Comparative enzymology of the adenosine triphosphate sulphurylases from leaf tissue of selenium-accumulator and non-accumulator plants

1. ATP sulphurylases were partially purified (20-40-fold) from leaf tissue of Astragalus bisulcatus, Astragalus racemosus (selenium-accumulator species) and Astragalus hamosus and Astragalus sinicus (non-accumulator species). Activity was measured by sulphate-dependent PP(i)-ATP exchange. The enzymes were separated from pyrophosphatase and adenosine triphosphatase activities. The properties of the Astragalus ATP sulphurylases were similar to the spinach enzyme. 2. The ATP sulphurylases from both selenium-accumulator and non-accumulator species catalysed selenate-dependent PP(i)-ATP exchange; selenate competed with sulphate. The ratio of V(selenate)/V(sulphate) and K(m)(selenate)/K(m)(sulphate) was approximately the same for the enzyme from each species. 3. Sulphate-dependent PP(i)-ATP exchange was inhibited by ADP, chlorate and nitrate. The kinetics of the inhibition for each enzyme were consistent with an ordered reaction mechanism, in which ATP is the first substrate to react with the enzyme and PP(i) is the first product released. 4. Synthesis of adenosine 5'-[(35)S]sulphatophosphate from [(35)S]sulphate was demonstrated by coupling the Astragalus ATP sulphurylases with Mg(2+)-dependent pyrophosphatase; the reaction was inhibited by selenate. An analogous reaction using [(75)Se]selenate as substrate could not be demonstrated.     

Adenosine diphosphate sulphurylase activity in leaf tissue

1. A new method is described for the assay of ADP sulphurylase. The method involves sulphate-dependent [(32)P]P(i)-ADP exchange; the method is simpler, more sensitive and more direct than the method involving adenosine 5'-sulphatophosphate-dependent uptake of P(i). 2. ADP sulphurylase activity was demonstrated in crude extracts of leaf tissue from a range of plants. Crude spinach extract catalysed the sulphate-dependent synthesis of [(32)P]ADP from [(32)P]P(i); spinach extracts did not catalyse sulphate-dependent AMP-P(i), ADP-PP(i) or ATP-P(i) exchange under standard assay conditions. ADP sulphurylase activity in spinach leaf tissue was associated with chloroplasts and was liberated by sonication. 3. Some elementary kinetics of crude spinach leaf and purified yeast ADP sulphurylases in the standard assay are described; addition of Ba(2+) was necessary to minimize endogenous P(i)-ADP exchange of the yeast enzyme and crude extracts of winter-grown spinach. 4. Spinach leaf ADP sulphurylase was activated by Ba(2+) and Ca(2+); Mg(2+) was ineffective. The yeast enzyme was also activated by Ba(2+). The activity of both enzymes decreased with increasing ionic strength. 5. Purified yeast and spinach leaf ADP sulphurylases were sensitive to thiol-group reagents and fluoride. The pH optimum was 8. ATP inhibited sulphate-dependent P(i)-ADP exchange. Neither selenate nor molybdate inhibited sulphate-dependent P(i)-ADP exchange and crude spinach extracts did not catalyse selenate-dependent P(i)-ADP exchange. 6. The presence of ADP sulphurylase activity jeopardizes the enzymic synthesis of adenosine 5'-sulphatophosphate from ATP and sulphate with purified ATP sulphurylase and pyrophosphatase.     

The distribution of l-asparagine synthetase in the principal organs of several mammalian and avian species

