Summary
Plasma contains enzymes capable of dephosphorylating ADP, ATP and AMP (adenosine di-, tri- and monophosphate). In platelet-rich plasma these enzymes are important for the regulation of the levels of (platelet-aggregating) ADP and (aggregation-inhibitory) adenosine.
Plasma ADPase and ATPase were studied at 1 (µM substrate concentration using an isotope technique. Both enzymes were precipitated from plasma at 45-65% saturation with (NH4)2S04 and emerged together by gel filtration on Sephadex G-200 and from DEAE-Sephadex (0.12-0.20 M Cl-, pH 8.2). In combination these procedures gave 1,500-1,800 times purification of ADPase relative to plasma. The purest fraction contained ATPase, ADPase and AMPase in a 0.17:1.00:2.92 proportion, quite different from their 5.34:1.00:5.34 proportion in plasma. Adenosine deaminase and adenylate kinase were not present in the purest fraction, whereas nucleoside diphosphokinase appeared to be present.
The purified ADPase was stimulated by low concentrations of Mg2+ and Mn2+, whereas high concentrations were inhibitory. This inhibition could not be explained by an increase in the ionic strength. Ca2+ and Zn2+ were inhibitory at all concentrations used (0-3 mM). Lineweaver-Burke plots were linear for both ADPase and ATPase in the 0−4 x 10-5 M substrate range, and both enzymes had Km = 1.1 x 10−5 M. Increase of the substrate concentration above 4 x 10−5M gave deviation from MichaelisMenten kinetics, and Eadie-Hofstee plots indicated the presence of “high-Km” ADPase and ATPase. The latter enzymes were not studied.
Déphosphorylation of 3H-ADP by purified “low-Km” ADPase was reduced by nonradioactive diphosphates of guanosine, inosine, cytidine and uridine in the same way as when nonradioactive ADP was used. Nonradioactive AMP also reduced dephosphorylation of 3H-ADP, whereas nonradioactive ATP did not.
Cyanide, cysteine and tartrate inhibited “low-Km” ADPase whereas p-chloromercuribenzoate, p-chloromercuribenzoesulphonate and N-ethylmaleimide had no effect. EDTA inhibited the enzyme activity in a way that could not be abolished by excess Mg2+. Purified plasma “low-Km” ADPase thus appears to be an unspecific enzyme, as one and the same active site does not seem to distinguish between the base moiety of nucleoside diphosphates, and catalyzes hydrolysis of phosphate esters as well as pyrophosphate bonds. The relation between plasma ADPase and ATPase remains unclear.
