Mg2+ adenosine triphosphatase from cell envelopes of free-living and bacteroid forms of Rhizobium lupini strain NZP2257.
A procedure for the purification of Mg2+ adenosine triphosphatase (EC 3.6.1.3) from free-living and bacteroid forms of Rhizobium lupini NZP2257 is described. The enzyme was released from cell envelopes using Triton X-100 and purified by gel filtration on Ultrogel AcA 22, followed by preparative gel electrophoresis on agarose. The purified ATPase had a molecular weight of about 355,000, as determined from sedimentation coefficients on sucrose gradients. Kinetic analysis of activity of the enzyme from free-living R. lupini showed it to be typical of F1-type Mg2+ ATPases from bacteria. Mg stimulated activity at pH 7.0, although, when present as the free ion, Mg caused non-competitive inhibition (K1 = 1.5 mM). Maximum activity with ATP occurred over a broad pH range from 6.0 to 10.5. ATP, GTP, and UTP, and, to a much lesser degree, CTP and ADP, were hydrolyzed by the enzyme. Hydrolysis of glucose 6-phosphate was not observed. The Km for ATP at pH 7.0 was 0.67 and for GTP 1.4 mM. ATPase activity was inhibited by ADP, and competitive with ATP (KI = 0.18 mM). Azide also caused inhibition but fluoride and DCCD had no effect. Native and sodium dodecyl sulfate-gel electrophoretic analysis revealed no obvious differences between ATPases from free-living and bacteroid forms of R. lupini.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D017301:Ca(2+) Mg(2+)-ATPase; D002462:Cell Membrane / Q000201:enzymology; D002473:Cell Wall / Q000201:enzymology; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D012231:Rhizobium / Q000201:enzymology*; D013379:Substrate Specificity
D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase