563rd MEETING, LONDON
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This suggests that the steps involving the binding (or release) of pyruvate may contribute towards the overall rate of the reaction. If this is true, it follows that, strictly, it is invalid to use the rapidequilibrium assumption to describe the rabbit muscle pyruvate kinase reaction, although in the forward reaction all the steady-state kinetic data available are consistent with this assumption. Of the other kinases studied, creatine kinase is truly rapid-equilibrium (Morrison & Cleland, 1966), whereas the sugar-sugar phosphate exchange of galactokinase (Gulbinsky & Cleland, 1968) and hexokinase (Fromm et af., 1964) is slower than the other exchanges by a factor of 1.5 to 2.5. Ainsworth, S. & MacFarlane, N. (1973) Biochem. J. 131,223-236 Cleland, W. W. (1967) Annu. Rev. Biochem. 36,77-112 Frornrn, H . J., Silverstein, E. & Boyer, P. D. (1964)J. Biol. Chem. 239,3645-3652 Giles, I. G., Poat, P. C. & Munday, K. A. (1975) Biochem. SOC.Trans. 3,312-314 Gulbinsky, J. S. & Cleland, W. W. (1968) Biochemistry 7 , 566-575 Kachmar, J. F. & Boyer, P. D. (1953) J. Biol. Chem. 200,669-682 Melchior, J. (1965) Biochemistry 4, 1518-1525 Mildvan, A. S . & Cohn, M. (1965)J. Biol. Chem. 240,238-246 Mildvan, A. S. & Cohn, N. (1966) J. Biol. Chem. 241, 1178-1193 Morrison, J. F. & Cleland, W. W. (1966)J. Biol. Chem. 241,673-683 Reynard,A. M.,Hass, L. F., Jacobsen,D. D. &Boyer P. D. (1961)J.Biol.Chem. 236,2277-2283
Collagenolytic Cathepsin Activity in Rabbit Peritoneal Polymorphonuclear Leucocytes WALTER T. GIBSON,* DAVID W. MILSOM,* FRANK S. STEVEN* and JOHNS. LOWEt *Department of Medical Biochemistry, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, U.K., and t Department of Biochemistry, I.C.I. Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire, U.K.
The discovery of collagenolytic cathepsins in rat and human liver (Milsom ef al., 1972), rat leucocyte granules (Anderson, 1971), post-partum rat uterus (Etherington, 1973) and bovine spleen (Etherington, 1976), as well as the demonstration of the collagenolytic activity of purified cathepsin B1 (Burleigh eta!., 1974; Etherington, 1974), suggest that acid proteinases play an important role in collagen degradation. Although collagenase has been found in rabbit polymorphonuclear leucocytes (Robertson er al., 1972), there has been no report of an acid proteinase with collagenolytic activity in these cells, although Cochrane & Aikin (1966) showed that extracts of rabbit polymorphonuclear leucocytes will release peptides from purified glomerular basement membrane when incubated at an acid pH. This activity was attributed to cathepsins D and E, which, in addition to cathepsin A, were detected in rabbit polymorphonuclear-leucocyte granules by Wasi e f al. (1966). No cathepsin B or C activitywas observed. In this study, we report that rabbit polymorphonuclear leucocytes contain an enzyme capable of degrading polymeric collagen at an acid pH. Rabbit peritoneal exudatepolymorphonuclear leucocytes were obtained by themethod of Cohn & Hirsch (1960). Lysates of these cells were capable of solubilizing polymeric collagen, isolated by the method