Characteristics of sarcoplasmic reticulum from slowly glycolysing and from rapidly glycolysing pig skeletal muscle post mortem

Abstract

The composition and function of fragmented sarcoplasmic reticulum from pig skeletal muscle was examined in the period immediately post mortem. Muscle was defined as being either slowly glycolysing or rapidly glycolysing on the basis of colour, pH and concentrations of glycogen and lactate. The microsomal fraction was separated on a discontinuous gradient of 35, 40 and 45% (w/v) sucrose into heavy and intermediate fractions which sedimented to the interfaces, and a light fraction which remained on the surface of the 35%-sucrose layer. The sarcoplasmic reticulum from rapidly glycolysing muscle had a lower buoyant density than had that from slowly glycolysing muscle. This was reflected in the consistent lack of material in the heavy fraction and a greater proportion in the light fraction. The latter material had significantly lower ratios (w/w) of protein to phospholipid (2.3:1 versus 3.8:1) and of protein to cholesterol (10.4:1 versus 15.6:1). There were no gross differences in phospholipid content or in fatty acid composition of individual phospholipid classes in the membranes from the two types of muscle. Analysis of membrane proteins by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed that ATPase (adenosine triphosphatase) was a major component of each fraction and that its contribution to the total protein content of the membrane was greater in rapidly glycolysing muscle, suggesting a loss of non-ATPase proteins. The two fractions of sarcoplasmic reticulum prepared from rapidly glycolysing muscle had approximately one-third the normal activities of Ca²⁺ binding and Ca²⁺ uptake in the presence of ATP and one-half the passive Ca²⁺-binding capacity in the absence of ATP of the fractions from slowly glycolysing muscle. However, the (Ca²⁺+Mg²⁺)-stimulated ATPase activities were similar. Efflux from actively loaded vesicles, after the addition of EDTA, consisted of a rapid and a slow phase. Vesicles from rapidly glycolysing muscle lost 60% of associated Ca²⁺(approx. 0.10μmol of Ca²⁺/mg of protein) during the rapid phase, compared with 30% (approx. 0.17μmol of Ca²⁺/mg of protein) in those from slowly glycolysing muscle. The efflux rate during the slower phase was comparable in both types of vesicles. Analysis of the temperature-dependence of (Ca²⁺+Mg²⁺)-stimulated ATPase activity revealed that a high-activation-energy process operating in the temperature range 31–45°C in the intermediate and light fractions from slowly glycolysing muscle was not apparent in vesicles from rapidly glycolysing muscle. Conditions that result in the prolonged activation of glycogenolysis in pig muscle post mortem primarily affect the protein components of the sarcoplasmic-reticular membrane, giving rise to a loss of loosely associated proteins. The function of the membranes observed under these conditions does not appear to be due to enhanced permeability of the membrane to Ca²⁺ and may be the result of a defect in the transport of Ca²⁺ into the vesicles.