1. Sulphate-dependent PP(i)-ATP exchange, catalysed by purified spinach leaf ATP sulphurylase, was correlated with the concentration of MgATP(2-) and MgP(2)O(7) (2-); ATP sulphurylase activity was not correlated with the concentration of free Mg(2+). 2. Sulphate-dependent PP(i)-ATP exchange was independent of PP(i) concentration, but dependent on the concentration of ATP and sulphate. The rate of sulphate-dependent PP(i)-ATP exchange was quantitatively defined by the rate equation applicable to the initial rate of a bireactant sequential mechanism under steady-state conditions. 3. Chlorate, nitrate and ADP inhibited the exchange reaction. The inhibition by chlorate and nitrate was uncompetitive with respect to ATP and competitive with respect to sulphate. The inhibition by ADP was competitive with respect to ATP and non-competitive with respect to sulphate. 4. ATP sulphurylase catalysed the synthesis of [(32)P]ATP from [(32)P]PP(i) and adenosine 5'-sulphatophosphate in the absence of sulphate; some properties of the reaction are described. Enzyme activity was dependent on the concentration of PP(i) and adenosine 5'-sulphatophosphate. 5. The synthesis of ATP from PP(i) and adenosine 5'-sulphatophosphate was inhibited by sulphate and ATP. The inhibition by sulphate was non-competitive with respect to PP(i) and adenosine 5'-sulphatophosphate; the inhibition by ATP was competitive with respect to adenosine 5'-sulphatophosphate and non-competitive with respect to PP(i). It was concluded that the reaction catalysed by spinach leaf ATP sulphurylase was ordered; expressing the order in the forward direction, MgATP(2-) was the first product to react with the enzyme and MgP(2)O(7) (2-) was the first product released. 6. The expected exchange reaction between sulphate and adenosine 5'-sulphatophosphate could not be demonstrated.     

The effect of starvation on insulin secretion and glucose metabolism in mouse pancreatic islets

1. Crude extracts of seeds of Pinus radiata catalysed acetate-, propionate-, n-butyrate- and n-valerate-dependent PP(i)-ATP exchange in the presence of MgCl(2), which was apparently due to a single enzyme. Propionate was the preferred substrate. Crude extracts did not catalyse medium-chain or long-chain fatty acid-dependent exchange. 2. Ungerminated dry seeds contained short-chain fatty acyl-CoA synthetase activity. The activity per seed was approximately constant for 11 days after imbibition and then declined. The enzyme was located only in the female gametophyte tissue. 3. The synthetase was purified 70-fold. 4. Some properties of the enzyme were studied by [(32)P]PP(i)-ATP exchange. K(m) values for acetate, propionate, n-butyrate and n-valerate were 4.7, 0.21, 0.33 and 2.1mm respectively. Competition experiments between acetate and propionate demonstrated that only one enzyme was involved and confirmed that the affinity of the enzyme for propionate was greater than that for acetate. CoA inhibited fatty acid-dependent PP(i)-ATP exchange. The enzyme catalysed fatty acid-dependent [(32)P]PP(i)-dATP exchange. 5. The enzyme also catalysed the fatty acyl-AMP-dependent synthesis of [(32)P]ATP from [(32)P]PP(i). Apparent K(m) (acetyl-AMP) and apparent K(m) (propionyl-AMP) were 57mum and 7.5mum respectively. The reaction was inhibited by AMP and CoA. 6. Purified enzyme catalysed the synthesis of acetyl-CoA and propionyl-CoA. Apparent K(m) (acetate) and apparent K(m) (propionate) were 16mm and 7.5mm respectively. The rate of formation of acetyl-CoA was enhanced by pyrophosphatase. 7. It was concluded that fatty acyl adenylates are intermediates in the formation of the corresponding fatty acyl-CoA.     

Tissue distribution of S-adenosylmethionine and S-adenosylhomocysteine in the rat. Effect of age, sex and methionine administration on the metabolism of S-adenosylmethionine, S-adenosylhomocysteine and polyamines

