Diphosphatidylglycerol-induced changes in the organization of mitochondrial ATPase

1. On submitochondrial particles from bovine heart, diphosphatidylglycerol produced a selective solubilization of ATPase. The solubilized enzyme was purified further by ammonium sulfate fractionation and shown to have the same reconstitutive activity as coupling factor F₁ (Pullman, M.E., Penefsky, H. S., Datta, A. and Racker, E. (1960) J. Biol. Chem. 235, 3322–3329).

2. Diphosphatidylglycerol-treated submitochondrial particles retained large amounts of the phospholipid and showed a decreased ATPase activity. Once the excess of phospholipid was removed, soluble ATPase could be again reincorporated in an oligomycin-sensitive complex.

3. On Mg-ATP particles the solubilization of ATPase induced by diphosphatidylglycerol was preceded by a stimulation of oligomycin-sensitive ATPase which indicated a dissociation of F₁ from the ATPase inhibitor (Pullman, M. E. and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762–3769). Magnesium was required to obtain the oligomycin-sensitive stimulation whereas in the absence of magnesium the solubilization of ATPase was prevalent.

4. It is concluded that the decreased association of F₁ with the ATPase inhibitor produced by diphosphatidylglycerol, causes a labilization of ATPase-membrane interaction. Under particular conditions, e.g. a high amount of phospholipid and a low concentration of magnesium, this is followed by the detachment of ATPase.     

Spermine binding to submitochondrial particles and activation of adenosine triphosphatase.

Studies on the effects of polyamines on oligomycin-sensitive ATPase activity of ox heart submitochondrial particles showed that, of the polyamines tested, only spermine affected the enzyme activity. Spermine within the physiological concentration range increased the Vmax. of the enzyme, but the Km for ATP was virtually unaffected. Binding studies of [14C]spermine to submitochondrial particles, under the same conditions as used for the ATPase assay, showed that the spermine binds to submitochondrial particles in a co-operative way; Hill plots of the data gave a Hill coefficient of 2 and a Kd of 8 microM. When submitochondrial particles were treated with trypsin, ATPase was not stimulated by spermine and the amount of spermine bound concomitantly was drastically decreased. The ATPase activity of isolated F1-ATPase was not affected by spermine. Removal of the natural protein ATPase inhibitor did not suppress either the stimulation of the ATPase activity by spermine or the spermine binding to the particles. The results obtained suggested that the polyamine binds and acts at the level of the liaison between the coupling factor F1 and the membrane sector F0 of the ATPase complex.     

Cleavage of type 2 adenovirus DNA BY HaeIII endonuclease. II. Map of HaeIII sites in EcoRI fragments C and E

1. A new method for the isolation of the oliogomycin-sensitive ATPase from beef-heart mitochondria is described. 2. A Triton-soluble ATPase complex was isolated as a by-product of the standard procedure, or as the main product when the submitochondrial particles were pretreated with 1% Triton. The ATPase activity of this complex is sensitive neither to oligomycin nor to dicyclohexylcarbodiimide. 3. The ATPase activity of the oligomycin-sensitive ATPase complex is nearly completely dependent on added phospholipids. The highest activation was found with asolectin. 4. The oligomycin-sensitive complex can be integrated into phospholipid vesicles resulting in an ATP- and Mg2+-dependent energization of the vesicles as monitored with the fluorescent dye 9-amino-6-chloro-2-methoxyacridine. 5. Aurovertin-binding studies based on fluorescence measurement reveal the presence of 1.5 mumol aurovertin-binding sites per g protein for the oligomycin-sensitive complex and about 2.2 mumol for the oligomycin-insensitive complex. 6. The preparation of the oligomycin-sensitive complex contains at least 6--7 polypeptides in addition to those derived from F1. One of these polypeptides, with an apparent molecular weight of 31 000, is virtually absent from the oligomycin-insensitive complex. 7. Some of these polypeptides have been identified and isolated.     

Resolution and reconstitution of mitochondrial nicotinamide nucleotide transhydrogenase.

Nicotinamide nucleotide transhydrogenase from bovine heart mitochondria was solubilized with cholate and partially purified by ammoniumsulphate fractionation and density gradient centrifugation. Compared to submitochondrial particles this preparation contained less than 10% of oligomycin-sensitive ATPase and cytochromes. When reconstituted with purified mitochondrial phosphatidylcholine, the enzyme catalyzed a reduction of NAD⁺ by NADPH that was stimulated by uncouplers and which showed a concomitent uncoupler-sensitive uptake of the lipophilic anion tetraphenylboron, indicating the generation of a membrane potential. It is concluded that transhydrogenase can energize the vesicles directly without the intervention of ATPase or cytochromes.     

