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Purification and Partial Characterization of Camel Anionic Chymotrypsin
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 348, No. 2, December 15, pp. 363–368, 1997 Article No. BB970376
Purification and Partial Characterization of Camel Anionic Chymotrypsin Abdulrahman Al-Ajlan and Graham S. Bailey1 Department of Biological Sciences, Central Campus, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom
Received July 18, 1997, and in revised form September 5, 1997
An anionic chymotrypsin-like enzyme was isolated from a crude extract of camel pancreas by a threestep procedure consisting of anion-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. The purified enzyme was homogeneous on native and SDS gel electrophoresis and on gel isoelectric focusing. Its molecular mass was estimated as 28.5 kDa and its isoelectric point was found to be 4.4. The enzyme differed markedly from bovine chymotrypsin A in its substrate specificity, showing considerably lower values of the specificity constant for its action on tyrosine, tryptophan, and phenylalanine esters. Its pH optimum was found to be 7.8. It showed lower kininase activity and was more susceptible to inhibition by a number of inhibitors than the bovine cationic chymotrypsin. On the other hand, the camel enzyme showed a much greater hydrolytic activity than the bovine enzyme toward a leucine ester. In terms of its size, charge, and substrate specificity the camel enzyme was very similar to anionic chymotrypsins that have been isolated from other species and thus appears to be a camel anionic chymotrypsin. q 1997 Academic Press
Key Words: camel; anionic chymotrypsin; purification; characterization; substrate specificity; kininase activity; inhibition; pH optimum; stability.
Numerous forms of chymotrypsinogens and chymotrypsins have been isolated from mammalian pancreas. For example, cationic chymotrypsinogen A and anionic chymotrypsinogens B and C have been recovered from porcine pancreas (1–3). Chymotrypsinogens A and B are two cationic forms of the zymogen that have been isolated from bovine pancreas (4). An anionic form of 1 To whom correspondence should be addressed. Fax: 01 206 87 25 92. E-mail: [email protected].
bovine chymotrypsinogen, corresponding to porcine chymotrypsinogen C, exists as a subunit of procarboxypeptidase A aggregates (5). The zymogens and their respective enzymes form two distinct subgroups on the basis of size, amino acid composition, and enzymatic properties (6–8). One characteristic feature of porcine chymotrypsin C and its bovine counterpart is a relatively high activity toward leucyl peptide bonds (2, 6). The one-humped camel (Camelius dromedarius) is the least studied of all domestic species of mammals despite being an essential source of food in many parts of the Arabian peninsula (9). Very few studies of camel pancreatic enzymes have been reported (10–12). We are currently studying enzymatic components of the kinin system of the camel to see if they have any special features that may contribute to the camel’s remarkable ability to withstand drought for many days (13). Kinins help to control water and electrolyte balance in mammals (14). Their levels are determined by the relative activities of kallikreins, enzymes that proteolytically release kinin from its kininogen precursor, and kininases, enzymes that render kinin biologically inactive by cleaving any of its peptide bonds (15). Bovine chymotrypsin is a potent kininase, at least in vitro (16), and in this paper we report on the purification and characterization of an anionic chymotrypsin-like enzyme from camel pancreas. It possesses only weak kininase activity and in many of its properties it appears to be the camel equivalent of porcine and bovine anionic chymotrypsins. MATERIALS AND METHODS
Materials Pancreas was obtained from a freshly slaughtered young camel at an abbatoir in Riyadh, Saudi Arabia. It was transported in dry ice to the laboratory where it was stored at 0207C until use. Substrates, inhibitors, bovine chymotrypsin A (a-chymotrypsin), other chemicals, and resins were mainly purchased from Sigma (Poole, United Kingdom). The substrate N-acetyl-L-leucine methyl ester 363
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(Ac-Leu-OMe)2 was obtained from Bachem (Saffron Walden, United Kingdom).
protein (mg/ml) Å 1.55 A280 0 0.76 A260 , to measurements of absorbances at 280 and 260 nm (24).
Enzyme Assays
Analytical Gel Electrophoresis
All assays were carried out at 257C. Fractions obtained at different stages of the purification procedure were routinely tested for the presence of chymotrypsin activity by a modification of a published spectrophotometric assay (17). A 1.07 mM stock solution of the substrate N-benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt) was made in 5.5% (v/v) methanol. The reaction cuvette contained 1.4 ml of the substrate solution, 1.5 ml 0.08 M Tris-HCl buffer, pH 7.8, containing 0.1 M CaCl2 and 0.1 ml of the sample to be tested. The reaction was followed by measuring the increase in absorbance at 256 nm. The esterolytic activity of the purified enzyme toward a number of potential substrates was measured at pH 7.8 by a titrimetric assay (18). Carboxypeptidase A activity was assayed at pH 7.5 with 1 mM hippuryl-L-phenylalanine (Bz-Gly-Phe) as substrate (19). Carboxypeptidase B activity was determined at pH 7.65 using 1 mM hippuryl-Larginine (Bz-Gly-Arg) as substrate (20). Values of the kinetic parameters kcat and Km at pH 7.8 over the concentration ranges 0.02 to 0.7 mM for Bz-Tyr-OEt, 0.3 to 0.7 mM for N-acetyl-L-tyrosine ethyl ester (Ac-Tyr-OEt), 0.6 to 2.0 mM for N-acetyl-L-phenylalanine ethyl ester (Ac-Phe-OEt), 0.2 to 1.0 mM for N-acetyl-L-tryptophan ethyl ester (Ac-Trp-OEt), and 0.08 to 1.0 mM for Ac-Leu-OMe were estimated for the pure enzyme using the direct linear plot analysis (21). For comparative purposes, the same analysis was carried out for bovine chymotrypsin A over the concentration ranges 0.02 to 0.2 mM (BzTyr-OEt), 0.28 to 1.87 mM (Ac-Tyr-OEt), 0.11 to 1.34 mM (Ac-PhOEt), 0.075 to 0.51 mM (Ac-Trp-OEt), and 1 to 5 mM (Ac-Leu-OMe). For all of these enzyme assays, the unit of enzymatic activity is expressed as mmol/min.The kininase activities of the camel enzyme and bovine chymotrypsin A were determined at pH 7.4 using 500 nM bradykinin as substrate. Residual substrate was measured by a specific and sensitive radioimmunoassay (22) with an interassay coefficient of variation of 4.5% and an intraassay coefficient of variation of 3%. The unit of activity was defined as the amount of enzyme that catalyzed the degradation of 1 nmol bradykinin/min. The pH optima of the activities of the camel and bovine enzymes toward Bz-Tyr-OEt were established using 0.05 M sodium phosphate (pH 5.0 to 7.0), 0.05 M Tris-HCl (pH 7.0 to 9.0), and 0.05 M glycineNaOH (pH 9.5 and 10.0) as buffers, containing 0.1 M CaCl2 . Aprotinin, soybean trypsin inhibitor (SBTI), lima bean trypsin inhibitor (LBTI), Na-p-tosyl-L-lysine chloromethyl ketone (TLCK), 1,10-phenanthroline, and EDTA were each tested for their ability to inhibit the camel enzyme. The pure enzyme and each potential inhibitor were preincubated at 257C for 15 min before assaying the residual enzymatic activity against 0.2 and 0.5 mM Bz-Tyr-OEt. The type of inhibition and values of the inhibition constants were determined through the use of Dixon plots (23). A similar analysis was carried out for bovine chymotrypsin A using 0.1 and 0.5 mM Bz-Tyr-OEt.
