ATPase Activity of Pea Cotyledon Submitochondrial Particles: ACTIVATION, SUBSTRATE SPECIFICITY, AND ANION EFFECTS

Submitochondrial particles freshly prepared by sonication from pea cotyledon mitochondria showed low ATPase activity. Activity increased 20-fold on exposure to trypsin. The pea cotyledon submitochondrial particle ATPase was also activated by “aging” in vitro. At pH 7.0 addition of 1 millimolar ATP prevented the activation. ATPase of freshly prepared pea cotyledon submitochondrial particles had a substrate specificity similar to that of the soluble ATPase from pea cotyledon mitochondria, with GTPase > ATPase. “Aged” or trypsin-treated particles showed equal activity with the two substrates. NaCl and NaHCO₃, which stimulate the ATPase but not the GTPase activity of the soluble pea enzyme, were stimulatory to both the ATPase and GTPase activities of freshly prepared submitochondrial particles. However, they were stimulatory only to the ATPase activity of trypsin-treated or “aged” submitochondrial particles. In contrast, the ATPase activity of rat liver submitochondrial particles was stimulated by HCO₃⁻, but inhibited by Cl⁻, indicating that Cl⁻ stimulation is a distinguishing property of the pea mitochondrial ATPase complex.     

Spegazzinine,a new inhibitor of mitochondrial oxidative phosphorylation

The effects of spegazzinine, a dihydroindole alkaloid, on mitochondrial oxidative phosphorylation were studied.Spegazzinine inhibited coupled respiration and phosphorylation in rat liver mitochondria. The I₅₀ was 120 μM. Uncouplers released the inhibition of coupled respiration. Arsenate-stimulated mitochondrial respiration was partially inhibited by spegazzinine. The stimulation of mitochondrial respiration by Ca²⁺ and the proton ejection associated with the ATP-dependent Ca²⁺ uptake were not affected by the alkaloid.Oxidative phosphorylation and the P_i-ATP exchange reaction of phosphorylating beef heart submitochondrial particles were strongly inhibited by spegazzinine (I₅₀, 50 μM) while the ATP-dependent reactions, reduction of NAD⁺ by succinate and the pyridine nucleotides transhydrogenase were less sensitive (I₅₀, 125 μM). Oxygen uptake by submitochondrial particles was not affected.The 2,4-dinitrophenol-stimulated ATPase activity of rat liver mitochondria was not affected by 300 μM spegazzinine, a concentration of alkaloid that completely inhibited phosphorylation. However, higher concentrations of spegazzinine did partially inhibit it. The ATPase activities of submitochondrial particles, insoluble and soluble ATPases were also partially inhibited by high concentrations of spegazzinine.The inhibitory properties of spegazzinine on energy transfer reactions are compared with those of oligomycin, aurovertin and dicyclohexylcarbodiimide. It is concluded that spegazzinine effects are very similar to the effects of aurovertin and that its site of action may be the same or near the site of aurovertin.     

Isolation and properties of OSCP and an F1-ATPase binding protein from rat liver mitochondria - evidence against OSCP as the linking "stalk" between F1 and the membrane.

Oligomycin Sensitivity Conferral Protein (OSCP) and an F₁-ATPase Binding Protein were isolated from F₁-depleted rat liver mitochondrial membrane. Their molecular weights on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea were 22,500 and 8,500 respectively. When incubated with liver TUA (trypsin, urea and ammonia-treated) submitochondrial particles, the binding protein was effective in the binding of F₁ to the particles with the resultant particle-bound ATPase activity not oligomycin sensitive. When OSCP was then incubated with the reconstituted membrane-bound ATPase, its activity became oligomycin sensitive. These results suggest that, first; the binding protein, but not OSCP, connects F₁-ATPase to the membrane of rat liver mitochondria and maybe to the “stalk”, if indeed there is a stalk in mitochondrial membrane ATPase complex; and second; the function of OSCP is solely to render the ATPase activity sensitive to oligomycin and other similar inhibitors.     

