Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Sep 15;318(Pt 3):897–901. doi: 10.1042/bj3180897

Autolysis parallels activation of mu-calpain.

A Baki 1, P Tompa 1, A Alexa 1, O Molnár 1, P Friedrich 1
PMCID: PMC1217702  PMID: 8836135

Abstract

The kinetics of autolysis and activation of mu-calpain were measured with microtubule-associated protein 2 (MAP2) as a very sensitive substrate. The initial rate of MAP2 hydrolysis was found to be a linear function of the autolysed 76 kDa form of mu-calpain large subunit at both 10 and 300 microM Ca2+, and both straight lines intersected the origin. This finding supports the view that native mu-calpain is an inactive proenzyme and that activation is accompanied by autolysis. The first-order rate constant of autolysis, K1(aut), was determined at different Ca2+ concentrations: the half-maximal value was at pCa2+ = 3.7 (197 microM Ca2+), whereas the maximal value was 1.52 s-1, at 30 degrees C. The Ca(2+)-induced activation process was then monitored by using our novel, continuous fluorimetric assay with labelled MAP2 as substrate. The first-order rate constant of activation, k1(act), was derived as the reciprocal of the lag phase ('transit time') at the initial part of the progress curve: half-maximum was at pCa2+ = 3.8 (158 microM Ca2+) and the maximum value was 2.15 s-1. The good agreement between the kinetic parameters of mu-calpain autolysis and activation is remarkable. We claim that this is the first kinetically correct determination of the rate constant of autolysis of mu-calpain. Pre-activated mu-calpain has a Ca2+ requirement that is almost three orders of magnitude smaller [half-maximal activation at pCa2+ = 6.22 (0.6 microM Ca2+)]. We cannot exclude the possibility that the activation process involves other mechanistic steps, e.g. the rapid dissociation of the mu-calpain heterodimer, but we state that in our conditions in vitro autolysis and activation run in close parallel.