Protein Concentration Dilute solutions were concentrated by ultrafiltration using Omega membranes (Flowgen, Sittingbourne, United Kingdom). Determinations of protein concentrations were made by applying the equation,
2 Abbreviations used: Ac-Leu-OMe, N-acetyl-L-leucine methyl ester; Bz-Tyr-OEt, N-benzoyl-L-tyrosine ethyl ester; Bz-Gly-Phe, hippuryl-L-phenylalanine; Bz-Gly-Arg, hippuryl-L-arginine; Ac-TyrOEt, N-acetyl-L-tyrosine ethyl ester; Ac-Phe-OEt, N-acetyl-phenylalanine ethyl ester; Ac-Trp-OEt, N-acetyl-L-tryptophan ethyl ester; SBTI, soybean trypsin inhibitor; LBTI, lima bean trypsin inhibitor; TLCK, Na-p-tosyl-L-lysine chloromethyl ketone; SDS, sodium dodecyl sulfate.
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The purified enzyme was treated with diisopropyl fluorophosphate prior to gel electrophoresis (25). Slab gel disc electrophoresis under native, nondenaturing conditions was carried out at pH 8.9 on 10% acrylamide gels (26). SDS–gel electrophoresis on 10 to 20% acrylamide gradient gels was performed using the discontinuous Tris– glycine buffer system (27). Samples were prepared for electrophoresis by boiling for 5 min in 0.0625 M Tris-HCl buffer, pH 6.8, containing 10% glycerol, 2% SDS and 5% mercaptoethanol. Sigma molecular mass standards (14 to 66 kDa) were used for calibration. Gels were stained by gentle shaking at room temperature for 1 h in 0.3% Coomassie blue R-250 in methanol:acetic acid:water (5:2:5, v/v).Gel isoelectric focusing was performed on commercial polyacrylamide plates (nominal range pH 3–10) according to the manufacturers instructions (Serva, Heidelberg, Germany) using a Serva set of isoelectric point markers (pH 3.5–10.65).
Purification Procedure All stages of the purification procedure were carried out at 47C. Preparation of crude extract. The camel pancreas was chopped into small pieces using a sterilized scalpel after removal of excess fat. The pieces of tissue (79 g) were washed with starting buffer (0.05 Tris-HCl, pH 7.4, containing 0.1 M NaCl) to remove blood and were then homogenized in that buffer (250 ml) using a Waring blender at low speed for 2 min and at high speed for 2 min. The resulting homogenate was filtered through muslin cloth and was then centrifuged at 4100g for 7 min. The supernatant was filtered through muslin cloth and centrifuged at 23,500g for 30 min. This resultant supernatant was filtered through muslin cloth and centrifuged at 23,500g for 1 h. The final supernatant was filtered through muslin cloth. Anion-exchange chromatography. The final supernatant (165 ml) was applied to a column (5 1 12 cm) of DEAE-Sephadex A-50 resin that had been equilibrated with the starting buffer. The column was washed at a flow rate of 45 ml/h with starting buffer (2150 ml) to remove cationic substances that did not bind to the resin. Bound substances were sequentially eluted by application of a linear salt gradient consisting of starting buffer (1000 ml), which included 0.1 M NaCl, and finishing buffer (1000 ml) composed of 0.05 M TrisHCl, pH 7.4, containing 0.7 M NaCl. The flow rate was maintained at 45 ml/h and fractions of 7.5 ml were collected every 10 min. Gel filtration. The most active anionic fraction from the anionexchange chromatography was further purified by gel filtration. This step was performed a number of times in order to process all of the relevant fraction. In a typical run, an aliquot (5 ml) of the fraction in 0.05 M Tris-HCl buffer, pH 7.4, containing 0.1 M NaCl, was passed through a column (2.6 1 80 cm) of Sephadex G150 resin at a flow rate of 6.75 ml/h, collecting fractions of 2.25 ml every 20 min. Hydrophobic interaction chromatography. The active fraction from the gel filtration was further purified by hydrophobic interaction chromatography. This step was carried out a number of times in order to process all of the relevant fraction. In a typical run an aliquot (6 ml) of the fraction was made to 0.6 M (NH4)2SO4 and was applied to a column (1.2 1 8 cm) of phenyl-Sepharose resin that had been equilibrated with 0.05 M Tris-HCl buffer, pH 7.4, containing 0.6 M (NH4)2SO4 and 0.1 M NaCl. Unadsorbed substances were eluted by washing the column with the forementioned buffer (80 ml) at a flow rate of 6 ml/h, collecting fractions of 2 ml every 20 min. A negative salt gradient, consisting of 30 ml of the forementioned buffer as the initial buffer and 30 ml of 0.05 M Tris-HCl, pH 7.4, as the final buffer, was then applied to the column. To complete the elution of
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CAMEL ANIONIC CHYMOTRYPSIN
FIG. 1. Gradient elution profile of crude extract of camel pancreas on anion-exchange chromatography. (—) Absorbance at 280 nm, (—) NaCl gradient.
bound substances the column was washed with 70 ml of 0.05 M TrisHCl, pH 7.4, buffer.
