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.
D000244:Adenosine Diphosphate; D001464:Barium; D002118:Calcium; D002736:Chloroplasts; D005459:Fluorides; D007700:Kinetics; D008274:Magnesium; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010944:Plants / Q000201:enzymology*; D011755:Pyrophosphatases; D012441:Saccharomyces cerevisiae; D013431:Sulfates; D013439:Sulfhydryl Reagents; D013455:Sulfur; D013462:Sulfur Radioisotopes
D010710:Phosphates; D010761:Phosphorus Radioisotopes; D013431:Sulfates; D013439:Sulfhydryl Reagents; D013462:Sulfur Radioisotopes; D001464:Barium; D000244:Adenosine Diphosphate; D013455:Sulfur; D009713:Nucleotidyltransferases; D011755:Pyrophosphatases; D008274:Magnesium; D005459:Fluorides; D002118:Calcium