THE POSSIBLE SELF-DOWN-REGULATION OF CALPAIN TRIGGERED BY CELL-MEMBRANES

In order to confirm whether the binding sites for mu-calpain on the inner surface of erythrocyte membranes are substrate proteins themselves, we examined the binding properties of mu-calpain to mu-calpain-pretreated inside-out membranes. When native mu-calpain was incubated with mu-calpain-pretreated membranes, however, newly added calpain was degraded rapidly in a time- and Ca2+-dependent manner. Although the degradation of mu-calpain was not inhibited by various proteinase inhibitors, it was strongly inhibited by digestible substrates for calpain that possess the ability to inhibit the binding of mu-calpain to erythrocyte membranes. On the other hand, when mu-calpain inactivated by N-ethylmaleimide was incubated with mu-calpain-pretreated membranes, no degradation was observed. These results indicate that the degradation of mu-calpain occurs on the surface of mu-calpain-modified membranes and that it depends on the autoproteolytic activity of mu-calpain itself. It seems likely that the autoproteolytic activity of mu-calpain is accelerated markedly by some component(s) exposed on the surface of membranes during the pretreatment with mu-calpain. The possibility is thus proposed that cell membranes possess the ability to down-regulate calpain to protect cell membranes from overdegradation by excessively bound calpain. The active factor(s) in the membranes that can accelerate the autoproteolytic degradation of mu-calpain could be almost completely removed from mu-calpain-modified membranes by treatment with Triton X-100.