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.     

Isolation and properties of anion-sensitive adenosine triphosphatase from erythrocyte membranes

Using the non-ionic detergent Triton X-100 and gel-chromatography, an anion-sensitive ATPase was isolated from rat and rabbit erythrocyte membranes. The ATPase preparations possess no Na, K- or Mg, Ca-ATPase activities. ATPase from rat erythrocyte membranes is made up of five subunits with molecular weights of 58 000 (alpha), 50 000 (beta), 36 000 (gamma), 25 000 (delta) and 12 000 (epsilon) and can be represented by the formula alpha 3 beta 3 gamma delta epsilon.     

Hydrodynamic properties of adenosine Ri receptors solubilized from rat cerebral-cortical membranes.

Adenosine Ri receptors and inhibitory guanine-nucleotide-regulatory components were solubilized from rat cerebral-cortical membranes with sodium cholate. (-)-N6-Phenylisopropyl[2,8-3H]adenosine [( 3H]PIA) binds with high affinity to the soluble receptors, which retain the pharmacological specificity of adenosine Ri receptors observed in membranes. The binding is regulated by bivalent cations and guanine nucleotides. Bivalent cations increase [3H]PIA binding by increasing both the affinity and the apparent number of receptors. Guanine nucleotides decrease agonist binding by increasing the dissociation of the ligand-receptor complex. Adenosine agonists stabilize the high-affinity form of the soluble receptor. The hydrodynamic properties of the adenosine receptor were determined with cholate extracts of membranes that were treated with [3H]PIA. Sucrose-gradient-centrifugation analysis indicates that the receptor has a sedimentation coefficient of 7.7 S. The receptor is eluted from Sepharose 6B columns with an apparent Stokes radius of 7.2 nm. Labelling of either sucrose-gradient or gel-filtration-column fractions with pertussis toxin and [32P]-NAD+ reveals that both the 41,000- and 39,000-Mr substrates overlap with the receptor activity. These studies suggest that the high-affinity adenosine-receptor-binding activity in the cholate extract represents a stable R1-N complex.     

Tightly bound magnesium in mitochondrial adenosine triphosphatase from beef heart.

Tightly bound magnesium was found in soluble, purified ATPase (F1) from beef heart mitochondria in the amount of 1 mol/mol of F1. Iron, zinc, cobalt, manganese, calcium, sodium, copper, and potassium were not tightly bound at stoichiometric levels. Removal of magnesium by chelating agents caused loss of ATPase activity. Removal of tightly bound nucleotide by gel filtration in 50% glycerol- or 60 mM K2SO4-containing buffers did not remove magnesium. Cold dissociation did release magnesium when complete denaturation was accomplished. The results suggest that magnesium is an integral part of F1, that it is required for activity, and that magnesium and nucleotides are tightly bound at separate sites. The idea that the tightly bound nucleotides are not complexed with cations suggests certain structural requirements at their binding sites which might account for the unusual properties of the sites.     

Activation of deoxycholate solubilized adenosine triphosphatase by ganglioside and asialoganglioside preparations.

ATPase was prepared from brain microsomes by solubilization with sodium deoxycholate and fractionated at different concentrations of ammonium sulfate. The Mg²⁺-ATPase was activated by total brain gangliosides, disialoganglioside, monosialo — ganglioside, hematoside, total brain gangliosides obtained from a patient with Tay-Sachs disease and asialoganglioside. The effect was smaller on Ca²⁺-ATPase and negligible on (Na⁺ + K⁺)-ATPase. Lactosyl-ceramide, glucosyl-ceramide, galactosyl-ceramide, ceramide and sialyl-lactose failed to produce activation.     

Mitochondrial adenosine triphosphatase from human placenta--purification and catalytic properties

1. The purification of ATPase (EC 3.6.1.3) from human placental mitochondria is described. The yield based on mitochondrial enzyme activity was about 70% and the purification was 380-fold. 2. The rate of Mg-ATP hydrolysis was 85 mumole per min per mg of protein under optimum conditions. 3. Nucleoside triphosphates were hydrolyzed by the purified enzyme at decreasing rates in the following order: GTP greater than ITP greater than ATP greater than epsilon-ATP greater than UTP greater than CTP in Tris-HCl buffer (pH 8.0), and in the order: ATP greater than GTP greater than or equal to ITP greater than epsilon-ATP greater than UTP greater than CTP in Tris-bicarbonate buffer at pH 8.0. 4. The values of kinetic parameters are reported. The ATPase reaction deviated from typical Michaelis-Menten kinetics in Tris-HCl buffer but not in Tris-bicarbonate. Eadie-Hofstee plots for Mg-ATP hydrolysis were biphasic in Tris-HCl (Km = 0.2 mM, 0.09 mM) and monophastic in Tris-becarbonate medium (Km = 0.16 mM). 5. In the presence of Mg-ITP or Mg-GTP as substrates no curvature of the reciprocal plots was observed. 6. The results presented reflect the fact that multiple conformations of the enzyme molecule do exist and are probably involved in its regulatory functions. 7. The existence of two kinetically distinct classes of catalytic sites and of an anion-binding site on the placental ATPase is proposed.     