In the presence of ATP and Mg2+, ATP sulphurylase from Saccharomyces cerevisiae catalysed the conversion of selenate into a compound with the electrophoretic and acid-lability properties of adenosine 5'-sulphatophosphate. Structural characterization, involving extensive purification of adenosine 5'-selenophosphate, proved impossible. However, we showed ATP-, Mg2+- and ATP sulphurylase-dependent, and inorganic pyrophosphatase-stimulated, production of elemental selenium from selenate in the presence of GSH (reduced glutathione). Since selenate was not reduced by GSH, this reaction proved that ATP sulphurylase had formed an active selenate. The enzyme catalysed formation of elemental selenium had the same kinetics and GSH-dependency as the non-enzymic reduction of selenite to elemental selenium by GSH. In the presence of inorganic pyrophosphatase, 2 mol of Pi was released for each mol of 'active selenate' formed. This was shown by a spectrophotometric assay for elemental selenium. The observed reactivity with thiols and the instability of the enzymic product were those predicted for selenium anhydrides. By analogy with the chemistry of sulphur, the product of the thiolytic cleavage of a selenium anhydride would be converted into selenite. The selenite would then be reduced by the thiol to elemental selenium. We conclude that ATP sulphurylase can catalyse the formation of adenosine 5'-selenophosphate. The anhydride can be reduced by thiols in a manner similar to the reduction of selenite. These results probably explain the ability of mammals, lacking a sulphate reductase system, to incorporate selenium from selenate into seleno-amino acids.     

The enzymology of short-chain fatty acyl-coenzyme A synthetase from seeds of Pinus radiata. Kinetic studies and a proposed reaction mechanism

1. Short-chain fatty acyl-CoA synthetase from seeds of Pinus radiata was examined by acetate- and propionate-dependent PP(i)-ATP exchange. Reaction mixtures came to equilibrium almost instantly as judged by rates of exchange and analysis of an incubation mixture. 2. The activity of the enzyme was correlated with the concentration of MgP(2)O(7) (2-) but not with the concentration of Mg(2+), as judged by PP(i)-ATP exchange and fatty acyl AMP-dependent synthesis of ATP in the presence of PP(i). In PP(i)-ATP exchange assays, no clear relationship between activity and any single species of ATP was apparent. 3. High concentrations of fatty acid inhibited PP(i)-ATP exchange. PP(i)-dATP exchange was less than PP(i)-ATP exchange at low concentrations of fatty acid, but at higher concentrations PP(i)-dATP exchange exceeded PP(i)-ATP exchange. The rate of synthesis of fatty acyl-CoA in the presence of dATP was less than with ATP. 4. ATP and propionate inhibited the synthesis of ATP from propionyl-AMP and PP(i). The inhibition by ATP was competitive with respect to propionyl-AMP and non-competitive with respect to PP(i). The inhibition by propionate was non-competitive with respect to propionyl-AMP and PP(i). 5. AMP was a competitive inhibitor of propionyl-AMP-dependent synthesis of ATP and competitively inhibited propionate-dependent PP(i)-ATP exchange when ATP was the variable substrate. 6. It was concluded that the first partial reaction catalysed by the enzyme is ordered; ATP is the first substrate to react with the enzyme and PP(i) is probably the only product released.     

Microsomal palmitoyl-coenzyme A synthetase from rat liver. Partial and exchange reactions

The partial and exchange reactions of long-chain fatty acid activation were determined by using purified microsomal long-chain fatty acyl-CoA synthetase (EC 6.2.1.3). No significant ATP formation from palmitoyl-AMP and PP(i), nor palmitoyl-AMP-dependent CoA disappearance could be demonstrated. Similarly, no palmitate-dependent [(32)P(2)]PP(i)-ATP exchange was catalysed by the pure enzyme. The above partial and exchange reactions were, however, catalysed by the parent microsomal fraction at a rate similar to that of the overall reaction. The implications of these results are discussed.     

