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. 1986 Oct;83(20):7588–7592. doi: 10.1073/pnas.83.20.7588

Endogenous inhibitor of nonlysosomal high molecular weight protease and calcium-dependent protease.

K Murakami, J D Etlinger
PMCID: PMC386766  PMID: 3020549

Abstract

An endogenous inhibitor of high molecular weight protease was purified from human erythrocytes and partially characterized. The inhibitor was isolated by DEAE-Sephacel ion-exchange chromatography followed by separation on a Bio-Gel A-0.5m column. The inhibitor displayed a native Mr of 240,000 and contained a single subunit of Mr 40,000 after NaDodSO4/polyacrylamide gel electrophoresis. The Mr 240,000 hexamer inhibited high molecular weight protease noncompetitively (Ki = 8.3 X 10(-8) M) and showed marked susceptibility to proteolytic digestion and heat treatment. The purified factor was also a potent inhibitor of calcium-dependent protease (Ki = 2.8 X 10(-8) M), whereas it had no effect on trypsin, chymotrypsin, or papain. Heat treatment (50-70 degrees C X 10 min) caused loss of inhibition against high molecular weight protease; however, inhibition of calcium-dependent protease was stable under the same conditions. This result is consistent with different domains on the inhibitor that interact with high molecular weight protease and calcium-dependent protease. Together with earlier studies in which repression of inhibitor by an ATP-ubiquitin-dependent process was proposed, the present results suggest a general mechanism for regulation of multiple nonlysosomal proteases that are complexed with endogenous inhibitors.

