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Development of Methods to Measure Activity of Polysomes and Cytoplasmic Enzymes from Bovine Skeletal Muscle in in Vitro Protein-Synthesis Assays^1,2,

Iowa State University^,6, Ames 50011

Abstract

A cell-free, protein-synthesis system containing components from bovine skeletal muscle was developed as prerequisite to attempts to learn whether polysomes or cytoplasmic enzymes limit rate of muscle protein synthesis in the current population of domestic animals. Amino acid incorporation into trichloroacetic acid-precipitable protein was optimal at pH 7.5 and, if Tris buffer was used, at K⁺ and Mg²⁺ concentrations of 40 mM and 4 mM, respectively. Optimal concentrations of compounds that provide energy for amino acid incorporation were 1 mM ATP, .2 mM GTP, 20 µg creatine phosphokinase/ml and 20 mM creatine phosphate; .03 mM of each of the 20 amino acids was required for assays lasting up to 60 min. Neither rate of tRNA acylation nor availability of aminoacyl-tRNA was rate-limiting in the cell-free system established; hence, the cytoplasmic enzyme fraction from bovine skeletal muscle contains ample tRNA and has aminoacyl-tRNA synthases that are sufficiently active to form aminoacyl-tRNA faster than these compounds are used to form polypeptides in the cell-free system developed. Reinitiation of ribosomes onto new mRNA occurred very slowly, if at all, in the proteinsynthesis system developed. Cytoplasmic enzymes were rate-limiting whenever cytoplasmic enzyme protein to polysomal protein ratios were 3.2:1 or lower in the cell-free system. Polysomes were rate-limiting whenever cytoplasmic enzyme protein to polysomal protein ratios were 400:1 or higher in the cell-free system. These ratios define the conditions needed to assay activity of cytoplasmic enzymes or polysomes from different animals quantitatively.

¹ Journal Paper No. J-11172 of the Iowa Agr. and Home Econ. Exp. Sta., Ames, Projects 2361 and 2127. This research was supported in part by Arizona Agr. Exp. Sta., Project 28, and by grants from the National Institutes of Health to DEG (AM-19864 and HL-20984) and to RMR (HL-15679), from the Muscular Dystrophy Assoc. (DEG and RMR), and the American Heart Assoc., Iowa Affiliate (RMR). This paper is a contribution from North Central Reg. Res. Proj. NC-131.

² We gratefully acknowledge the technical assistance of Jacqueline Harvey Martin and Darlene Markley and the help of Elizabeth Hutton, Nancy Fahringer, and Joan Andersen with the manuscript.

³ Baylor College of Medicine, Dept. of Med., Houston, TX 77030.

⁴ Send reprint requests to Darrel E. Goll, Muscle Biology Group, Univ. of Arizona, Tucson 85721.

⁵ Muscle Biology Group, Iowa State Univ., Ames 50011.

⁶ Dept. of Anim. Sci., Biochem. and Biophys. and Food Technol., Cooperating.