Adenosine 5′-triphosphate sulphurylase from Saccharomyces cerevisiae

1. ATP sulphurylase from Saccharomyces cerevisiae was purified 140-fold by using heat treatment, DEAE-cellulose chromatography and Sepharose 6B gel filtration. 2. The enzyme was stable at -15 degrees C, optimum reaction velocity was between pH7.0 and 9.0, and the activation energy was 62kJ/mol (14.7kcal/mol). 3. The substrate was shown to be the MgATP(2-) complex, free ATP being inhibitory. 4. Double-reciprocal plots from initial-velocity studies were intersecting and the K(m) of each substrate was determined at infinite concentration of the other (K(m) MgATP(2-), 0.07mm; MoO(4) (2-), 0.17mm). 5. Radio-isotopic exchange between the substrate pairs, adenosine 5'-[(35)S]sulphatophosphate and SO(4) (2-), (35)SO(4) (2-) and adenosine 5'-sulphatophosphate, occurred only in the presence of either MgATP(2-) or PP(i). This suggests, along with the initial-velocity data, a sequential reaction mechanism in which both substrates bind before any product is released. 6. The enzyme reaction was specific for ATP and was not inhibited by l-cysteine, l-methionine, SO(3) (2-), S(2)O(3) (2-) (all 2mm) nor by p-chloromercuribenzoate (1mm). 7. Competitive inhibition of the enzyme with respect to MoO(4) (2-) was produced by SO(4) (2-) (K(i)=2.0mm) and non-competitive inhibition by sulphide (K(i)=3.4mm). 8. Adenosine 5'-sulphatophosphate inhibited strongly and concentrations as low as 0.02mm altered the normal hyperbolic velocity-substrate curves with both MgATP(2-) and MoO(4) (2-) to sigmoidal forms.     

Some properties of adenosine kinase from Ehrlich ascites-tumour cells

1. Adenosine kinase was measured in dialysed extracts from Ehrlich ascites-tumour cells by a chromatographic procedure. 2. In the absence of added Mg(2+) the K(m) values for ATP and adenosine were 0.22mm and 2.8mum respectively. 3. The maximum velocity of adenosine kinase with free ATP was about three times that with the Mg(2+)-ATP complex. Free Mg(2+) was a non-competitive inhibitor of the reaction. A small amount of added Mg(2+), Mn(2+) or Ca(2+) was required for maximum adenosine kinase activity after cation bound to the enzyme had been released by treatment with p-chloromercuribenzoate and then removed by dialysis. 4. GTP, ITP, deoxy-ATP, deoxy-GTP, CTP, xanthosine triphosphate, UTP and thymidine triphosphate could partially or completely replace ATP as a phosphate donor. 5. The reaction of ATP with adenosine kinase was competitively inhibited by AMP, GMP, IMP, ADP, deoxy-ADP and IDP (K(i) 0.2, 1.1, 5.9, 1.2, 0.5 and 0.78mm respectively). Enzymic activity was markedly affected by the relative concentrations of AMP, ADP and ATP in assay mixtures. 6. The results are discussed in terms of possible mechanisms regulating the rate of adenosine kinase in vivo.     

Cloning of the ATP sulphurylase gene of Schizosaccharomyces pombe by functional complementation

The ATP sulphurylase gene of Schizosaccharomyces pombe has been cloned by complementation of cysteine auxotrophy of a selenate-resistant mutant, which supposedly had a defect in ATP sulphurylase. A sulphate nonutilizing (cysteine auxotrophic) and selenate-resistant mutant of S. pombe was transformed with a wild-type S. pombe genomic library and sulphate-utilizing clones were isolated. The open reading frame encoding the ATP sulphurylase enzyme was found to be responsible for the restoration of sulphate assimilation. Transformants became as sensitive for selenate as the wild-type strain and produced a comparable amount of ATP sulphurylase as the prototrophic strains. The cloned ATP sulphurylase gene (sua1) proved to be an efficient selection marker in an ARS vector, when different isogenic or nonisogenic S. pombe selenate-resistant mutants were used as cloning hosts. Complementation of sua1- mutations by sua1-bearing multicopy vectors functions as a useful dual positive and negative selection marker. The cloned sua1 gene also complemented the met3 (ATP sulphurylase deficient) mutation in Saccharomyces cerevisiae.     

