Abstract
Recent studies have demonstrated that a calcium-sensitive protease converts Ca2+/phospholipid-dependent protein kinase C to a Ca2+/phospho-lipid-independent form during the activation of human neutrophils. In this paper, the results of the purification and characterization of a calcium-dependent cytosolic protease from neutrophils is reported. Calcium-dependent protease has been purified 1062-fold from human neutrophils and behaves as a single species on native polyacrylamide gels. The protease is active in the neutral pH range with no observable activity at pH values greater than 8.0, has an absolute requirement for calcium for expression of activity with half-maximal activity observed at 12 /μM free calcium, and has an apparent molecular weight of 110,000 based on gel filtration. The protease requires the presence of dithiothreitol for activity and is inhibited by sulfhydryl inhibitors, leupeptin, and antipain but not by serine protease inhibitors, pepstatin, or orthophenanthroline. The protease is also susceptible to inactivation by autoproteolysis. Based on the similarities of this calcium-dependent protease with calpains from a variety of other mammalian tissues, the protease isolated from human neutrophils appears to be a calpain I.
Similar content being viewed by others
References
Cooke, E., andM. B. Hallett. 1985.Biochem J. 232:323–327.
Wolfson, M., L. C. McPhail, V. N. Nassallah, andR. Snyderman. 1985.J. Immunol. 135:2057–2062.
Gennuno, R., C. Florio, andD. Romeo. 1986.Biochem. Biophys. Res. Commun. 134:305–312.
Cox, J. A., A. Y. Jeng, N. A. Sharkey, P. M. Blumbero, andA. I. Tauber. 1985.J. Clin. Invest. 76:1932–1938.
Papini, E., M. Grzeskowiak, P. Bellavite, andF. Rossi. 1985.FEBS. Lett. 190:204–208.
Huang, C.-K., J. M. Hill, Jr., J.-M. Borman, W. M. Mackin, andE. L. Becker. 1983.Biochim. Biophys. Acta 670:126–135.
Inoue, M., A. Kishimoto, Y. U. Takai, andY. Nishizuka. 1977.J. Biol. Chem. 252:7610–7616.
Melloni, E., S. Pontremoli, M. Michetti, O. Sacco, B. Sparatore, F. Salamino, andB. L. Horecker. 1985.Proc. Natl. Acad. Sci. U.S.A. 82:6435–6439.
Melloni, E., S. Pontremoli, M. Michette, O. Sacco, B. Sparatore, andB.L. Horecker. 1986.J. Biol. Chem. 261:4101–4105.
Legendre, J. L.., andH. P. Jones. 1983.J. Reticuloendothel. Soc. 34:89–97.
Rice, R., andG. Means. 1971.J. Biol. Chem. 246:831–832.
Bradford, M.. 1976.Anal. Biochem. 72:248–254.
Perrin, D., andI. Sayce. 1967.Talanta 14:833–842.
Kishimoto, A. 1983.J. Biol. Chem. 258:1156–1164.
Pant, H., P. E. Gallant, R. Gould, andH. Gainer. 1982.J. Neurosci. 2:1578–1587.
Murakami, T., M. Hatanaka, andT. Murachi. 1981.J. Biochem. 90:1809–1816.
Suzuki, K. 1983.J. Biochem. 93:1305–1312.
Mellgren, R., A. Repetti, T. C. Muck, andJ. Easly. 1982.J. Biol. Chem. 257:7203–7209.
Yanagisawa, K. 1983.Neurochem. Res. 8:1285–1293.
Yoshimura, N., T. Kikuchi, T. Sasaki, A. Kitahara, M. Hatanaka, andT. Murachi. 1983.J. Biol. Chem. 258:8883–8889.
Wheelock, M. 1982.J. Biol. Chem. 257:12471–12474.
Huston, R., andE. G. Krebs. 1968.Enzyme Biochem. 7:2116–2121.
Dayton, W. R. 1982.Biochim. Biophys. Acta 709:166–172.
DeMartino, G., andD. Blumenthal. 1982.Biochemistry 21:4297–4303.
A. Kitahara, T. Sasaki, T. Kikuchi, N. Yumoto, N. Yoshimura, M. Hatanka, andT. Murachi. 1984.J. Biochem. 95:1759–1766.
Dayton, W., J. V. Schollmeyer, R. A. Lepley, andL. R. Cortes. 1981.Biochim. Biophys. Acta 659:48–61.
Murachi, T., K. Tanaka, M. Hatanaka, andT. Murakami. 1981.Adv. Enzyme Regul. 19:407–424.
Dayton, W. R., W. J. Reville, D. E. Goll, andM. H. Stromer. 1976.Biochemistry 15:2159–2167.
Inomata, M., M. Nomoto, M. Hayashi, M. Nakamura, K. Imahori, andS. Kawashima. 1984.J. Biochem 95:1661–1670.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Legendre, J.L., Jones, H.P. Purification and characterization of calpain from human polymorphonuclear leukocytes. Inflammation 12, 51–65 (1988). https://doi.org/10.1007/BF00915892
Issue Date:
DOI: https://doi.org/10.1007/BF00915892