Loose binding of testicular mitochondrial ATPase to the inner membrane

Rat testis mitochondrial ATPase was not inhibited by oligomycin at pH 7.5. It was inhibited only at higher alkaline pH's, and showed a lower sensitivity both to oligomycin and N,N′-dicyclohexylcarbodiimide and a higher one to efrapeptin. In submitochondrial particles, testis ATPase was only slightly inhibited by oligomycin, ossamycin, and efrapeptin. The possibility of a loose binding of F₁ to the membrane was supported by its recovery from the supernatant of the submitochondrial particles. Furthermore, by electron microscopy, after hypoosmotic shock and negative staining of the mitochondrial preparations, most of the inner mitochondrial membranes showed only a few “knobs” or none at all. The capacity of the testis mitochondrial preparation to produce ATP was tested and compared to that from liver. ATP synthetase/ATPase activity ratio was $\text{30}\text{1}$ in liver mitochondria, whereas in the testis it was $\text{3}\text{1}$. In spite of this large difference, at least part of the testis ATPase must be firmly bound to the membrane, since it is able to form ATP. The rest seems to be loosely bound and its functional significance is still unknown.     

Interaction between ubiquinone and ATPase in mitochondrial membranes

The extraction of ubiquinone from mitochondrial membranes produces alterations of ATPase activity including a reversible loss of oligomycin sensitivity which is restored by long-chain Q-homologs. Short-chain ubiquinones like Q₃ produce a loss of oligomycin and dicyclohexylcarbodiimide (DCCD) sensitivity in submitochondrial particles. The effect shows uncompetitive or noncompetitive kinetics with respect to oligomycin or DCCD respectively. Long-chain ubiquinones have a competitive effect with Q₃, thus restoring oligomycin sensitivity; they behave, however, in about the same way as Q₃ in lowering the DCCD sensitivity in submitochondrial particles. On the basis of these observations we suggest that ubiquinone may be a physiological modulator of ATPase activity in the mitochondrial membrane.Abbreviations used: BHM, beef heart mitochondria; DCCD, dicyclohexylcarbodiimide; ETP, electron transfer particles (submitochondrial particles); Q, ubiquinone.     

Studies on the immunological properties of complex V (mitochondrial ATP synthetase complex)

Antibody raised in rabbits against Complex V (miochondrial ATP synthetase complex) purified from beef heart mitochondria cross-reacted with Complex V and submitochondrial particles from beef heart, beef adrenals, and rat liver as shown by double-diffusion and rocket immunoelectrophoresis analysis. Of the various isolated and purified components of Complex V, only the oligomycin sensitivity-conferring protein showed strong reactivity with the anti-Complex V antibody, soluble F₁-ATPase reacted very faintly, while F₆ and ATPase inhibitor protein showed no precipitin lines. Crossed immunoelectrophoresis indicated that antigenic determinants recognized by the antibody were present on OSCP and possibly on the dicyclohexylcarbodiimide-binding protein. The components of Complex V could be precipitated from beef heart submitochondrial particles dissolved in Triton X-100 and pretreated with control IgG. When the composition of the immunoprecipitate was compared to that of purified Complex V, all the constituent polypeptides of the latter were present in the immunoprecipitate, except for one polypeptide in the low-molecular-weight region. Incubation of Complex V or submitochondrial particles with the anti-Complex V antibody in the absence of Triton X-100 caused inhibition of ATP-P_i exchange but not of ATPase activity. In the presence of Triton X-100, oligomycin sensitivity of Complex V was lost and the antibody was able to inhibit also the ATPase activity. The enzymic activity of soluble F₁-ATPase was unaffected by the antibody in the absence or presence of Triton X-100. These results suggest that the anti-Complex V antibody might be a useful tool for identifying and probing the role of Complex V components involved in energy transduction.     

Presence of two enzymes, different from the F1F0-ATPase, hydrolyzing nucleotides in human term placental mitochondria

The hydrolysis of ATP, ADP or GTP was characterized in mitochondria and submitochondrial particles since a tightly-bound ATPase associated with the inner mitochondrial membrane from the human placenta has been described. Submitochondrial particles, which are basically inner membranes, were used to define the location of this enzyme. Mitochondria treated with trypsin and specific inhibitors were also used. The oxygen consumption stimulated by ATP or ADP was 100% inhibited in intact mitochondria by low concentrations of oligomycin (0.5 microgram/mg) or venturicidine (0.1 microgram/mg), while the hydrolysis of ATP or ADP was insensitive to higher concentrations of these inhibitors but it was inhibited by vanadate. Oligomycin or venturicidine showed a different inhibition pattern in intact mitochondria in relation to the hydrolysis of ATP, ADP or GTP. When submitochondrial particles were isolated from mitochondria incubated with oligomycin or venturicidine, no further inhibition of the nucleotide hydrolysis was observed, contrasting with the partial inhibition observed in the control. By incubating the placental mitochondria with trypsin, a large fraction of the hydrolysis of nucleotides was eliminated. In submitochondrial particles obtained from mitochondria treated with trypsin or trypsin plus oligomycin, the hydrolysis of ATP was 100% sensitive to oligomycin at low concentrations, resembling the oxygen consumption; however, this preparation still showed some ADP hydrolysis. Native gel electrophoresis showed two bands hydrolyzing ADP, suggesting at least two enzymes involved in the hydrolysis of nucleotides, besides the F1F0-ATPase. It is concluded that human placental mitochondria possesses ADPase and ATP-diphosphohydrolase activities (247).     

