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Oligomycin interacts with the Na,K-ATPase by increasing the apparent Na+ affinity in the non-phosphorylated state of the enzyme. This property is used to estimate rate constants attributed to oligomycin binding and dissociation reactions with Na,K-ATPase. The rate constants are determined indirectly, employing stop-flow fluorimetry of eosin, the fluorescence of which is a marker for the E1 state of the enzyme, i.e. for Na+ binding. The second-order rate constants derived for oligomycin binding are in the range (6-12).10(4) M-1 s-1 at 6 degrees C for both shark rectal gland and pig kidney enzyme. Rate constants for dissociation of the enzyme-oligomycin complex are about 0.05 s-1 at 6 degrees C. The slow rates of binding and dissociation suggest that oligomycin acts from within the membrane lipid phase rather than from the aqueous phase. The dissociation constant at 6 degrees C for the enzyme-oligomycin complex can be calculated to be about 1 microM for shark enzyme and about 2 microM for kidney enzyme, at pH 7.0 in 2 mM NaCl.
Studies to establish the structure/function relationships of oligomycin sensitivity-conferring protein (OSCP) of mitochondrial ATP synthase were carried out using genetic engineering and biochemical approaches. A full-length cDNA clone encoding OSCP was isolated from a bovine heart cDNA library, and the mature form of OSCP was expressed in Escherichia coli using plasmid expression vector pKP1500. Recombinant OSCP was found to accumulate in the cytoplasmic inclusion bodies, by virtue of which the recombinant protein could be purified to greater than 85% purity by simple low speed centrifugation of cell lysates. Recombinant OSCP was found to be indistinguishable from OSCP isolated from mitochondria with respect to (i) apparent molecular mass on sodium dodecyl sulfate gel electrophoresis, (ii) immunological reactivity to anti-OSCP serum, (iii) biological activity in restoring oligomycin-sensitive ATPase and Pi-ATP exchange activities to OSCP-depleted ATP synthase complexes, and (iv) insensitivity of the biological activity to sulfhydryl-directed alkylating reagents. The amino-terminal sequence of the recombinant protein revealed that the initiating methionine was not removed by E. coli, although that apparently did not affect protein folding or its biological activity. Data on nested deletion mutations starting from the carboxyl terminus in OSCP demonstrated that, in each instance, the mutant form was expressed and the protein product was sequestered in cytoplasmic inclusion bodies, similar to the wild-type form. However, none of the variants, including the one in which only the last 10 residues were deleted, was able to restore cold-stable oligomycin-sensitive ATPase or Pi-ATP exchange activity in OSCP-depleted complexes. Taken together, these data suggest that amino acid residues 181-190 (or some of the residues in this region) in the OSCP sequence may be important for OSCP-F1 interactions.
Oligomycin F, a new homologue of oligomycin A (1), was isolated from a Streptomyces species and structure 2 was deduced by NMR methods. Compound 2 is highly active against plant pathogenic fungi and is an extremely potent suppressive agent for various immunological systems.
