Purification and properties of muscle phosphoglycerate kinase

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Abstract
A partial purification of the enzyme, phosphoglycerate kinase, from an aqueous extract of rabbit skeletal muscle has been achieved. The purified enzyme was not homogeneous electrophoretically and contained 2 components. It was free of most of the enzymes of the anaerobic glycolytic pathway but contained triose phosphate isomerase. A new assay method for studying the phosphoglycerate-kinase reaction in the forward direction was developed. The method has the advantage that it can be used with partially purified enzyme preparations containing triose phosphate isomerase and [alpha]-glycerophosphate dehydrogenase. The assay method also facilitated an independent study of inhibitors and metal ions in the enzyme reactions. Phosphoglycerate kinase is activated by Mg2+ and Mn2+ ions, and by no other metal ions tested. It reacts specifically with adenosine and di-and tri-phosphate, but with no other nucleotides. The study of the effect of various thiol-binding agents on the muscle and yeast phosphoglycerate kinases revealed that the muscle phosphoglycerate kinase is a sulphydryl enzyme whereas the yeast enzyme is not. The Michaelis constants for the various substrates and activators obtained with the muscle enzyme are similar to the values obtained with the yeast enzyme. These values are also in good agreement with the data originally reported by BCtcher (1947). The Michaelis constants of the muscle and yeast enzymes for phosphoglycerly phosphate have been determined independently by an isotopic method.