Glycine biosynthesis in rat and sheep muscle
Glycine is the third most abundant of the amino acids released by muscle. Perfused rat hind-limb and sheep diaphragm preparations were employed to study the origin of glycine produced by non-ruminant and ruminant muscle. Neither the degradation of muscle and erythrocyte glutathione nor the 'leaching out' of the intracellular glycine pool contributed to the glycine released by either muscle. When the perfusions were carried out with the medium free of amino acids, the proteolysis accounted for 57% of the total glycine release by the rat hind-limb and 38% by the sheep diaphragm. Minimum de novo synthesis of glycine was 12.3 umol/3 h/30 g in the rat muscle and 10.3 umol/3 h/30 g in the sheep muscle. Addition of serine to the perfusion medium stimulated significantly both the rate of glycine efflux and total glycine production in the rat hind-limb. Similar results were obtained with the sheep diaphragm; however, the increases were not statistically significant. Addition of 5-formyl tetrahydrofolate, a specific inhibitor of serine hydroxymethyltransferase, SHMT (EC 220.127.116.11) significantly decreased the rate of glycine efflux from both the muscles. The observations using cold serine were confirmed with the experiments employing radioisotopes. Up to 40% of total glycine produced by the rat hind-limb was derived from serine, whereas in the sheep diaphragm it was only 4%. In both the muscles synthesis of glycine from serine was by SHMT and not glycine synthase (EC 18.104.22.168). Synthesis of glycine from threonine was negligible in both the muscles. SHMT activity increased in liver, diaphragm and hind-limb muscle of female rats treated with trenbolone acetate or testosterone, anabolic agents. Both the muscles incorporated 14C from (U-14C) serine and (3-14C) serine to methionine, cystine, alanine, aspartate and glutamate + glutamine. The label from (U-14C) glucose was recovered in serine and glycine in the rat hind-limb but not in the sheep diaphragm. A 'serine-glycine' cycle involving kidney and muscle is proposed. The possible significance of glycine released by muscle is discussed. Development of a system for the perfusion of sheep diaphragm with erythrocyte-free medium, and a method for the determination of radioactivity in C-2 of glycine also form a part of the thesis.