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1. Oligomycin and dicyclohexylcarbodiimide-sensitive ATPase was isolated from beef-heart mitochondria and treated with 3.5 M NaBr in order to remove F1. The residue, called F0, was found to consist of seven components. Five of these are stained by Coomassie blue after dodecylsulfate-polyacrylamide-gel electrophoresis. Two of them correspond to the oligomycin-sensitivity-conferring protein and coupling factor F6, with apparent molecular weights of 21,000 and 9,400, respectively. Three additional polypeptides of molecular weights 23,000, 10,500 and 8,600 were not identified with known proteins. Two components not stained with Coomassie blue were detected by autoradiography of the gels of F0 preincubated with [14C]dicyclohexylcarbodiimide. These two components probably represent monomeric and oligomeric forms of the dicyclohexylcarbodiimide-binding protein. 2. F0 induced an oligomycin and dicyclohexylcarbodiimide-sensitive enhancement of K+ + valinomycin-driven proton translocation across the membrane of artificial phospholipid vesicles. 3. The interaction of F0 with purified, soluble beef heart F1 was investigated. F0 was capable of binding F1 and conferring oligomycin and dicyclohexylcarbodiimide sensitivity and cold stability on its ATPase activity. Furthermore F0 was found to diminish the specific activity of F1-ATPase. A comparison of these effects at varying F0/F1 ratios shows that F0 binds F1 in both an oligomycin-sensitive and an oligomycin-insensitive manner, and that both types of binding involve a conferral of cold stability and a decrease in specific activity. High F0/F1 ratios favoured in oligomycin-sensitive type of binding, indicating that F1 binds preferentially to oligomycin-sensitivity-conferring sites. Treatment of ATPase complex with trypsin resulted in an F0 with a decreased proportion of oligomycin-sensitivity-conferring binding sites and a diminished ability to lower the specific activity an cold lability of F1. 4. Reconstitution of F0 treated with trypsin and F1, oligomycin-sensitivity-conferring protein and F6 showed that at a constant amount of F1 bound, both oligomycin-sensitivity-conferring protein and F6 increased the oligomycin sensitivity of ATPase activity. It was therefore concluded that both of these coupling factors are involved in the conferral of oligomycin sensitivity. 5. The effect of the order of addition of F1, oligomycin-sensitivity-conferring protein and F6 to F0 on the reconstitution of oligomycin-sensitive ATPase activity, and of F1 and oligomycin-sensitivity-conferring protein to submitochondrial particles on the reconstitution of respiratory control, was investigated. The highest values of oligomycin sensitivity and respiratory control were obtained when F1 was added as the first component, indicating that F1 plays a directing role in the organisation of the components.
The inheritance of oligomycin resistance was studied in three mouse L-cell mutants, OLI 2, OLI 4, and OLI 14. All three mutants had previously been shown to have oligomycin-resistant mitochondrial ATPase activity. In addition, OLI 14 has DCCD-resistant mitochondrial ATPase activity and an altered DCCD-binding protein. Oligomycin-resistant cells were enucleated and fused with oligomycin-sensitive cells under a variety of selective regimes designed to allow growth of oligomycin-resistant cybrids. No transfer of oligomycin resistance via the cytoplasm of OLI 2, OLI 4, or OLI 14 was detected. In contrast, oligomycin resistance was transferred with the karyoplasts of OLI 14 in karyoplast-cell fusions. Fusions between OLI 14 cells and oligomycin-sensitive cells also produced oligomycin-resistant hybrids. Transfer of oligomycin resistance in the karyoplast-cell and cell-cell fusions were demonstrated at the level of the mitochondrial ATPase. These results indicate that oligomycin resistance in OLI 14 is most likely under nuclear control. Furthermore, nuclear inheritance of oligomycin resistance in a mutant with a modified DCCD-binding protein suggests that the gene for the DCCD-binding protein is encoded in the nucleus of mammalian cells.
The effects on phosphoenzyme (E-P) formation of ligands which activate Electrophorus (Na,K)-ATPase were investigated in the presence of oligomycin. When the enzyme was allowed to bind oligomycin in the presence of NaCl and MgCl2, subsequent addition of ATP plus KCl produced a monoexponential time course of E-P formation with a rate of 56 s-1, similar to the rate obtained in the uninhibited enzyme phosphorylated by ATP in the absence of KCl. Pi liberation under these conditions was slow and showed no initial burst phase, consistent with the inhibitory effect oligomycin has on the E1-P to E2-P conformational transition. Addition to KCl to a preincubation medium containing oligomycin, NaCl, and MgCl2 had no further effect on E-P formation. However, equilibration with oligomycin, KCl, and MgCl2 prior to the addition of NaCl plus ATP gave a much slower rate of E-P formation (5 s-1) and resulted in an initial rapid release of Pi similar to that found in the uninhibited enzyme. The slow increase in E-P level observed after incubation with oligomycin, KCl, and MgCl2 may be due to secondary formation of an inhibition complex following rapid binding of oligomycin. In contrast to the monophasic behavior which resulted from pre-exposure to NaCl or KCl, preincubation with oligomycin in the presence of MgCl2 plus Tris or Tris alone gave a biphasic pattern of E-P formation in which about 50% of the intermediate accumulated at a rate of 56 s-1 and the remainder at a rate of 5 s-1. In addition, the Pi burst amplitude was reduced, indicating partial inhibition of the enzyme. These results suggest that in the absence of Na+ and K+ only half of the enzyme is inhibited by oligomycin while the remainder undergoes inhibition subsequent to initiation of phosphorylation. Since the oligomycin concentration was saturating, the partial inhibition reflected in the biphasic pattern of E-P formation may be due to half-of-the-sites reactivity in which only half of the subunits bind oligomycin in the absence of monovalent cations.
