Autolysis, Ca2+ Requirement, and Heterodimer Stability in m-Calpain
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 Ca 2+ requirement, and in reducing its stability have been investigated with heterodimeric recombinant calpains containing modified subunits. Both autolysis and [Ca 2+...
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Veröffentlicht in: | The Journal of biological chemistry 1997-04, Vol.272 (17), p.11268-11275 |
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Sprache: | eng |
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Zusammenfassung: | 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 Ca 2+ requirement, and in reducing its stability have been investigated with heterodimeric recombinant calpains containing modified
subunits. Both autolysis and [Ca 2+ ] 0.5 were influenced by the ionic strength of the buffers, which accounts for the wide variations in previous reports. Autolysis
of the small subunit (from 28 to 20 kDa) was complete within 1 min but did not alter either the Ca 2+ requirement ([Ca 2+ ] 0.5 ) or the 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 [Ca 2+ ] 0.5 from 364 to 187 μ M . The lower value of [Ca 2+ ] 0.5 is therefore a property of the Î9-80k 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 [Ca 2+ ] 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 high ionic strength. Calpain in which the
large subunit autolysis site had been mutated was fully active but did not undergo a drop in [Ca 2+ ] 0.5 , showing that m-calpain is active prior to autolysis. The main physiological importance of autolysis of calpain is probably
to generate an active but unstable enzyme, thus limiting the in vivo duration of calpain activity. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.272.17.11268 |