RESULTS AND DISCUSSION
The crude extract of camel pancreatic tissue showed no activity toward Bz-Tyr-OEt even after standing for 24 h at room temperature, reflecting the presence and stability of the chymotrypsinogens, so the zymogens were activated by incubating a sample of the crude extract with bovine trypsin (protein:trypsin Å 68:1 w/ w) for various time periods at 257C. The activated sample showed a maximum activity of 2.6 units/mg protein after 30 min incubation. The gradient elution profile of the crude extract on anion-exchange chromatography is shown in Fig 1. The fractions were pooled as indicated. None of them showed any direct activity toward Bz-Tyr-OEt but after activation by exogenous trypsin the pregradient, cationic fraction PG and two of the anionic fractions, fractions 2 and 3, showed considerable chymotrypsin-like
FIG. 2. Elution profile of F2 on gel filtration.
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FIG. 3. Elution profile of GF3 on hydrophobic interaction chromatography. The resin was initially washed with 0.05 M Tris-HCl buffer, pH 7.4, containing 0.6 M (NH4)2SO4 and 0.1 M NaCl. From tube No. 41 to tube No. 70 it was washed with a linear decreasing gradient of 0.6 M (NH4)2SO4 and 0.1 M NaCl to zero salt in the forementioned buffer. From tube 71 onward it was washed with buffer alone.
activity towards that substrate (see Table I). Cationic and anionic forms of chymotrypsinogen and chymotrypsin have been isolated from porcine and bovine pancreas (2, 6), so it is reasonable to assume that fraction PG contained camel cationic chymotrypsinogen
FIG. 4. SDS–gel electrophoretic pattern of the purified enzyme.Lane 1, purified enzyme. Lane 2, standard marker proteins with molecular masses in kDa denoted to the right.
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AL-AJLAN AND BAILEY TABLE I
Purification of Anionic Chymotrypsin from Camel Pancreas
Fraction
Total protein (mg)
Total activity (units)a
Specific activity (units/mg)
Recovery (%)/
Purification
Crude extract PG (cationic) F2 (anionic) F3 (anionic) GF3 H3
5577 2632 286 93 94 21
14,500 6,054 4,090 577 3,243 1,730
2.6 2.3 14.3 6.2 34.5 82.4
(100)
(1)
65
12.6
51 27
30.5 72.9
a b
Measured using 0.5 mM Bz-Tyr-OEt as substrate. Calculated by taking into account the existence of different cationic and anionic chymotrypsins in the crude extract.
whereas fractions 2 and 3 contained the corresponding anionic zymogen. Considering the total enzymatic activities toward Bz-Tyr-OEt recovered in the activated pregradient and gradient fractions, it can be suggested that anionic chymotrypsin represented 43.5% of the total chymotryptic activity of the activated crude extract of camel pancreas. The anionic chymotrypsin was further purified by gel filtration. A typical elution profile is shown in Fig 2. Chymotryptic activity was found to be present in fraction GF3. The level of activity did not increase on incubation with exogenous trypsin, thus indicating that the measured activity was due to the presence of the active enzyme. None of the other fractions showed activity toward Bz-Tyr-OEt either before or after incubation with exogenous trypsin. Fraction GF3 was subjected to hydrophobic interaction chromatography. A representative elution profile is shown in Fig 3. Only fraction H3 showed chymotryptic activity. Assuming that anionic chymotrypsin represented 43.5% of the total chymotryptic activity of the activated crude extract it can be calculated that the purification factor for the anionic enzyme was 72.9fold with a 27% recovery of activity. A summary of the overall purification is presented in Table I. Fraction H3 was seen to be homogeneous on slab gel
electrophoresis under nondenaturing conditions and on gel isoelectric focusing (data not shown). Its isoelectric point was estimated to be 4.4. It also showed a single component on SDS–gel electrophoresis (Fig. 4) and its molecular mass was estimated to be 28.5 kDa. A molecular mass of 29 kDa has been determined for porcine chymotrypsinogen C from amino acid composition and by ultracentrifugation (3), and a value of 28 kDa found by SDS–gel electrophoresis for the corresponding bovine zymogen (8). The purified enzyme showed no catalytic activity toward Tos-Arg-OMe and Bz-Arg-OEt, thus showing a lack of trypsin-like activity. It was not active toward Bz-Gly-Phe or Bz-Gly-Arg, thus showing an absence of carboxypeptidase A and carboxypeptidase B activities, respectively. In contrast, it was active toward a number of substrates hydrolyzed by chymotrypsin-like enzymes (Table II). It can be seen that the values of the specificity constant (kcat/Km) of the camel enzyme for its action on the tyrosine, tryptophan, and phenylalanine esters were considerably less that the corresponding values of bovine chymotrypsin A. However, the camel enzyme showed considerably more activity than the bovine enzyme toward the leucine ester. Similar findings have previously been reported for porcine anionic chymotrypsin C and its bovine counterpart, subunit II, com-
TABLE II
Kinetic Constants of Camel Anionic Chymotrypsin and Bovine Chymotrypsin A Camel anionic chymotrypsin
Substrate
Km (mM)
kcat (s01)
Bz-Tyr-OEt Ac-Tyr-OEt Ac-Trp-OEt Ac-Phe-OEt Ac-Leu-OMe
0.64 2.00 1.12 1.39 1.00
57.5 25.4 9.3 5.3 22.1
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Bovine chymotrypsin A kcat/Km (M01 s01)
8.99 1.27 0.83 0.38 2.21
1 1 1 1 1
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104 104 104 104 104
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Km (mM)
kcat (s01)
0.13 1.27 0.17 1.04 7.27
76.6 126.0 48.4 52.4 6.6
kcat/Km (M01 s01) 5.82 0.99 2.85 0.50 0.09
1 1 1 1 1
105 105 105 105 104
CAMEL ANIONIC CHYMOTRYPSIN
