39] Arginine kinase (arthropod muscle)

Abstract

Publisher Summary This chapter describes the assay, purification, and properties of arginine kinase. The production of adenosine diphosphate (ADP) in the reaction is measured with a coupled assay system using phosphoenolpyruvate, pyruvate kinase, nicotinamide adenine dinucleotide phosphate (NADH), and lactic dehydrogenase. The oxidation of NADH is followed spectrophotometrically at 340mμ. Other assay methods for arginine kinase include measurement of the reverse reaction by following the formation of free arginine from phosphoarginine colorimetrically and the measurement of phosphoarginine production as phosphate liberated by a 90-second hydrolysis. The enzyme is stable at 4° for six months as a crystalline suspension in ammonium sulfate. However, after further storage, the crystals become inactive. The enzyme recovers full activity if the crystals are diluted with a buffer containing mercaptoethanol and allowed to stand at room temperature for 10–15 minutes. The enzyme is inhibited by sulfhydryl reagents, such as iodoacetamide and p-mercuribenzoate. L-Arginine completely protects the enzyme from the action of iodoacetamide while ADP and adenosine triphosphate (ATP) give partial protection. Arginine kinase has also been purified from the muscle of the Hungarian marsh crab, European lobster, sea crayfish, hermit crab, and the blue crab. Ammonium sulfate fractionation proves to be a useful step in these procedures, although the exact concentration of ammonium sulfate required to precipitate the enzymes varies from species to species.