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Adenosine diphosphate sulphurylase activity in leaf tissue.

ABSTRACT: 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.

SUBMITTER: Burnell JN

PROVIDER: S-EPMC1177721 | biostudies-other | 1973 Jul

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

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

Project description: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.

| S-EPMC1178578 | 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

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

Adenosine 5'-pyrophosphate sulphurylase in baker's yeast.

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| S-EPMC1173816 | biostudies-other

Adenosine 5'-triphosphate sulphurylase from Saccharomyces cerevisiae.

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| S-EPMC1177733 | biostudies-other

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P2X receptors regulate adenosine diphosphate release from hepatic cells.

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Poly Adenosine Diphosphate-Ribose Polymerase (PARP) Inhibitors in Pancreatic Cancer.

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Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in Escherichia coli.

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| S-EPMC35479 | biostudies-literature

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