The electrophilic and leaving group phosphates in the catalytic mechanism of yeast pyrophosphatase

Binding of pyrophosphate or two phosphate molecules to the pyrophosphatase (PPase) active site occurs at two subsites, P1 and P2. Mutations at P2 subsite residues (Y93F and K56R) caused a much greater decrease in phosphate binding affinity of yeast PPase in the presence of Mn(2+) or Co(2+) than mutations at P1 subsite residues (R78K and K193R). Phosphate binding was estimated in these experiments from the inhibition of ATP hydrolysis at a sub-K(m) concentration of ATP. Tight phosphate binding required four Mn(2+) ions/active site. These data identify P2 as the high affinity subsite and P1 as the low affinity subsite, the difference in the affinities being at least 250-fold. The time course of five "isotopomers" of phosphate that have from zero to four (18)O during [(18)O]P(i)-[(16)O]H(2)O oxygen exchange indicated that the phosphate containing added water is released after the leaving group phosphate during pyrophosphate hydrolysis. These findings provide support for the structure-based mechanism in which pyrophosphate hydrolysis involves water attack on the phosphorus atom located at the P2 subsite of PPase.     

Nucleoside pyrophosphatase activity associated with pig kidney alkaline phosphatase

1. A study was made of the hydrolysis, at pH9.0, of ATP and ADP catalysed by pig kidney alkaline phosphatase. Both of these nucleoside pyrophosphates are substrates for the enzyme; K(m) values are 4x10(-5)m for ATP and 6.3x10(-5)m for ADP. V(max.) for ADP is approximately double that of ATP. 2. Above 0.1mm approximately, both ATP and ADP are inhibitory, but the inhibition is reversible by the addition of Mg(2+) ions to form MgATP(2-) or MgADP(-) complexes. The complexes, besides being non-inhibitory, are also substrates for the enzyme with K(m) values identical with those of the respective free nucleotides. 3. Mg(2+) ions are inhibitory when present in excess of ATP or ADP. The degree of inhibition is greater with ATP as substrate, but with both ATP and ADP a mixed competitive-non-competitive type of inhibition is observed. 4. It is suggested that under normal conditions the enzyme is inhibited by cellular concentrations of ATP plus ADP but that an increase in the concentration of Mg(2+) ions stimulates activity by relieving nucleoside pyrophosphate inhibition. The properties may be of importance in the regulation of the transport of bivalent cations.     

Characterization and properties of the pyruvate phosphorylation system of Acetobacter xylinum

The enzyme responsible for the direct phosphorylation of pyruvate during gluconeogenesis in Acetobacter xylinum has been purified 46-fold from ultrasonic extracts and freed from interfering enzyme activities. The enzyme was shown to catalyze the reversible Mg(2+) ion-dependent conversion of equimolar amounts of pyruvate, adenosine triphosphate (ATP), and orthophosphate (P(i)) into phosphoenolpyruvate (PEP), adenosine monophosphate (AMP), and pyrophosphate (PP). The optimal pH for PEP synthesis was pH 8.2; for the reversal it was pH 6.5. The ratio between the initial rates of the reaction in the forward and reverse directions was 5.1 at pH 8.2 and 0.45 at pH 6.5. The apparent K(m) values of the components of the system in the forward reaction were: pyruvate, 0.2 mm; ATP, 0.4 mm; P(i), 0.8 mm; Mg(2+), 2.2 mm; and for the reverse reaction: PEP, 0.1 mm; AMP, 1.6 mum; PP, 0.067 mm; Mg(2+), 0.87 mm. PEP formation was inhibited by AMP and PP. The inhibition by AMP was competitive with regard to ATP (K(i) = 0.2 mm). The reverse reaction was inhibited competitively by ATP and noncompetitively by pyruvate. The enzyme was strongly inhibited by p-hydroxymercuribenzoate. The inhibition was reversed by dithiothreitol and glutathione. The properties of the enzyme are discussed in relation to the regulation of the opposing enzymatic activities involved in the interconversion of PEP and pyruvate in A. xylinum.     

The active centre of triose phosphate isomerase

1. Cystamine (2,2'-diaminodiethyl disulphide) caused an unmasking of mitochondrial adenosine triphosphatase and a leakage of Mg(2+) from the mitochondria, and decreased the stimulation of adenosine triphosphatase by 2,4-dinitrophenol. When Mg(2+) was added, cystamine potentiated the activation of adenosine triphosphatase by 2,4-dinitrophenol. 2. Cystamine was without effect on the adenosine triphosphatase of disrupted mitochondria. 3. Cystamine was moderately potent as an uncoupling agent and as an inhibitor of the [(32)P]P(i)-ATP exchange reaction. 4. Cysteamine (2-aminoethanethiol) was without the above effects, when special precautions were taken to counteract its autoxidation. 5. The effects of cystamine should probably be ascribed to its disulphide group, since the diamine cadaverine protected slightly against the loss of Mg(2+) and the decrease of 2,4-dinitrophenol-stimulated adenosine-triphosphatase activity caused by aging of the mitochondria. It is suggested that cystamine acts by a breakdown of mitochondrial permeability barriers.     

Evidence for a Mg2+-induced conformational change at the ATP-binding site of (Na+ + K+)-ATPase demonstrated with a photoreactive ATP-analogue

1. The 3'-ribosyl ester of ATP with 2-nitro-4-azidophenyl propionic acid has been prepared and its ability to act as a photoaffinity label of (Na+ + K+)-ATPase has been tested. 2. In the dark 3'-O-[3-(2-nitro-4-azidophenyl)-propionyl]adenosine triphosphate (N3-ATP) is a substrate of (Na+ + K+)-ATPase and a competitive inhibitor of ATP hydrolysis. 3. Upon irradiation by ultraviolet light, N3-ATP photolabels the high-affinity ATP-binding site and is covalently attached to the alpha-subunit and an approximately 12000-Mr component. 4. Photolabeling of the alpha-subunit by N3-ATP irreversibly inactivates (Na+ + K+)-ATPase. 5. Photoinactivation is strictly Mg2+-dependent. Na+ enhances the inactivation. ATP or ADP and K+ protect the enzyme against inactivation. 6. Mg2+, in concentrations required for photoinactivation, protects (Na+ + K+)-ATPase against inactivation by tryptic digestion under controlled conditions. 7. It is assumed that a conformational change of the ATP-binding site of (Na+ + K+)-ATPase occurs upon binding of Mg2+ to a low-affinity site.     

The concentration and control of cytoplasmic free inorganic pyrophosphate in rat liver in vivo

The concentration of cytoplasmic free pyrophosphate was calculated in freeze-clamped livers of rats from the measured concentration of reactants and K(eq.) of the UDP-glucose pyrophosphorylase reaction (UDP-alpha-d-glucose 1-phosphate uridylyltransferase, EC 2.7.7.9). The K(eq.) of the UDP-glucose pyrophosphorylase reaction was redetermined at 38 degrees C, pH7.0, I=0.25mol/l and free [Mg(2+)]=1mm, and was 4.55 in the direction of glucose 1-phosphate formation. The activity of UDP-glucose pyrophosphorylase in rat liver was between 46 and 58mumol of glucose 1-phosphate formed/min per g fresh wt. in the four dietary conditions studied. A fluorimetric assay with enzymic cycling was developed for the measurement of glucose 1-phosphate in HClO(4) extracts of rat liver. The calculated free cytoplasmic PP(i) concentration in nmol/g fresh wt. of liver was 2.3+/-0.3 in starved, 3.8+/-0.4 in fed, 4.9+/-0.6 in meal-fed and 5.2+/-0.4 in sucrose-re-fed animals. These values agree well with recently determined direct measurements of total PP(i) in rat liver and suggest that there is not a large amount of bound or metabolically inert PP(i) in rat liver. The cytoplasmic [ATP]/[AMP][PP(i)] ratio is 10(3) times the cytoplasmic [ATP]/[ADP][P(i)] ratio and varies differently with dietary state. The reaction PP(i)+H(2)O-->2P(i) catalysed by inorganic pyrophosphatase (EC 3.6.1.1) does not attain near-equilibrium in vivo. PP(i) should be considered as one of the group of small inorganic ions which is metabolically active and capable of exerting a controlling function in a number of important metabolic reactions.     