Full Text

The Full Text of this article is available as a PDF (484.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. Cong J., Goll D. E., Peterson A. M., Kapprell H. P. The role of autolysis in activity of the Ca2+-dependent proteinases (mu-calpain and m-calpain). J Biol Chem. 1989 Jun 15;264(17):10096–10103. [PubMed] [Google Scholar]
  3. Coolican S. A., Haiech J., Hathaway D. R. The role of subunit autolysis in activation of smooth muscle Ca2+-dependent proteases. J Biol Chem. 1986 Mar 25;261(9):4170–4176. [PubMed] [Google Scholar]
  4. Coolican S. A., Hathaway D. R. Effect of L-alpha-phosphatidylinositol on a vascular smooth muscle Ca2+-dependent protease. Reduction of the Ca2+ requirement for autolysis. J Biol Chem. 1984 Oct 10;259(19):11627–11630. [PubMed] [Google Scholar]
  5. Crawford C., Brown N. R., Willis A. C. Studies of the active site of m-calpain and the interaction with calpastatin. Biochem J. 1993 Nov 15;296(Pt 1):135–142. doi: 10.1042/bj2960135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DeMartino G. N., Huff C. A., Croall D. E. Autoproteolysis of the small subunit of calcium-dependent protease II activates and regulates protease activity. J Biol Chem. 1986 Sep 15;261(26):12047–12052. [PubMed] [Google Scholar]
  7. Frieden C. Slow transitions and hysteretic behavior in enzymes. Annu Rev Biochem. 1979;48:471–489. doi: 10.1146/annurev.bi.48.070179.002351. [DOI] [PubMed] [Google Scholar]
  8. Goll D. E., Thompson V. F., Taylor R. G., Zalewska T. Is calpain activity regulated by membranes and autolysis or by calcium and calpastatin? Bioessays. 1992 Aug;14(8):549–556. doi: 10.1002/bies.950140810. [DOI] [PubMed] [Google Scholar]
  9. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  10. Inomata M., Hayashi M., Nakamura M., Imahori K., Kawashima S. Hydrolytic and autolytic behavior of two forms of calcium-activated neutral protease (CANP). J Biochem. 1985 Aug;98(2):407–416. doi: 10.1093/oxfordjournals.jbchem.a135295. [DOI] [PubMed] [Google Scholar]
  11. Inomata M., Imahori K., Kawashima S. Autolytic activation of calcium-activated neutral protease. Biochem Biophys Res Commun. 1986 Jul 31;138(2):638–643. doi: 10.1016/s0006-291x(86)80544-7. [DOI] [PubMed] [Google Scholar]
  12. Inomata M., Kasai Y., Nakamura M., Kawashima S. Activation mechanism of calcium-activated neutral protease. Evidence for the existence of intramolecular and intermolecular autolyses. J Biol Chem. 1988 Dec 25;263(36):19783–19787. [PubMed] [Google Scholar]
  13. James P., Vorherr T., Carafoli E. Calmodulin-binding domains: just two faced or multi-faceted? Trends Biochem Sci. 1995 Jan;20(1):38–42. doi: 10.1016/s0968-0004(00)88949-5. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Malik M. N., Ramaswamy S., Tuzio H., Shiekh A. M., Fenko M. D., Wisniewski H. M., Howard R. G. Micromolar Ca2+ requiring protease from human platelets: purification, partial characterization and effect on the cytoskeletal proteins. Life Sci. 1987 Feb 9;40(6):593–604. doi: 10.1016/0024-3205(87)90374-2. [DOI] [PubMed] [Google Scholar]
  16. Mellgren R. L. Proteolysis of nuclear proteins by mu-calpain and m-calpain. J Biol Chem. 1991 Jul 25;266(21):13920–13924. [PubMed] [Google Scholar]
  17. Melloni E., Pontremoli S. The calpains. Trends Neurosci. 1989 Nov;12(11):438–444. doi: 10.1016/0166-2236(89)90093-3. [DOI] [PubMed] [Google Scholar]
  18. Ohno S., Emori Y., Imajoh S., Kawasaki H., Kisaragi M., Suzuki K. Evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calcium-binding protein? Nature. 1984 Dec 6;312(5994):566–570. doi: 10.1038/312566a0. [DOI] [PubMed] [Google Scholar]
  19. Pintér M., Stierandova A., Friedrich P. Purification and characterization of a Ca(2+)-activated thiol protease from Drosophila melanogaster. Biochemistry. 1992 Sep 8;31(35):8201–8206. doi: 10.1021/bi00150a012. [DOI] [PubMed] [Google Scholar]
  20. Pontremoli S., Melloni E., Sparatore B., Salamino F., Michetti M., Sacco O., Horecker B. L. Role of phospholipids in the activation of the Ca2+-dependent neutral proteinase of human erythrocytes. Biochem Biophys Res Commun. 1985 Jun 14;129(2):389–395. doi: 10.1016/0006-291x(85)90163-9. [DOI] [PubMed] [Google Scholar]
