Autolysis parallels activation of μ-calpain

A. Baki, P. Tompa, A. Alexa, O. Molnar, P. Friedrich

Research output: Contribution to journalArticle

90 Citations (Scopus)

Abstract

The kinetics of autolysis and activation of μ-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 μ-calpain large subunit at both 10 and 300 μM Ca2+, and both straight lines intersected the origin. This finding supports the view that native μ-calpain is an inactive proenzyme and that activation is accompanied by autolysis. The first-order rate constant of autolysis, κ(I)(aut), was determined at different Ca2+ concentrations: the half-maximal value was at pCa2+ = 3.7 (197 μM Ca2+), whereas the maximal value was 1.52 s-1, at 30°C. The Ca2+-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, κ(I)(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 μM Ca2+) and the maximum value was 2.15 s-1. The good agreement between the kinetic parameters of μ-calpain autolysis and activation is remarkable. We claim that this is the first kinetically correct determination of the rate constant of autolysis of μ-calpain. Pre-activated μ-calpain has a Ca2+ requirement that is almost three orders of magnitude smaller [half-maximal activation at pCa2+ = 6.22 (0.6 μM Ca2+)]. We cannot exclude the possibility that the activation process involves other mechanistic steps, e.g. the rapid dissociation of the p-calpain heterodimer, but we state that in our conditions in vitro autolysis and activation run in close parallel.

Original languageEnglish
Pages (from-to)897-901
Number of pages5
JournalBiochemical Journal
Volume318
Issue number3
Publication statusPublished - 1996

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Autolysis
Calpain
Chemical activation
Microtubule-Associated Proteins
Rate constants
Enzyme Precursors
Substrates
Kinetic parameters
Hydrolysis
Assays
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Baki, A., Tompa, P., Alexa, A., Molnar, O., & Friedrich, P. (1996). Autolysis parallels activation of μ-calpain. Biochemical Journal, 318(3), 897-901.

Autolysis parallels activation of μ-calpain. / Baki, A.; Tompa, P.; Alexa, A.; Molnar, O.; Friedrich, P.

In: Biochemical Journal, Vol. 318, No. 3, 1996, p. 897-901.

Research output: Contribution to journalArticle

Baki, A, Tompa, P, Alexa, A, Molnar, O & Friedrich, P 1996, 'Autolysis parallels activation of μ-calpain', Biochemical Journal, vol. 318, no. 3, pp. 897-901.
Baki A, Tompa P, Alexa A, Molnar O, Friedrich P. Autolysis parallels activation of μ-calpain. Biochemical Journal. 1996;318(3):897-901.
Baki, A. ; Tompa, P. ; Alexa, A. ; Molnar, O. ; Friedrich, P. / Autolysis parallels activation of μ-calpain. In: Biochemical Journal. 1996 ; Vol. 318, No. 3. pp. 897-901.
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