ENZYMATIC PHOSPHORYLATION OF ADENOSINE AND 2,6-DIAMINOPURINE RIBOSIDE

Abstract

PHOSPHORYLATION(5) 2-Aminoadenosine + 3 phosphopyruvate -+ 2-aminoATP + 3 pyruvate Of seventeen nucleosides tested, the enzyme reacts only with adenosine and 2-aminoadenosine and the transfer of phosphate is limited to the 5' carbon of the nucleoside. Methods Materials-ATPand ADP were purified from commercial products (Sigma) by ion exchange chromatography (5).Adenosine (recrystallized twice from water), guanosine, uridine, cytidine, yeast adenylic acid, and Dribose were products of the Nutritional Biochemicals Corporation.Desoxyadenosine was prepared from desoxyadenylic acid1 and inosine from inosinic acid by quantitative removal of the phosphate with lyophilized snake venom (Crotalus adamanteus), which contains a 5'-nucleotidase ( 6).2,6-Diaminopurine riboside and other synthetic nucleosides were generously provided by Dr. J. Davoll and Dr. G. B. Brown.Reduced diphosphopyridine nucleotide (DPNHZ) (purity 0.68) was prepared by Ohlmeyer's method (7).Triphosphopyridine nucleotide (TPN) (purity 0.86) was obtained by ion exchange chromatography of a crude liver fraction.2Phosphopyruvic acid was prepared by Ohlmeyer's modification of Kiessling's method and kindly supplied to us by Dr. P. Ohlmeyer.Pyruvate phosphokinase, the enzyme catalyzing the transfer of phosphate from phosphopyruvate to ADP to form pyruvate and ATP, was purified from rabbit muscle by a modification of the procedure described by Kubowitz and Ott for human muscle (8).(a) Assay-The incubation mixture (3.0 cc.) for enzyme assay contained 0.1 cc. of phosphopyruvate (0.005 M), 0.1 cc. of MgCL (0.1 M), 0.1 cc. of DPNH2 (0.002 M), 0.2 cc. of ADP (0.0015 M), 0.2 cc. of phosphate buffer (0.5 M, pH 7.0), and 0.02 cc. of lactic dehydrogenase (final fraction diluted 1:40).A unit of enzyme activity was defined as the amount producing 1 PM of pyruvate (determined as the oxidation of 1 I.IM of DPNH2 at 340 mr) per minute at Zl-23", corrected when necessary for a blank value owing to the contamination of the lactic dehydrogenase preparation by this activity.(6) Purification-Ground rabbit muscle (631 gm.) was stirred at 2" for 30 minutes with 3 volumes of 0.1 M KHzPOd and then centrifuged.The turbid extract (1730 cc., 30 units per cc., 3.3 units per mg. of protein) was fractionated with acetone (8).The acetone fraction (Acetone I, 71 cc., 800 units per cc., 11.5 units per mg. of protein) was stable for months when stored at -15'.(Fractional precipitation with nucleic acid at this point, as in the method of Kubowitz and Ott, was not successful.)