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Selected References

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  1. CRESTFIELD A. M., MOORE S., STEIN W. H. The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J Biol Chem. 1963 Feb;238:622–627. [PubMed] [Google Scholar]
  2. Charette M. F., Henderson G. W., Markovitz A. ATP hydrolysis-dependent protease activity of the lon (capR) protein of Escherichia coli K-12. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4728–4732. doi: 10.1073/pnas.78.8.4728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chung C. H., Goldberg A. L. The product of the lon (capR) gene in Escherichia coli is the ATP-dependent protease, protease La. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4931–4935. doi: 10.1073/pnas.78.8.4931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ciehanover A., Hod Y., Hershko A. A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes. Biochem Biophys Res Commun. 1978 Apr 28;81(4):1100–1105. doi: 10.1016/0006-291x(78)91249-4. [DOI] [PubMed] [Google Scholar]
  5. Cottin P., Vidalenc P. L., Ducastaing A. Ca2+-dependent association between a Ca2+-activated neutral proteinase (CaANP) and its specific inhibitor. FEBS Lett. 1981 Dec 28;136(2):221–224. doi: 10.1016/0014-5793(81)80622-9. [DOI] [PubMed] [Google Scholar]
  6. Cottin P., Vidalenc P. L., Merdaci N., Ducastaing A. Evidence for non-competitive inhibition between two calcium-dependent activated neutral proteinases and their specific inhibitor. Biochim Biophys Acta. 1983 Mar 16;743(2):299–302. doi: 10.1016/0167-4838(83)90227-3. [DOI] [PubMed] [Google Scholar]
  7. Dahlmann B., Kuehn L., Reinauer H. Identification of three high molecular mass cysteine proteinases from rat skeletal muscle. FEBS Lett. 1983 Aug 22;160(1-2):243–247. doi: 10.1016/0014-5793(83)80975-2. [DOI] [PubMed] [Google Scholar]
  8. Dayton W. R., Reville W. J., Goll D. E., Stromer M. H. A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Partial characterization of the purified enzyme. Biochemistry. 1976 May 18;15(10):2159–2167. doi: 10.1021/bi00655a020. [DOI] [PubMed] [Google Scholar]
  9. DeMartino G. N., Goldberg A. L. Identification and partial purification of an ATP-stimulated alkaline protease in rat liver. J Biol Chem. 1979 May 25;254(10):3712–3715. [PubMed] [Google Scholar]
  10. Desautels M., Goldberg A. L. Demonstration of an ATP-dependent, vanadate-sensitive endoprotease in the matrix of rat liver mitochondria. J Biol Chem. 1982 Oct 10;257(19):11673–11679. [PubMed] [Google Scholar]
  11. Etlinger J. D., Goldberg A. L. A soluble ATP-dependent proteolytic system responsible for the degradation of abnormal proteins in reticulocytes. Proc Natl Acad Sci U S A. 1977 Jan;74(1):54–58. doi: 10.1073/pnas.74.1.54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Etlinger J. D., Speiser S., Wajnberg E., Glucksman M. J. ATP-dependent proteolysis in erythroid and muscle cells. Acta Biol Med Ger. 1981;40(10-11):1285–1291. [PubMed] [Google Scholar]
  13. Eytan E., Hershko A. Relevance of protease "inhibitor" to the ATP-ubiquitin proteolytic system. Biochem Biophys Res Commun. 1984 Jul 18;122(1):116–123. doi: 10.1016/0006-291x(84)90447-9. [DOI] [PubMed] [Google Scholar]
  14. Goldberg A. L., St John A. C. Intracellular protein degradation in mammalian and bacterial cells: Part 2. Annu Rev Biochem. 1976;45:747–803. doi: 10.1146/annurev.bi.45.070176.003531. [DOI] [PubMed] [Google Scholar]
  15. Goll D. E., Edmunds T., Kleese W. C., Sathe S. K., Shannon J. D. Some properties of the Ca2+-dependent proteinase. Prog Clin Biol Res. 1985;180:151–164. [PubMed] [Google Scholar]
  16. Haas A. L., Murphy K. E., Bright P. M. The inactivation of ubiquitin accounts for the inability to demonstrate ATP, ubiquitin-dependent proteolysis in liver extracts. J Biol Chem. 1985 Apr 25;260(8):4694–4703. [PubMed] [Google Scholar]
  17. Haas A. L., Warms J. V., Hershko A., Rose I. A. Ubiquitin-activating enzyme. Mechanism and role in protein-ubiquitin conjugation. J Biol Chem. 1982 Mar 10;257(5):2543–2548. [PubMed] [Google Scholar]
  18. Hershko A., Ciechanover A., Heller H., Haas A. L., Rose I. A. Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1783–1786. doi: 10.1073/pnas.77.4.1783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hershko A., Ciechanover A. Mechanisms of intracellular protein breakdown. Annu Rev Biochem. 1982;51:335–364. doi: 10.1146/annurev.bi.51.070182.002003. [DOI] [PubMed] [Google Scholar]
  20. Hershko A., Heller H., Eytan E., Kaklij G., Rose I. A. Role of the alpha-amino group of protein in ubiquitin-mediated protein breakdown. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7021–7025. doi: 10.1073/pnas.81.22.7021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hirado M., Iwata D., Niinobe M., Fujii S. Purification and properties of thiol protease inhibitor from rat liver cytosol. Biochim Biophys Acta. 1981 Jun 29;669(1):21–27. doi: 10.1016/0005-2795(81)90218-x. [DOI] [PubMed] [Google Scholar]
  22. Hough R., Pratt G., Rechsteiner M. Ubiquitin-lysozyme conjugates. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte lysates. J Biol Chem. 1986 Feb 15;261(5):2400–2408. [PubMed] [Google Scholar]
