JBC, Vol. 250, Issue 3, 819-825, Feb, 1975
Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase. Proteolytic and thermal inactivation of the membrane component
R. R. Fisher, S. A. Rampey, A. Sadighi and K. Fisher
Pyridine dinucleotide transhydrogenase of the Rhodospirillum rubrum
chromatophore membrane was readily resolved by a washing procedure into two
inactive components, a soluble transhydrogenase factor protein and an
insoluble membrane-bound factor. Transhydrogenation was reconstituted on
reassociation of these components. The capacity of the membrane factor to
reconstitute enzymatic activity was lost after proteolysis of soluble
transhydrogenase factor-depleted membranes with trypsin. NADP+ or NADPH,
but neither NAD+ nor NADH, stimulated by several fold the rate of
trypsin-dependent inactivation of the membrane factor. Substantial
protection of the membrane factor from proteolytic inactivation was
observed in the presence of Mg2+ ions, an inhibitor of transhydrogenation,
or when the soluble transhydrogenase factor was bound to the membrane.
Coincident with the loss of enzymatic reconstitutive capacity of the
membrane factor was a loss in the ability of the membranes to bind the
soluble transhydrogenase factor in a stable complex. The membrane component
was inactivated by preincubating soluble transhydrogenase factor-depleted
membranes at temperatures above 45 degrees. NADP+, NADPH, or Mg2+ ions, but
neither NAD+ nor NADH, protected against inactivation. These studies
indicate that (a) the binding of NADP+ or NADPH to the membrane factor
promotes a conformational alteration in the protein such that its
themostability and susceptibility to proteolysis are increased, and (b) the
inhibitory Mg2+ ion-binding site resides in the membrane component.
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