On the mechanism of action of alkylguanidines in oxidative phosphorylation: their action on soluble F1.

Octylguanidine inhibits the adenosine triphosphatase (ATPase) activity of bovine heart submitochondrial particles and soluble F₁. The characteristics of the inhibition as a function of octylguanidine and Mg²⁺ concentrations and pH are very similar in submitochondrial particles and soluble F₁. Only those guanidines that possess an alkyl chain of more than six carbons inhibit the ATPase activity of submitochondrial particles and F₁. The inhibiting action of octylguanidine on F₁ is fully reversible. Octylguanidine prevents the cold-induced inactivation of F₁ at concentrations similar to those that inhibit ATPase activity. Guanidines that inhibit ATPase activity also prevent the cold-induced inactivations of F₁.     

Isolation of totally inverted submitochondrial particles by sonication of beef heart mitochondria

A novel procedure for isolating totally inverted preparations of submitochondrial particles by sonication of beef heart mitochondria is described. The procedure involves only differential centrifugation in 0.25 M sucrose containing 0.15 M KCl. The submitochondrial particles have 96% of their cytoplasmic face cytochromec-binding sites sequestered within the particles. Mild sonication exposes cytochromec-binding sites to the medium. The oligomycin-sensitive ATPase of sonic-derived submitochondrial particles, like that of electron transport particles, is inhibited 98% by exogenous isolated ATPase inhibitor protein. NADH oxidase activity in these particles is inhibited by oligomycin. The respiratory control index (uncoupled rate/oligomycin-inhibited rate) is approximately 3.4 and can be increased by washing the particles with medium containing bovine serum albumin.     

Cation requirement for the reconstitution of oligomycin-sensitive ATPase by means of soluble F1-ATPase and F1-depleted submitochondrial particles

Bovine heart submitochondrial particles depleted of F₁ by treatment with urea (‘F₁-depleted particles’) were incubated with soluble F₁-ATPase. The binding of F₁ to the particles and the concomitant conferral of oligomycin sensitivity on the ATPase activity required the presence of cations in the incubation medium. NH⁺₄, K⁺, Rb⁺, Cs⁺, Na⁺ and Li⁺ promoted reconstitution maximally at 40–74 mM, guanidinium⁺ and Tris⁺ at 20–30 mM, and Ca²⁺ and Mg²⁺ at 3–5 mM. The particles exhibited a negative ζ-potential, as determined by microelectrophoresis, and this was neutralized by mono- and divalent cations in the same concentration range as that needed to promote F₁ binding and reconstitution of oligomycin-sensitive ATPase. It is concluded that the cations act by neutralizing negative charges on the membrane surface, mainly negatively charged phospholipids. These results are discussed in relation to earlier findings reported in the literature with F₁-depleted thylakoid membranes and with submitochondrial particles depleted of both F₁ and the coupling proteins F₆ and oligomycin sensitivity-conferring protein.     

Resolution and Reconstitution of a Mammalian Membrane

The problem of the resolution and reconstitution of the inner mitochondrial membrane has been approached at three levels. (1) Starting with phosphorylating submitochondrial particles, a "resolution from without" can be achieved by stripping of surface components. The most extensive resolution was recently obtained with the aid of silicotungstate. Such particles require for oxidative phosphorylation the addition of several coupling factors as well as succinate dehydrogenase. (2) Starting with submitochondrial particles that have been degraded by trypsin and urea a resolution of the inner membrane proper containing an ATPase has been achieved. These experiments show that at least five components are required for the reconstitution of an oligomycin-sensitive ATPase: a particulate component, F ₁, Mg⁺⁺, phospholipids, and F_c. Morphologically, the reconstituted ATPase preparations resemble submitochondrial particles. (3) Starting with intact mitochondria individual components of the oxidation chain have been separated from each other. The following components were required for the reconstitution of succinoxidase: succinate dehydrogenase, cytochrome b\, cytochrome c ₁, cytochrome c, cytochrome oxidase, phospholipids and Q ₁₀. The reconstituted complex had properties similar to those of phosphorylating submitochondrial particles; i.e., the oxidation of succinate by molecular oxygen was highly sensitive to antimycin.     