The reversible delipidation of a solubilized sodium-plus-potassium ion-dependent adenosine triphosphatase from the salt gland of the spiny dogfish.

A microsomal fraction rich in Na+, K+-ATPase (sodium-plus-potassium ion-dependent adenosine triphosphatase) and the corresponding K+-dependent p-nitrophenyl phosphatase from the rectal salt gland of the spiny dogfish was solubilized by treatment with deoxycholate at high ionic strength. On gel filtration through Sepharose 6B, the ATPase apoenzyme could be separated, in apparently soluble form, from the tissue-fraction phospholipids and was almost free of enzymic activity (2% of the p-nitrophenyl phosphatase activity and 0.2% of the ATPase activity being recovered). On mixing the apoenzyme with an activator consisting of cooked ox brain, a large proportion of the original enzymic activity was obtained. Specific activities of the re-activated enzyme were somewhat higher than in the material before gel filtration: values of 1300-1450 mumol and 250-290 mumol/h per mg of protein were obtained for the hydrolysis of ATP and of p-nitrophenyl phosphate respectively. The activity was inhibitible by ouabain.     

Properties of mitochondrial adenosine triphosphatase isolated from rat liver.

Soluble, oligomysin-insensitive ATPase was isolated from liver mitochondria by a new technique [Drahota and Houst?k 1977]. Study of the resultant enzyme preparation provided further evidence that the isolated protein displays properties typical of mitochondrial ATPase (specific activity about 100 U/mg, optimum pH 8.2, activation by various bivalent cations and reassociation with membranes deprived of ATPase).     

Anion-sensitive adenosine triphosphatase from membranes of rat red blood cells]

Anion-sensitive ATPase was solubilized from membranes to rat red blood cells. The effect of bicarhonate, sulfite and perchlorate on the activity of ATPase was studied. Close resemblance of the properties of ATPase of rat red blood cells and of mitochondrial ATPase was observed.     

The effect of phospholipase C on the activity of adenosine triphosphatase and acetylcholinesterase in synaptic membranes isolated from the cerebral cortex of squirrel monkey

A synaptic-membrane fraction rich in junctional components and Na-K ATPase and AChE activity was isolated from the cerebral cortex of the squirrel monkey. Incubation of membrane preparations with phospholipase C decreased the activity of Na-K ATPase by 50 per cent but had no effect on the activity of AChE. Analysis of the membrane fraction showed that phospholipase C cleaved both choline phosphoglyceride and the diacyl type of ethanolamine phosphoglyceride from membrane lipids. Addition of egg lecithin at low concentrations partially restored the activity of Na-K ATPase. Kinetic studies revealed that treatment with phospholipase C may produce a non-competitive type of inhibition as a result of the cleavage of a charged phosphorylated nitrogen base from membrane lipids.     

The partial reactions in the catalytic cycle of the calcium-dependent adenosine triphosphatase purified from erythrocyte membranes

A preparation of purified erythrocyte membrane ATPase whose activation by Ca2+ is or is not dependent on calmodulin depending on the enzyme dilution was used in the low dilution state for these studies. In appropriate conditions, the purified ATPase in the absence of calmodulin exhibited a Ca2+ concentration dependence identical to that of the native enzyme in the erythrocyte membrane ghost in the presence of calmodulin. Accordingly, an apparent Kd approximately equal to 1 X 10(-7) M was derived for cooperative calcium binding to the activating and transport sites of the nonphosphorylated enzyme. The kinetics of enzyme phosphorylation in the transient state following addition of ATP to enzyme activated with calcium were then resolved by rapid kinetic methods, demonstrating directly that phosphoenzyme formation precedes Pi production, consistent with the phosphoenzyme role as an intermediate in the catalytic cycle. Titration of a low affinity site (Kd approximately equal to 2 X 10(-3) M) with calcium produced inhibition of phosphoenzyme cleavage and favored reversal of the catalytic cycle, indicating that calcium dissociation from the transport sites precedes hydrolytic cleavage of the phosphoenzyme. The two different calcium dissociation constants of the nonphosphorylated and phosphorylated enzyme demonstrate that a phosphorylation-induced reduction of calcium affinity is the basic coupling mechanism of catalysis and active transport, with an energy expenditure of approximately 6 kcal/mol of calcium in standard conditions. From the kinetic point of view, a rate-limiting step is identified with the slow dissociation of calcium from the phosphoenzyme; another relatively slow step following hydrolytic cleavage and preceding recycling of the enzyme is suggested by the occurrence of a presteady state phosphoenzyme overshoot.