Inhibition of chloroplast adenosine triphosphatase activity by adenosine triphosphatase inhibitor from beef heart mitochondria.

A new amino-acid sequence is proposed for silk fibroin peptide Cp, after automatic Edman degradation studies. The proposed sequence is: Gly-Ala-Gly-Ala-Gly-Ser-Gly-Ala-Ala-Gly-(Ser-Gly-(Ala-Gly)_n)₈ Tyr, where n is usually 2.     

Deficiency of uncoupler-stimulated adenosine triphosphatase activity in yeast mitochondria

Oligomycin-sensitive ATPase activity was studied in isolated yeast mitochondria. The protonophore CCCP, at a concentration which completely inhibited ATP synthesis, induced only a low rate of hydrolysis of externally added ATP, and the extent of hydrolysis was dependent upon phosphate (Pi) concentration. CCCP promoted hydrolysis of intramitochondrial ATP. However, hydrolysis of externally added ATP was total in a medium containing potassium phosphate plus valinomycin. Without ionophores, ATPase activity was only observed at high external pH or with detergent-treated mitochondria. Under state 4 conditions, external ATP had access to the catalytic nucleotide site of ATPase as shown by 32Pi-ATP exchange experiments. These results are discussed in terms of a limitation of the translocase-mediated ATP/ADP exchange in uncoupled mitochondria.     

Inhibition of mitochondrial adenosine triphosphatase activity by traponin comporent, TN-I

One of the troponin components, TN-I, strongly inhibits the ATPase activity of AS-particles obtained from mitochondria, while troponin and the other components, TN-C and TN-T, do not. The inhibition of the ATPase activity by Component TN-I is effective only in the presence of Mg²⁺ and ATP. Component TN-I digested with trypsin completely loses the inhibitory action of the ATPase activity. Adding tropomyosin does not affect the inhibitory effect of Component TN-I on the ATPase activity.     

Lipid-protein interactions. The mitochondrial complex III-phosphatidylcholine-water system

Bovine heart mitochondrial complex III (ubiquinol-cytochrome-c reductase) has been reconstituted into phosphatidylcholine bilayers and the effect of varying lipid/protein ratios on the structure and function of the protein has been examined. Electron microscopy, differential scanning calorimetry and Arrhenius plots of enzyme activity provide evidence that the protein is incorporated in an active conformation into pure phosphatidylcholine bilayers. At low lipid/protein ratios (e.g. 80:1 molar ratio) the protein exists in the form of aggregates. As the lipid proportion is increased, electron microscopy reveals the gradual formation of lipid bilayers; structures with the appearance of closed vesicles are seen at or above 300:1 phospholipid/protein molar ratios. Changes in enzyme activity as a function of lipid contents reveal a progressive increase in activity as more lipid is added, with a tendency to reach a saturation point. From the experimental data, a kinetic model is proposed, according to which the protein has an indefinite number of unspecific, independent and identical binding sites for phospholipids, the latter acting as essential enzyme activators. Varying lipid/protein ratios induce structural changes in complex III; visible spectra indicate changes in the polarity of the heme group environment, while Fourier-transform infrared spectroscopy suggests a change in the secondary structure of the protein as the lipid proportion is increased.     

Effects of triphenylsulphonium ions on mitochondria. Inhibition of adenosine triphosphatase activity.

Triphenylsulphonium ions inhibit mitochondrial oxidative phosphorylation and adenosine triphosphatase activity. The site of action is on the soluble F1 adenosine triphosphatase component. Triphenylsylphonium ions also inhibit electron transfer in the NAD-cytochrome b region of the respiratory chain. In both types of inhibition, triphenylsulphonium ions are effective at low concentrations, half-maximal inhibition being produced by a concentration of about 20-30 muM. These effects resemble the effects of alkylguanidines on mitochondria and are discussed in relation to the effects of alkylguanidines and other lipophilic cations such as ethidium and dibenzyldimethylammonium ions. A modification of the purification procedure for the soluble mitochondrial adenosine triphosphatase [Beechey, Hubbard, Linnett, Mitchell & Munn (1975) Biochem. J. 148, 533-537] IS DESCRIBED, WHICH YIELDS A PREPARATION WITH A HIGHER SPECIFIC ACTIVITY AND SHOWING FEWER BANDS IN GEL ELECTROPHORESIS.     

Electron transport dependent adenosine triphosphatase activity in castor bean endosperm mitochondria

The release of inorganic phosphate from ATP by mitochondriaisolated from endosperms of castor bean (Ricinus communis) wasstimulated by Mg⁺⁺, but not by Ca⁺⁺. EDTA, succinate, NADH₂or oligomycin depressed the reaction. The depression by succinatewas removed by KCN, antimycin A or anoxia. DNP alone did notaffect activity but did stimulate the Pi release in the presenceof succinate under aerobic conditions. Enhanced Pi release inthe presence of succinate and DNP was cancelled by KCN, antimycinA, oligomycin or anoxia. On the basis of these results, themechanism of ATPase action in castor bean endosperm mitochondriais discussed. (Received January 27, 1969; )     

Stimulation of rat liver mitochondrial adenosine triphosphatase by anions.