Extraction of mitochondrial protein-lipid complexes into organic solvents: an approach to study the interaction between the ATPase and the mitochondrial ATPase-inhibitor protein

Protein-lipid complexes were transferred directly from mitochondria and submitochondrial particles into hexane and ether. The protein-lipid residue left after solvent removal from these extracts was used to form liposomes which display low-temperature-resistant ATPase activity. Centrifugation experiments indicate that the ATPase activity is associated to the vesicles. Most of the F1-ATPases appear to be accessible to the external water phase of the liposomes. The ATPase activity of these particles was insensitive to dicyclohexylcarbodiimide and oligomycin. Incubation of these vesicles at room temperature activated (4--10-fold) the ATPase through a process that is partially sensitive to phenylmethylsulfonyl fluoride. The results with purified ATPase-inhibitor protein and (F1--ATPase)-inhibitor complex indicate that the activation process in the liposomes is due to the abolition of the inhibitory action of the inhibitor protein bound to a large fraction of the extracted ATPases. Liposomes prepared from hexane extracts obtained from submitochondrial particles having different levels of ATPase activity displayed an activation ratio which correlated with the number of ATPases that are inhibited by the inhibitor protein in the submitochondrial particles. The extraction of mitochondrial ATPase and its incorporation into liposomes followed by activity measurements may be used to judge the number of ATPases that in a given preparation contain the inhibitor protein in its inhibiting site.     

The extent of mitochondrial F1-ATPase and adenine nucleotide carrier activity with ϵ-ATP

1. The use of 1,N⁶-ethenoadenosine 5′-triphosphate (?-ATP), a synthetic, fluorescent analog of ATP, by whole rat liver mitochondria and by submitochondrial particles produced via sonication has been studied.2. Direct [³H]adenine nucleotide uptake studies with isolated mitochondria, indicate the ?-[³H]ATP is not transported through the inner membrane by the adenine nucleotide carrier and is therefore not utilized by the 2,4-dinitrophenol-sensitive F₁-ATPase (EC 3.6.1.3) that functions in oxidative phosphorylation. However, ?-ATP is hydrolyzed by a Mg²⁺-dependent, 2,4-dinitrophenol-insensitive ATPase that is characteristic of damaged mitochondria.3. ?-ATP can be utilized quite well by the exposed F₁-ATPase of sonic submitochondrial particles. This ?-ATP hydrolysis activity is inhibited by oligomycin and stimulated by 2,4-dinitrophenol. The particle F₁-ATPase displays similar K_m values for both ATP and ?-ATP; however, the V with ATP is approximately six times greater than with ?-ATP.4. Since ?-ATP is a capable substrate for the submitochondrial particle F₁-ATPase, it is proposed that the fluorescent properties of this ATP analog might be employed to study the submitochondrial particle F₁-ATPase complex, and its response to various modifiers of oxidative phosphorylation.     

Oxidative phosphorylation in pea cotyledon submitochondrial particles

Mitochondria and submitochondrial particles (SMP) from pea cotyledons were shown to catalyze oxidative phosphorylation as measured by ³²Pi uptake into phosphate esters. ATP synthesis was sensitive to the electron transport inhibitor KCN, the uncoupler carbonyl cyanide m-chlorophenylhydrazone, and the coupling factor inhibitor oligomycin. Experiments with the adenine nucleotide translocator inhibitor atractyloside indicated the SMP were inside-out. Mersalyl completely inhibited ATP synthesis by SMP, and a separate experiment indicated that mersalyl has a direct effect on the ATPase complex. The kinetics of ATP synthesis indicated a high affinity for phosphate (K_m = 0.18 millimolar). ADP kinetics gave a biphasic curve with K_m values of about 4.8 and 160 micromolar. O₂ uptake and ATP synthesis had a pH maximum of 7.6 while the ratio of micromoles phosphate esterified to microatoms O₂ taken up was highest at pH 7.2. Sodium chloride inhibited both ATP synthesis and O₂ uptake but stimulated the ATPase reaction. The SMP also catalyzed a slow ATP-phosphate exchange reaction.     

