Mitochondrial oligomycin-resistance mutations affecting the proteolipid subunit of the mitochondrial adenosine triphosphatase.

Two independently isolated oligomycin resistant mutants of Saccharomyces cerevisiae have been studied. The oligomycin resistance is conferred in each case by a single mutation at an oliA locus. In both strains the proteolipid subunit of the mitochondrial ATPase (subunit 9) shows an apparent increase in molecular weight as judged by its mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis. Variable effects are seen on other subunits. These results suggest that oliA loci may play some role in the determination of proteolipid ATPase subunit.     

Localization on mitochondrial DNA of mutations leading to a loss of rutamycin-sensitive adenosine triphosphatase.

Four cytoplasmic mutants of Saccharomyces cerevisiae showing loss of mitochondrial rutamycin-sensitive ATPase activity but having significant cytochrome oxidase and NADH-cytochrome c reductase have been isolated. Genetic studies indicate the mutations to be closely linked to each other and have been assigned to a new locus, PHO1. The mutations show a low frequency of recombination with the OL12 locus, suggesting a linkage to this marker. They are not, however, linked to the OLI1 locus. Linkage of the ATPase mutations to the OLI2 locus is also indicated by restoration of wild-type diploids by sigma- clones that retain the segment of mitochondrial DNA carrying OLI2. Based on the recombinants issued from crosses of the mutants with a triple drug-resistant strain and an analysis of the resistance markers present in sigma- clones that are effective in restoring a wild-type phenotype, the PHO1 locus has been placed in the segment of DNA located between PAR1 and OLI2.     

Erythrocyte membrane proteins and adenosine triphosphatase activity

Erythrocyte membrane was treated from outside with crude mold protease, and the membrane proteins were analyzed on acrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate. Band — III protein, in the region of an intermediate of Na-K-ATPase, was digested almost completely, although approximately 80% of the ATPase activity was retained by the treated membranes. It is concluded that band — III protein is not the Na-K-ATPase intermediate, although they co-electrophorese.     

MgATP-induced inhibition of the adenosine triphosphatase activity of the chloroform-released mitochondrial adenosine triphosphatase.

1. Preincubation of the ox heart chloroform-released mitochondrial ATPase with MgATP results in a time-dependent inhibition of ATPase activity. No re-activation occurs when MgATP remains in the preincubation medium. The enzyme activity returns when all the MgATP in the preincubation system has been hydrolysed. 2. The mechanism of the MgATP-induced inhibition was examined. Inhibition occurs on incubation with MgATP or other hydrolysable nucleotides. Incubation with MgADP or Pi does not cause any inhibition. Neither freshly bound adenine nucleotide nor Pi is associated with inhibited enzyme. The rate of MgATP-induced inhibition correlates with the rate of ATP hydrolysis in the preincubation medium. Changing the rate of ATP hydrolysis at a fixed concentration of ATP also changes the rate of MgATP-induced inhibition by the same proportion. The inhibition is thus related to the ATP-hydrolysis process itself. 3. We propose that intermediate enzyme species of the ATP-hydrolytic sequence can undergo a conformational change to form inhibited species. The kinetics of the inhibition suggest that a substrate-activation step is involved in ATP hydrolysis and MgATP-induced inhibition. 4. The effects of the nature of the preincubation medium on the process of MgATP-induced inhibition and its reversal were examined.     

Lipid requirement of the membrane sodium-plus-potassium ion-dependent adenosine triphosphatase system.

The dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) on lipid has been examined in a number of different ways, with the use of various preparations from kidney tissue. The main findings were as follows. (1) The ATPase activities of the preparations examined were closely correlated with their total phospholipid content. (2) Extraction of the ATPase with deoxycholate or Lubrol W, combined with suitable salt-fractionation and washing procedures, removed phospholipid, cholesterol and enzymic activity in parallel; but activity was completely lost before all lipid had been removed. (3) The loss of activity could not be attributed to inhibition by residual detergent. (4) No selective removal of any particular phospholipid class by detergent could be detected. (5) Consistent reactivation of the Lubrol-extracted enzymes was obtained by adding dispersions of exogenous phospholipid, but only some, bearing a net negative charge, such as phosphatidylserine and phosphatidylglycerol, were effective. (6) The degree of reactivation was correlated with the amount of residual activity remaining after lipid depletion. (7) Partial purification of the ATPase, giving a 50-fold increase in specific activity, was not accompanied by selective enhancement of any particular class of phospholipid. We conclude that although the ATPase is dependent on phospholipid, only the reactivation results provide evidence for specificity.     

Mitochondrial adenosine triphosphatase from yeast, Saccharomyces cerevisiae. Purification, subunit structure and kinetics.

1. A procedure for the purification of ATPase extracted by chloroform from baker's yeast (Saccharomyces cerevisiae) is reported. The yield based on submitochondrial particles was 55% and the purification was 100-fold. The isolated complex was homogenous as assessed by gel filtration, ion-exchange chromatography, sedimentation in sucrose gradient and in the analytical ultracentrifuge. The molecular weight determined by gel filtration was 400000 +/- 20000. Ultracentrifugation yielded s020,w = 12.50 +/- 0.13 S and the laser light scattering study gave a diffusion coeficient of D20w - 2.92 X 10(-7) cm2 s-1. The amino acid composition as well as absorption, fluorescence, and circular dichroism spectra, from which the helicity of 39% was evaluated, are given. 2. On polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, six components with molecular weights of 58500(alpha), 55000 (beta), 42000, 34000 (gamma), 10000(delta), and 8600 (epsilon) were observed with a stoichiometry of 3:3:1:1:1:1. The amino acid composition is given for alpha + beta and gamma as well as delta and epsilon components. 3. The maximum specific activity of the enzyme was 200 U/mg under the optimum conditions. The enzyme was inactivated by incubation at 0 degrees C and strongly inhibited by the antibiotic Dio-9 but not by oligomycin and N, N'-dicyclohexyl-carbodiimide. The effects of kinetic parameters and anions on the enzyme are reported. Two active sites for Mg-ATP with Km values of 0.045mM and 0.37mM and a single activie site for Mg-ITP with Km = 0.179mM were found. A study of the temperature dependence of the maximum activity revealed a straight line in the Arrhenius plots with an activation energy of 11.0 kcal/mol (= 46 kH/mol).     

The crystallization of beef heart mitochondrial adenosine triphosphatase.

Conditions are described under which crystals are formed with the ATPase enzyme from beef heart mitochondria. Enzyme activity is retained during the crystallization process. Some unit cell parameters have been determined by electron microscopy of negatively stained crystals; comparison with the unit cell crystalline matris inclusions indicates that such inclusions could be ATPase crystals.