Continuous enzymatic assay for phosphorylase kinase in a monocascade enzyme system.
A turbidimetric method for continuous monitoring of the enzymatic reaction catalyzed by rabbit skeletal muscle phosphorylase kinase has been developed. The reaction mixture contained the substrates of glycogen phosphorylase a, i.e., glycogen and glucose 1-phosphate (or P(i)), in addition to the usual components of the kinase reaction. The kinetics of the cascade enzyme system were followed by the change in glycogen concentration over time, as measured by the absorbance of the reaction medium at 360 nm. The reliability of this turbidimetric method for measuring phosphorylase kinase activity was proven by comparison with a commonly used radiochemical assay. We present here a newly developed method for calculating the initial rate of phosphorylase kinase reaction in our conjugated system. We demonstrate that our procedure is applicable for investigating the hysteretic properties of phosphorylase kinase.
Livanova
N B
NB
Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia. inbio@glas.apc.org
Andreeva
I E
IE
Makeeva
V F
VF
Kurganov
B I
BI
D000818:Animals; D004789:Enzyme Activation; D006003:Glycogen / Q000032:analysis; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology; D009391:Nephelometry and Turbidimetry; D010761:Phosphorus Radioisotopes; D010764:Phosphorylase Kinase / Q000032:analysis* / Q000737:chemistry; D010762:Phosphorylase a / Q000737:chemistry; D010763:Phosphorylase b / Q000737:chemistry; D011817:Rabbits
D010761:Phosphorus Radioisotopes; D006003:Glycogen; D010762:Phosphorylase a; D010763:Phosphorylase b; D010764:Phosphorylase Kinase