Reconstitution of Oxidative Phosphorylation and of Oligomycin-Sensitive ATPase by Five- and Six-Subunit Forms of Pea Mitochondrial F(1)-ATPase

Five- and six-subunit forms of F₁-ATPase were purified from pea (Pisum sativum L. cv Homesteader) cotyledon submitochondrial particles. Apart from the usual complement of five subunits, the six-subunit enzyme contained an additional 26,500-dalton protein. Both forms of the F₁-ATPase were used to reconstitute oxidative phosphorylation in F₁-depleted (ASU) as well as in F₁ and oligomycin-sensitivity conferring protein (OSCP)-depleted (ASUA) bovine mitochondrial membranes. The six-subunit enzyme was considerably more efficient in reconstituting the ATP synthesis than the five-subunit enzyme. Both forms of the enzyme were also able to reconstitute the ATPase activity in ASU- as well as in ASUA-particles. There were substantial differences, however, in the oligomycin sensitivity of the ATPase bound to the ASUA-particles: 20 and 60% inhibition by oligomycin was obtained in the case of the five-subunit and six-subunit enzyme, respectively. We conclude, that the 26,500-dalton protein present in the six-subunit F₁-ATPase is responsible for the increase in oligomycin sensitivity of the bound enzyme and functions, therefore, as the plant OSCP.     

Effect of cetyltrimethylammonium on ATP hydrolysis and proton translocation in the F0-F1 H+-ATP synthase of mitochondria

The amphiphylic alkyl cation cetyltrimethylammonium inhibits the catalytic activity of soluble and membrane-bound F₁ in a noncompetitive fashion. In sonic submitochondrial particles the Dixon plot showed a peculiar pattern with upward deviation at cetyltrimethylammonium concentration higher than 80µM. In membrane-bound F₁ the inhibition by cetyltrimethylammonium was potentiated by the F₀ inhibitor ologomycin. Cetyltrimethylammonium also inhibited the oligomycin-sensitive proton conductivity in F₁-containing particles but was without any effect in F₁-depleted particles. Also this inhibitory effect was potentiated by oligomycin. These results indicate functional cooperative interactions between F₀ and F₁.     

Isolation,characterization, and reconstitution of a solubilized fraction containing the hydrophobic sector of the mitochondrial proton pump

The hydrophobic sector of the mitochondrial ATPase complex was purified by sequential extraction with cholate and octylglucoside, by further differential solubilization with guanidine and cholate in the presence of phosphatidylcholine, and by fractionation with ammonium sulfate. A polypeptide with a mass of 28,000 dalton was present in the purified hydrophobic section which was cleaved by trypsin, resulting in loss of reconstitution activity. In contrast, dicyclohexylcarbodiimide-binding proteolipid remained unimpaired after exposure to trypsin. The³²P_i-ATP exchange activity of the reconstituted ATPase complex was inhibited byp-hydroxymercuribenzoate, which reacted primarily with the 28,000-dalton protein, as monitored by acrylamide gel electrophoresis with¹⁴C-labeled inhibitor. The function of a 22,000-dalton polypeptide and of some minor components in the region of the proteolipid remains unknown. An examination of the phospholipid requirements for reconstitution of an active complex revealed an unexpected discrepancy. With an excess of phosphatidylethanolamine, optimal reconstitution of³²P_i-ATP exchange and ATP synthesis in the presence of bacteriorhodopsin and light was achieved; at a high phosphatidylcholine:phosphatidylethanolamine ratio, the rate of ATP synthesis remained high, but the rate of³²P_i-ATP exchange dropped precipitously. A new procedure is described for the reconstitution of the ATPase complex with purified phospholipids which is stable for at least 15 days.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - STE-DTT buffer sucrose (250 mM), Tricine-KOH (50 mM), EDTA (5 mM), DTT (5 mM), pH 8.0 - F _o a membranous preparation from mitochondria conferring oligomycin (or rutamycin) sensitivity to F₁ - F₁F₆ coupling factors 1 (ATPase) and 6 - OSCP oligomycin-sensitivity-conferring protein - BSA bovine serum albumin - SDS sodium dodecyl sulfate - DTT dithiothreitol - STE buffer sucrose (250 mM), Tricine-KOH (50 mM), EDTA (5 mM) - TUA particles submitochondrial particles prepared by stepwise exposure of light-layer submitochondrial particles to trypsin and urea, then sonic oscillation in the presence of dilute ammonia (pH 10.4) - OG-cholate buffer glycerol (20%), Tricine (50 mM), MgSO₄ (5 mM), DTT (5mM), cholate (0.5%), octylglucoside (0.5%), pH 8.0 - p-HMB p-hydroxymercuribenzoate     

Oligomycin effects on ATPase and photophosphorylation of pea chloroplast thylakoid membranes

Oligomycin inhibited the membrane-bound, Ca²⁺-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 Mg²⁺-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.     

Resolution and Reconstitution of H+-ATPase Complex from Beef Heart Mitochondria

Mitochondrial H⁺-ATPase complex, purified by the lysolecithin extraction procedure, has been resolved into a “membrane” (NaBr-F₀) and a “soluble” fraction by treatment with 3.5 M sodium bromide. The NaBr-F₀ fraction is completely devoid of p, 8, and e subunits of the F, ATPase and largely devoid of α and γ subunits of F₁, where F₀ is used to denote the membrane fraction and F₁, coupling factor 1. This is confirmed by complete loss of ATPase and P₁-ATP exchange activities. The addition of F₁ (400 μg · mg^?1 F₀) results in complete restoration of oligomycin sensitivity without any reduction in the F₁-ATPase activity. Presumably, this is due to release of ATPase inhibitor protein from the F₁-F₀ complex consequent to sodium bromide extraction. Restoration of Pf-ATP exchange and H⁺-pumping activities require coupling factor B in addition to FpATPase. The oligomycin-sensitive ATPase and 32P₁ATP exchange activities in reconstituted Fr F₀ have the same sensitivity to uncouplers and energy transfer inhibitors as in starting submitochondrial particles from the heavy layer of mitochondria and F₁-F₀ complex. The data suggest that the altered properties of NaBr-F₀ observed in other laboratories are probably inherent to their F₁F₀ preparations rather than to sodium bromide treatment itself.

The H⁺-ATPase (F₁-F₀) complex of all known prokaryotic (3, 8, 9, 10, 21, 32, 34) and eukaryotic (11, 26, 30, 33, 35–37) phosphorylating membranes contain two functionally and structurally distinct entities. The hydrophilic component F₁, composed of five unlike subunits, shows ATPase activity that is cold labile as well as uncoupler-and oligomycin-insensitive. The membrane-bound hydrophobic component F₀, having no energy-linked catalytic activity of its own, is indirectly assayed by its ability to regain oligomycin sensitive ATPase and P₁-ATP exchange activities on binding to F¹-ATPase (33). The purest preparations of bovine heart mitochondrial F₀ show seven or eight major components in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate or SDS-PAGE (1, 2, 12, 14), ranging from 6 to 54 ku in molecular weight (12). The precise structure and polypeptide composition of mitochondrial Fo is not known.

The F₀ preparations from bovine heart reported so far have been derived from H⁺-ATPase preparations isolated in the presence of cholate and deoxycholate (11, 33, 36, 37). The ATPase and P₁-ATP exchange activity of the preparations so obtained are low, dependent upon additional phospholipids and coupling factors; they show altered sensitivity to energy transfer inhibitors as compared to submitochondrial particles from the heavy layer of the mitochondria or ETPh (1. 2, 12, 14, 29, 33). Recently, lysolecithin has been successfully employed to extract highly active H⁺-ATPase from beef (17, 19, 28) and pig (24) heart mitochondria. The beef heart H⁺-ATPase preparation has the same ratio of ATPase to PrATP exchange activity and apparently the same sensitivity to energy transfer inhibitors as submitochondrial particles (17). The present communication describes resolution of this F₁-F₀ preparation using sodium bromide (NaBr) and reconstitution of ATPase and Pr ATP exchange activities. The NaBr-F₀ prepared from this preparation shows no dependence on lipids, and the same or increased sensitivity to energy transfer inhibitors when reconstituted with F₁-ATPase. Furthermore, F₁ ATPase activity does not decrease on binding of F₁ to NaBr-F₀, even though the reconstituted ATPase activity is 99% sensitive to oligomy-cin and dicyclohexylcarbodiimide. These properties are in contrast to the properties of F₀ reported by other workers (12, 14).