Location of an oligomycin-insensitive and magnesium ion-stimulated adenosine triphosphatase in rat spleen mitochondria.

1. When rat spleen mitochondria are incubated with oxidizable substrates, added MgCl2 (greater than 150 muM free concentration) markedly stimulates state-4 respiration and lowers both the respiratory control and ADP/O ratios; this effect is reversible on addition of excess of EDTA. 2. With [gamma-32P]ATP as substrate, an Mg2+-stimulated ATPase (adenosine triphosphate) was identified in the atractyloside-insensitive and EDTA-accessible space of intact rat spleen mitochondria. 3. Oligomycin has no effect on the activity of the Mg2+-stimulated ATPase at a concentration (2.0mug/mg of protein) that completely inhibits the atractyloside-sensitive reaction. Of the two ATPase activities, only the atracytoloside sensitive reaction is stimulated (approx. 40%) by dinitrophenol. 4. On digitonin fractionation the atractyloside-insensitive Mg2+-stimulated ATPase co-purifies with the outer membrane-fraction of rat spleen mitochondria, whereas (as expected) the atractylosidesensitive activity co-purifies with the inner-membrane plus matrix fraction. 5. Stoicheiometric amounts of ADP and Pi are produced as the end products of ATP hydrolysis by purified outer-membrane fragments; no significant AMP production is detected during the time-course of the reaction. 6. The outer-membrane ATPase is present in rat kidney cortex and heart mitochondria as well as in spleen, but is absent from rat liver, thymus, brain, lung, diaphragm and skeletal muscle.     

Oxidative phosphorylation in Escherichia coli K 12. Mutations affecting magnesium ion- or calcium ion-stimulated adenosine triphosphatase

1. Two mutants of Escherichia coli K 12 were isolated which, although able to grow on glucose, are unable to grow with succinate or d-lactate as the sole source of carbon. 2. Genetic mapping of these mutants showed that they both contain a mutation in a gene (designated uncA) mapping at about minute 73.5 on the E. coli chromosome. 3. The uncA(-) alleles were transferred by bacteriophage-mediated transduction into another strain of E. coli and the transductants compared with the parent strain to determine the nature of the biochemical lesion in the mutants. 4. The mutants gave low aerobic growth yields when grown on limiting concentrations of glucose, but oxidase activities in membranes from both the mutants and the normal strain were similar. 5. Measurement of P/O ratios with d-lactate as substrate indicated that a mutation in the uncA gene causes uncoupling of phosphorylation associated with electron transport. 6. Determination of the Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activities in the mutant and normal strains indicated that the uncA gene is probably the structural gene for Mg(2+),Ca(2+)-stimulated adenosine triphosphatase. 7. Mg(2+),Ca(2+)-stimulated adenosine triphosphatase therefore appears to be essential for oxidative phosphorylation in E. coli.     

Reconstitution of the energy-linked transhydrogenase activity in membranes from a mutant strain of Escherichia coli K12 lacking magnesium ion- or calcium ion-stimulated adenosine triphosphatase

1. We have isolated a mutant of Escherichia coli K12 (strain AN295) that forms de-repressed amounts of Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase. 2. The Mg²⁺,Ca²⁺-stimulated triphosphatase activity was separated from membrane preparations from strain AN295 by extraction with 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, resulting in a loss of the ATP-dependent transhydrogenase activity. The non-energy-linked transhydrogenase activity remained in the membrane residue. 3. The solubilized Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase activity from strain AN295 was partially purified by repeated gel filtration. The addition of the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase to the membrane residue from strain AN295 reactivated the ATP-dependent transhydrogenase activity. 4. Strain AN296, lacking Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase activity, was derived by transducing the mutant allele, uncA401, into strain AN295. The ATP-dependent transhydrogenase activity was lost but the non-energy linked transhydrogenase was retained. 5. The ATP-dependent transhydrogenase activity in membrane preparations from strain AN296 (uncA⁻) could not be re-activated by the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase from strain AN295. However, after extraction by 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, the ATP-dependent transhydrogenase activity could be re-activated by the addition of the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase from strain AN295 to the membrane residue from strain AN296 (uncA⁻).     