Yeast submitochondrial particles, in a Pi- and NADH-dependent reaction, produced low concentrations of free ATP in the absence of added ADP. This formation of free ATP, as measured by the luciferin-luciferase method, was strongly stimulated by oligomycin. For maximal stimulation, oligomycin was to be added not earlier than 5-10 min after the addition of NADH. Upon addition of antimycin or FCCP the system was completely inhibited. The amount of free ATP formed corresponded to one-third of the amount of bound ATP in submitochondrial particles. The stimulatory effect of oligomycin disappeared if the submitochondrial particles were spun down after oligomycin stimulation and then resuspended in the reaction medium, whereas submitochondrial particles with no oligomycin added initially were stimulated by oligomycin after the same procedure. A different picture emerged with addition of ADP. If the submitochondrial particles were preenergized with NADH in the presence of oligomycin before the addition of ADP the formation of free ATP upon subsequent addition of ADP was inhibited by oligomycin. In the presence of oligomycin, but lacking preenergization with NADH, a stimulation of free ATP formation was achieved with added ADP. A possible explanation for the stimulating effect of oligomycin on ATP formation in the absence of added ADP is that it enhances the release of bound ATP in an energy-requiring process. The release of only about one-third of the bound ATP could indicate that one of three nucleotide-binding subunits involved in the mechanism of ATP formation by ATP synthase is in a state suitable for such an energy-dependent release of ATP.
The mechanism whereby oligomycin occludes Na+ within Na/K-ATPase was investigated to study Na+ and K+ transport mechanisms. Oligomycin stimulated Na+ binding to Na/K-ATPase but inhibited Na-K and Na-Na exchange. The oligomycin concentration required to stimulate Na+ binding to half-maximal was 4.5 microM, which was close to the concentration that reduced Na-Na and Na-K exchange and ATPase activity to half-maximal, suggesting that Na/K-ATPase possesses an oligomycin binding site responsible for stimulating Na+ binding and reducing ion exchange and ATPase activity. In contrast, neither K+ binding nor K+ transport was affected by oligomycin. Limited tryptic digestion of Na/K-ATPase showed that, unlike Na+, K+, and ouabain, oligomycin treatment did not result in a specific digestion pattern. Oligomycin appeared to inhibit ouabain binding in a noncompetitive manner, whereas it did not affect ATP binding. Na/K-ATPase isoforms with low and high sensitivities to ouabain were equally sensitive to oligomycin. These results suggest that the oligomycin binding site is located on the extracellular side of Na/K-ATPase, at a different position from the ouabain binding site, and this antibiotic did not induce a conformational change of Na/K-ATPase. We propose that oligomycin interacts with the Na+ occlusion site from the extracellular side of Na/K-ATPase, which delays Na+ release to the extracellular side without inducing a conformational change, suggesting that the pathways responsible for Na+ and K+ transport differ.
Oligomycin inhibited the membrane-bound, Ca(2+)-dependent ATPase of pea (Pisum sativum var. Progress No. 9) chloroplasts up to 50%, but only after treating the membranes with trypsin, whether or not the trypsin step was needed for full activity. The energy-linked Mg(2+)-dependent (light- and dithiothreitol (DTT)-activated) ATPase of pea thylakoids could be inhibited up to 100% under specified conditions. The data indicate that oligomycin does not interfere with activation processes, and it failed to inhibit the ATPase of solubilized chloroplast coupling factor 1 under any circumstances. Photophosphorylation, previously thought insensitive to oligomycin, was inhibited 30% in the case of pea chloroplasts, and this increased to 50% inhibition after pretreating the chloroplasts with either trypsin or DTT. The nature of inhibition of phosphorylation was complex, with apparent small components of electron transport inhibition and uncoupling, as well as energy transfer inhibition.Sensitivity of isolated pea thylakoid Mg(2+)-dependent ATPase to oligomycin was found during summer months but not during the winter. This was traced to a seasonal variation in relative humidity with values of 20% or less during the winter, leading to lack of inhibition in the isolated chloroplasts. The effect of low relative humidity during growth could be reversed by placing plants for 17 to 20 hours in a chamber at 50% relative humidity. The optimum humidity range for sensitivity to oligomycin was between 40 and 60%.With chloroplasts potentially sensitive to oligomycin due to "permissive" growth conditions, the inhibition was modulated by a number of parameters during chloroplast activation or ATPase assay. Preincubation in high concentrations of KCl diminished oligomycin sensitivity, and this effect was reversed by valinomycin. The greatest sensitivity, as well as the highest ATPase rates, occurred only if thylakoids were activated by light and DTT at a relatively high temperature (35 to 40 degrees C). Controlling the levels of uncouplers and running the reaction in darkness rather than in the light, both of which diminished the degree of membrane energization, increased the sensitivity to oligomycin. It is possible that accessibility of the oligomycin binding site is diminished by membrane energization. These results are contrasted with those from studies of variability in sensitivity of yeast mitochondria to oligomycin.
Oligomycin inhibition of the maximal hydrolysis activity of ox brain Na+/K(+)-ATPase was studied at varying NaCl concentrations and it was found that for a given amount of live enzyme, the observed inhibition of a particular total oligomycin concentration decreased as the amount of added, (heat-) denatured enzyme increased. In the present article we derive a scale factor for the oligomycin concentration, i.e., the fraction of the total concentration of oligomycin which is free in solution, as a function of the enzyme concentration used. This fraction decreased linearly with the protein concentration and may attain quite small values. We also study the Na(+)-dependence of the hydrolysis rate at saturating substrate concentrations ([Mg2+] = [ATP] = 3 mM), in the presence as well as the absence of KCl, at various concentrations of oligomycin. These data may be explained if it is assumed that the sole effect of oligomycin is to confer upon the enzyme an increased affinity for Na+, i.e., oligomycin merely enhances the inhibitory effect of Na+ on the (maximal) activity seen at high Na(+)-concentrations. The increased Na(+)-affinity in the presence of oligomycin should result in activation of the hydrolysis rate measured under conditions where Na(+)-activation is predominant, i.e., at low Na(+)-concentration and sub-saturating substrate concentrations. This prediction is verified for both Na(+)-ATPase and for Na+/K(+)-ATPase. This proposed action of oligomycin seems to be corroborated also by other evidence discussed in the text.
