Specific spectrophotometric assay for the M isoenzyme of pyruvate kinase in plasma samples containing mixtures of the muscle (M) and liver (L) isoenzymes

Abstract

Plasma pyruvate kinase (EC 2.7.2.40) activity contains two major isoenzymes. the 'L' type originates from liver (erythrocytes contain a similar isoenzyme), the 'M' type predominantly from skeletal muscle. The diagnostic use of pyruvate kinase as an index of muscle leakage requires an assay of only the M type. An M-type-selective assay for mixtures of the two isoenzymes was devised, based on the differences that the L form has a higher K(m) for phosphoenolpyruvate and is inhibited about 95% by a high concentration of ADP. All relevant conditions of the assay have been examined and the reaction system has been optimized at 30°C. The assay yields the expected K(m) values for M type pyruvate kinase. An apparent increase activity in hemolysed plasma samples was found to be derived from leukocytes.