Properties of the sarcolemmal calcium ion-stimulated adenosine triphosphatase of hamster skeletal muscle

1. A sarcolemmal fraction was isolated from hamster hind-leg skeletal muscles by successive treatment with lithium bromide and potassium chloride. The membranous fraction was observed to contain a highly active Ca(2+)-stimulated ATPase (adenosine triphosphatase), a Mg(2+)-stimulated ATPase, and an Na(+)+K(+)-stimulated Mg(2+)-dependent ouabain-sensitive ATPase. 2. The Ca(2+)-stimulated ATPase activity was pH-dependent, the optimum being pH7.6. 3. Optimum activation of this enzyme was obtained with 3-4mm-Ca(2+) when 4mm-ATP was present as a substrate, and was not influenced by Na(+), K(+) or ouabain, whereas 2,4-dinitrophenol, sodium azide, oligomycin, sodium fluoride and ethanedioxybis(ethylamine)tetra-acetate were inhibitory. 4. The Ca(2+)-stimulated ATPase was markedly inhibited by thiol-blocking reagents, and cysteine was able to reverse this inhibition. 5. Various bivalent cations stimulated ATP hydrolysis by the sarcolemmal fraction in the following decreasing order of potency: Mg(2+), Ca(2+), Mn(2+), Co(2+), Sr(2+), Ba(2+), Zn(2+), Cu(2+).     

Preparation and general properties of a soluble adenosine triphosphatase from mitochondria

1. The purification of an adenosine triphosphatase present in aqueous extracts of acetone-dried ox-heart mitochondria is described. 2. No evidence was found for the presence of more than one protein having adenosine-triphosphatase activity in these extracts. 3. The enzyme is less stable at 0 degrees than at 25 degrees but is stabilized by glycerol. 4. The activity is dependent on the presence of Mg(2+) or certain other bivalent metal cations. 5. The adenosine-triphosphatase activity of the Mg(2+)-activated enzyme is enhanced by 2,4-dinitrophenol. 6. The kinetics of Mg(2+) activation indicate that the ATP-Mg(2+) complex is the important substrate: free ATP and Mg(2+) are inhibitory. 7. This preparation of mitochondrial adenosine triphosphatase has many properties in common with the adenosine triphosphatase coupling factor from mitochondria (Racker, 1961).     

Isolation and partial characterization of magnesium ion-and calcium ion-dependent adenosine triphosphatase activity from bovine brain microsomal fraction

Microsomal fraction was prepared by ultracentrifugation of homogenates of cortical tissue from bovine brains. The preparation displayed ATPase (adenosine triphosphatase) activity in the presence of Mg(2+) (6.4mumol of P(i)/h per mg of protein) and Ca(2+) (3.4mumol of P(i)/h per mg of protein). Kinetic analysis of the activation of the enzyme preparation by Ca(2+) resulted in the demonstration of two apparent K(m) values for Ca(2+) (6.0x10(-8)m and 1.2x10(-6)m). Treatment of the microsomal membranes with Triton X-100 resulted in solubilization of the ATPase, though with some loss of activity. The solubilized microsomal proteins were incorporated into liposomes. By incubation of the liposomes in media containing (45)Ca(2+) an ATP-dependent uptake of Ca(2+) was demonstrated. The solubilized preparation was subjected to preparative isoelectric focusing in granulated gel beds. Two distinct peaks of Mg(2+)- and Ca(2+)-dependent ATPase activity were observed at pH4.8 (peak 4.8) and at pH6.3 (peak 6.3). The material isolated in peaks 4.8 and 6.3 was focused in polyacrylamide gel with pH gradients. The material corresponding to peak 4.8 consisted of a single protein, whereas peak 6.3 contained one major and at least one minor protein. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis confirmed these results and indicated that the major component of peak 4.8 and the protein of peak 6.3 both had a molecular weight of 105000. The material in peaks 4.8 and 6.3 was assayed for ATPase activity in the presence of various concentrations of Ca(2+). Kinetic analysis of the results for peak 4.8 demonstrated an apparent K(m) value for Ca(2+) of 4.1x10(-8)m. The enzyme isolated at pH6.3 had an apparent K(m) value of 3.8x10(-6)m. However, when the material from peak 4.8 was incubated in the presence of 1mm-Mg(2+) the ATPase could not be activated by Ca(2+).     