Properties of ATPase activity associated with peroxisomes of rat and bovine liver.

1. Peroxisomes were isolated from bovine and rat liver by use of differential and density gradient centrifugations. 2. In the final density gradient (Nycodenz) a distinct peak of ATPase activity codistributed with the peroxisome marker catalase and was well separated from the bulk of the ATPase activity and from markers for other subcellular organelles. 3. The peroxisome-associated ATPase had a pH optimum of 7.5 and was inhibited by N-ethylmaleimide, by N,N'-dicyclohexylcarbodiimide and by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, but was unaffected by up to 30 microM n-tributyltin chloride. 4. Prolonged incubation with oligomycin at high concentrations indicated that 50% of peroxisomal ATPase was resistant to this inhibitor. The oligomycin-sensitive ATPase activity required at least a four-fold higher ratio of inhibitor to protein for inhibition than mitochondrial ATPase did. It was concluded that oligomycin-sensitive and oligomycin-resistant ATPase may be associated with liver peroxisomes.     

Reconstitution of mitochondrial oligomycin and dicyclohexylcarbodiimide-sensitive ATPase

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.     

Physiological and genetic modifications of the expression of the yeast mitochondrial adenosine triphosphatase inhibitor.

1. The oligomycin-sensitive ATPase activity of submitochondrial particles of the glycerol-grown "petite-negative" yeast: Schizosaccharomyces pombe is markedly stimulated by incubation at 40 degrees C and by trypsin activations are treatment. Both increased in Triton-X 100 extracts of the submitochondrial particles. 2. A trypsin-sensitive inhibitory factor of mitochondrial ATPase with properties similar to that of beef heart has been extracted and purified from glycerol-grown and glucose-grown S. pombe wild type, from the nuclear pleiotropic respiratory-deficient mutant S. pombe M126 and from Saccharomyces cerevisiae. 3. ATPase activation by heat is more pronounced in submitochondrial particles isolated from glycerol-grown than from glucose-grown S. pombe. An activation of lower extent is observed in rat liver mitochondrial particles but is barely detectable in the "petite-positive" yeast: S. cerevisiae. No activation but inhibition by heat is observed in the pleitotropic respiratory-deficient nuclear mutant S. pombe M126. 4. The inhibition of S. pombe ATPase activity by low concentrations of dicyclohexylcarbodiimide dissapears at inhibitor concentrations above 25 muM. In Triton-extract of submitochondrial particles net stimulation of ATPase activity is observed at 100 muM dicyclohexylcarbodiimide. The pattern of stimulation of ATPase activity by dicyclohexylcarbodiimide in different genetic and physiological conditions parallels that produced by heat and trypsin. A similar mode of action is therefore proposed for the three agents: dissociation or inactivation of an ATPase inhibitory factor. 5. We conclude that "petite-positive" and "petite-negative" yeasts contain an ATPase inhibitor factor with properties similar to those of the bovine mitochondrial ATPase inhibitor. The expression of the ATPase inhibitor, measured by ATPase activation by heat, trypsin or high concentrations of dicyclohexylcarbodiimide, is sensitive to alterations of the hydrophobic membrane environment and dependent on both physiological state and genetic conditions of the yeast cells.     

Increased content of natural ATPase inhibitor in tumor mitochondria

The ATPase activity of Zajdela hepatoma and Yoshida sarcoma submitochondrial particles was several times lower than the enzyme activity in rat heart and rat liver submitochondrial particles. The content of F1-ATPase in the tumor mitochondria was found not to be very different from that in mitochondria of rat liver. Immunochemical determination of the amount of the natural ATPase inhibitor revealed that the tumor mitochondria contain 2-3-times more ATPase inhibitor than control mitochondria. It is concluded that the low ATPase activity of the tumor mitochondria results from the inhibition of the enzyme activity by the natural ATPase inhibitor.     

