Inhibition of sarcoplasmic reticulum calcium pump by cytosolic protein(s) endogenous to heart and slow skeletal muscle but not fast skeletal muscle

Abstract

Cytosol from rabbit heart and slow and fast skeletal muscles was fractionated using (NH 4) 2SO 4 to yield three cytosolic protein fractions, viz., CPF-I (protein precipitated at 30% saturation), CPF-II (protein precipitated between 30 and 60% saturation), and cytosol supernatant (protein soluble at 60% saturation). The protein fractions were dialysed and tested for their effects on ATP-dependent, oxalate-supported Ca 2+ uptake by sarcoplasmic reticulum from heart and slow and fast skeletal muscles. CPF-I from heart and slow muscle, but not from fast muscle, caused marked inhibition (up to 95%) of Ca 2+ uptake by sarcoplasmic reticulum from heart and from slow and fast muscles. Neither unfractionated cytosol nor CPF-II or cytosol supernatant from any of the muscles altered the Ca 2+ uptake activity of sarcoplasmic reticulum. Studies on the characteristics of inhibition of sarcoplasmic reticulum Ca 2+ uptake by CPF-I (from heart and slow muscle) revealed the following: (a) Inhibition was concentration- and temperature-dependent (50% inhibition with approx. 80 to 100 μg CPF-I; seen only at temperatures above 20°C). (b) The inhibitor reduced the velocity of Ca 2+ uptake without appreciably influencing the apparent affinity of the transport system for Ca 2+. (c) Inhibition was uncompetitive with respect to ATP. (d) Sarcoplasmic reticulum washed following exposure to CPF-I showed reduced rates of Ca 2+ uptake, indicating that inhibition results from an interaction of the inhibitor with the sarcoplasmic reticulum membrane. (e) Concomitant with the inhibition of Ca 2+ uptake, CPF-I also inhibited the Ca 2+-ATPase activity of sarcoplasmic reticulum. (f) Heat-treatment of CPF-I led to loss of inhibitor activity, whereas exposure to trypsin appeared to enhance its inhibitory effect. (g) Addition of CPF-I to Ca 2+-preloaded sarcoplasmic reticulum vesicles did not promote Ca 2+ release from the vesicles. These results demonstrate the presence of a soluble protein inhibitor of sarcoplasmic reticulum Ca 2+ pump in heart and slow skeletal muscle but not in fast skeletal muscle. The characteristics of the inhibitor and its apparently selective distribution suggest a potentially important role for it in the in vivo regulation of sarcoplasmic reticulum Ca 2+ pump, and therefore in determining the duration of Ca 2+ signal in slow-contracting muscle fibers.