Loss of Ca2+-dependent regulation in glycerinated skeletal muscle contraction.

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Abstract
Glycerinated muscle fiber from rabbit psoas muscle often lost Ca2+-dependent regulation of its contraction with long-term extraction in a 50% glycerol solution containing 5 mM EGTA [ethyleneglycol bis-(.beta.-amino-ethyl-ether)-N,N,N'',N''-tetraacetic acid] at -20.degree. C, designated as Ca2+-insensitive muscle fiber (CaIS-fiber). About 30 or 40% of glycerinated muscle fibers were CaIS-fibers after 1-3 mo. in the glycerol solution. The cause of the loss of Ca2+-sensitivity of the glycerinated muscle fiber was investiaged by tension mechanogram and SDS [sodium dodecyl sulfate] polyacrylamide gel electrophoresis. This natural CaIS-fiber showed a new band of 30K daltons peptide on SDS gels. Ca2+-sensitive fiber (CaS-fiber) changed to CaIS-fiber by trypsin digestion for 40 s. The tryptic CaIS-fiber had no troponin C and 30K daltons peptide bands in the electrophoretograms. Incubating with 2 mM CaCl2 for 40 h at 25.degree. C, CaS-fiber changed easily to CaIS-fiber which had 30K daltons peptide and faint troponin T and I bands, as in natural CaIS-fiber. All CaIS-fibers could recover their Ca2+-dependent regulation by incubating with native tropomyosin from rabbit skeletal muscle for 2 days at 4% C. Evidently, the loss of Ca2+-dependent regulation of glycerinated muscle fiber is due to degradation of regulatory protein systm by endogenous Ca2+-activated proteolytic enzymes.