shown in Fig. 3. It is clear from the elution pattern that both carboxypeptidases A and B are present in extracts of pyloric caeca of this starfish and that both enzymes occur in zymogen form. Although the carboxypeptidase A and carboxypeptidase B activities were not completely resolved by this DEAE-cellulose chromatography, the two activities were completely separated in the succeeding step. No further attempt was made to purify the starfish carboxypeptidase A. The procarboxypeptidase B activity was further purified by isoelectric focusing
1.80
I
I
I
I
I
I
16,0
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16.0
1.40
14.0
1.20
120 ~ 0.
<~ LO0
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0.60
6.0 )-
o
0.60
6.0 w
0.40
4.0
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20 30 FRACTION
40 NUMBER
50
60
Fig. 4. Isoelectric focusing of procarboxypeptidase B after DEAE-cellulose chromatography (see text). Linear pH gradient from pH 3 to 12. Absorbance at 280 nm, @; carboxypeptidase B activity (units/ml) before activation with trypsin, A, and carboxypeptidase B activity after activation with. trypsin, O.
on a linear pH gradient from pH 3 to 12 (Fig. 4). This gave a product free of carboxypeptidase A, chymotrypsin and trypsin activities. The molecular weight of both the purified active enzyme and the purified zymogen from isoelectric focusing were determined by gel filtration on a calibrated
266 I
I
I
I
\
70,000
\
[3
60,000 t.- 50,000 "r
o_
IJ.I 40°000 n.-
~ 30,000 U J 0 20,000
12,000
I
I
I
I0
50
75
ELUTION
VOLUME
I
I00
I
t25
ml)
Fig. 5. Estimation of molecular weight of D. imbricata carboxypeptidase B and procarboxypeptidase B by gel filtration on Sephadex G-100. Bovine serum albumin, [] ; procarboxypeptidase B from D. imbricata, A; ovalbumin, I ; carboxypeptidase B from D. imbricata, ~ ; bovine pancreas carboxypeptidase B, O; cytochrome c, 0 .
I00
I
I
2
4
I
I
l
6
8
10
- -
>. 80
~ 60
-J w ~
zo
0
pH
Fig. 6. Effect of pH on the activity of bovine pancreas carboxypeptidase B, ©, and carboxypeptidase B from D. imbricata, 0. Each enzyme was incubated for 1 h at room temperature at the appropriate p H and the enzymatic activity remaining was determined in the standard assay.
267 column of Sephadex G-100 according to the method of Andrews [9] as detailed under Experimental Procedure. The results are presented in Fig. 5.
p H optima and enzyme stability The pH versus activity profile for the enzyme as well as that for bovine carboxypeptidase B (Fig. 6) shows that both have an activity maximum in the region pH 7-7.5. Both enzymes also show a second peak in activity at the high pH end of the profile. However, this may be an artifact due to the spontaneous hydrolysis of substrate at high pH values. The enzymes seem to be quite stable, as little loss of activity was observed on incubation for 15 min at temperatures up to 50 °C. Incubation at 60 and 70 °C for this period of time resulted in losses o f 56 and 85 ~o respectively. Enzyme specificity The enzyme was tested for enzymatic activity against a variety of synthetic substrates. As shown in Table I, the greatest activity was shown toward the substrates hippuryl-L-lysine and hippuryl-L-arginine. As reported for bovine carboxypeptidase B [8], incubation of the D. imbricata enzyme with 10 -3 M 1,10-phenanthroline for one hour at 0 °C destroys all of its activity toward the substrate hippuryl-L-arginine. This loss of activity is not restored with removal of the chelating agens by dialysis. The enzyme was found to retain 100 ~o of its activity when incubated with 10 -3 M EDTA under the same conditions. DISCUSSION The present experiments have demonstrated the existence, in the pyloric caeca of the starfish D. imbricata, of a carboxypeptidase enzyme which has many properties in common with the carboxypeptidase B of bovine pancreas. On the basis of its substrate specificity, particularly its high activity toward synthetic substrates having either lysine or arginine as the carboxy-terminal residue, it has been called