A CONTINUOUS SPECTROPHOTOMETRIC ASSAY FOR PHOSPHORYLASE-KINASE

A continuous spectrophotometric assay for the determination of the ini tial rate of the phosphorylase kinase catalyzed reaction at pH 7.0 is presented. The assay incorporates two coupling enzyme systems: (a) rec ombinant rabbit skeletal muscle type 1 protein phosphatase catalytic s ubunit which dephosphorylates the phosphorylase a product of the phosp horylase kinase reaction, and (b) the system of Webb (Proc. Natl. Acad , Sci. USA 89, 4884-4887, 1992), which uses purine nucleoside phosphor ylase and its chromophoric substrate, 7-methyl-6-thioguanosine, for th e quantitation of the resultant inorganic phosphate. The effects of re action components on the enzyme activities were studied. The system wa s standardized and validated, The continuous coupled enzyme system was used for the kinetic analysis of nonactivated phosphorylase kinase at pH 7.0. K-m and k(cat) values of 15.36 +/- 0.2 mu M (phosphorylase b monomer) and 21 +/- 1.12 s(-1), respectively, were determined. (C) 199 5 Academic Press, Inc.