pared to porcine chymotrypsin A and bovine chymotrypsins A and B which are cationic enzymes (2, 6). The camel enzyme showed a kininase activity of 11.4 units/mg, considerably lower than the value of 76.4 units/mg recorded for bovine chymotrypsin A under the same experimental conditions. Understanding the physiological importance, if any, of the kininase activity of the camel chymotrypsin-like enzyme must await a full characterization of other components of the kinin system, particularly those that reside in the kidney. However, it should be pointed out that chymotrypsin and other pancreatic proteolytic enzymes have been detected in the blood stream of other mammals (28, 29). Furthermore, an enzymatically active tissue kallikrein has been isolated from human plasma (30). Also, it is probable that plasma carboxypeptidase N (kininase 1) does not function in vivo as a kininase (31), so there has to be an enzyme in plasma other than kininase 1 that is responsible for the removal of the C terminal arginine residue (32). Bovine chymotrysin in vitro rapidly cleaves the C terminal peptide bond of bradykinin (33, 34). It is possible that in certain circumstances chymotrypsin could act as a physiological inactivator of locally generated kinin. The esterolytic activity of the camel enzyme toward Bz-Tyr-OEt was not inhibited by a 1000-fold molar excess of the metalloproteinase inhibitors EDTA and 1,10-phenanthroline or by a 2000-fold molar excess of TLCK, an irreversible inhibitor of trypsin-like enzymes. However, it was competitively inhibited by several chymotrypsin inhibitors (Table III). It can be seen that the camel enzyme was more susceptible than bovine chymotrypsin A to inhibition by SBTI and LBTI. The pH optimum for the hydrolysis of Bz-Tyr-OEt by camel anionic chymotrypsin was found to be pH 7.8 (Fig. 5), the same value as found for bovine chymotrypsin A (data not shown). The esterolytic activity of the camel enzyme at pH 7.4 was stable for at least 10 h at 257C, in both the presence and the absence of 0.1 M CaCl2 . Under the same conditions bovine chymotrypsin A was stable in the presence of 0.1 M CaCl2 but exhibited a 27% loss of activity in its absence. Thus,on the basis of physiochemical properties, substrate specificity, and interaction with potential inhibitors, it can be
TABLE III
Inhibition of Camel Anionic Chymotrypsin and Bovine Chymotrypsin A
Inhibitor
Camel chymotrypsin Ki (nM)
Bovine chymotrypsin A Ki (nM)
Aprotinin SBTI LBTI
15.4 3.5 18.0
3.5 250.0 42.5
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FIG. 5. Effect of pH on the activity of the purified enzyme toward Bz-Tyr-OEt.
concluded that the purified enzyme is camel anionic chymotrypsin. It is the camel equivalent of porcine chymotrypsin C and its bovine counterpart (2, 6). It is a relatively weak kininase. REFERENCES 1. Charles, M., Gratecos, D., Rovery, M., and Desnuelle, P. (1967) Biochim. Biophys. Acta 140, 395–409. 2. Folk, J. E., and Schirmer, W. E. (1965) J. Biol. Chem. 240, 181– 192. 3. Gratecos, D., Guy, O., Rovery, M., and Desnuelle, P. (1969) Biochim. Biophys. Acta 175, 82–96. 4. Wilcox, P. E. (1970) Methods Enzymol. 19, 64–112. 5. Pe´tra, P. H. (1970) Methods Enzymol. 19, 460–503. 6. Keil-Dlouha, V., Puigserver, A., Marie, A., and Keil, B. (1972) Biochim. Biophys. Acta 276, 531–535. 7. Peanasky, R. J., Gratecos, D., Baratti, J., and Rovery, M. (1969) Biochim. Biophys. Acta 181, 82–92. 8. Puigserver, A., Vaugoyeau, H., and Desnuelle, P. (1972) Biochim. Biophys. Acta 276, 519–530. 9. Hussein, M. F., Al-Momen, A. K., and Gader, A. M. A. (1992) Comp. Haematol. Int. 2, 92–96. 10. Bricteux-Gregoire, S., Schyns, R., and Florkin M. (1972) Comp. Biochem. Physiol. 42B, 23–39. 11. Rafiq, A., and Bailey, G. S. (1996) Comp. Biochem. Physiol. 115B, 363–367. 12. Welling, G. W., Groen, G., and Beintema, J. J. (1975) Biochem. J. 147, 505–511. 13. Yagil, R. (1985) The Desert Camel, Karger, Basel. 14. Damas, J. (1993) Arch. Int. Physiol. Biochim. Biophys. 101, 227– 232. 15. Bhoola, K. D., Figueroa, C. D., and Worthy, D. (1992) Pharmacol Rev. 44, 1–80. 16. Schacter, M. (1969) Physiol. Rev. 49, 509–547. 17. Hummel, B. C. W. (1959) Can. J. Biochem. Physiol. 37, 1391– 1399. 18. Walsh, K. A., and Wilcox, P. E. (1970) Methods Enzymol. 19, 31– 41. 19. Folk, J. E., and Schirmer, E. W. (1963) J. Biol. Chem 238, 3884– 3894.
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20. Folk, J. E., Piez, K. A., Carroll, W. R., and Gladner, J. (1960) J. Biol. Chem. 235, 2272–2277. 21. Eisenthal, R., and Cornish-Bowden, A. (1960) Biochem. J. 139, 715–720. 22. Bailey, G. S., Al-Joufi, A., Rawat, S., and Smith, D. C. (1991) Toxicon 29, 777–781. 23. Dixon, M. (1952) Biochem. J. 55, 170–171. 24. Stoscheck, C. M. (1990) Methods Enzymol. 82, 50–68. 25. Cohen, J. A., Oosterbaan, R. A., and Berends, F. (1967) Methods Enzymol. 11, 686–702. 26. Blackspear, P. J. (1984) Methods Enzymol. 104, 237–255. 27. Laemmli, U. K. (1970) Nature 227, 680–685.
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28. Largman, C. Brodrick, J. W., and Geokas, M. C. (1981) Methods Enzymol. 74, 272–290. 29. Geokas, M. C., Largman, C., Brodrick, J. W., Johnson, J. H., and Fassell, M. (1979) J. Biol. Chem. 254, 2775–2781. 30. Geiger, R., Clausnitzer, B., Fink, E., and Fritz, H. (1980) HoppeSeyler’s Z. Physiol. Chem. 361, 1795–1803. 31. Ryan, J. W. (1988) Methods Enzymol. 163, 186–194. 32. Sheikh, I. A., and Kaplan, A. P. Biochem.Pharm. 35, 1957–1963. 33. Elliott, D. F., Lewis, G. P., and Horton, E. W. (1960) Biochem. Biophys. Res. Commun. 3, 87–91. 34. Sampaio, C. A. M., Nunes, S. T., Graca, M. D., Mazzacoratti, N., and Prado, J. L. (1976) Biochem. Pharm. 25, 2391–2394.
