Concentration of skeletal and cardiac muscle pyruvate kinase determined by specific radioimmunoassay

Abstract

A specific radioimmunoassay has been developed for rabbit skeletal muscle pyruvate kinase (ATP:pyruvate 2- O-phosphotransferase, EC 2.7.1.40) and applied to extracts of skeletal and cardiac muscle from rabbits subjected to differing dietary states and alloxan diabetes. In animals fed Purina Checkers ® rabbit chow the concentration was 11.1 ± 3.3 μM is skeletal and 3.1 ± 0.6 μM (μmol/1000 g wet weight) in heart muscle. Compared to the chow-fed animals the enzyme concentration increased approximately 2-fold in both tissues after a 4 day fast or after fat feeding, without any change in enzyme activity. Using slab gel electrophoresis, we demonstrated that fat feeding and fasting evidently cause the muscle cell to accumulate inactive or less active, tetrameric pyruvate kinase. Alloxan diabetes had no influence on the concentration of the enzyme in chow-fed animals. Pyruvate kinase activity was measurement in the same extracts by a conventional assay. From the data specific activities were calculated: for example, 40 units/nmol skeletal muscle enzyme and 19 units/nmol heart muscle enzyme in chow-fed animals. It is only by the combined application of activity and chemical concentration assays to tissue extracts could such data be obtained. Thus, in addition to measuring enzyme concentration an approach is provided to detect differences in the catalytic state of the enzyme. Our data show that assayable activity of this enzyme does not always reflect enzyme concentration and that regulation of enzyme concentration and enzyme activity can occur independently.