A temperature-dependent structural change of mitochondrial ATPase

The temperature dependence of the intrinsic tryptophan fluorescence in either bovine heart submitochondrial particles or oligomycin-sensitive ATPase isolated therefrom shows a discontinuity at near 25°C, which coincides with the temperature where a break in the Arrhenius plot of ATPase activity is found. Addition of n-butanol to submitochondrial particles induces a decrease of tryptophan fluorescence in the whole temperature range. The discontinuity is interpreted as a temperature-dependent structural change and related to a viscosity-induced phase separation of the intrinsic mitochondrial proteins.     

Inactivation of the mitochondrial ATPase inhibitor protein by chemical modification with diethylpyrocarbonate

Modification of histidine residue(s) by diethylpyrocarbonate treatment of submitochondrial particles obtained by sonication results in inhibition of ATPase activity and stimulation of oligomycin-sensitive H+ conduction. The inhibition of the ATPase (EC 3.6.1.3) activity persisted in F1 isolated from diethylpyrocarbonate-treated submitochondrial particles, which exhibited the absorbance spectrum of modified histidine. Thus the inhibition of the ATPase activity results from histidine modification in F1 subunits. Removal of the natural inhibitor protein from submitochondrial particles resulted in stimulation of proton conduction. After removal of F1 inhibitor protein from the particles the stimulatory effect exerted by diethylpyrocarbonate treatment on proton conduction was lost. Reconstitution experiments showed that purified F1 inhibitor protein lost, after histidine modification, its capacity to inhibit the ATPase activity and proton conduction. These observations show that the stimulation of proton conduction by the ATPase complex effected by diethylpyrocarbonate treatment results from histidine modification in F1 inhibitor protein.     

Energy-linked nicotinamide-nucleotide transhydrogenase. Characterization of reconstituted ATP-driven transhydrogenase from beef heart mitochondria

The interaction between pure transhydrogenase and ATPase (Complex V) from beef heart mitochondria was investigated with transhydrogenase-ATPase vesicles in which the two proteins were co-reconstituted by dialysis or dilution procedures. In addition to phosphatidylcholine and phosphatidylethanolamine, reconstitution required phosphatidylserine and lysophosphatidylcholine. Transhydrogenase-ATPase vesicles catalyzed a 20-30-fold stimulation of the reduction of NADP+ or thio-NADP+ by NADH and a 70-fold shift of the apparent equilibrium expressed as the nicotinamide nucleotide ratio [NADPH][NAD+]/[NADP+][NADH]. In both of these respects, the transhydrogenase-ATPase vesicles were severalfold more efficient than beef heart submitochondrial particles. By measuring the ATP-driven transhydrogenase and the oligomycin-sensitive ATPase activities simultaneously and under the same conditions at low ATP concentrations, i.e. below 15 microM, the ATP-driven transhydrogenase/oligomycin-sensitive ATPase activity ratio was found to be about 3. This value is consistent with the stoichiometries of three protons translocated per ATP hydrolyzed and one proton translocated per NADPH formed and with a mechanism where the two enzymes interact through a delocalized proton-motive force.     

A novel mechanism of enzymic ester hydrolysis. Inversion of configuration and carbon-oxygen bond cleavage by secondary alkylsulphohydrolases from detergent-degrading micro-organisms.

Ligand-binding studies with labelled triethyltin on yeast mitochondrial membranes showed the presence of high-affinity sites (KD = 0.6 micronM; 1.2 +/- 0.3 nmol/mg of protein) and low-affinity sites (KD less than 45 micronM; 70 +/- 20 nmol/mg of protein). The dissociation constant of the high-affinity site is in good agreement with the concentration of triethyltin required for inhibition of mitochondrial ATPase (adenosine triphosphatase) and oxidative phosphorylation. The high-affinity site is not competed for by oligomycin or venturicidin, indicating that triethyltin reacts at a different site from these inhibitors of oxidative phosphorylation. Fractionation of the mitochondrial membrane shows a specific association of the high-affinity sites with the ATP synthase complex. During purification of ATP synthase (oligomycin-sensitive ATPase) there is a 5-6-fold purification of oligomycin- and triethyltin-sensitive ATPase activity concomitant with a 7-9-fold increase in high-affinity triethyltin-binding sites. The purified yeast oligomycin-sensitive ATPase complex contains approximately six binding sites for triethyltin/mol of enzyme complex. It is concluded that specific triethyltin-binding sites are components of the ATP synthase complex, which accounts for the specific inhibition of ATPase and oxidative phosphorylation by triethyltin.     

