Purification and properties of 5'-nucleotidase from the membrane of Vibrio costicola, a moderately halophilic bacterium.
Two different Mg2+-dependent adenosine 5'-triphosphate-hydrolyzing activities were detected in membranes of Vibrio costicola, a novel 5'-nucleotidase and an N,N'-dicyclohexylcarbodiimide-sensitive adenosine triphosphatase. The former and the latter had different requirements for Mg2+ and were selectively assayed in the membranes by using, respectively, 20 and 2 mM Mg2+. The two enzymes were extracted with a combination of Triton X-100 and octylglucoside, separated on a diethylaminoethyl cellulose column, and purified on glycerol gradients. The purified 5'-nucleotidase consisted of one major polypeptide of 70,000 daltons when analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate. The purified 5'-nucleotidase was similar in substrate specificities, divalent cation specificities, and pH profiles to the membrane-bound N,N'-dicyclohexylcarbodiimide-insensitive nucleotide-phosphohydrolyzing activity. The enzyme hydrolyzed nucleoside 5'-tri, 5'-di, and 5'-monophosphates at comparable rates. Inorganic pyrophosphate, p-nitrophenyl phosphate, glucose 6-phosphate, beta-glycerophosphate, adenosine 5'-diphosphate glucose, adenosine 3'-monophosphate, and cyclic adenosine 3',5'-monophosphate were not hydrolyzed, either im membranes or by the purified 5'-nucleotides. Actions of NaCl and KCl on the activity of the 5'-nucleotidase were studied. The enzyme was activated by both NaCl and KCl; the activation profiles however, were different for the membrane-bound and purified 5'-nucleotidase. The purified enzyme, unlike the membrane-bound enzyme, was markedly stimulated by high concentrations of NaCl (up to 3 M).
D015720:5'-Nucleotidase; D000251:Adenosine Triphosphatases / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D002848:Chromatography, DEAE-Cellulose; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004586:Electrophoresis; D009708:Nucleotidases / Q000302:isolation & purification* / Q000378:metabolism; D006180:Proton-Translocating ATPases; D013379:Substrate Specificity; D014461:Ultracentrifugation; D014733:Vibrio / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure
D004024:Dicyclohexylcarbodiimide; D009708:Nucleotidases; D015720:5'-Nucleotidase; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases