The preparation and characterization of subcellular fractions of rat myocardium

Abstract

An investigation of methods of disruption of rat myocardium has shown that using the teflon/glass grinder, no difference in overall fibre disruption was obtained when the composition of the suspension medium was varied. The efficiency of fibre disintegration was markedly increased when teflon homogenization was followed by manual rubbing in a dialysis sac and then by sonication, greater than 90 per cent break-up of fibres being obtained as judged by the liberation of sarcosomes. This enabled reassessment of the proportions of myofibrils, sarcosomes, “sarcotubular” elements, and non-granular components in heart muscle. Maximum concentration of cytochrome oxidase was found in the sarcosomes, of RNA and of antimycin-resistant NADH-cytochrome c reductase in the “sarcotubular” fraction, and of esterase in the supernatant. The myofibrillar fraction, which contributed 34 per cent of the total cellular protein, contained only 6 per cent of the total cytochrome oxidase activity, was virtually free of esterase, and still retained a high content of RNA. The prepared sarcosomal fraction contained 88 per cent of the total cytochrome oxidase activity and 21 per cent of the RNA. The “sarcotubular” fraction (85,000 × g for 75 min) resembled in some respects “microsomal” material derived from liver cells. The “intermediate” fraction (60,000 × g for 40 min) appeared to consist of “sarcotubular” elements contaminated with sarcosomal fragments. The results are compared with those of previous investigators concerned with the characteristics of subcellular fractions of heart muscle.