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Vol. 348, No. 2, December 15, pp. 363–368, 1997 Article No. BB970376
Purification and Partial Characterization of Camel Anionic Chymotrypsin Abdulrahman Al-Ajlan and Graham S. Bailey1 Department of Biological Sciences, Central Campus, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom
Received July 18, 1997, and in revised form September 5, 1997
An anionic chymotrypsin-like enzyme was isolated from a crude extract of camel pancreas by a threestep procedure consisting of anion-exchange chromatography, gel filtration, and hydrophobic interaction chromatography. The purified enzyme was homogeneous on native and SDS gel electrophoresis and on gel isoelectric focusing. Its molecular mass was estimated as 28.5 kDa and its isoelectric point was found to be 4.4. The enzyme differed markedly from bovine chymotrypsin A in its substrate specificity, showing considerably lower values of the specificity constant for its action on tyrosine, tryptophan, and phenylalanine esters. Its pH optimum was found to be 7.8. It showed lower kininase activity and was more susceptible to inhibition by a number of inhibitors than the bovine cationic chymotrypsin. On the other hand, the camel enzyme showed a much greater hydrolytic activity than the bovine enzyme toward a leucine ester. In terms of its size, charge, and substrate specificity the camel enzyme was very similar to anionic chymotrypsins that have been isolated from other species and thus appears to be a camel anionic chymotrypsin. q 1997 Academic Press
Key Words: camel; anionic chymotrypsin; purification; characterization; substrate specificity; kininase activity; inhibition; pH optimum; stability.
Numerous forms of chymotrypsinogens and chymotrypsins have been isolated from mammalian pancreas. For example, cationic chymotrypsinogen A and anionic chymotrypsinogens B and C have been recovered from porcine pancreas (1–3). Chymotrypsinogens A and B are two cationic forms of the zymogen that have been isolated from bovine pancreas (4). An anionic form of 1 To whom correspondence should be addressed. Fax: 01 206 87 25 92. E-mail: [email protected].
bovine chymotrypsinogen, corresponding to porcine chymotrypsinogen C, exists as a subunit of procarboxypeptidase A aggregates (5). The zymogens and their respective enzymes form two distinct subgroups on the basis of size, amino acid composition, and enzymatic properties (6–8). One characteristic feature of porcine chymotrypsin C and its bovine counterpart is a relatively high activity toward leucyl peptide bonds (2, 6). The one-humped camel (Camelius dromedarius) is the least studied of all domestic species of mammals despite being an essential source of food in many parts of the Arabian peninsula (9). Very few studies of camel pancreatic enzymes have been reported (10–12). We are currently studying enzymatic components of the kinin system of the camel to see if they have any special features that may contribute to the camel’s remarkable ability to withstand drought for many days (13). Kinins help to control water and electrolyte balance in mammals (14). Their levels are determined by the relative activities of kallikreins, enzymes that proteolytically release kinin from its kininogen precursor, and kininases, enzymes that render kinin biologically inactive by cleaving any of its peptide bonds (15). Bovine chymotrypsin is a potent kininase, at least in vitro (16), and in this paper we report on the purification and characterization of an anionic chymotrypsin-like enzyme from camel pancreas. It possesses only weak kininase activity and in many of its properties it appears to be the camel equivalent of porcine and bovine anionic chymotrypsins. MATERIALS AND METHODS
Materials Pancreas was obtained from a freshly slaughtered young camel at an abbatoir in Riyadh, Saudi Arabia. It was transported in dry ice to the laboratory where it was stored at 0207C until use. Substrates, inhibitors, bovine chymotrypsin A (a-chymotrypsin), other chemicals, and resins were mainly purchased from Sigma (Poole, United Kingdom). The substrate N-acetyl-L-leucine methyl ester 363
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AL-AJLAN AND BAILEY
(Ac-Leu-OMe)2 was obtained from Bachem (Saffron Walden, United Kingdom).
protein (mg/ml) Å 1.55 A280 0 0.76 A260 , to measurements of absorbances at 280 and 260 nm (24).
Enzyme Assays
Analytical Gel Electrophoresis
All assays were carried out at 257C. Fractions obtained at different stages of the purification procedure were routinely tested for the presence of chymotrypsin activity by a modification of a published spectrophotometric assay (17). A 1.07 mM stock solution of the substrate N-benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt) was made in 5.5% (v/v) methanol. The reaction cuvette contained 1.4 ml of the substrate solution, 1.5 ml 0.08 M Tris-HCl buffer, pH 7.8, containing 0.1 M CaCl2 and 0.1 ml of the sample to be tested. The reaction was followed by measuring the increase in absorbance at 256 nm. The esterolytic activity of the purified enzyme toward a number of potential substrates was measured at pH 7.8 by a titrimetric assay (18). Carboxypeptidase A activity was assayed at pH 7.5 with 1 mM hippuryl-L-phenylalanine (Bz-Gly-Phe) as substrate (19). Carboxypeptidase B activity was determined at pH 7.65 using 1 mM hippuryl-Larginine (Bz-Gly-Arg) as substrate (20). Values of the kinetic parameters kcat and Km at pH 7.8 over the concentration ranges 0.02 to 0.7 mM for Bz-Tyr-OEt, 0.3 to 0.7 mM for N-acetyl-L-tyrosine ethyl ester (Ac-Tyr-OEt), 0.6 to 2.0 mM for N-acetyl-L-phenylalanine ethyl ester (Ac-Phe-OEt), 0.2 to 1.0 mM for N-acetyl-L-tryptophan ethyl ester (Ac-Trp-OEt), and 0.08 to 1.0 mM for Ac-Leu-OMe were estimated for the pure enzyme using the direct linear plot analysis (21). For comparative purposes, the same analysis was carried out for bovine chymotrypsin A over the concentration ranges 0.02 to 0.2 mM (BzTyr-OEt), 0.28 to 1.87 mM (Ac-Tyr-OEt), 0.11 to 1.34 mM (Ac-PhOEt), 0.075 to 0.51 mM (Ac-Trp-OEt), and 1 to 5 mM (Ac-Leu-OMe). For all of these enzyme assays, the unit of enzymatic activity is expressed as mmol/min.The kininase activities of the camel enzyme and bovine chymotrypsin A were determined at pH 7.4 using 500 nM bradykinin as substrate. Residual substrate was measured by a specific and sensitive radioimmunoassay (22) with an interassay coefficient of variation of 4.5% and an intraassay coefficient of variation of 3%. The unit of activity was defined as the amount of enzyme that catalyzed the degradation of 1 nmol bradykinin/min. The pH optima of the activities of the camel and bovine enzymes toward Bz-Tyr-OEt were established using 0.05 M sodium phosphate (pH 5.0 to 7.0), 0.05 M Tris-HCl (pH 7.0 to 9.0), and 0.05 M glycineNaOH (pH 9.5 and 10.0) as buffers, containing 0.1 M CaCl2 . Aprotinin, soybean trypsin inhibitor (SBTI), lima bean trypsin inhibitor (LBTI), Na-p-tosyl-L-lysine chloromethyl ketone (TLCK), 1,10-phenanthroline, and EDTA were each tested for their ability to inhibit the camel enzyme. The pure enzyme and each potential inhibitor were preincubated at 257C for 15 min before assaying the residual enzymatic activity against 0.2 and 0.5 mM Bz-Tyr-OEt. The type of inhibition and values of the inhibition constants were determined through the use of Dixon plots (23). A similar analysis was carried out for bovine chymotrypsin A using 0.1 and 0.5 mM Bz-Tyr-OEt.