Chemical modification of a cerebral sodium-plus-potassium ion-stimulated adenosine triphosphatase preparation

1. A particulate Na(+)+K(+)-stimulated adenosine triphosphatase preparation obtained by treatment of bovine cerebral microsomes with a sodium iodide reagent has been further treated with acid anhydrides likely to convert amino groups into acidic derivatives. 2. The extent of acylation of amino groups was determined by reaction of the remaining amino groups with 2,4,6-trinitrobenzenesulphonic acid. The unmodified preparation contains about 1.2 muequiv. of amino groups/mg of protein of which only about 0.5 muequiv. are accounted for by protein amino groups. Kinetics of the trinitrobenzenesulphonic acid reaction with the unmodified preparation are complex and are altered by ATP or ouabain. 3. The compounds examined cause loss of Na(+)+K(+)-stimulated adenosine triphosphatase activity when relatively few amino groups are modified but ATP was found to afford partial protection against inactivation by methylmaleic anhydride. Na(+)+K(+)-stimulated adenosine triphosphatase activity is partly restored to the dimethylmaleylated preparation by hydrolysis of the dimethylmaleyl-amide bonds but not if more than about 20% of the amino groups have been acylated. 4. Supernatants obtained by high-speed centrifugation of the dimethylmaleylated preparation contained up to 45% of the total protein with less than 10% of the total phospholipid. Methylmaleyl and benzenetricarboxylyl derivatives of the enzyme preparation behaved similarly but tetrafluorosuccinylated material was almost entirely deposited by centrifugation.     

Studies on the microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase system in rat ventral prostate

A Mg(2+)+Na(+)+K(+)-stimulated adenosine triphosphatase (ATPase) preparation was isolated from rat ventral prostate by flotation of microsomal membranes in high-density sucrose solutions. The reaction medium for optimum Na(+)+K(+)-stimulated ATPase activity was found to be: Na(+), 115mm; K(+), 7-10mm; Mg(2+), 3mm; ATP, 3mm; tris buffer, pH7.4 at 38 degrees , 20mm. The average DeltaP(i) (Mg(2+)+Na(+)+K(+) minus Mg(2+)+Na(+)) was 9mumoles/mg. of protein/hr., representing a 30% increase over the Mg(2+)+Na(+)-stimulated ATPase activity. At high concentrations, K(+) was inhibitory to the enzyme activity. Half-maximal inhibition of Na(+)+K(+)-stimulated ATPase activity was elicited by ouabain at 0.1mm. The preparation exhibited phosphatase activity towards ribonucleoside triphosphates other than ATP. However, stimulation of P(i) release by Na(+)+K(+) was observed only with ATP as substrate. The apparent K(m) for ATP for Na(+)+K(+)-stimulated activity was about 0.3x10(-3)m. Ca(2+) inhibited only the Na(+)+K(+)-stimulated ATPase activity. Mg(2+) could be replaced by Ca(2+) but then no Na(+)+K(+) stimulation of ATPase activity was noticed. The addition of testosterone or dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) in vitro at 0.1-10mum under a variety of experimental conditions did not significantly increase the Na(+)+K(+)-stimulated ATPase activity. The enzyme preparations from prostates of orchidectomized rats, however, exhibited a drastic decrease in the specific activity of Na(+)+K(+)-stimulated ATPase; these changes were prevented in the orchidectomized rats by injection of testosterone propionate.     

