Rye grass 3′-nucleotidase has been purified to apparent homogeneity on Sephadex A-25 and CM-cellulose columns and shown to hydrolyze 2′-O-methyladenosine 3′-monophohate and 2′-deoxyadenosine 3′-monophosphate 35.8 and 542 times more slowly than the normal substrate (3′-AMP), verifying the importance of the 2′-β-OH group of the substrate in the overall hydrolysis process. Although neither was hydrolyzed as rapidly as 3′-AMP, both the 2′-O-methyl and 2′-deoxy analogs acted as competitive inhibitors of the hydrolysis of 3′-AMP (Km= 0.12 mM), with apparent Ki's of 0.39 and 0.51 mM, respectively. In order to determine the possible susceptibility of naturally occurring ribonucleoside 3′-diphosphates, such as guanosine tetraphosphate (ppGpp), to 3′-phosphohydrolase activities, the 3′-nucleotidase was also employed in the attempted pyrophosphorolysis of adenosine 3′-diphosphate and guanosine tetraphosphate. Neither adenosine 3′-diphosphate nor guanosine tetraphosphate was degraded at a significant rate by the nucleotidase, relative to the normal substrate.
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