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

1972 ◽  
Vol 127 (1) ◽  
pp. 237-247 ◽  
Author(s):  
W. H. Shaw ◽  
J. W. Anderson
Keyword(s):  
Substrate Specificity ◽  
Adenosine Triphosphate ◽  
Leaf Tissue ◽  
Coupled System ◽  
Thiol Group ◽  
Ph Optimum ◽  
Spinach Leaf ◽  
Inorganic Sulphur ◽  
Atp Sulphurylase

1. ATP sulphurylase was purified up to 1000-fold from spinach leaf tissue. Activity was measured by sulphate-dependent [32P]PPi–ATP exchange. The enzyme was separated from Mg2+-requiring alkaline pyrophosphatase (which interferes with the PPi–ATP-exchange assay) and from other PPi–ATP-exchange activities. No ADP sulphurylase activity was detected. 2. Sulphate was the only form of inorganic sulphur that catalysed PPi–ATP exchange; Km (sulphate) was 3.1mm, Km (ATP) was 0.35mm and the pH optimum was 7.5–9.0. The enzyme was insensitive to thiol-group reagents and required either Mg2+ or Co2+ for activity. 3. The enzyme catalysed [32P]PPi–dATP exchange; Km (dATP) was 0.84mm and V (dATP) was 30% of V (ATP). Competition between ATP and dATP was demonstrated. 4. Selenate catalysed [32P]PPi–ATP exchange and competed with sulphate; Km (selenate) was 1.0mm and V (selenate) was 30% of V (sulphate). No AMP was formed with selenate as substrate. Molybdate did not catalyse PPi–ATP exchange, but AMP was formed. 5. Synthesis of adenosine 5′-[35S]sulphatophosphate was demonstrated by coupling purified ATP sulphurylase and Mg2+-dependent alkaline pyrophosphatase (also prepared from spinach) with [35S]sulphate and ATP as substrates; adenosine 5′-sulphatophosphate was not synthesized in the absence of pyrophosphatase. Some parameters of the coupled system are reported.

scholarly journals Adenosine diphosphate sulphurylase activity in leaf tissue

1973 ◽  
Vol 133 (3) ◽  
pp. 417-428 ◽  
Author(s):  
Jim N. Burnell ◽  
John W. Anderson
Keyword(s):  
Leaf Tissue ◽  
Thiol Group ◽  
Adenosine Diphosphate ◽  
Ph Optimum ◽  
Elementary Kinetics ◽  
Crude Extracts ◽  
Standard Assay ◽  
Spinach Leaf ◽  
Kinetics Of ◽  
Assay Conditions

1. A new method is described for the assay of ADP sulphurylase. The method involves sulphate-dependent [32P]Pi–ADP exchange; the method is simpler, more sensitive and more direct than the method involving adenosine 5′-sulphatophosphate-dependent uptake of Pi. 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 [32P]ADP from [32P]Pi; spinach extracts did not catalyse sulphate-dependent AMP–Pi, ADP–PPi or ATP–Pi 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 Ba2+ was necessary to minimize endogenous Pi–ADP exchange of the yeast enzyme and crude extracts of winter-grown spinach. 4. Spinach leaf ADP sulphurylase was activated by Ba2+ and Ca2+; Mg2+ was ineffective. The yeast enzyme was also activated by Ba2+. 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 Pi–ADP exchange. Neither selenate nor molybdate inhibited sulphate-dependent Pi–ADP exchange and crude spinach extracts did not catalyse selenate-dependent Pi–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.

scholarly journals Adenosine 5′-sulphatophosphate kinase activity in spinach leaf tissue

1973 ◽  
Vol 134 (2) ◽  
pp. 565-579 ◽  
Author(s):  
J. N. Burnell ◽  
J. W. Anderson
Keyword(s):  
Kinase Activity ◽  
Leaf Tissue ◽  
Nucleotidase Activity ◽  
Specific Enzyme ◽  
Ph Optimum ◽  
Spinach Leaf ◽  
Hydrolysis Of ◽  
Isolated Chloroplasts ◽  
Atp Sulphurylase

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 PPi. The pH optimum of the enzyme was 7.5; Km (3′-AMP) was approx. 0.8mm and Km (3′-CMP) was approx. 3.3mm. 3′-Nucleotidase activity was not associated with chloroplasts. Purified Mg2+-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.

scholarly journals The enzymology of adenosine triphosphate sulphurylase from spinach leaf tissue. Kinetic studies and a proposed reaction mechanism

1974 ◽  
Vol 139 (1) ◽  
pp. 27-35 ◽  
Author(s):  
W. H. Shaw ◽  
J. W. Anderson
Keyword(s):  
Enzyme Activity ◽  
Steady State ◽  
Exchange Reaction ◽  
Initial Rate ◽  
Leaf Tissue ◽  
Kinetic Studies ◽  
Forward Direction ◽  
Sequential Mechanism ◽  
Spinach Leaf ◽  
Atp Sulphurylase

