Purification and properties of NADP pyrophosphatase from Proteus vulgaris

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Abstract 1. 1. NADP pyrophosphatase was purified about 700-fold from Proteus vulgaris by a procedure consisting of solubilization by detergents, fractionation by ammonium sulfate and cold ethanol, and column chromatography using DEAE-cellulose, hydroxylapatite and phosphocellulose. 2. 2. The purified enzyme was slightly activated by Mg 2+ and Mn 2+ and inhibited by Co 2+ and Zn 2+ . When the enzyme was treated with 5 mM EDTA and dialyzed against 10 mM Tris-HCl buffer, pH 7.0, the activity almost disappeared and the enzyme was reactivated strongly by Co 2+ and slightly by Mn 2+ . 3. 3. The optimum pH is 7.0. K m value for NADP + in Tris-HCl buffer at pH 7.0 was about 25 μM. 4. 4. Both NADP + and NADPH were cleaved rapidly at almost the same rate. NADH was also cleaved at half the rate of NADP + cleavage, while NAD + was cleaved at only 7% of this rate. ADP and UDP were also cleaved to release inorganic orthophosphate. 5. 5. NADP + cleavage by the enzyme was competitively inhibited by CoA, FAD, ATP, GTP, UTP and CTP. Among them CoA, GTP and CTP were very effective inhibitors. RNA, DNA and denatured DNA also inhibited NADP pyrophosphatase activity as well as boiled extracts of P. vulgaris , but these inhibitors were not competitive with NADP + .

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