A continuous spectrophotometric assay for phosphorylase kinase.
A continuous spectrophotometric assay for the determination of the initial rate of the phosphorylase kinase catalyzed reaction at pH 7.0 is presented. The assay incorporates two coupling enzyme systems: (a) recombinant rabbit skeletal muscle type 1 protein phosphatase catalytic subunit which dephosphorylates the phosphorylase a product of the phosphorylase kinase reaction, and (b) the system of Webb (Proc. Natl. Acad. Sci. USA 89, 4884-4887, 1992), which uses purine nucleoside phosphorylase and its chromophoric substrate, 7-methyl-6-thioguanosine, for the quantitation of the resultant inorganic phosphate. The effects of reaction components on the enzyme activities were studied. The system was standardized and validated. The continuous coupled enzyme system was used for the kinetic analysis of nonactivated phosphorylase kinase at pH 7.0. Km and kcat values of 15.36 +/- 0.2 microM (phosphorylase b monomer) and 21 +/- 1.12 s-1, respectively, were determined.
Wang
Z X
ZX
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing, People's Republic of China.
Cheng
Q
Q
Killilea
S D
SD
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D007700:Kinetics; D010749:Phosphoprotein Phosphatases / Q000032:analysis*; D010764:Phosphorylase Kinase / Q000032:analysis*; D006005:Phosphorylases / Q000737:chemistry; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry; D011817:Rabbits; D012044:Regression Analysis; D015203:Reproducibility of Results; D013053:Spectrophotometry / Q000379:methods*
D006005:Phosphorylases; D011683:Purine-Nucleoside Phosphorylase; D010764:Phosphorylase Kinase; D010749:Phosphoprotein Phosphatases