Titration of the binding sites for the oligomycin-sensitivity conferring protein in beef heart submitochondrial particles

The binding parameters of the oligomycin-sensitivity conferring protein (OSCP) in inside-out particles from beef heart mitochondria have been tested by means of two assays, the oligomycin-sensitive ATP-Pi exchange, and the oligomycin-sensitive ATP hydrolysis. The total number of OSCP binding sites in A particles was equal to 220 pmol/mg particle protein. Each mole of ATPase active site was able to bind 1.1 +/- 0.5 mol OSCP with Kd 1.7 nM.     

Purification and properties of adenosine triphosphatase solubilized from beef heart mitochondria by chloroform

Summary Soluble, oligomycin-insensitive ATPase released from beef heart mitochondria by chloroform extraction can be further purified by Sepharose 6B gel filtration. This purification increases enzyme activity 4–5 times (100–130 U/mg). According to specific activity, high purity and ability to reconstitute oligomycin-sensitive complex, isolated ATPase is quite comparable with enzyme preparations isolated by other methods.     

Physiological and genetic modifications of the expression of the yeast mitochondrial adenosine triphosphatase inhibitor

1. The oligomycin-sensitive ATPase activity of submitochondrial particles of the glycerol-grown “petite-negative” yeast: Schizosaccharomyces pombe is markedly stimulated by incubation at 40°C and by trypsin activations are treatment. Both increased in Triton-X 100 extracts of the submitochondrial particles.

2. A trypsin-sensitive inhibitory factor of mitochondrial ATPase with properties similar to that of beef heart has been extracted and purified from glycerolgrown and glucose-grown S. pombe wild type, from the nuclear pleiotropic respiratory-deficient mutant S. pombe M126 and from Saccharomyces cerevisiae.

3. ATPase activation by heat is more pronounced in submitochondrial particles isolated from glycerol-grown than from glucose-grown S. pombe. An activation of lower extent is observed in rat liver mitochondrial particles but is barely detectable in the “petite-positive” yeast: S. cerevisiae. No activation but inhibition by heat is observed in the pleitotropic respiratory-deficient nuclear mutant S. pombe M126.

4. The inhibition of S. pombe ATPase activity by low concentrations of dicyclohexylcarbodiimide dissapears at inhibitor concentrations above 25 μM. In Triton-extract of submitochondrial particles net stimulation of ATPase activity is observed at 100 μM dicyclohexylcarbodiimide. The pattern of stimulation of ATPase activity by dicyclohexylcarbodiimide in different genetic and physiological conditions parallels that produced by heat and trypsin. A similar mode of action is therefore proposed for the three agents: dissociation or inactivation of an ATPase inhibitory factor.

5. We conclude that “petite-positive” and “petite-negative” yeasts contain an ATPase inhibitor factor with properties similar to those of the bovine mitochondrial ATPase inhibitor. The expression of the ATPase inhibitor, measured by ATPase activation by heat, trypsin or high concentrations of dicyclohexylcarbodiimide, is sensitive to alterations of the hydrophobic membrane environment and dependent on both physiological state and genetic conditions of the yeast cells.     

Negatively charged phospholipid requirement of the oligomycin-sensitive mitochondrial ATPase

The highly-purified, oligomycin-sensitive mitochondrial adenosine triphosphatase has been reconstituted with phosphatidylserine. Treatment of the phosphatidylserine-reconstituted ATPase with phosphatidylserine decarboxylase produced a 3-fold decrease in the specific activity of the resulting phosphatidylethanolamine-enriched ATPase complex. Subsequent control experiments indicated that the resulting phosphatidylethanolamine was responsible for the lowered ATPase specific activity. These observations indicate that acidic phospholips do more than facilitate an interaction between the highly-purified, lipid-depleted ATPase and phospholipid. The negatively charged phospholipid appears to be essential for maintaining high levels of oligomycin-sensitive activity even after the initial interaction between phospholipid and the ATPase complex has occurred.     

Immunological and reconstitution studies on the adenosine triphosphatase complex from Rhodospirillum rubrum.

