The stable, inactive but reactivatable, unfolding intermediate of rat muscle sarcoplasmic reticulum Ca(2+)-ATPase differs from the age-modified form of this protein.

Abstract

Ca(2+)-ATPase from rat skeletal-muscle sarcoplasmic-reticulum has been reported to be less stable in old animals. These changes were suggested to be due to age-related modifications in the membrane. In the present study, the guanidinium-chloride (GuHCl)-induced inactivation, and reactivation by dilution of the denaturant, of this enzyme have been investigated. The cooperativity of the inactivation-transition was found to increase when the membrane was dissolved in the non-ionic detergent polyoxyethylene 10 laurylether. There is an inactive unfolding intermediate in equilibrium with the native state at low concentrations of GuHCl. This intermediate can be reactivated, even after as long a time as 19 h. The stability of this intermediate is only slightly affected by detergent solubilization of the enzyme. Hence, the membrane probably only plays a minor role in stabilizing the intermediate.