The hydrolysis of MgATP by isolated rat liver mitochondrial ATPase (EC 3.6.1.3) at pH 8.0 was stimulated by various anions. The rate of hydrolysis was increased from 18 to 170 mumol per min per mg, a 9.4-fold stimulation, by HSeO3 at 1 mM MgATP. In the absence of a stimulatory anion, reciprocal plots of initial velocity studies with MgATP as the variable substrate were curved (Hill coefficient approximately 0.5). With the addition of anion, the reciprocal plots became linear. When the substrate was MgITP or MgGTP with the isolated enzyme or MgATP with submitochondrial particles, no curvature of the reciprocal plots was observed. With purified ATPase, anions stimulated the hydrolysis of MgITP, MgGTP, MgUTP or MgCTP only slightly. With submitochondrial particles the stimulation by anions of MgATP hydrolysis was limited to approximately 2-fold. These data are interpreted to indicate the existence of two substrate sites for MgATP and an anion-binding site on the isolated enzyme.     

Effect of clofibrate on the adenosine triphosphatase activity of rat liver mitochondria.

The antihypercholesterolemic drug clofibrate (ethyl-α-p-chlorophenoxyisobutyrate) stimulated the latent ATPase activity and “superstimulated” the uncoupler-induced ATPase activity of rat-liver mitochondria. Addition of clofibrate decreased the turbidity of mitochondrial suspensions and released considerable amount of mitochondrial protein into solution. In these properties it closely resembled detergents like Triton X-100 and deoxycholate. However, unlike the detergents, clofibrate required the presence of a permeant cation for its disruptive action. Also, it was without any such effect on sonic submitochondrial particles. The drug enhanced the uptake of both Mg² and Cl^? by mitochondria suggesting that osmotic swelling precedes lysis. Sonic submitochondrial particles prepared in the presence of clofibrate showed a greater yield and comparable ATPase activity.     

Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase.

Beef heart mitochondrial ATPase (F1) exhibited a single binding site for Pi. The interaction with Pi was reversible, partially dependent on the presence of divalent metal ions, and characterized by a dissociation constant at pH 7.5 of 80 micronM. A variety of substances known to influence oxidative phosphorylation or the activity of the soluble ATPase (F1) also influenced Pi binding by the enzyme. Thus aurovertin, an inhibitor of oxidative phosphorylation, which was bound tightly by F1 and inhibited ATPase activity, enhanced Pi binding via a 4-fold increase in the affinity of the enzyme for Pi (KD = 20 micronM) but did not alter binding stoichiometry. Anions such as SO4(2-), SO3(2-), chromate, and 2,4-dinitrophenolate, which stimulated ATPase activity of F1, also enhanced Pi binding. Inhibitors of ATPase activity such as nickel/bathophenanthroline and the protein ATPase inhibitor of Pullman and Monroy (Pullman, M. E., and Monroy, G. C. (1963) J. Biol. Chem. 238, 3762-3769) inhibited Pi binding. The adenine nucleotides ADP, ATP, and the ATP analog adenylyl imidodiphosphate as well as the Pi analog arsenate, also inhibited Pi binding. The observations suggest that the Pi binding site was located in or near an adenine nucleotide binding site on the molecule.     

Regulation of adenosine triphosphatase activity in mitochondria, chloroplasts and erythrocytes by anions, adenosine diphosphate and alcohols

The effects of sulfite, bicarbonate, thiocyanate, methanol, ethanol, glycerol, dimethy sulfoxide and ADP on the ATPase activity of the coupling factor from liver mitochondria (F1) and pea chloroplasts (CF1) and of the anion-sensitive ATPase from rat erythrocytes were investigated. Under steady-state conditions of ATP hydrolysis catalyzed by F1, CF1, and erythrocyte ATPase, three Km values for each of the enzymes, three activation constants for sulfite and three inhibition constants for thiocyanate were determined. The efficiency and direction of the effects of anions, alcohols and ADP strongly depend on temperature and substrate (Mg-ATP) concentration. The mechanisms of modification by anions and alcohols of the ATPase activities are discussed.     

Molecular interactions of adenosine triphosphatase with the mitochondrial membrane as revealed by a spin label study.

Mitochondrial ATPase complex has been spin-labeled in the membrane using the inhibitor N-(2,2,6,6-tetramethylpeperidyl-1-OXYL)-N(cyclohexyl)carbodiimide (nccd). the amount of NCCD bound to mitochondrial fragments is 0.5 nmol/mg and cannot be dialyzed or extracted with ether, chloroform, or methanol. The electron paramagnetic resonance spectrum of NCCD bound to fragments is pH-sensitive, a greater label immobilization occurring at pH values lower or higher than 7. Ether extraction removes the ATPase inhibition by NCCD without detaching the label. This effect appears to be the consequence of the dislocation of some components of the ATPase complex. Removal of F1 natural inhibitor or of F1 does not affect the spectrum of NCCD bound to fragments, while the removal of oligomycin sensitivity-conferring protein produces an increase in the extreme splitting. Oligomycin sensitivity-conferring protein may thus interact with the NCCD binding component of the membrane. The isolation of the NCCD-binding proteolipid results in a large increase in the mobility of the label, but addition of dipalmitoyllecithin decreases the mobility of the label to the original level. Phospholipids are thus necessary to keep the NCCD-binding proteolipid in the native conformation.