Inhibition of Escherichia coli H+-ATPase by venturicidin, oligomycin and ossamycin

The antibiotics venturicidin, oligomycin and ossamycin were investigated as potential inhibitors of the Escherichia coli H+-ATPase. It was found that venturicidin strongly inhibited ATP-driven proton transport and ATP hydrolysis, while oligomycin weakly inhibited these functions. Inhibition of the H+-ATPase by venturicidin and oligomycin was correlated with inhibition of F0-mediate proton transport. Both inhibitors were found to interfere with the covalent reaction between dicyclohexyl[14C]carbodiimide and the F0 subunit c (uncE protein). Ossamycin had no direct inhibitory effect on E. coli F0 or F1; rather, it was found to uncouple ATP hydrolysis from proton transport.     

Studies of leukotriene B4-specific binding and function in rat polymorphonuclear leukocytes: absence of a chemotactic response

Rat PMN isolated from peripheral blood show a small amount of high-affinity (specific) binding of [3H]-LTB4 at nanomolar concentrations. This binding is reversible and has a stereospecificity similar to rat PMN aggregation in response to several LTB4 analogs. This population of binding sites shares many characteristics with a population of high-affinity binding sites in human PMN; however, human PMN bind a significantly greater amount of [3H]-LTB4 to a second population of specific binding sites that is not present in rat PMN. The aggregation responses of human and rat peripheral blood PMN to LTB4 are similar in magnitude and specificity, but unlike human PMN, LTB4 fails to elicit a chemotactic response in rat PMN at concentrations from 10(-10) M to 10(-6) M. Rat PMN also fail to metabolize exogenous LTB4 when compared with human PMN. These data suggest that different PMN functions, such as chemotaxis and aggregation, may involve different classes of specific receptors. The finding that rat PMN do not exhibit chemotaxis to LTB4 calls for a reevaluation of the relevance to inflammation in humans of studies of inflammation performed in rat models.     

Catalytic sites ofEscherichia coli F1-ATPase

The catalytic site ofEscherichia coli F₁-ATPase is reviewed in terms of structure and function. Structural prediction, biochemical analyses, and mutagenesis experiments suggest that the catalytic site is formed primarily by residues 137–335 of -subunit. Subdomains of the site involved in phosphate-bond cleavage/synthesis and adenine-ring binding are discussed. Ambiguities inherent in steady-state catalytic measurements due to catalytic site cooperativity are discussed, and the advantages of pre-steady-state (unisite) techniques are emphasized. The emergence of a single high-affinity catalytic site occurs as a result of F₁-oligomer assembly. Measurements of unisite catalysis rate and equilibrium constants, and their modulation by varied pH, dimethylsulfoxide, and mutations, are described and conclusions regarding the nature of the high-affinity catalytic site and mechanism of catalysis are presented.     

ATP hydrolysis by multidrug-resistance protein from Chinese hamster ovary cells

ATPase activity of multidrug-resistance protein (P-glycoprotein, Pgp) from Chinese hamster ovary cells was studied. Catalytic characteristics were established for Pgp both in its natural plasma membrane environment and in purified reconstituted protein. Generally the two preparations of Pgp behaved similarly, and demonstrated low affinity for MgATP, low nucleotide specificity, preference for Mg-nucleotide, and pH optimum near 7.5. A high-affinity binding site involved in catalysis was not apparent. Effective covalent inactivators were NBD-C1, NEM, 8-azido-ATP, and 2-azido-ATP. DCCD, FITC, and pyridoxal phosphate were only weakly inhibitory. Lipid composition was found to affect the degree of drug stimulation of ATPase in purified reconstituted Pgp, suggesting that the lipid environment affects coupling between drug-binding and catalytic sites, and that Pgp expressed in different tissues could show different functional characteristics.     

Value of thallium-201 imaging in detecting adverse cardiac events after myocardial infarction and thrombolysis: a follow up of 100 consecutive patients.

OBJECTIVE: To determine the prognostic role of thallium-201 imaging compared with that of exercise electrocardiography in patients with acute myocardial infarction treated by thrombolysis. DESIGN: Patients who remained free of adverse cardiac events six weeks after myocardial infarction had stress and rest 201TI imaging and exercise electrocardiography and were followed up for 8-32 months. Adverse cardiac events (death, reinfarction, unstable angina, and congestive heart failure) were documented. SETTING: Large district general hospital, Middlesex. SUBJECTS: 100 consecutive male and female patients who were stable six weeks after thrombolysis for myocardial infarction. MAIN OUTCOME MEASURES: Prediction of occurrence of adverse cardiac events after myocardial infarction by exercise cardiography and 201TI myocardial perfusion imaging. RESULTS: Reversible ischaemia on 201TI imaging predicted adverse cardiac events in 33 out of 37 patients with such events during follow up (hazard ratio 8.1 (95% confidence interval 2.7 to 23.8), P < 0.001). Exercise electrocardiography showed reversible ischaemia in 33 patients, of whom 13 had subsequent events, and failed to predict events in 24 patients (hazard ratio 1.1 (0.56 to 2.2), P = 0.8). CONCLUSION: 201TI imaging is a sensitive predictor of subsequent adverse cardiac events in patients who have received thrombolysis after acute myocardial infarction, whereas exercise electrocardiography fails to predict outcome.     

