AUTOLYSIS, CA2-CALPAIN( REQUIREMENT, AND HETERODIMER STABILITY IN M)

The roles of N-terminal autolysis of the large (80 kDa) and small (28 kDa) subunits in activation of rat m-calpain, in lowering its Ca2+ req uirement, and in reducing its stability have been investigated with he terodimeric recombinant calpains containing modified subunits. Both au tolysis and [Ca2+](0.5) were influenced by the ionic strength of the b uffers, which accounts for the wide variations in previous reports. Au tolysis of the small subunit (from 28 to 20 kDa) was complete within 1 min but did not alter either the Ca2+ requirement ([Ca2+](0.5)) or th e stability of the enzyme. Autolysis of the NHis(10)-80k large subunit at Ala(9)-Lys(10) is visible on gels, was complete within 1 min, and caused a drop in [Ca2+](0.5) from 364 to 187 mu M. The lower value of [Ca2+](0.5) is therefore a property of the Delta 9-80k large subunit. Autolysis at Ala(9)-Lys(10) of the unmodified 80-kDa large subunit is not detectable on gels but was assayed by means of the fall in [Ca2+]( 0.5). This autolysis was complete in 3.5 min and was inhibited by high [NaCl]. The autolysis product of these calpains, which is essentially identical to that of natural m-calpain, was unstable in buffers of hi gh ionic strength. Calpain in which the large subunit autolysis site h ad been mutated was fully active but did not undergo a drop in [Ca2+]( 0.5), showing that m-calpain is active prior to autolysis. The main ph ysiological importance of autolysis of calpain is probably to generate an active but unstable enzyme, thus limiting the in vivo duration of calpain activity.