  21. Pontremoli S., Sparatore B., Melloni E., Michetti M., Horecker B. L. Activation by hemoglobin of the Ca2+-requiring neutral proteinase of human erythrocytes: structural requirements. Biochem Biophys Res Commun. 1984 Aug 30;123(1):331–337. doi: 10.1016/0006-291x(84)90417-0. [DOI] [PubMed] [Google Scholar]
  22. Saido T. C., Nagao S., Shiramine M., Tsukaguchi M., Sorimachi H., Murofushi H., Tsuchiya T., Ito H., Suzuki K. Autolytic transition of mu-calpain upon activation as resolved by antibodies distinguishing between the pre- and post-autolysis forms. J Biochem. 1992 Jan;111(1):81–86. doi: 10.1093/oxfordjournals.jbchem.a123723. [DOI] [PubMed] [Google Scholar]
  23. Saido T. C., Sorimachi H., Suzuki K. Calpain: new perspectives in molecular diversity and physiological-pathological involvement. FASEB J. 1994 Aug;8(11):814–822. [PubMed] [Google Scholar]
  24. Saido T. C., Suzuki H., Yamazaki H., Tanoue K., Suzuki K. In situ capture of mu-calpain activation in platelets. J Biol Chem. 1993 Apr 5;268(10):7422–7426. [PubMed] [Google Scholar]
  25. Saito K., Elce J. S., Hamos J. E., Nixon R. A. Widespread activation of calcium-activated neutral proteinase (calpain) in the brain in Alzheimer disease: a potential molecular basis for neuronal degeneration. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2628–2632. doi: 10.1073/pnas.90.7.2628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Salamino F., De Tullio R., Mengotti P., Viotti P. L., Melloni E., Pontremoli S. Site-directed activation of calpain is promoted by a membrane-associated natural activator protein. Biochem J. 1993 Feb 15;290(Pt 1):191–197. doi: 10.1042/bj2900191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sorimachi H., Saido T. C., Suzuki K. New era of calpain research. Discovery of tissue-specific calpains. FEBS Lett. 1994 Apr 18;343(1):1–5. doi: 10.1016/0014-5793(94)80595-4. [DOI] [PubMed] [Google Scholar]
  28. Suzuki K., Imajoh S., Emori Y., Kawasaki H., Minami Y., Ohno S. Calcium-activated neutral protease and its endogenous inhibitor. Activation at the cell membrane and biological function. FEBS Lett. 1987 Aug 17;220(2):271–277. doi: 10.1016/0014-5793(87)80828-1. [DOI] [PubMed] [Google Scholar]
  29. Suzuki K., Sorimachi H., Yoshizawa T., Kinbara K., Ishiura S. Calpain: novel family members, activation, and physiologic function. Biol Chem Hoppe Seyler. 1995 Sep;376(9):523–529. doi: 10.1515/bchm3.1995.376.9.523. [DOI] [PubMed] [Google Scholar]
  30. Suzuki K., Tsuji S., Ishiura S. Effect of Ca2+ on the inhibition of calcium-activated neutral protease by leupeptin, antipain and epoxysuccinate derivatives. FEBS Lett. 1981 Dec 21;136(1):119–122. doi: 10.1016/0014-5793(81)81227-6. [DOI] [PubMed] [Google Scholar]
  31. Suzuki K., Tsuji S., Kubota S., Kimura Y., Imahori K. Limited autolysis of Ca2+-activated neutral protease (CANP) changes its sensitivity to Ca2+ ions. J Biochem. 1981 Jul;90(1):275–278. doi: 10.1093/oxfordjournals.jbchem.a133463. [DOI] [PubMed] [Google Scholar]
  32. Tompa P., Schád E., Baki A., Alexa A., Batke J., Friedrich P. An ultrasensitive, continuous fluorometric assay for calpain activity. Anal Biochem. 1995 Jul 1;228(2):287–293. doi: 10.1006/abio.1995.1352. [DOI] [PubMed] [Google Scholar]
  33. Tsien R., Pozzan T. Measurement of cytosolic free Ca2+ with quin2. Methods Enzymol. 1989;172:230–262. doi: 10.1016/s0076-6879(89)72017-6. [DOI] [PubMed] [Google Scholar]
  34. Yoshizawa T., Sorimachi H., Tomioka S., Ishiura S., Suzuki K. Calpain dissociates into subunits in the presence of calcium ions. Biochem Biophys Res Commun. 1995 Mar 8;208(1):376–383. doi: 10.1006/bbrc.1995.1348. [DOI] [PubMed] [Google Scholar]
  35. Zimmerman U. J., Schlaepfer W. W. Two-stage autolysis of the catalytic subunit initiates activation of calpain I. Biochim Biophys Acta. 1991 Jun 24;1078(2):192–198. doi: 10.1016/0167-4838(91)99009-h. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES