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Purification, properties and substrate specificity of adenosine triphosphate sulphurylase from spinach leaf tissue.

ABSTRACT: 1. ATP sulphurylase was purified up to 1000-fold from spinach leaf tissue. Activity was measured by sulphate-dependent [(32)P]PP(i)-ATP exchange. The enzyme was separated from Mg(2+)-requiring alkaline pyrophosphatase (which interferes with the PP(i)-ATP-exchange assay) and from other PP(i)-ATP-exchange activities. No ADP sulphurylase activity was detected. 2. Sulphate was the only form of inorganic sulphur that catalysed PP(i)-ATP exchange; K(m) (sulphate) was 3.1mm, K(m) (ATP) was 0.35mm and the pH optimum was 7.5-9.0. The enzyme was insensitive to thiol-group reagents and required either Mg(2+) or Co(2+) for activity. 3. The enzyme catalysed [(32)P]PP(i)-dATP exchange; K(m) (dATP) was 0.84mm and V (dATP) was 30% of V (ATP). Competition between ATP and dATP was demonstrated. 4. Selenate catalysed [(32)P]PP(i)-ATP exchange and competed with sulphate; K(m) (selenate) was 1.0mm and V (selenate) was 30% of V (sulphate). No AMP was formed with selenate as substrate. Molybdate did not catalyse PP(i)-ATP exchange, but AMP was formed. 5. Synthesis of adenosine 5'-[(35)S]sulphatophosphate was demonstrated by coupling purified ATP sulphurylase and Mg(2+)-dependent alkaline pyrophosphatase (also prepared from spinach) with [(35)S]sulphate and ATP as substrates; adenosine 5'-sulphatophosphate was not synthesized in the absence of pyrophosphatase. Some parameters of the coupled system are reported.

SUBMITTER: Shaw WH

PROVIDER: S-EPMC1178578 | biostudies-other | 1972 Mar

REPOSITORIES: biostudies-other

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The enzymology of adenosine triphosphate sulphurylase from spinach leaf tissue.

Project description: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.

| S-EPMC1166247 | biostudies-other

Adenosine diphosphate sulphurylase activity in leaf tissue.

Project description: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.

| S-EPMC1177721 | biostudies-other

Adenosine 5'-sulphatophosphate kinase activity in spinach leaf tissue.

Project description:1. An F(-)-insensitive 3'-nucleotidase was purified from spinach leaf tissue; the enzyme hydrolysed 3'-AMP, 3'-CMP and adenosine 3'-phosphate 5'-sulphatophosphate but not adenosine 5'-nucleotides nor PP(i). The pH optimum of the enzyme was 7.5; K(m) (3'-AMP) was approx. 0.8mm and K(m) (3'-CMP) was approx. 3.3mm. 3'-Nucleotidase activity was not associated with chloroplasts. Purified Mg(2+)-dependent pyrophosphatase, free from F(-)-insensitive 3'-nucleotidase, catalysed some hydrolysis of 3'-AMP; this activity was F(-)-sensitive. 2. Adenosine 5'-sulphatophosphate kinase activity was demonstrated in crude spinach extracts supplied with 3'-AMP by the synthesis of the sulphate ester of 2-naphthol in the presence of purified phenol sulphotransferase; purified ATP sulphurylase and pyrophosphatase were also added to synthesize adenosine 5'-sulphatophosphate. Adenosine 5'-sulphatophosphate kinase activity was associated with chloroplasts and was released by sonication. 3. Isolated chloroplasts synthesized adenosine 3'-phosphate 5'-sulphatophosphate from sulphate and ATP in the presence of a 3'-nucleotide; the formation of adenosine 5'-sulphatophosphate was negligible. In the absence of a 3'-nucleotide the synthesis of adenosine 3'-phosphate 5'-sulphatophosphate was negligible, but the formation of adenosine 5'-sulphatophosphate was readily detected. Some properties of the synthesis of adenosine 3'-phosphate 5'-sulphatophosphate by isolated chloroplasts are described. 4. Adenosine 3'-phosphate 5'-sulphatophosphate, synthesized by isolated chloroplasts, was characterized by specific enzyme methods, electrophoresis and i.r. spectrophotometry. 5. Isolated chloroplasts catalysed the incorporation of sulphur from sulphate into cystine/cysteine; the incorporation was enhanced by 3'-AMP and l-serine. It was concluded that adenosine 3'-phosphate 5'-sulphatophosphate is an intermediate in the incorporation of sulphur from sulphate into cystine/cysteine.

| S-EPMC1177844 | biostudies-other

Adenosine 5'-triphosphate sulphurylase from Saccharomyces cerevisiae.

Project description: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.

| S-EPMC1177733 | biostudies-other

Activation of selenate by adenosine 5'-triphosphate sulphurylase from Saccharomyces cerevisiae.

Project description: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.

| S-EPMC1164733 | biostudies-other

ADENOSINE 5'-TRIPHOSPHATE-ARGININE PHOSPHOTRANSFERASE FROM LOBSTER MUSCLE: PURIFICATION AND PROPERTIES.

Project description:1. A simple chromatographic method is described for the purification of arginine kinase from lobster (Homarus vulgaris) muscle. 2. Some physical properties and the effects on enzyme activity of ionic strength, pH, buffer salts, metal ions and substrates are reported. 3. The kinetic parameters, evaluated by variation of the concentration of one of the substrates, are dependent on the concentration of the other substrate. 4. The properties of the enzyme are discussed in relation to previous findings about phosphagen phosphotransferases.

| S-EPMC1206586 | biostudies-other

Cryo electron tomography of spinach leaf tissue

Project description: Not available

| EMPIAR-10302 | biostudies-other

Supplementation of exogenous adenosine 5'-triphosphate enhances mechanical properties of 3D cell-agarose constructs for cartilage tissue engineering.

Project description:Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.

| S-EPMC3761430 | biostudies-literature

Effects of adenine nucleotides and phosphate on adenosine triphosphate sulphurylase from Anabaena cylindrica.

Project description:Production of adenosine 5'-[35S]sulphatophosphate by a partially purified ATP sulphurylase from Anabaena cylindrica was inhibited by AMP, ADP and P1. Decreases in enzyme activity in the presence of these inhibitors were reversed by increasing the concentrations of ATP. The adenine nucleotides inhibited the enzyme competitively with respect to ATP. In the presence of P1, ATP showed a positive co-operative effect on enzyme activity. The inhibition by P1 was enhanced by increasing concentrations of MG2+. The effects of the adenine nucleotides and the interaction of P1 and Mg2+ on ATP sulphurylase activity are discussed in relation to the regulation of sulphate assimilation via the energy metabolism of the alga.

| S-EPMC1164770 | biostudies-other

Effects of adenine nucleotides on rice-root adenosine triphosphate sulphurylase activity in vitro.

Project description:The presence of ATP sulphurylase activity in the 30000g supernatant fraction of rice-root homogenate has been demonstrated. Studies of the effects of adenosine and its nucleotides on the enzymic activity showed that AMP activated but that ADP and adenosine inhibited the enzyme.

| S-EPMC1165619 | biostudies-other

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