Effect of sulfur on nitrate reductase and ATP sulfurylase Activities in groundnut (Arachis hypogea L.)

Field experiments were conducted to determine the effect of sulfur (S) and Nitrogen (N) on nitrate reductase (NR) and ATP-sulfurylase activities in groundnut cultivars (Arachis hypogea L. cv. Ambar and Kaushal). Two combinations of S (in kg ha^-1): OS (-S) and 20S (+S) were used with 20 kg ha^-1 N. The application of S enhanced the NR and ATP-sulfurylase activities in both the cultivars at all the growth stages. The application of S also increased soluble protein and chlorophyll content in the all growth stages of both the cultivars. NR and ATP-sulfurylase activities in the leaves were measured at various growth stages as the two enzymes catalyze the rate limiting steps of the assimilatory pathways of nitrate and sulfate, respectively.     

Species specific release of sulfate from adenylyl sulfate by ATP sulfurylase or ADP sulfurylase in the green sulfur bacteria Chlorobium limicola and Chlorobium vibrioforme

High activities of ATP sulfurylase were found in the soluble protein fraction of two Chlorobium limicola strains, whereas ADP sulfurylase was absent. ATP sulfurylase was partially purified and characterized. It was a stable soluble enzyme with a molecular weight of 230,000, buffer-dependent pH optima at 8.6 and 7.2 and an isoelectric point at pH 4.8. No physiological inhibitor was found. Inhibition was observed with p-CMB and heavy metals. Sulfur compounds had no effect on enzyme activity. The stoichiometry of the reaction was proven. In contrast, an ADP sulfurylase, but no ATP sulfurylase, was found in Chlorobium vibrioforme. This enzyme was very labile with a molecular weight of about 120,000 and buffer-dependent pH optima at 9.0 and 8.5. Under test conditions the apparent K _m value was determined to be 0.28 mM for adenylyl sulfate and 8.0 mM for phosphate.Abbreviations APS adenylyl sulfate - p-CMB parachloromercuribenzoate - PP_i inorganic pyrophosphate     

Cloning of two cDNAs encoding a family of ATP sulfurylase from Camellia sinensis related to selenium or sulfur metabolism and functional expression in Escherichia coli.

ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of two cDNAs encoding ATP sulfurylase (APS1 and APS2) from Camellia sinensis. They were isolated by RT-PCR and RACE-PCR reactions. The expression of APS1 and APS2 are correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1415-bp cDNA with an open reading frame predicted to encode a 360-amino acid, 40.5kD protein; APS2 is a 1706-bp cDNA with an open reading frame to encode a 465-amino acid, 51.8kD protein. The predicted amino acid sequences of APS1 and APS2 have high similarity to ATP sulfurylases of Medicago truncatula and Solanum tuberosum, with 86% and 84% identity respectively. However, they share only 59.6% identity with each other. The enzyme extracts prepared from recombinant Escherichia coli containing Camellia sinensis APS genes had significant enzyme activity.     

Changes in ATP sulfurylase and adenosine 5'-phosphosulfate sulfotransferase activity during autumnal senescence of beech leaves

The decrease in extractable activity of ribuloscbisphosphate carboxylase (EC 4.1.1.39), ATP sulfurylase (EC 2.7.7.4) and adenosine 5'-phosphosulfate sulfotransferase and the content in chlorophyll and protein was compared in leaves of cloned beech trees ( Fagus sylvatica L.) during autumnal senescence. Leaves excised at the same time but containing different amounts of chlorophyll gave extracts with correspondingly varying amounts of ribulosebisphosphate carboxylase activity. Leaves which had almost completely lost this enzyme activity contained still appreciable ATP sulfurylase and adenosine 5'-phosphosulfate sulfotransferase activity and soluble protein. For all components determined, there was a period lasting until mid or end of October during which there was no or only a small decrease. They were then all lost rapidly from the leaves. The specific activity of ribulosebisphosphate carboxylase decreased during this phase of rapid loss, whereas it remained essentially constant for ATP sulfurylase and adenosine 5'-phosphosulfate sulfotransferase. During this period, the mean half life of ribulosebisphosphate carboxylase was shorter than the one of ATP sulfurylase and of adenosine 5'-phosphosulfate sulfotransferase. These experiments clearly show that ribulosebisphosphate carboxylase was preferentially lost from beech leaves during autumnal senescence as compared to ATP sulfurylase and adenosine 5'-phosphosulfate sulfotransferase.     

Temperature effects on the allosteric transition of ATP sulfurylase from Penicillium chrysogenum

The effects of temperature on the initial velocity kinetics of allosteric ATP sulfurylase from Penicillium chrysogenum were measured. The experiments were prompted by the structural similarity between the C-terminal regulatory domain of fungal ATP sulfurylase and fungal APS kinase, a homodimer that undergoes a temperature-dependent, reversible dissociation of subunits over a narrow temperature range. Wild-type ATP sulfurylase yielded hyperbolic velocity curves between 18 and 30 degrees C. Increasing the assay temperature above 30 degrees C at a constant pH of 8.0 increased the cooperativity of the velocity curves. Hill coefficients (n(H)) up to 1.8 were observed at 42 degrees C. The bireactant kinetics at 42 degrees C were the same as those observed at 30 degrees C in the presence of PAPS, the allosteric inhibitor. In contrast, yeast ATP sulfurylase yielded hyperbolic plots at 42 degrees C. The P. chrysogenum mutant enzyme, C509S, which is intrinsically cooperative (n(H) = 1.8) at 30 degrees C, became more cooperative as the temperature was increased yielding n(H) values up to 2.9 at 42 degrees C. As the temperature was decreased, the cooperativity of C509S decreased; n(H) was 1.0 at 18 degrees C. The cumulative results indicate that increasing the temperature increases the allosteric constant, L, i.e., promotes a shift in the base-level distribution of enzyme molecules from the high MgATP affinity R state toward the low MgATP affinity T state. As a result, the enzyme displays a true "temperature optimum" at subsaturating MgATP. The reversible temperature-dependent transitions of fungal ATP sulfurylase and APS kinase may play a role in energy conservation at high temperatures where the organism can survive but not grow optimally.     

