ON THE PREPARATION OF ADENOSINE TRIPHOSPHATE-γ-P32* AND ADENOSINE DIPHOSPHATE-β-P32

Abstract

ATP-gamma-P32 is synthesized by incubating (pH 7.4, 30[degree]C, 30 min.) a mixture of Li carbamyl phos-phate-p32; adenosine diphosphate (ADP) and carbamyl phosphate kinase purified from Streptococcus faecalis (strain 8022 ATCC) in the presence of Mg2+, followed by Dowex 1 column chromatography at pH 8.0 by using 0.02 [image] NaCl-0.01 NHC1 as an eluant (yield 72%). ATP-beta-p32 is synthesized upon incubation of ADP-P32, crystalline rabbit muscle py-ruvate kinase and phosphoenol pyruvate in the presence of Mg2+, K+, F" and ethylenediamine tetraacetate at pH 7.5, 37[degree] C for 30 min. which followed by the above column chromatographic procedure (yield 70%). Secondary formation of ATP-beta-P32 from ATP-gamma-P32 by contaminating adenylate kinase can be prevented by use of a relatively high amount of ADP to carbamyl phosphate (80 to 14 micromoles). Ratios of beta- and gamma-P32 are determined as 1 to 210 and 70 to 1 with ATP-gamma-p32 and ATP-beta-p32; respectively.