Unisite ATP hydrolysis by soluble Rhodospirillum rubrum F1-ATPase is accelerated by Ca2+

At saturating concentrations of ATP, soluble F1 from the Rhodospirillum rubrum (RF1) exhibits a higher rate of hydrolysis with Ca2+ than with Mg2+. The mechanisms involved in the expression of a higher catalytic activity with Ca2+ were explored by measuring the ATPase activity of RF1 at substiochiometric concentrations of ATP (unisite conditions). At a ratio of 0.25 [gamma-32P]ATP per RF1, the enzyme exhibited a 50 times higher hydrolytic rate with Ca2+ than with Mg2+. The rate of [gamma-32P]ATP binding to RF1 was in the same range with the two divalent metal ions. Centrifugation-filtration of RF1 exposed to substoichiometric [gamma-32P]ATP concentrations and Mg2+ through Sephadex columns yielded an enzyme that contained [gamma-32P]ATP and [32P]phosphate in a stoichiometry that was close to one. In the presence of Ca2+, the eluted enzyme did not contain [gamma-32P]ATP nor [32P]phosphate. This indicated that the rate of product release was faster with Ca2+ than with Mg2+. It was also observed that the ratio of multisite to unisite hydrolysis rates was of similar magnitude with both divalent cations. This suggests that they do not affect differently the cooperative mechanisms that may exist between catalytic sites. In consequence, the higher ATPase activity of RF1 in presence of Ca2+ strongly suggests that the retention time of products is decreased in the presence of this cation. Copyright 1998 Elsevier Science B.V.

Reverse Micelle Systems Composed of Water, Triton X-100, and Phospholipids in Organic Solvents

Catalysis, stability, and thermostability of yeast hexokinase were determined in the microenvironments of two organic solvent/Triton X-100/phospholipids systems. In the abscence of enzyme, phase diagrams showed two transparent/turbid transitions, and reverse micelles were only observed in the second region of transparency (T2), where particle size as a function of water content shows a minima (see previous paper in this issue). In the present work, enzyme activity was detected throughout the four regions of the phase diagrams of these systems. Catalysis increased with water content; nevertheless, the maximum activities that were reached in the toluene and propylbenzene systems were 30 and 1.6%, respectively, of the activity in all aqueous media. Because in the T2 region in the propylbenzene system, micelles are much smaller than in toluene (see preceding paper), it would appear that expression of catalysis depends on the size of the micelles. However, a comparison of the dimensions of hexokinase and those of reverse micelles in the T2 region, suggests that in this region, hexokinase entrapment increases the inner volume of the micelle. High enzyme thermostability was only observed in the first transparent region (T1) of the system that contained phospholipids. In this region, hexokinase induced the formation of reverse micelles from dispersed surfactant monomers. There is a striking similarity in the dimensions of hexokinase entrapped in reverse micelles as determined by dynamic light scattering measurements in the T1 region with those of hexokinase as obtained from X ray diffraction studies of the enzyme in a crystalline environment. This suggest that high thermostability, and low catalytic rates result from restrictions in mobility imposed by a low water environment. Copyright 1998 Academic Press. Copyright 1998Academic Press

A Column Centrifugation Method for the Reconstitution in Liposomes of the Mitochondrial F0F1 ATP Synthase/ATPase

A method for reconstitution of membrane proteins into unilamellar liposomes is described. The model enzyme was the F0F1 ATP synthase from mitochondria when in complex or free from its inhibitor protein. The enzymes were first solubilized with either of two detergents, i.e., n-dodecyl-beta-D maltoside or lauryldimethylamine oxide. After solubilization, the enzymes were passed through a column of Sepharose-AH using an ADP/sodium cholate selective elution buffer. The enzymes recovered from the column were subsequently passed through a centrifuge column of Sephadex G-50 fine. The eluate contained liposomes in which the F0F1 complex (with and without inhibitor protein) had been reconstituted. The reconstituted enzymes were capable of hydrolyzing ATP with formation of electrochemical H+ gradients. They also catalyzed the ATP-Pi exchange reactions. Thus the F0F1 complex which is formed by 18 subunits can be rapidly reconstituted into liposomes in a fully functional state. Moreover the data show that the interactions between the enzyme and its inhibitor protein are not perturbed in the reconstitution procedure.