Autolysis parallels activation of mu-calpain.
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.
Baki
A
A
Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary.
Tompa
P
P
Alexa
A
A
Molnár
O
O
Friedrich
P
P
D001329:Autolysis; D002154:Calpain / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000097:blood / Q000737:chemistry / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D015394:Molecular Structure; D011487:Protein Conformation
D004792:Enzyme Precursors; D008869:Microtubule-Associated Proteins; D002154:Calpain; C098484:mu-calpain proenzyme