Protein Concentration Dilute solutions were concentrated by ultrafiltration using Omega membranes (Flowgen, Sittingbourne, United Kingdom). Determinations of protein concentrations were made by applying the equation,
2 Abbreviations used: Ac-Leu-OMe, N-acetyl-L-leucine methyl ester; Bz-Tyr-OEt, N-benzoyl-L-tyrosine ethyl ester; Bz-Gly-Phe, hippuryl-L-phenylalanine; Bz-Gly-Arg, hippuryl-L-arginine; Ac-TyrOEt, N-acetyl-L-tyrosine ethyl ester; Ac-Phe-OEt, N-acetyl-phenylalanine ethyl ester; Ac-Trp-OEt, N-acetyl-L-tryptophan ethyl ester; SBTI, soybean trypsin inhibitor; LBTI, lima bean trypsin inhibitor; TLCK, Na-p-tosyl-L-lysine chloromethyl ketone; SDS, sodium dodecyl sulfate.
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The purified enzyme was treated with diisopropyl fluorophosphate prior to gel electrophoresis (25). Slab gel disc electrophoresis under native, nondenaturing conditions was carried out at pH 8.9 on 10% acrylamide gels (26). SDS–gel electrophoresis on 10 to 20% acrylamide gradient gels was performed using the discontinuous Tris– glycine buffer system (27). Samples were prepared for electrophoresis by boiling for 5 min in 0.0625 M Tris-HCl buffer, pH 6.8, containing 10% glycerol, 2% SDS and 5% mercaptoethanol. Sigma molecular mass standards (14 to 66 kDa) were used for calibration. Gels were stained by gentle shaking at room temperature for 1 h in 0.3% Coomassie blue R-250 in methanol:acetic acid:water (5:2:5, v/v).Gel isoelectric focusing was performed on commercial polyacrylamide plates (nominal range pH 3–10) according to the manufacturers instructions (Serva, Heidelberg, Germany) using a Serva set of isoelectric point markers (pH 3.5–10.65).
Purification Procedure All stages of the purification procedure were carried out at 47C. Preparation of crude extract. The camel pancreas was chopped into small pieces using a sterilized scalpel after removal of excess fat. The pieces of tissue (79 g) were washed with starting buffer (0.05 Tris-HCl, pH 7.4, containing 0.1 M NaCl) to remove blood and were then homogenized in that buffer (250 ml) using a Waring blender at low speed for 2 min and at high speed for 2 min. The resulting homogenate was filtered through muslin cloth and was then centrifuged at 4100g for 7 min. The supernatant was filtered through muslin cloth and centrifuged at 23,500g for 30 min. This resultant supernatant was filtered through muslin cloth and centrifuged at 23,500g for 1 h. The final supernatant was filtered through muslin cloth. Anion-exchange chromatography. The final supernatant (165 ml) was applied to a column (5 1 12 cm) of DEAE-Sephadex A-50 resin that had been equilibrated with the starting buffer. The column was washed at a flow rate of 45 ml/h with starting buffer (2150 ml) to remove cationic substances that did not bind to the resin. Bound substances were sequentially eluted by application of a linear salt gradient consisting of starting buffer (1000 ml), which included 0.1 M NaCl, and finishing buffer (1000 ml) composed of 0.05 M TrisHCl, pH 7.4, containing 0.7 M NaCl. The flow rate was maintained at 45 ml/h and fractions of 7.5 ml were collected every 10 min. Gel filtration. The most active anionic fraction from the anionexchange chromatography was further purified by gel filtration. This step was performed a number of times in order to process all of the relevant fraction. In a typical run, an aliquot (5 ml) of the fraction in 0.05 M Tris-HCl buffer, pH 7.4, containing 0.1 M NaCl, was passed through a column (2.6 1 80 cm) of Sephadex G150 resin at a flow rate of 6.75 ml/h, collecting fractions of 2.25 ml every 20 min. Hydrophobic interaction chromatography. The active fraction from the gel filtration was further purified by hydrophobic interaction chromatography. This step was carried out a number of times in order to process all of the relevant fraction. In a typical run an aliquot (6 ml) of the fraction was made to 0.6 M (NH4)2SO4 and was applied to a column (1.2 1 8 cm) of phenyl-Sepharose resin that had been equilibrated with 0.05 M Tris-HCl buffer, pH 7.4, containing 0.6 M (NH4)2SO4 and 0.1 M NaCl. Unadsorbed substances were eluted by washing the column with the forementioned buffer (80 ml) at a flow rate of 6 ml/h, collecting fractions of 2 ml every 20 min. A negative salt gradient, consisting of 30 ml of the forementioned buffer as the initial buffer and 30 ml of 0.05 M Tris-HCl, pH 7.4, as the final buffer, was then applied to the column. To complete the elution of
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CAMEL ANIONIC CHYMOTRYPSIN
FIG. 1. Gradient elution profile of crude extract of camel pancreas on anion-exchange chromatography. (—) Absorbance at 280 nm, (—) NaCl gradient.
bound substances the column was washed with 70 ml of 0.05 M TrisHCl, pH 7.4, buffer.