a carboxypeptidase B-like enzyme. The enzyme co-chromatographs with bovine carboxypeptidase B on gel filtration and the apparent molecular weight of 34 000 is similar to the molecular weights reported for carboxypeptidase B from pancreatic tissues of the lungfish [15], the dogfish [3], pig [6], cow [8], and the white shrimp [4]. As shown in Table I, the carboxypeptidase B of D. imbricata is not absolutely specific for arginine or lysine at the carboxy-terminus, but shows a low level of activity toward the synthetic substrate hippuryl-L-phenylalanine. This low level of activity toward a substrate for carboxypeptidase A has also been reported for the dogfish carboxypeptidase B [3]. The presence of a zymogen which gave this carboxFpeptidase B-like activity when activated with trypsin was detected when the original homogenate was treated with Dip-Fa to prevent activation by trypsin during ion-exchange chromatography. The zymogen of D. imbricata carboxypeptidase B is activated by bovine pancreatic trypsin. Pyloric caeca extracts, not treated with Dip-F3, show a very marked increase in carboxypeptidase B activity upon incubation and in this case the zymogen is presumably activated by the trypsin-like enzymes known to be present in the pyloric caeca of this starfish [l 1].
268 TABLE I RELATIVE ACTIVITY OF CARBOXYPEPTIDASE B-LIKE ENZYME D. 1MBRICATA AGAINST SYNTHETIC SUBSTRATES Substrate
7oo relative activity
Hippuryl-L-arginine Hippuryl-L-lysine Hippuryl-L-glycine Hippuryl-L-alanine Hippuryl-L-phenylalanine Benzoyl-oL-arginine-p-nitroanilide N-Carbobenzoxy-L-serine
100 129 10 0 18 0 0
The molecular weight of the procarboxypeptidase of D. imbricata, as determined by gel filtration, is approximately 40 (3(30. This is similar to the reported molecular weights for the bovine, lungfish [15] and dogfish [3] zymogens. These reported values are 42 000-47 000, 45 000 and 45 000, respectively. The similarities between the procarboxypeptidase B and carboxypeptidase B of the starfish D. imbricata and the reported properties of these enzymes from both higher organisms and from the white shrimp strongly suggest that they are of common evolutionary origin. While Gates and Travis [4] did not specifically investigate the existence of zymogen forms of the carboxypeptidases in the white shrimp Penaeus setiferus, the increase in total activity they observed, following chromatography, over that of the applied sample is suggestive of the presence of pro-enzymes in that species also. An earlier report [16] indicated that a single carboxypeptidase possessing both A and B activities was present in the crayfish. Gates and Travis [4] have suggested that the inability to separate these two activities in the crayfish might have been due to the similar ionic properties of the two enzymes since these latter workers were able to clearly demonstrate separate proteins with A and B activities in the related crustacean, P. setiferus. They propose that the separation of the two carboxypeptidases from a single precursor may have occurred far back in evolutionary history, possibly long before the arthropods. Our finding of separable carboxypeptidases A and B in another invertebrate further supports that view. Histological and physiological studies by Anderson [17, 18] and Mauzey [19] suggested that the pyloric caeca in starfish serve many of the functions of an exocrine pancreas. Biochemical evidence is accumulating that this is indeed the case. The isolation of trypsin-like enzymes has now been reported from pyloric caeca extracts of several starfish [11, 20, 21]. Chymotryptic-like activity has been demonstrated in extracts of D. imbricata [10], and we now report the presence of carboxypeptidase-like enzymes in this species. ACKNOWLEDGEMENTS
This work was supported in part by grants from the Clayton Foundation for Research in Biochemistry and from the National Institutes of Health (HD02889).
269 REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
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