1. Sulphate-dependent PPi–ATP exchange, catalysed by purified spinach leaf ATP sulphurylase, was correlated with the concentration of MgATP2− and MgP2O72−; ATP sulphurylase activity was not correlated with the concentration of free Mg2+. 2. Sulphate-dependent PPi–ATP exchange was independent of PPi concentration, but dependent on the concentration of ATP and sulphate. The rate of sulphate-dependent PPi–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 [32P]ATP from [32P]PPi and adenosine 5′-sulphatophosphate in the absence of sulphate; some properties of the reaction are described. Enzyme activity was dependent on the concentration of PPi and adenosine 5′-sulphatophosphate. 5. The synthesis of ATP from PPi and adenosine 5′-sulphatophosphate was inhibited by sulphate and ATP. The inhibition by sulphate was non-competitive with respect to PPi and adenosine 5′-sulphatophosphate; the inhibition by ATP was competitive with respect to adenosine 5′-sulphatophosphate and non-competitive with respect to PPi. It was concluded that the reaction catalysed by spinach leaf ATP sulphurylase was ordered; expressing the order in the forward direction, MgATP2− was the first product to react with the enzyme and MgP2O72− was the first product released. 6. The expected exchange reaction between sulphate and adenosine 5′-sulphatophosphate could not be demonstrated.

scholarly journals Plant holo-(acyl carrier protein) synthase

1988 ◽  
Vol 252 (1) ◽  
pp. 39-45 ◽  
Author(s):  
S A Elhussein ◽  
J A Miernyk ◽  
J B Ohlrogge
Keyword(s):  
Spinacia Oleracea ◽  
Acyl Carrier Protein ◽  
Ricinus Communis ◽  
Plant Cells ◽  
Gel Permeation Chromatography ◽  
Carrier Protein ◽  
Improved Method ◽  
Ph Optimum ◽  
Spinach Leaf

1. An improved method was developed for the assay of plant holo-(acyl carrier protein) synthase activity, using Escherichia coli acyl-(acyl carrier protein) synthetase as a coupling enzyme. 2. Holo-(acyl carrier protein) synthase was partially purified from spinach (Spinacia oleracea) leaves by a combination of (NH4)2SO4 fractionation and anion-exchange and gel-permeation chromatography. 3. The partially purified enzyme had a pH optimum of 8.2 and Km values of 2 microM, 72 microM and 3 mM for apo-(acyl carrier protein), CoA and Mg2+ respectively. Synthase activity was inhibited in vitro by the reaction product 3′,5′-ADP. 4. Results from the fractionation of spinach leaf and developing castor-oil-seed (Ricinus communis) endosperm cells were consistent with a cytosolic localization of holo-(acyl carrier protein) synthase activity in plant cells.

scholarly journals Glycolate kinase activity in human red cells

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 480-483 ◽  
Author(s):  
S Fujii ◽  
E Beutler
Keyword(s):  
Pyruvate Kinase ◽  
Adenosine Triphosphate ◽  
Kinase Activity ◽  
Maximum Velocity ◽  
Red Cells ◽  
Half Maximum ◽  
Ph Optimum ◽  
Pyruvate Kinase Deficiency ◽  
Low Activity ◽  
Human Red Cells

Abstract Human red cells manifest glycolate kinase activity. This activity copurifies with pyruvate kinase and is decreased in the red cells of subjects with hereditary pyruvate kinase deficiency. Glycolate kinase activity was detected in the presence of FDP or glucose-1,6-P2. In the presence of 1 mmol/L FDP, the Km for adenosine triphosphate (ATP) was 0.28 mmol/L and a half maximum velocity for glycolate was obtained at 40 mmol/L. The pH optimum of the reaction was over 10.5 With 10 mumol/L FDP, 500 mumol/L glucose-1,6-P2, 2 mmol/L ATP, 5 mmol/L MgCl2, and 50 mmol/L glycolate at pH 7.5, glycolate kinase activity was calculated to be approximately 0.0013 U/mL RBC. In view of this low activity even in the presence of massive amounts of glycolate, the glycolate kinase reaction cannot account for the maintenance of the reported phosphoglycolate level in human red cells.

scholarly journals Phosphoenolpyruvate Carboxylase from Spinach Leaf Tissue

1974 ◽  
Vol 53 (6) ◽  
pp. 829-834 ◽  
Author(s):  
S. K. Mukerji ◽  
S. F. Yang
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Leaf Tissue ◽  
Spinach Leaf

scholarly journals Glyoxalase III from Escherichia coli: a single novel enzyme for the conversion of methylglyoxal into d-lactate without reduced glutathione

1995 ◽  
Vol 305 (3) ◽  
pp. 999-1003 ◽  
Author(s):  
K Misra ◽  
A B Banerjee ◽  
S Ray ◽  
M Ray
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Carbonyl Compounds ◽  
Reduced Glutathione ◽  
Thiol Group ◽  
Glyoxalase I ◽  
Ph Optimum ◽  
Native Form ◽  
Wide Range ◽  
Glyoxalase Iii

A single novel enzyme, glyoxalase III, which catalyses the conversion of methylglyoxal into D-lactate without involvement of GSH, has been detected in and purified from Escherichia coli. Of several carbonyl compounds tested, only the alpha-ketoaldehydes methylglyoxal and phenylglyoxal were found to be substrates for this enzyme. Glyoxalase III is active over a wide range of pH with no sharp pH optimum. In its native form it has an M(r) of 82000 +/- 2000, and it is composed of two subunits of equal M(r). Glutathione analogues, which are inhibitors of glyoxalase I, do not inhibit glyoxalase III. Glyoxalase III is found to be sensitive to thiol-blocking reagents. The p-hydroxymercuribenzoate-inactivated enzyme could be almost completely re-activated by dithiothreitol and other thiol-group-containing compounds, indicating the possible involvement of thiol group(s) at or near the active site of the enzyme.

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