of Steven (1967), at pH4.0 and 37°C. The collagen preparations showed a small but variable susceptibility to degradation by trypsin, sometimes exceeding 10%. Activity in cell lysates and isolated granules usually exceeded that of trypsin on the same preparation of collagen, and, as further indication of its ability to degrade native collagen, the enzyme was shown to be able to solubilize polymeric collagen that had been pretreated with trypsin. Optimal activity of the collagenolytic cathepsin was found at around pH3, but assays were generally performed at pH4.0 to minimize the risk of denaturation during incubation at 37°C. There was no increase in the trypsin susceptibility of polymeric collagen
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BIOCHEMICAL SOCIETY TRANSACTIONS
after 18h at this pH and temperature (showing that no denaturation of the collagen occurred). L-Cysteine and EDTA were both included in the assay, because in their absence the activity was found to be low and variable. EDTA had little effect by itself, but enhanced the activation by L-cysteine. The high-molecular-weight products of polymeric collagen degradation were studied by sodium dodecyl sulphate/polyacrylamide-gelelectrophoresis (Fiirthmayr & Timpl, 1971). This revealed the presence of a, Band y chains in the digest, suggesting that both intra- and inter-molecular cross-linking regions are attacked. In view of the absence of cathepsin B1 from rabbit polymorphonuclear leucocytes, first reported by Wasi et al. (1966) and confirmed by our own studies, it seems probable that the collagenolytic cathepsin in these cells is similar to the enzyme in bovine spleen, which Etherington (1976) has shown to be separable from cathepsin B1. W. T. G. is grateful for the provision of an S.R.C. studentship, and financial assistance from I.C.I. Ltd. We are grateful for the expert technical assistance of Mrs. Susan Aston and MIS. Hilary Mabelis. Anderson, A. J. (1971) Ann. Rheum. Dis. 30,299-302 Burleigh, M. C., Barrett, A. J. & Lazarus, G. S. (1974) Biochem. J. 137,387-398 Cochrane, C. G. & Aikin, B. S. (1966)J. Exp. Med. 124,733-752 Cohn, Z . A. & Hirsch, J. G. (1960)J. Exp. Med. 112,983-1004 Etherington, D. J. (1973) Eur. J. Biochem. 32, 126128 Etherington, D. J. (1974) Biochem. J. 137, 547-557 Etherington, D. J. (1976) Biochem. J . 153, 199-209 Fiirthmayr, H. & Timpl, R. (1971) Anal. Biochem. 41, 510-516 Milsorn, D. W., Steven, F. S., Hunter, J. A. A., Thomas, H. &Jackson, D. S. (1972) Connect. Tissue Res. 1,251-265 Robertson, P. B., Ryel, R. B., Taylor, R. E., Shyu, K. W. & Fullmer, H. M. (1972) Science 177, 64-65 Steven, F. S . (1967) Biochim. Biophys. Acta 140,522-528 Wasi, S . , Murray, R. K., MacMorine, 0. R. L. & Movat, H. Z. (1966) Br. J. Exp. Pathol. 47, 411-422
The Subcellular Location of a Collagenolytic Cathepsin in Rabbit Peritoneal Polymorphonuclear Leucocytes WALTER T. GIBSON,* DAVID W. MILSOM,* FRANKS. STEVEN* and JOHNS. LOWET *Department of Medical Biochemistry, Unicersity of Manchester, Stopford Building, Oxford Road, Manchester M 13 9PT, U.K., and ?Department of Biochemistry, I.C.I. Pharmaceriticals Division, AIdcrley Park, Macclesfield, Cheshire, U.K.
