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1. Membrane preparations from both uncA(-) and uncB(-) mutant strains of Escherichia coli K12, in which electron transport is uncoupled from phosphorylation, were fractionated by washing with a low-ionic-strength buffer. The fractionation gave a ;5mm-Tris wash' and a ;membrane residue' from each strain. This technique, applied to membranes from normal cells, separates the Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activity from the membrane-bound electron-transport chain and the non-energy-linked transhydrogenase activity. 2. Reconstitution of both oxidative phosphorylation and the ATP-dependent transhydrogenase activity was obtained by a combination of the ;membrane residue' from strain AN249 (uncA(-)) with the ;5mm-Tris wash' from strain AN283 (uncB(-)). 3. Valinomycin plus NH(4) (+) inhibited oxidative phosphorylation both in membranes from a normal strain of E. coli and in the reconstituted membrane system derived from the mutant strains. 4. The electron-transport-dependent transhydrogenase activity was located in the membrane residue and was de-repressed in both the mutant strains. 5. The spatial and functional relationships between the proteins specified by the uncA and uncB genes and the transhydrogenase protein are discussed.

Exponentially growing Chinese hamster cells are found to contain two major phosphokinase activities with specificity for the phosphorylation of F1 (lysine-rich) histone. These two activities, designated KI and KII, were extracted with 0.35 M NaCl and fractionated in 0.2 M NaCl by Sephadex G-200 gel filtration. KI, which is similar to the ubiquitous cyclic 3',5'-adenosine monophosphate (cAMP)-dependent phosphokinase, differs from KII by several criteria. KII is mol wt 90,000, cAMP independent, rapidly turned over in vivo, low K(m) for ATP, and phosphorylates F1 histone at several unique sites. Comparative examination of metaphase-arrested (M) and counterpart interphase (I) cells for these two activities reveals that KII is responsible for the overall high activity in M-arrested cells. Pulse labeling of cells with (32)P during traverse of the G(2)-M phase of the cell cycle reveals an in vivo tryptic-phosphopeptide pattern in whole unfractionated F1 which is unique to M cells. Seven major phosphopeptides derived by in vitro phosphorylation of F1 with the KII enzyme correspond to these M cell-specific phosphorylation sites observed in vivo. It is suggested that KII activity predominates during the G(2)-M transition and that F1 is its natural in vivo substrate.