The phosphotransferase activity of phosphatases. 1. Spectrophotometric methods for the estimation of some phosphate esters and other compounds

Abstract

Methods are described for kinetic study of the phosphotransferase reaction. The phosphate esters synthesized by phosphate transfer are estimated spectrophotometrically with substrate-specific enzymes. The methods avoid the separation of the phosphate esters from the other components of the reaction mixtures. Slater''s (Slater, E.C. (1953). Biochem. J. 53, 157) procedures were used for estimation of triose phosphates and hexose phosphates. Extensions of these procedures for differentiations of fructose diphosphate, glycer-aldehyde, phosphate and dihydroxyacetone phosphate are described. Methods for estimation of L-[alpha]-glycerophosphate, succinate and L-lactate are described. Cytochrome c is used as the hydrogen acceptor with the appropriate cytochrome c-linked dehydrogenase system, or with the appropriate diphosphopyridine nucleotide-linked dehydrogenase together with reduced diphosphopyridine nucleotide-cytochrome c reductase.