Dual coenzyme activities of high-Km aldehyde dehydrogenase from rat liver mitochondria

Various kinetic approaches were carried out to investigate kinetic attributes for the dual coenzyme activities of mitochondrial aldehyde dehydrogenase from rat liver. The enzyme catalyses NAD(+)- and NADP(+)-dependent oxidations of ethanal by an ordered bi-bi mechanism with NAD(P)+ as the first reactant bound and NAD(P)H as the last product released. The two coenzymes presumably interact with the kinetically identical site. NAD+ forms the dynamic binary complex with the enzyme, while the enzyme-NAD(P)H complex formation is associated with conformation change(s). A stopped-flow burst of NAD(P)H formation, followed by a slower steady-state turnover, suggests that either the deacylation or the release of NAD(P)H is rate limiting. Although NADP+ is reduced by a faster burst rate, NAD+ is slightly favored as the coenzyme by virtue of its marginally faster turnover rate.     

Cytoplasmic oestradiol-binding sites and their relationship to oestradiol content in the endometrium of cattle.

Twenty-three cows and heifers were killed at known times during the oestrous cycle or during the first 35 days of pregnancy. Duplicate cytosol preparations were made from the endometrium of each uterine horn and both the binding-site concentration and the oestradiol level were determined for each sample. During the cycle, the oestradiol concentration was only 0-2 to 1-7% of the concentration of binding sites which varied considerably between Days 19 and 5 (47,665 +/- 7538 sites/cell, mean +/- S.E.M.) and Days 6 to 18 (7060 +/- 444 sites/cell). The concentration of binding sites remained low in pregnant animals (6689 +/- 492), although the oestradiol concentration was high about 20 days after insemination, resulting in almost 14% of the sites being occupied. Five inseminated animals in which no conceptus was found when they were slaughtered 19 to 22 days later had low concentrations of binding sites but two animals had high levels of oestradiol with 13% and 15%, respectively, of their cytoplasmic sites being occupied. It is suggested that these animals had recently lost their conceptuses. Two ovariectomized cows and one non-cyclic animal contained high concentrations of oestradiol-binding sites in the uterine cytoplasm. No significant difference was found between the uterine horn adjacent to the ovary with the CL and the contralateral horn in early pregnancy or during the luteal phase of the oestrous cycle. An animal killed 1 week after parturition contained fourfold more sites in the involuting horn than in the opposite horn. It is suggested that progesterone plays a major role in regulating oestrogen-induced replacement of cytoplasmic binding sites.     

On the relationship between the oligomycin-sensitivity conferring protein and other mitochondrial coupling factors

1. The oligomycin-sensitivity conferring protein (OSCP) has been further investigated and by modifying the purification procedure, the protein has been obtained free of minor contaminants. The single protein has the properties of both an energy-transfer factor and an oligomycin-sensitivity conferring factor when assayed with suitable depleted submitochondrial particles.
2. Several physical and chemical properties of OSCP have been examined. The protein has an isoelectric point of around 9·3 and is not inhibited by sulfhydryl reagents such as iodoacetate, iodoacetamide or parachloromeruriphenyl sulphonate, or by high concentrations of iodine.
3. Knowledge of those and other physical properties of OSCP allows comparison of OSCP with several other previously described coupling factors.

     

Catalytic properties of the F1-adenosine triphosphatase from Escherichia coli K-12 and its genetic variants as revealed by 18O exchanges

We have examined intermediate Pi-water oxygen exchange during [gamma-18O]ATP hydrolysis by the F1 adenosine triphosphatase from Escherichia coli K-12. Water oxygen incorporation into each Pi released was increased as ATP concentration was lowered as observed previously for the same reaction catalyzed by the enzyme from eukaryotic sources. Heterogeneous distributions of 18O in product Pi were produced by coexisting epsilon subunit-replete and epsilon subunit-depleted enzyme molecules. The epsilon-replete enzyme showed a much higher probability for oxygen exchange. These data imply that the epsilon subunit inhibits net ATP hydrolysis by imposing conformational constraints which reduce the cooperative conformational interactions that promote ADP and Pi release. Four enzyme variants altered in alpha or beta subunit structure with reduced net hydrolytic activity showed sharply increased oxygen exchange during ATP hydrolysis. Heterogeneity was apparent in the 18O distribution of the product Pi, however. That behavior could reflect hindered conformational interactions and/or increased affinity of the alpha 3 beta 3 gamma delta complex for the epsilon subunit. In contrast, enzyme from mutant uncA401 showed very little oxygen exchange accompanying hydrolysis of 20 microM ATP. This is the only enzyme so far reported with this unusual property. Its rate limitation appears to be in the hydrolytic rather than the product release step of the catalytic sequence.