  23. Imajoh S., Suzuki K. Reversible interaction between Ca2+-activated neutral protease (CANP) and its endogenous inhibitor. FEBS Lett. 1985 Jul 22;187(1):47–50. doi: 10.1016/0014-5793(85)81211-4. [DOI] [PubMed] [Google Scholar]
  24. Ismail F., Gevers W. A high-molecular-weight cysteine endopeptidase from rat skeletal muscle. Biochim Biophys Acta. 1983 Jan 26;742(2):399–408. doi: 10.1016/0167-4838(83)90327-8. [DOI] [PubMed] [Google Scholar]
  25. Jentoft N., Dearborn D. G. Labeling of proteins by reductive methylation using sodium cyanoborohydride. J Biol Chem. 1979 Jun 10;254(11):4359–4365. [PubMed] [Google Scholar]
  26. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  27. Lenney J. F., Tolan J. R., Sugai W. J., Lee A. G. Thermostable endogenous inhibitors of cathepsins B and H. Eur J Biochem. 1979 Nov 1;101(1):153–161. doi: 10.1111/j.1432-1033.1979.tb04227.x. [DOI] [PubMed] [Google Scholar]
  28. Lepley R. A., Pampusch M., Dayton W. R. Purification of a high-molecular-weight inhibitor of the calcium-activated proteinase. Biochim Biophys Acta. 1985 Mar 22;828(1):95–103. doi: 10.1016/0167-4838(85)90014-7. [DOI] [PubMed] [Google Scholar]
  29. Mellgren R. L. Canine cardiac calcium-dependent proteases: Resolution of two forms with different requirements for calcium. FEBS Lett. 1980 Jan 1;109(1):129–133. doi: 10.1016/0014-5793(80)81326-3. [DOI] [PubMed] [Google Scholar]
  30. Murakami K., Voellmy R., Goldberg A. L. Protein degradation is stimulated by ATP in extracts of Escherichia coli. J Biol Chem. 1979 Sep 10;254(17):8194–8200. [PubMed] [Google Scholar]
  31. Murakami T., Hatanaka M., Murachi T. The cytosol of human erythrocytes contains a highly Ca2+-sensitive thiol protease (calpain I) and its specific inhibitor protein (calpastatin). J Biochem. 1981 Dec;90(6):1809–1816. doi: 10.1093/oxfordjournals.jbchem.a133659. [DOI] [PubMed] [Google Scholar]
  32. Nakamura M., Inomata M., Hayashi M., Imahori K., Kawashima S. Purification and characterization of 210,000-dalton inhibitor of calcium-activated neutral protease from rabbit skeletal muscle and its relation to 50,000-dalton inhibitor. J Biochem. 1985 Sep;98(3):757–765. doi: 10.1093/oxfordjournals.jbchem.a135333. [DOI] [PubMed] [Google Scholar]
  33. Pontremoli S., Melloni E., Salamino F., Sparatore B., Michetti M., Horecker B. L. Endogenous inhibitors of lysosomal proteinases. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1261–1264. doi: 10.1073/pnas.80.5.1261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Reddy M. K., Etlinger J. D., Rabinowitz M., Fischman D. A., Zak R. Removal of Z-lines and alpha-actinin from isolated myofibrils by a calcium-activated neutral protease. J Biol Chem. 1975 Jun 10;250(11):4278–4284. [PubMed] [Google Scholar]
  35. Rieder R. F., Ibrahim A., Etlinger J. D. A particle-associated ATP-dependent proteolytic activity in erythroleukemia cells. J Biol Chem. 1985 Feb 25;260(4):2015–2018. [PubMed] [Google Scholar]
  36. Rose I. A., Warms J. V., Hershko A. A high molecular weight protease in liver cytosol. J Biol Chem. 1979 Sep 10;254(17):8135–8138. [PubMed] [Google Scholar]
  37. Roth M., Hoechst M., Afting E. G. Controlled intracellular proteolysis during postpartal involution of the uterus: characterization and regulation of an alkaline proteinase. Acta Biol Med Ger. 1981;40(10-11):1357–1363. [PubMed] [Google Scholar]
  38. Spanier A. M., Bird J. W. Endogenous cathepsin B inhibitor activity in normal and myopathic red and white skeletal muscle. Muscle Nerve. 1982 Apr;5(4):313–320. doi: 10.1002/mus.880050407. [DOI] [PubMed] [Google Scholar]
  39. Speiser S., Etlinger J. D. ATP stimulates proteolysis in reticulocyte extracts by repressing an endogenous protease inhibitor. Proc Natl Acad Sci U S A. 1983 Jun;80(12):3577–3580. doi: 10.1073/pnas.80.12.3577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Takahashi-Nakamura M., Tsuji S., Suzuki K., Imahori K. Purification and characterization of an inhibitor of calcium-activated neutral protease from rabbit skeletal muscle. J Biochem. 1981 Dec;90(6):1583–1589. doi: 10.1093/oxfordjournals.jbchem.a133632. [DOI] [PubMed] [Google Scholar]
  41. Takano E., Murachi T. Purification and some properties of human erythrocyte calpastatin. J Biochem. 1982 Dec;92(6):2021–2028. doi: 10.1093/oxfordjournals.jbchem.a134134. [DOI] [PubMed] [Google Scholar]
  42. Waxman L., Fagan J. M., Tanaka K., Goldberg A. L. A soluble ATP-dependent system for protein degradation from murine erythroleukemia cells. Evidence for a protease which requires ATP hydrolysis but not ubiquitin. J Biol Chem. 1985 Oct 5;260(22):11994–12000. [PubMed] [Google Scholar]
  43. Waxman L., Krebs E. G. Identification of two protease inhibitors from bovine cardiac muscle. J Biol Chem. 1978 Sep 10;253(17):5888–5891. [PubMed] [Google Scholar]
  44. Zeman R. J., Kameyama T., Matsumoto K., Bernstein P., Etlinger J. D. Regulation of protein degradation in muscle by calcium. Evidence for enhanced nonlysosomal proteolysis associated with elevated cytosolic calcium. J Biol Chem. 1985 Nov 5;260(25):13619–13624. [PubMed] [Google Scholar]

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