In the preceding paper, we demonstrated that rabbit peritoneal polymorphonuclear leucocytes contain a collagenolytic cathepsin capable of degrading native polymeric collagen. It is known that the granules of rabbit polymorphonuclear leucocytes contain enzymes and other agents that are capable of causing tissue injury and promoting inflammatory responses (Henson, 1972), and the presence of a collagenolytic cathepsin in these granules would be an important factor in such processes. In this study, therefore, we have investigated the subcellular distribution of collagenolytic cathepsin in comparison with other acid hydrolases. The subcellular distribution of this enzyme after differential centrifugation is shown inTable 1, in comparison with two granule marker enzymes, N-acetyl-B-glucosaminidase, and arylsulphatases A and B. Fraction 11 contains the highest relative specific activity of all three enzymes, and fraction 111 contains significant amounts of collagenolytic but little arylsulphatase activity. The acticathepsin and N-acetyl-P-glucosaminidase, vities in fraction I probably reflect the presence of unbroken cells. 1976
627
This suggests that the steps involving the binding (or release) of pyruvate may contribute towards the overall rate of the reaction. If this is true, it follows that, strictly, it is invalid to use the rapidequilibrium assumption to describe the rabbit muscle pyruvate kinase reaction, although in the forward reaction all the steady-state kinetic data available are consistent with this assumption. Of the other kinases studied, creatine kinase is truly rapid-equilibrium (Morrison & Cleland, 1966), whereas the sugar-sugar phosphate exchange of galactokinase (Gulbinsky & Cleland, 1968) and hexokinase (Fromm et af., 1964) is slower than the other exchanges by a factor of 1.5 to 2.5. Ainsworth, S. & MacFarlane, N. (1973) Biochem. J. 131,223-236 Cleland, W. W. (1967) Annu. Rev. Biochem. 36,77-112 Frornrn, H . J., Silverstein, E. & Boyer, P. D. (1964)J. Biol. Chem. 239,3645-3652 Giles, I. G., Poat, P. C. & Munday, K. A. (1975) Biochem. SOC.Trans. 3,312-314 Gulbinsky, J. S. & Cleland, W. W. (1968) Biochemistry 7 , 566-575 Kachmar, J. F. & Boyer, P. D. (1953) J. Biol. Chem. 200,669-682 Melchior, J. (1965) Biochemistry 4, 1518-1525 Mildvan, A. S . & Cohn, M. (1965)J. Biol. Chem. 240,238-246 Mildvan, A. S. & Cohn, N. (1966) J. Biol. Chem. 241, 1178-1193 Morrison, J. F. & Cleland, W. W. (1966)J. Biol. Chem. 241,673-683 Reynard,A. M.,Hass, L. F., Jacobsen,D. D. &Boyer P. D. (1961)J.Biol.Chem. 236,2277-2283
Collagenolytic Cathepsin Activity in Rabbit Peritoneal Polymorphonuclear Leucocytes WALTER T. GIBSON,* DAVID W. MILSOM,* FRANK S. STEVEN* and JOHNS. LOWEt *Department of Medical Biochemistry, University of Manchester, Stopford Building, Oxford Road, Manchester M13 9PT, U.K., and t Department of Biochemistry, I.C.I. Pharmaceuticals Division, Alderley Park, Macclesfield, Cheshire, U.K.
The discovery of collagenolytic cathepsins in rat and human liver (Milsom ef al., 1972), rat leucocyte granules (Anderson, 1971), post-partum rat uterus (Etherington, 1973) and bovine spleen (Etherington, 1976), as well as the demonstration of the collagenolytic activity of purified cathepsin B1 (Burleigh eta!., 1974; Etherington, 1974), suggest that acid proteinases play an important role in collagen degradation. Although collagenase has been found in rabbit polymorphonuclear leucocytes (Robertson er al., 1972), there has been no report of an acid proteinase with collagenolytic activity in these cells, although Cochrane & Aikin (1966) showed that extracts of rabbit polymorphonuclear leucocytes will release peptides from purified glomerular basement membrane when incubated at an acid pH. This activity was attributed to cathepsins D and E, which, in addition to cathepsin A, were detected in rabbit polymorphonuclear-leucocyte granules by Wasi e f al. (1966). No cathepsin B or C activitywas observed. In this study, we report that rabbit polymorphonuclear leucocytes contain an enzyme capable of degrading polymeric collagen at an acid pH. Rabbit peritoneal exudatepolymorphonuclear leucocytes were obtained by themethod of Cohn & Hirsch (1960). Lysates of these cells were capable of solubilizing polymeric collagen, isolated by the