25] Pyruvate kinase isozymes from rat

Abstract

Publisher Summary This chapter describes the tissue distribution, methods of assay, electrophoretic behavior, purification, and characterization of pyruvate kinase (PK) isozymes. The isozymes are called, L, R, M 1 , and M 2 . Two different assay methods are presented in the chapter: one is the simple colorimetric 2,4-dinitrophenylhydrazone method, in which pyruvic acid formed from phosphoenol pyruvate (PEP) by PK is determined with the reagent for carbonyl residues, 2,4-dinitrophenylhydrazine. The other method is the NADH–LDH coupled method in which PK activity is measured by following the oxidation of NADH at 340 nm using a recording spectrophotometer with a coupled assay system. M 1 is the main type in specifically differentiated tissues, such as adult skeletal muscle, heart, and brain. L is predominant in gluconeogenic tissues, especially liver, but is a minor type in kidney, while R is present in erythrocytes and hematopoietic tissues. The four types of PK differ clearly in electrophoretic mobilities. However, L and R, and M 1 and M 2 , respectively, are closely similar in certain properties, including their immunological and kinetic properties, phosphorylations by cAMP-dependent protein kinase, amino acid compositions, and peptide maps of limited proteolysis products.