ABSTRACT
Homogeneous glycogen phosphorylase from human leukocytes has been obtained. A one-step bioluminescent procedure for the enzyme activity assay has been developed. This method is based on a continuous recording of the product of the glycogen phosphorylase-catalyzed reaction using a coimmobilized multienzyme system (phosphoglucomutase, glucose-6-phosphate dehydrogenase, NADH:FMN oxidoreductase and bacterial luciferase). The method sensitivity is 10 times as high compared to earlier described methods. The Km values for glycogen (0.2 mg/ml) and phosphate (3.9 mM) at pH 7.9 were determined. AMP was shown to be the enzyme effector.
Subject(s)
Leukocytes/enzymology , Phosphorylases/blood , Animals , Catalysis , Humans , Kinetics , Muscles/enzymology , Phosphorylase a/blood , Phosphorylase a/isolation & purification , Phosphorylase a/metabolism , Phosphorylase b/blood , Phosphorylase b/isolation & purification , Phosphorylase b/metabolism , Phosphorylases/isolation & purification , Phosphorylases/metabolism , Rabbits , Substrate SpecificityABSTRACT
Co-immobilization methods have been developed for a bienzymatic system of luminescent Beneckea harveyi bacteria with formate dehydrogenase, glucose-6-phosphate dehydrogenase, and phosphoglucomutase. Bioluminescent assays have been devised for NADH, NAD, FMN, glucose 6-phosphate, and glucose 1-phosphate using the co-immobilized enzyme preparation. The lowest detection limits were in the picomole range with the bacterial extract and in the femtomole range with the partially purified enzymes, bacterial luciferase, and NADH:FMN oxidoreductase.
