Autolysis parallels activation of μ-calpain

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

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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
DOIs
Publication statusPublished - Jan 1 1996

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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