DETERIORATION OF CONNECTIN TITIN AND NEBULIN FILAMENTS BY AN EXCESS OF PROTEASE INHIBITORS/

We studied the effect of protease inhibitors at a high concentration a n connectin and nebulin filaments in myofibrils. Calpastatin domain I at 0.1 mM bound to connectin and nebulin filaments, and deteriorated t heir physico-chemical properties; the calcium-binding ability of conne ctin and nebulin filaments was suppressed, the susceptibility of both filaments to trypsin was markedly decreased, and the resting tension o f mechanically skinned fibers was increased by 2.5 times that of the c ontrol at a sarcomere length of 3.6 mu m. This indicates that the conn ectin filaments were made more rigid. The same phenomenon was observed from the treatment of skinned fibers with 1 mhs leupeptin whose resti ng tension was increased to 2 times the control value. Microscopically , both protease inhibitors induced dense aggregation and disappearance of the regular striation of myofibrils due to their non-specific bind ing to many myofibriilar proteins. The use of excess calpastatin domai n I and leupeptin should therefore be avoided in physiological and bio chemical studies on connectin and nebulin filaments, as well as on myo fibrils.