Abstract
Publisher Summary This chapter describes the purification procedure of adenosine monophosphate (AMP) (dAMP) kinase from human erythrocytes. AMP (dAMP) kinase or adenylate kinase phosphotransferase, exists in the human erythrocyte in multiple molecular forms and comprises about 0.005% of the cell protein. Although most of the enzyme is soluble, significant activity is retained with the stroma at low ionic strength. The stroma-associated enzyme is released in 0.3 M KCl and does not differ from the soluble enzyme forms. Adenylate kinase activity can be determined either in the forward or back reaction by measuring adenosine triphosphate (ATP) or adenosine diphosphate (ADP) production or consumption. Rates of ATP or ADP production are most conveniently determined by one-step combined assays at high sensitivity by fluorometric measurements involving enzyme-coupled nicotinamide adenine dinucleotide phosphate (NADP) reduction or NADH oxidation, respectively. Overall yield is usually between 10–20% with most of the loss attributable to instability associated with fractionation sequences requiring low ionic strengths.
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