Studies on the Sarcoplasmic Reticulum of Normal and Dystrophic Animals ∗

Abstract

The presence of increased amounts of free lysosomal hydrolytic enzymes and decreased protein concentration within the muscle cell of dystrophic animals is well established (1–4). The role of these enzymes in the mechanism of the disease is not clear (5, 6). Barany et al. found that among the cellular proteins proportionally the sarcoplasmic proteins were decreased most in the dystrophic muscle cell (7).

Sreter et al. discovered that the Ca²⁺ transport function of the sarcoplasmic reticulum was impaired in dystrophic animals (8–10). These observations led us to investigate some characteristics of the sarcoplasmic proteins and lipids and to study the effects of protease and phospholipase C on fragmented sarcoplasmic reticulum using both normal and dystrophic animals.

Materials and Methods. Dystrophic chickens were generously given by Dr. D. W. Peterson, Department of Poultry Husbandry, University of California, Davies, California. Dystrophic mice (129 B6F1-dy) were purchased from The Jackson Laboratory, Bar Harbor, Maine.

Fragmented sarcoplasmic reticulum of various sources was prepared according to the method of Martonosi and Feretos (11). Great care was taken to remove the contaminant proteins by repeated washings with 0.6 M KCl. For the determination of the amino acid composition of sarcoplasmic protein about 30–60 mg (wet weight) of sarcoplasmic reticulum was dissolved in 1 ml 90% phenol solution and the sarcoplasmic protein was precipitated by the addition of 6 ml ice-cold acetone. The dried and well washed protein was hydrolyzed by 6 M HCl at 110° for 2 days and the amino acid composition determined with a Phoenix Model M-7800 automatic amino acid analyzer using an automated version of the Moore and Stein technique (12). The yield of the phenol-acetone extraction procedure was 0.3 mg protein per 1.0 mg (wet weight) sarcoplasmic reticulum.