The lipid-free particulate preparations of the mitochondrial ATPase require phospholipid for activity and can be inhibited by oligomycin, as has been demonstrated previously. In this communication a steady state analysis of the activation of a particulate preparation of the ATPase by phospholipids and its subsequent inhibition by oligomycin has been carried out. The relative affinity of the ATPase for purified phospholipids has been determined by measuring the Km for activation (Ka) for several phospholipids. The Ka values varied from 30 to 100 mum. The Vmax in the presence of phosphatides varies from 0.29 to 1.11 mumol ATP hydrolyzed/min/mg of protein; no correlation is noted between the relative affinity of the enzyme for a phospholipid and the V max value. Higher V max values are noted with the more acidic phospholipids, however. Sodium dodecyl sulfate and monoolein also activate with Ka values of 25 and 800 mum, respectively. Diglycerides, however, do not activate. With all lipids the ATPase activity stimulated is oligomycin-sensitive. The Ki values for oligomycin range from 0.1 to 0.6 mum. Oligomycin is a competitive inhibitor with respect to all the phospholipids tested except phosphatidylethanolamine and phosphatidyglycerol. It is also competitive with respect to sodium dodecyl sulfate (k-i equals 0.94 mum). In reciprocal plots of activity versus ATP concentration, with and without oligomycin, an intercept consistent with either mixed or partial noncompetitive inhibition kinetics is noted. Comparable K-i values for oligomycin are obtained when calculated assuming either mixed or partial noncompetitive inhibition. The Km for ATP is the same in the unactivated and the lipid activated particulate ATPase; the value obtained is slightly lower than the Km for ATP in the solubilized, purified ATPase. Using a spectrophotometric assay the time required for activation with phospholipid and inhibition with oligomycin has also been determined. This investigation suggests the possibility that activation of the ATPase is due a position to interact with the water-soluble substrate. Consistent with the above suggestion is the supposition that the lipids do not necessarily confer inhibitor sensitivity to the ATPase, but rather allow an oligomycin-sensitive activity to be expressed.
The catabolism of hexose sugars and glycerol by the bloodstream form of Trypanosoma brucei brucei incubated with oligomycin was investigated. Oligomycin at a concentration of 10 micrograms/10(8) trypanosomes inhibited the catabolism of fructose, glucose and mannose by 70-80%, but not that of glycerol. Permeabilization of the trypanosome membranes by digitonin did not reverse the inhibition by oligomycin. Oligomycin did not inhibit pyruvate production in digitonin-permeabilized trypanosomes which were catabolizing exogenous glycolytic intermediates. It is concluded that the oligomycin-sensitive glycolysis is dependent on trypanosome membrane integrity. Oligomycin caused a rapid increase in the levels of hexose phosphates and some triose phosphates, but a decrease in the levels of glycerate 2-phosphate and phosphoenolpyruvate. There was a crossover point in the sequence of reactions between the formation of glycerol 3-phosphate and glycerate 2-phosphate during catabolism of the hexoses. Addition of the same concentration of oligomycin caused no change in the levels of glycolytic intermediates during the catabolism of glycerol. It is proposed that the catabolism of hexose sugars requires the transport of glycerol 3-phosphate from the glycosome via a glycerol 3-phosphate carrier which is probably inhibited by a hexose-sugar derivative formed on inhibition of the mitochondrial Mg(2+)-ATPase by oligomycin.
Effects of oligomycin on carotid chemoreceptor responses to O2 and CO2 were investigated using an in situ perfusion technique. Cats were anesthetized, paralyzed, and artificially ventilated. To avoid a possible reaction between an oligomycin-ethanol mixture and blood, we administered oligomycin to the carotid body via cell- and protein-free perfusate. Except for the perfusion periods, the carotid body received its own natural blood supply. Responses to O2, CO2, sodium cyanide, and nicotine of the same carotid chemoreceptor afferents were studied before and after each perfusion. An appropriate low dose of oligomycin completely blocked carotid chemoreceptor response to O2 while preserving the CO2 response. At the same time cyanide response was attenuated leaving nicotine response intact. Additional doses of oligomycin attenuated carotid chemoreceptor response to CO2 as well. Perfusion with a blank solution containing ethanol did not change the carotid body chemoreceptor responses. These effects of oligomycin on carotid chemoreceptor responses to O2 and CO2 were reversible, and restoration of the response to CO2 preceded that to O2. In addition, oligomycin administered into the blood with close intra-arterial injection produced similar differential blockade of O2 and CO2 chemoreception, preserving the nicotine and dopamine effects. This study confirmed the previous findings and provided new evidence showing that 1) the responses of carotid chemoreceptor to O2 and CO2 were separable by oligomycin due to the inhibition of oxidative phosphorylation and 2) the responses to nicotine and dopamine were intact even after blockade of O2 response.