method of Steven (1967), at pH4.0 and 37°C. The collagen preparations showed a small but variable susceptibility to degradation by trypsin, sometimes exceeding 10%. Activity in cell lysates and isolated granules usually exceeded that of trypsin on the same preparation of collagen, and, as further indication of its ability to degrade native collagen, the enzyme was shown to be able to solubilize polymeric collagen that had been pretreated with trypsin. Optimal activity of the collagenolytic cathepsin was found at around pH3, but assays were generally performed at pH4.0 to minimize the risk of denaturation during incubation at 37°C. There was no increase in the trypsin susceptibility of polymeric collagen
Vol. 4
628
BIOCHEMICAL SOCIETY TRANSACTIONS
after 18h at this pH and temperature (showing that no denaturation of the collagen occurred). L-Cysteine and EDTA were both included in the assay, because in their absence the activity was found to be low and variable. EDTA had little effect by itself, but enhanced the activation by L-cysteine. The high-molecular-weight products of polymeric collagen degradation were studied by sodium dodecyl sulphate/polyacrylamide-gelelectrophoresis (Fiirthmayr & Timpl, 1971). This revealed the presence of a, Band y chains in the digest, suggesting that both intra- and inter-molecular cross-linking regions are attacked. In view of the absence of cathepsin B1 from rabbit polymorphonuclear leucocytes, first reported by Wasi et al. (1966) and confirmed by our own studies, it seems probable that the collagenolytic cathepsin in these cells is similar to the enzyme in bovine spleen, which Etherington (1976) has shown to be separable from cathepsin B1. W. T. G. is grateful for the provision of an S.R.C. studentship, and financial assistance from I.C.I. Ltd. We are grateful for the expert technical assistance of Mrs. Susan Aston and MIS. Hilary Mabelis. Anderson, A. J. (1971) Ann. Rheum. Dis. 30,299-302 Burleigh, M. C., Barrett, A. J. & Lazarus, G. S. (1974) Biochem. J. 137,387-398 Cochrane, C. G. & Aikin, B. S. (1966)J. Exp. Med. 124,733-752 Cohn, Z . A. & Hirsch, J. G. (1960)J. Exp. Med. 112,983-1004 Etherington, D. J. (1973) Eur. J. Biochem. 32, 126128 Etherington, D. J. (1974) Biochem. J. 137, 547-557 Etherington, D. J. (1976) Biochem. J . 153, 199-209 Fiirthmayr, H. & Timpl, R. (1971) Anal. Biochem. 41, 510-516 Milsorn, D. W., Steven, F. S., Hunter, J. A. A., Thomas, H. &Jackson, D. S. (1972) Connect. Tissue Res. 1,251-265 Robertson, P. B., Ryel, R. B., Taylor, R. E., Shyu, K. W. & Fullmer, H. M. (1972) Science 177, 64-65 Steven, F. S . (1967) Biochim. Biophys. Acta 140,522-528 Wasi, S . , Murray, R. K., MacMorine, 0. R. L. & Movat, H. Z. (1966) Br. J. Exp. Pathol. 47, 411-422
The Subcellular Location of a Collagenolytic Cathepsin in Rabbit Peritoneal Polymorphonuclear Leucocytes WALTER T. GIBSON,* DAVID W. MILSOM,* FRANKS. STEVEN* and JOHNS. LOWET *Department of Medical Biochemistry, Unicersity of Manchester, Stopford Building, Oxford Road, Manchester M 13 9PT, U.K., and ?Department of Biochemistry, I.C.I. Pharmaceriticals Division, AIdcrley Park, Macclesfield, Cheshire, U.K.
In the preceding paper, we demonstrated that rabbit peritoneal polymorphonuclear leucocytes contain a collagenolytic cathepsin capable of degrading native polymeric collagen. It is known that the granules of rabbit polymorphonuclear leucocytes contain enzymes and other agents that are capable of causing tissue injury and promoting inflammatory responses (Henson, 1972), and the presence of a collagenolytic cathepsin in these granules would be an important factor in such processes. In this study, therefore, we have investigated the subcellular distribution of collagenolytic cathepsin in comparison with other acid hydrolases. The subcellular distribution of this enzyme after differential centrifugation is shown inTable 1, in comparison with two granule marker enzymes, N-acetyl-B-glucosaminidase, and arylsulphatases A and B. Fraction 11 contains the highest relative specific activity of all three enzymes, and fraction 111 contains significant amounts of collagenolytic but little arylsulphatase activity. The acticathepsin and N-acetyl-P-glucosaminidase, vities in fraction I probably reflect the presence of unbroken cells. 1976
