Membrane reconstitution in chl-r mutants of Escherichia coli K 12. VII. Purification of the soluble ATPase of supernatant extracts and kinetics of incorporation into reconstituted particles.
Membrane-bound ATPase (EC 3.6.1.3) of Escherichia coli K 12 is released in a soluble form by the mechanical treatments applied to the cells in order to break them. The purification of the soluble enzyme is described. The purified protein gives a single band in 7.5% polyacrylamide gel electrophoresis. The molecular weight is estimated to be 350 000. The enzyme is cold-labile, Mg-2+ dependent, insensitive to inhibition by N, N'-dicyclohexylcarbodiimide and specific for ATP and ADP. Membranes depleted of their ATPase activity by dilution in a buffer of low ionic strength and without Mg-2+ are able to incorporate the purified ATPase only in the presence of 2-6 mM Mg-2+. ATPase binds to particles formed by complementation between supernatant extracts of chl A and chl B mutants. There are three kinds of particles of different buoyant densities (1.10, 1.18 and 1.23); ATPase binds only to the 1.10 and 1.18 particles. The kinetics of incorporation have been studied. ATPase begins to be incorporated into the 1.10 particles after 10 min of incubation up to a maximum at 20 min: from 30 min, ATPase is incorporated only into 1.18 particles and the amount of incorporated ATPase increased in proportion with the peak of 1.18 particles. These kinetics have a hyperbolic pattern. In order to explain the mechanism of assembly involved in complementation, two hypotheses are proposed.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D002848:Chromatography, DEAE-Cellulose; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D005816:Genetic Complementation Test; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009154:Mutation; D012995:Solubility; D014461:Ultracentrifugation
D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases; D008274:Magnesium