Studies on restoration of membrane-bound adenosinetriphosphatase (ATP phosphohydrolase, EC 3.6.1.3) from Rhodospirillum rubrum show that the delta-subunit is capable of binding to the F1 factor or to the F0 moiety of the F0-F1 ATPase complex. This subunit is thus likely involved in linking the F0 and F1 factor. During solubilization of the oligomycin-sensitive F0-F1 ATPase complex with Triton X-100 the detergent becomes specifically associated with the lipophilic F0 part of the enzyme complex. Crossed immunoelectrophoresis, agglutination tests, and kinetic studies with anti-F1 ATPase antibodies reveal a reaction of immunological identity of membrane-bound ATPase, F0-F1 ATPase, and F1 ATPase.     

Selective incorporation of membrane proteins into proteoliposomes of different compositions.

1. Cytochrome oxidase was incorporated into preformed liposomes containing phosphatidylserine. When confronted with a mixture of liposomes, some containing phosphatidylserine and some without it, the enzyme was incorporated only into the phosphatidylserine-containing liposomes. 2. The hydrophobic proteins of the oligomycin-sensitive ATPase incubated in the presence of a mixture of liposomes with and without cytochrome oxidase were preferentially incorporated into cytochrome oxidase-containing liposomes. This selectivity was abolished by either cytochrome c or ascorbate. 3. Cytochrome oxidase incubated in the presence of a mixture of liposomes with and without the hydrophobic proteins of the ATPase was preferentially incorporated into liposomes that did not contain the hydrophobic proteins. 4. Cytochrome oxidase and the oligomycin-sensitive ATPase were preferentially incorporated into pure liposomes over bacteriorhodopsin-containing vesicles. 5. Reduced coenzyme Q (QH2)-cytochrome c reductase was incorporated randomly when incubated in the presence of a mixture of pure liposomes and liposomes containing the hydrophobic proteins of the ATPase complex. 6. The significance of the incorporation procedure as a model for membrane biogenesis is discussed.     

Reconstitution of mitochondrial F0.F1-ATPase with phosphatidylcholine using the nonionic detergent, octylglucoside

A reconstitution procedure has been developed for the incorporation of the mitochondrial F0.F1-ATPase into the bilayer of egg phosphatidylcholine vesicles. The nonionic detergent, octylglucoside, egg phosphatidylcholine, and the lipid-deficient, oligomycin-sensitive F0.F1-ATPase (Serrano, R., Kanner, B., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) were combined in a 4770:320:1 detergent/phospholipid/protein molar ratio and then centrifuged on a discontinuous sucrose gradient to isolate the F0.F1-phosphatidylcholine complex. The specific activity of the reconstituted F0.F1-ATPase was as high as 14.5 mumol/min/mg protein, whereas with no added lipid the activity ranged between 1.4 and 2.2 mumol/min/mg protein. This reconstituted preparation exhibited greater than 90% oligomycin sensitivity which demonstrated the intactness of the multisubunit enzyme complex. The phosphatidylcholine/protein molar ratio of the reconstituted F0.F1 was 250:1 with less than 0.4% of the added octylglucoside remaining. Titrations with both phosphatidylcholine and octylglucoside demonstrated that the specific activity and oligomycin sensitivity were highly dependent on the concentrations of both phospholipid and detergent in the original reconstitution mixture. Analysis of the reconstituted ATPase by electron microscopy demonstrated that the catalytic portion of the enzyme complex projected from the phospholipid bilayer with an orientation similar to that observed with submitochondrial particles. The F0.F1-phosphatidylcholine complex was able to trap inulin, which suggests a vesicular structure impermeable to macromolecules. The electrophoretic mobility of the complex was identical to that for liposomes of egg phosphatidylcholine alone. The reconstitution conditions utilized give rise to an enzyme-phospholipid complex with very low ionic charge that demonstrates high oligomycin-sensitive ATPase activity.     

Ionic charge on phospholipids and their interaction with the mitochondrial adenosine triphosphatase

The activity of the lipid-depleted, oligomycin-sensitive mitochondrial ATPase has been measured in the presence of liposomes prepared from mixtures of phosphatidylglycerol and phosphatidylglycerol lysine. Enzyme activity increased linearly with an increase in the negative charge of liposomes prepared from the phosphatidylglycerol-phosphatidylglycerol lysine mixtures. The electrophoretic mobility and activating capacity of liposomes of several other phospholipids were determined. A linear relationship between electrophoretic mobility of the liposomes and oligomycin-sensitive activity was again apparent. These observations demonstrate that the activity of the ATPase is directly proportional to the ionic charge on phospholipid activators if the acyl chain composition of the phosphoglycerides is relatively constant.