ATP binding properties of the nucleotide-binding folds of SUR1

Pancreatic beta cell ATP-sensitive potassium (K(ATP)) channels regulate glucose-induced insulin secretion. The activity of the K(ATP) channel, composed of SUR1 and Kir6.2 subunits, is regulated by intracellular ATP and ADP, but the molecular mechanism is not clear. To distinguish the ATP binding properties of the two nucleotide-binding folds (NBFs) of SUR1, we prepared antibodies against NBF1 and NBF2, and the tryptic fragment of SUR1 was immunoprecipitated after photoaffinity labeling with 8-azido-[(32)P]ATP. The 35-kDa fragment was strongly labeled with 5 microM 8-azido-[(32)P]ATP even in the absence of Mg(2+) and was immunoprecipitated with the antibody against NBF1. The 65-kDa fragment labeled with 100 microM 8-azido-[alpha-(32)P]ATP in the presence of Mg(2+) was immunoprecipitated with anti-NBF2 and anti-C terminus antibodies. These results indicate that NBF1 of SUR1 binds 8-azido-ATP strongly in a magnesium-independent manner and that NBF2 binds 8-azido-ATP weakly in a magnesium-dependent manner. Furthermore, the 65-kDa tryptic fragment was not photoaffinity-labeled with 8-azido-[gamma-(32)P]ATP at 37 degrees C, whereas the 35-kDa tryptic fragment was, suggesting that NBF2 of SUR1 may have ATPase activity and that NBF1 has none or little.     

Pig liver pyruvate carboxylase. The reaction pathway for the carboxylation of pyruvate

1. The reaction pathway for the carboxylation of pyruvate, catalysed by pig liver pyruvate carboxylase, was studied in the presence of saturating concentrations of K(+) and acetyl-CoA. 2. Free Mg(2+) binds to the enzyme in an equilibrium fashion and remains bound during all further catalytic cycles. MgATP(2-) binds next, followed by HCO(3) (-) and then pyruvate. Oxaloacetate is released before the random release, at equilibrium, of P(i) and MgADP(-). 3. This reaction pathway is compared with the double displacement (Ping Pong) mechanisms that have previously been described for pyruvate carboxylases from other sources. The reaction pathway proposed for the pig liver enzyme is superior in that it shows no kinetic inconsistencies and satisfactorily explains the low rate of the ATP[unk][(32)P]P(i) equilibrium exchange reaction. 4. Values are presented for the stability constants of the magnesium complexes of ATP, ADP, acetyl-CoA, P(i), pyruvate and oxaloacetate.     

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.     

Purification and characterization of (Na+ + K+)-ATPase from toad kidney.

This report describes the partial purification and the characteristics of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from an amphibian source. Toad kidney microsomes were solubilized with sodium deoxycholate and further purified by sodium dodecyl sulphate treatment and sucrose gradient centrifugation, according to the methods described by Lane et al. [(1973) J. Biol. Chem. 248, 7197--7200], J?rgensen [(1974) Biochim. Biophys. Acta 356, 36--52] and Hayashi et al. [(1977) Biochim. Biophys. Acta 482, 185--196]. (Na+ + K+)-ATPase preparations with specific activities up to 1000 mumol Pi/mg protein per h were obtained. Mg2+-ATPase only accounted for about 2% of the total ATPase activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed three major protein bands with molecular weights of 116 000, 62 000 and 26 000. The 116 000 dalton protein was phosphorylated by [gamma-32P]ATP in the presence of sodium but not in the presence of potassium. The 62 000 dalton component stained for glycoproteins. The Km for ATP was 0.40 mM, for Na+ 12.29 mM and for K+ 1.14 mM. The Ki for ouabain was 35 micron. Temperature activation curves showed two activity peaks at 37 degrees C and at 50 degrees C. The break in the Arrhenius plot of activity versus temperature appeared at 15 degrees C.