5-bromo-2''-deoxyuridine-stimulated calcium ion- or magnesium ion-dependent ecto-(adenosine triphosphatase) activity of cultured hamster cardiac cells.

1. Treatment of hamster heart cells in primary culture with 5-bromo-2'-deoxyuridine resulted in the greatly increased activity of a particulate Ca2+- or Mg2+-dependent ATPase (adenosine triphosphatase). 2. 5-Bromo-2'-deoxyuridine exerted these effects only when it was incorporated into cellular DNA, and then in a concentration-dependent manner. 3. Serially replated cells contained less of the activity (expressed as a function of total cell protein) than did the primary cultures, but the stimulation caused by 5-bromo-2'-deoxyuridine addition was much greater. 4. The affected enzyme was apparently localized in the plasma membrane of the cells with its active centre exposed to the outer environment [ecto-(ATPase) dependent on Ca2+ or Mg2+].5. The activity was unaffected by treatment with p-chloromercuriphenylsulphonate, ouabain andverapamil. 6. Ecto (5'-nucleotidase) activity was not increased by 5-bromo-2'-deoxyuridine treatment of cells, and ecto-(p-nitrophenyl phosphatase) activity was only slightly enhanced.     

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.     

Kinetic properties of soluble adenosine triphosphatase of escherichia coli

Summary Bound and solubilized ATPase from Escherichia coli show similar kinetic properties. The saturation curves for MgATP are hyperbolic with both preparations. The straight lines in the Line-weaver–Burk plot indicate that MgATP is the true substrate, that one molecule MgATP is bound per enzyme molecule, and that there is no cooperativity. Presence of EDTA leads to sigmoidal saturation curves. This effect could be reversed by adding MgCl₂ stoichiometrically to EDTA.Different results in other publications, especially in that ofCarreira andMuñoz can be explained as being primarily the consequence of complexing agent contaminations in the assay.     

The effects of calcium and magnesium ions on the adenosine triphosphatase and inosine triphosphatase activities of myosin A

1. The effects of Ca(2+) and Mg(2+) on the enzymic activity of myosin were studied with myosin preparations treated by the ion-exchange resin Chelex-100. A reaction mixture containing 0.05m-potassium chloride was chosen in which the effects of univalent ions such as K(+), Na(+) and Cl(-) do not change significantly with small variations in their concentrations. 2. The relationship between the rate of hydrolysis of ATP or ITP and the concentration of Ca(2+) suggests that a relatively weak binding of Ca(2+) either to myosin or to the substrate nucleotide is responsible for the activation of the enzymic activity. According to the experiments with an ultrafiltration technique, the binding of Ca(2+) to myosin proceeds in at least two steps, the first occurring at one site on every 500000 atomic mass units of myosin with an apparent association constant, K(app.), 1.3x10(6)m(-1), and the second seeming to be so weak that its binding parameters cannot be determined by the method used. The first type of Ca(2+) binding is not observable with N-ethylmaleimide-modified myosin, yet this modified myosin shows activation by Ca(2+) of its adenosine triphosphatase and inosine triphosphatase. 3. The inhibition by Mg(2+) can be related to a binding reaction of Mg(2+) with myosin having K(app.) approximately 10(6)m(-1). Mg(2+) replaces the Ca(2+) bound tightly to myosin. The K(app.) for Mg(2+)-myosin binding calculated by assuming a competition between Ca(2+) and Mg(2+) for the same site is 2.1x10(5)-3.0x10(5)m(-1). When myosin is modified with a thiol reagent (p-mercuribenzoate) at a certain ratio to myosin, the inhibition by Mg(2+) becomes unobservable. 4. The behaviour of the hydrolytic activity of myosin on ATP or ITP in the presence of both Ca(2+) and Mg(2+) is consistent with the explanation that the inhibition by Mg(2+) is due to the tight binding of Mg(2+) to myosin, whereas the activation by Ca(2+) is caused either by a weak binding of Ca(2+) to myosin or by CaATP(2-) or by both.