Enzymatic Properties of 5′-AMP Deaminase in Platelet Lysates

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Abstract
Metabolic ATP is converted to hypoxanthine during platelet secretion, metabolic shock and slowly in the resting state. This conversion involves deamination of 5′-AMP to 5′-IMP which has been studied by incubating 5′-AMP (3–40 μM) with human platelet lysates and quantifying the metabolites formed. Deamination occurred only when EDTA was present or endogenous ATP absent, showing that 5’-AMP deaminase (EC 3.5.4.6) was the only enzyme attacking AMP. EDTA stimulated AMP deamination, probably by removal of endogenous heavy metals which were powerful inhibitors of deamination. The experiments were therefore performed with EDTA. 5′-AMP deaminase was soluble, and had optimal activity at pH 6.5; however, the rate of AMP deamination was highly dependent on the type of buffer used. Km was 0.92 × 10–3M and Vmax was 0.26 μmoles/min x mg protein. The deamination required presence of monovalent cation with Li+ = Na+ >K+ >NH4 +, a sequence distinctly different from that seen with erythrocyte and muscle deaminase. 50 mM Na+ gave maximal activity with [math]. Tris+ and K+ were competitive inhibitors with respect to Na+, a feature not reported for the enzyme from other tissues. Above 60 mM K+ there was no effect by Na+ (0–100 mM). PPi, GTP, ITP, CTP and UTP gave greater inhibition than Pi and phospho-esters. Unlike AMP deaminase from other tissues, the platelet enzyme was insensitive to fluoride. ATP counteracted Pi and GTP inhibition and activated with or without Na+; ATP-and Na+-activation were additive. The activity increased as the adenylate energy charge was lowered, but was linearly related to the AMP concentration, thus in sharp contrast to deaminase from the liver. It is suggested that in the intact platelet AMP deamination is regulated chiefly through variations in the AMP level and not likely through variations in the ATP/Pi ratio. * AMP, ADP and ATP = 5′-adenosine monophosphate, diphosphate and triphosphate, respectively. IMP and ITP = 5′-inosine mono- and triphosphate. GDP and GTP = 5′-guanosine diphosphate and -triphosphate. UTP = 5′-uridine triphosphate; CTP = 5′-cytidine triphosphate. Pi = inorganic orthophosphate; PPi = inorganic pyrophosphate; EDTA = ethylene diaminotetraacetate.