RESULTS AND DISCUSSION
The crude extract of camel pancreatic tissue showed no activity toward Bz-Tyr-OEt even after standing for 24 h at room temperature, reflecting the presence and stability of the chymotrypsinogens, so the zymogens were activated by incubating a sample of the crude extract with bovine trypsin (protein:trypsin Å 68:1 w/ w) for various time periods at 257C. The activated sample showed a maximum activity of 2.6 units/mg protein after 30 min incubation. The gradient elution profile of the crude extract on anion-exchange chromatography is shown in Fig 1. The fractions were pooled as indicated. None of them showed any direct activity toward Bz-Tyr-OEt but after activation by exogenous trypsin the pregradient, cationic fraction PG and two of the anionic fractions, fractions 2 and 3, showed considerable chymotrypsin-like
FIG. 2. Elution profile of F2 on gel filtration.
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FIG. 3. Elution profile of GF3 on hydrophobic interaction chromatography. The resin was initially washed with 0.05 M Tris-HCl buffer, pH 7.4, containing 0.6 M (NH4)2SO4 and 0.1 M NaCl. From tube No. 41 to tube No. 70 it was washed with a linear decreasing gradient of 0.6 M (NH4)2SO4 and 0.1 M NaCl to zero salt in the forementioned buffer. From tube 71 onward it was washed with buffer alone.
activity towards that substrate (see Table I). Cationic and anionic forms of chymotrypsinogen and chymotrypsin have been isolated from porcine and bovine pancreas (2, 6), so it is reasonable to assume that fraction PG contained camel cationic chymotrypsinogen
FIG. 4. SDS–gel electrophoretic pattern of the purified enzyme.Lane 1, purified enzyme. Lane 2, standard marker proteins with molecular masses in kDa denoted to the right.
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AL-AJLAN AND BAILEY TABLE I
Purification of Anionic Chymotrypsin from Camel Pancreas
Fraction
Total protein (mg)
Total activity (units)a
Specific activity (units/mg)
Recovery (%)/
Purification
Crude extract PG (cationic) F2 (anionic) F3 (anionic) GF3 H3
5577 2632 286 93 94 21
14,500 6,054 4,090 577 3,243 1,730
2.6 2.3 14.3 6.2 34.5 82.4
(100)
(1)
65
12.6
51 27
30.5 72.9
a b
Measured using 0.5 mM Bz-Tyr-OEt as substrate. Calculated by taking into account the existence of different cationic and anionic chymotrypsins in the crude extract.
whereas fractions 2 and 3 contained the corresponding anionic zymogen. Considering the total enzymatic activities toward Bz-Tyr-OEt recovered in the activated pregradient and gradient fractions, it can be suggested that anionic chymotrypsin represented 43.5% of the total chymotryptic activity of the activated crude extract of camel pancreas. The anionic chymotrypsin was further purified by gel filtration. A typical elution profile is shown in Fig 2. Chymotryptic activity was found to be present in fraction GF3. The level of activity did not increase on incubation with exogenous trypsin, thus indicating that the measured activity was due to the presence of the active enzyme. None of the other fractions showed activity toward Bz-Tyr-OEt either before or after incubation with exogenous trypsin. Fraction GF3 was subjected to hydrophobic interaction chromatography. A representative elution profile is shown in Fig 3. Only fraction H3 showed chymotryptic activity. Assuming that anionic chymotrypsin represented 43.5% of the total chymotryptic activity of the activated crude extract it can be calculated that the purification factor for the anionic enzyme was 72.9fold with a 27% recovery of activity. A summary of the overall purification is presented in Table I. Fraction H3 was seen to be homogeneous on slab gel
electrophoresis under nondenaturing conditions and on gel isoelectric focusing (data not shown). Its isoelectric point was estimated to be 4.4. It also showed a single component on SDS–gel electrophoresis (Fig. 4) and its molecular mass was estimated to be 28.5 kDa. A molecular mass of 29 kDa has been determined for porcine chymotrypsinogen C from amino acid composition and by ultracentrifugation (3), and a value of 28 kDa found by SDS–gel electrophoresis for the corresponding bovine zymogen (8). The purified enzyme showed no catalytic activity toward Tos-Arg-OMe and Bz-Arg-OEt, thus showing a lack of trypsin-like activity. It was not active toward Bz-Gly-Phe or Bz-Gly-Arg, thus showing an absence of carboxypeptidase A and carboxypeptidase B activities, respectively. In contrast, it was active toward a number of substrates hydrolyzed by chymotrypsin-like enzymes (Table II). It can be seen that the values of the specificity constant (kcat/Km) of the camel enzyme for its action on the tyrosine, tryptophan, and phenylalanine esters were considerably less that the corresponding values of bovine chymotrypsin A. However, the camel enzyme showed considerably more activity than the bovine enzyme toward the leucine ester. Similar findings have previously been reported for porcine anionic chymotrypsin C and its bovine counterpart, subunit II, com-
TABLE II
Kinetic Constants of Camel Anionic Chymotrypsin and Bovine Chymotrypsin A Camel anionic chymotrypsin
Substrate
Km (mM)
kcat (s01)
Bz-Tyr-OEt Ac-Tyr-OEt Ac-Trp-OEt Ac-Phe-OEt Ac-Leu-OMe
0.64 2.00 1.12 1.39 1.00
57.5 25.4 9.3 5.3 22.1
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Bovine chymotrypsin A kcat/Km (M01 s01)
8.99 1.27 0.83 0.38 2.21
1 1 1 1 1
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104 104 104 104 104
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Km (mM)
kcat (s01)
0.13 1.27 0.17 1.04 7.27
76.6 126.0 48.4 52.4 6.6
kcat/Km (M01 s01) 5.82 0.99 2.85 0.50 0.09
1 1 1 1 1
105 105 105 105 104
CAMEL ANIONIC CHYMOTRYPSIN
