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Inside-out sealed membrane vesicles were prepared from strains of Escherichia coli engineered to be lacking in the major phospholipid of this organism, phosphatidylethanolamine (DeChavigny, A., Heacock, P. N., and Dowhan, W. (1991) J. Biol. Chem. 266, 5323-5332). The energy transducing properties, namely the ability to generate a proton gradient directed inward and to transport electrons to molecular oxygen, were compared to those of membranes isolated from wild type cells containing normal levels of phosphatidylethanolamine. Membranes from both cell types were equal in their ability to oxidize succinate and lactate as well as hydrolyze ATP with the generation of proton gradients of similar magnitude, thus establishing the structural integrity of the membrane barrier and basic functionality of the energy transducing systems in the mutant membranes. However, mutant membranes were reduced by about 80% in their type II NADH dehydrogenase-dependent oxidase activity which resulted in a reduced ability to generate a proton gradient using NADH as an energy source. Use of artificial electron acceptors indicated that the level of type II NADH dehydrogenase activity was normal. Whole chain NADH oxidase activity could be restored by addition of short chain analogs of the naturally occurring Q8, even though the level of the Q8 pool in both cell types was the same. These results suggest that the function of Q8 in linking type II NADH dehydrogenase with the terminal oxidase(s) is dependent on the phosphatidylethanolamine content of the surrounding phospholipid matrix.

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The F1-ATPase from Micrococcus lysodeikticus is isolated in the absence of exogenous nucleotides. After removing loosely bound nucleotides from the isolated enzyme by gel permeation chromatography, analysis for tightly bound nucleotides revealed in 14 experiments 0.4 +/- 0.1 mol ADP, 0.5 +/- 0.2 mol GDP, and 0.8 +/- 0.2 mol ATP per mol of F1. Incubation of the isolated enzyme with Mg2+ or Ca2+ did not alter the endogenous nucleotide composition of the enzyme, indicating that endogenous ATP is not bound to a catalytic site. Incubation of the enzyme with P(i) decreased the amount of tightly bound ADP and GDP but did not effect the ATP content. Hydrolysis of MgATP in the presence of sulfite raised the tightly bound ADP and lowered tightly bound GDP on the enzyme. In the reciprocal experiment, hydrolysis of MgGTP in the presence of sulfite raised tightly bound GDP and lowered tightly bound ADP. Turnover did not affect the content of tightly bound ATP on the enzyme. These results suggest that endogenous ADP and GDP are bound to exchangeable catalytic sites, whereas endogenous ATP is bound to noncatalytic sites which do not exchange. The presence of endogenous GDP on catalytic sites of isolated F1 suggests that the F0F1-ATP synthase of M. lysodeikticus might synthesize both GTP and ATP under physiological conditions. In support of this hypothesis, we have found that plasma membrane vesicles derived from M. lysodeikticus synthesize [32P]GTP from [32P]P(i) using malate as electron donor for oxidative phosphorylation.

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The H+-ATPase complex has been isolated from the membranes of the anaerobic bacterium Lactobacillus casei by two independent methods. 1. The crossed-immunoelectrophoresis of the 14C-labelled ATPase complex against antibodies to a highly purified soluble ATPase has been used. The subunit composition of the complex has been established by autoradiography. The soluble part of L. casei ATPase, in contrast to coupling factor F1-ATPases of aerobic bacteria, chloroplasts and mitochondria which include two kinds of large subunit (alpha and beta), consists of one kind of large subunit with a molecular mass of 43 kDa. Moreover, a minor polypeptide of 25 kDa has been found in the soluble ATPase. Factor F0 of L. casei ATPase complex consists of a 16-kDa subunit and two subunits with molecular masses less than 14 kDa. 2. A dicyclohexylcarbodiimide-sensitive ATPase complex has been isolated from L. casei membranes by treating them with a mixture of octyl glucoside and sodium cholate. The complex, purified by centrifugation on a sucrose density gradient, contains the main subunits with molecular masses of 43 kDa, 25 kDa and 16 kDa and a dicyclohexylcarbodiimide-binding subunit with a molecular mass less than 14 kDa.

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Radiation inactivation analysis gave the target sizes of 176 +/- 5 kDa and 275 +/- 33 kDa for ATPase from anaerobic Lactobacillus casei and aerobic Micrococcus luteus bacteria respectively. The values are close to the known molecular masses of the enzymes. Thus, to function the L. casei ATPase, like the F1-ATPases, requires a complete structure composed of all the enzyme subunits. L. casei ATPase is inhibited by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole owing to modification of an amino acid residue(s) with pK greater than 8.5. L. casei ATPase consists of six identical subunits and differs from alpha 3 beta 3 gamma delta epsilon-type F1-ATPases in a number of catalytic properties. Namely, ATP hydrolysis under the 'unisite' conditions proceeds at a relatively high rate suggesting the absence of cooperative interactions between the catalytic sites. Contrary to mitochondrial F1-ATPase. L. casei ATPase does not form an inactive complex with ADP. These findings imply essential differences in the operating mechanism for L. casei ATPase and F1 ATPase.

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Treatment of phosphorylating fragments of bacterial membrane from Micrococcus lysodeikticus with trypsin leads to increase ATPase activity. As a result of this treatment, the membrane fragments acquire the ability to transform the ATP energy into transmembrane difference in potential. Dithiothreitol has a similar effect to that of trypsin on the membrane fragments from M. lysodeikticus. Dicyclohexylcarbodimide inhibits ATPase of the membrane fragments of M. lysodeikticus, and also the ATPase-reaction-coupled generation of membrane potential. It has been suggested that the increased ATPase activity of membranes from M. lysodeikticus during treatment with trypsin and dithiothreitol is connected with the effect of these agents on the protein inhibitor of ATPase.

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A preparation of ATPase with a high specific activity was isolated from the membrane of M. lysodeikticus. The enzyme was studied using UV-spectroscopy and circular dichroism. The homogeneity of the protein preparation was shown by gel electrophoresis. The catalytic properties of the enzyme were studied using steady state kinetic methods. The values of Km app. and kcat were determined to be 6-10(-4) and 6 mumoles/mg/min respectively. It is shown that ADP is an effective inhibitor of the ATPase reaction, and the inhibition activity increases in the presence of an excess of Ca2+. The nature of the rate dependence of the ATPase reaction on the concentration of the substrate and on Ca-ADP corresponds to a competitive type of inhibition with binding several molecules of Ca-ADP in the active site of the enzyme.

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