pared to porcine chymotrypsin A and bovine chymotrypsins A and B which are cationic enzymes (2, 6). The camel enzyme showed a kininase activity of 11.4 units/mg, considerably lower than the value of 76.4 units/mg recorded for bovine chymotrypsin A under the same experimental conditions. Understanding the physiological importance, if any, of the kininase activity of the camel chymotrypsin-like enzyme must await a full characterization of other components of the kinin system, particularly those that reside in the kidney. However, it should be pointed out that chymotrypsin and other pancreatic proteolytic enzymes have been detected in the blood stream of other mammals (28, 29). Furthermore, an enzymatically active tissue kallikrein has been isolated from human plasma (30). Also, it is probable that plasma carboxypeptidase N (kininase 1) does not function in vivo as a kininase (31), so there has to be an enzyme in plasma other than kininase 1 that is responsible for the removal of the C terminal arginine residue (32). Bovine chymotrysin in vitro rapidly cleaves the C terminal peptide bond of bradykinin (33, 34). It is possible that in certain circumstances chymotrypsin could act as a physiological inactivator of locally generated kinin. The esterolytic activity of the camel enzyme toward Bz-Tyr-OEt was not inhibited by a 1000-fold molar excess of the metalloproteinase inhibitors EDTA and 1,10-phenanthroline or by a 2000-fold molar excess of TLCK, an irreversible inhibitor of trypsin-like enzymes. However, it was competitively inhibited by several chymotrypsin inhibitors (Table III). It can be seen that the camel enzyme was more susceptible than bovine chymotrypsin A to inhibition by SBTI and LBTI. The pH optimum for the hydrolysis of Bz-Tyr-OEt by camel anionic chymotrypsin was found to be pH 7.8 (Fig. 5), the same value as found for bovine chymotrypsin A (data not shown). The esterolytic activity of the camel enzyme at pH 7.4 was stable for at least 10 h at 257C, in both the presence and the absence of 0.1 M CaCl2 . Under the same conditions bovine chymotrypsin A was stable in the presence of 0.1 M CaCl2 but exhibited a 27% loss of activity in its absence. Thus,on the basis of physiochemical properties, substrate specificity, and interaction with potential inhibitors, it can be
TABLE III
Inhibition of Camel Anionic Chymotrypsin and Bovine Chymotrypsin A
Inhibitor
Camel chymotrypsin Ki (nM)
Bovine chymotrypsin A Ki (nM)
Aprotinin SBTI LBTI
15.4 3.5 18.0
3.5 250.0 42.5
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FIG. 5. Effect of pH on the activity of the purified enzyme toward Bz-Tyr-OEt.
concluded that the purified enzyme is camel anionic chymotrypsin. It is the camel equivalent of porcine chymotrypsin C and its bovine counterpart (2, 6). It is a relatively weak kininase. REFERENCES 1. Charles, M., Gratecos, D., Rovery, M., and Desnuelle, P. (1967) Biochim. Biophys. Acta 140, 395–409. 2. Folk, J. E., and Schirmer, W. E. (1965) J. Biol. Chem. 240, 181– 192. 3. Gratecos, D., Guy, O., Rovery, M., and Desnuelle, P. (1969) Biochim. Biophys. Acta 175, 82–96. 4. Wilcox, P. E. (1970) Methods Enzymol. 19, 64–112. 5. Pe´tra, P. H. (1970) Methods Enzymol. 19, 460–503. 6. Keil-Dlouha, V., Puigserver, A., Marie, A., and Keil, B. (1972) Biochim. Biophys. Acta 276, 531–535. 7. Peanasky, R. J., Gratecos, D., Baratti, J., and Rovery, M. (1969) Biochim. Biophys. Acta 181, 82–92. 8. Puigserver, A., Vaugoyeau, H., and Desnuelle, P. (1972) Biochim. Biophys. Acta 276, 519–530. 9. Hussein, M. F., Al-Momen, A. K., and Gader, A. M. A. (1992) Comp. Haematol. Int. 2, 92–96. 10. Bricteux-Gregoire, S., Schyns, R., and Florkin M. (1972) Comp. Biochem. Physiol. 42B, 23–39. 11. Rafiq, A., and Bailey, G. S. (1996) Comp. Biochem. Physiol. 115B, 363–367. 12. Welling, G. W., Groen, G., and Beintema, J. J. (1975) Biochem. J. 147, 505–511. 13. Yagil, R. (1985) The Desert Camel, Karger, Basel. 14. Damas, J. (1993) Arch. Int. Physiol. Biochim. Biophys. 101, 227– 232. 15. Bhoola, K. D., Figueroa, C. D., and Worthy, D. (1992) Pharmacol Rev. 44, 1–80. 16. Schacter, M. (1969) Physiol. Rev. 49, 509–547. 17. Hummel, B. C. W. (1959) Can. J. Biochem. Physiol. 37, 1391– 1399. 18. Walsh, K. A., and Wilcox, P. E. (1970) Methods Enzymol. 19, 31– 41. 19. Folk, J. E., and Schirmer, E. W. (1963) J. Biol. Chem 238, 3884– 3894.
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20. Folk, J. E., Piez, K. A., Carroll, W. R., and Gladner, J. (1960) J. Biol. Chem. 235, 2272–2277. 21. Eisenthal, R., and Cornish-Bowden, A. (1960) Biochem. J. 139, 715–720. 22. Bailey, G. S., Al-Joufi, A., Rawat, S., and Smith, D. C. (1991) Toxicon 29, 777–781. 23. Dixon, M. (1952) Biochem. J. 55, 170–171. 24. Stoscheck, C. M. (1990) Methods Enzymol. 82, 50–68. 25. Cohen, J. A., Oosterbaan, R. A., and Berends, F. (1967) Methods Enzymol. 11, 686–702. 26. Blackspear, P. J. (1984) Methods Enzymol. 104, 237–255. 27. Laemmli, U. K. (1970) Nature 227, 680–685.
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28. Largman, C. Brodrick, J. W., and Geokas, M. C. (1981) Methods Enzymol. 74, 272–290. 29. Geokas, M. C., Largman, C., Brodrick, J. W., Johnson, J. H., and Fassell, M. (1979) J. Biol. Chem. 254, 2775–2781. 30. Geiger, R., Clausnitzer, B., Fink, E., and Fritz, H. (1980) HoppeSeyler’s Z. Physiol. Chem. 361, 1795–1803. 31. Ryan, J. W. (1988) Methods Enzymol. 163, 186–194. 32. Sheikh, I. A., and Kaplan, A. P. Biochem.Pharm. 35, 1957–1963. 33. Elliott, D. F., Lewis, G. P., and Horton, E. W. (1960) Biochem. Biophys. Res. Commun. 3, 87–91. 34. Sampaio, C. A. M., Nunes, S. T., Graca, M. D., Mazzacoratti, N., and Prado, J. L. (1976) Biochem. Pharm. 25, 2391–2394.
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