Effect of reactive oxygen species on the metabolism of tryptophan in rat brain: Influence of age

In an earlier study, oxidation of tryptophan hydroxylase was implicated as its affinity was decreased with aging in rat brain. To establish any potential link between its oxidative damage and aging, we have determined the activities of antioxidant enzymes in midbrain, pons and medulla of 2, 12 and 24 month old Fisher 344 BNF1 rats. The results obtained suggest that the activities of antioxidant enzymes varied considerably with age and brain regions studied. Activities of Cu/Zn superoxide dismutase and glutathione peroxidase were found to increase from 2 to 12 months and then decrease in 24 month old rats. However catalase activity decreased consistently with the age. A parallel increase in the carbonyl content was observed in these brain regions indicating the oxidation of proteins. Reactive oxygen species when included in the incubation mixture decreased the activity of tryptophan hydroxylase in a concentration dependent manner. The loss of tryptophan hydroxylase activity induced by hydrogen peroxide and superoxide anion was prevented by catalase. However superoxide dismutase did not provide such protection. Sulfhydryl agents, cysteine, glutathione and dithiothreitol partially prevented the loss of activity. These studies suggest an involvement of reactive oxygen species for sulfhydryl oxidation of tryptophan hydroxylase in aging.     

Studies on the metabolism of lipid molecular species in immature soybean cotyledons

Metabolism of lipid molecular species in soybean cotyledons (Glycine max [L.] Merr. var. “Harosoy 63”) was determined from incorporation studies with radioactive acetate and glycerol. Lipid synthetic activity was highest in immature cotyledons at 30 days after flowering. Distinct differences in labeling patterns of molecular species within lipid classes demonstrated that selective utilization of diglyceride intermediates occurred in complex lipid biosynthesis in soybean. The phospholipid molecular species in this tissue that displayed the highest turnover rates had the following acyl combinations: saturate-linoleic and dioleic in phosphatidic acid; saturate-oleic in phosphatidylinositol and phosphatidylethanolamine; dioleic in phosphatidylcholine; oleic-dilinoleic in N-acylphosphatidylethanolamine. Saturate-dilinoleic, oleic-dilinoleic, trioleic, and trilinoleic structures were rapidly synthesized species of triglyceride in immature soybean cotyledons.     

Influence of mouse age and erythrocyte age on glutathione metabolism.

In order to determine whether the biological age of a mouse influences erythrocyte metabolism and erythrocyte aging in vivo, blood samples were collected from male C57/BL6J mice of different biological ages ranging from mature (10 months) to "very old" (37 months). In the very old mouse, compared with the mature mouse, the erythrocyte survival time was decreased, erythrocyte densities were increased, the concentrations of total free thiol and reduced glutathione, and glutathione reductase activity were decreased. Erythrocytes were separated into different density (age) groups by phthalate ester two-phase centrifugation or by albumin density-gradient centrifugation. The density-age relationship of erythrocytes was established by pulse-labelling with 59Fe in vivo and by subsequent determinations of specific radioactivity of erythrocyte fractions of different densities prepared during a chase period of 60 days. The age of erythrocytes in mice of all ages was directly related to density. Also, in older erythrocytes compared with younger erythrocytes, decreased concentrations of total free thiol and reduced glutathione, and decreased glutathione reductase activity were observed. These were the lowest in the old erythrocytes of very old mice. These results in aging erythrocytes from aging mice suggest that the glutathione status the erythrocyte may be an index of aging, not only of the cell but also of the organism.     

Regulation of nitrogenase activity in soybean nodules by ATP and energy charge

Nonphosphorylating condition under anaerobiosis stopped nitrogenase activity in nodules of soybean ($\text{Glycine}$$\text{max}$ var. Chippewa) in less than three minutes and aeration quickly activated the enzyme. This stop-and-go treatment can be repeatedly applied on excised nodules, and a concomitant low-and-high ATP and energy charge (EC) was observed. After 2 minutes under anaerobiosis, nodule ATP and EC were decreased, respectively, to 20 and 40% of the control. These decreases were not as great with longer anaerobic treatments, and there was no change in the content of total adenosine phosphates. Oxygen enrichment (40%) stimulated the activity of nitrogenase by 2.5 fold in four minutes with a concomitant increase of ATP and EC by 40% and 14%, respectively, and an exhaustion of AMP. Longer treatments of oxygen enrichment lessened the initial effects. These findings indicate that ATP and energy charge probably regulate the activity of nitrogenase $\text{in}$$\text{vivo}$ and an active adenylate kinase must be operating in the nodules to maintain an energy supply for the basal metabolism and for the nitrogenase under temporary stressed conditions.     

Kinetic and stability properties of Penicillium chrysogenum ATP sulfurylase missing the C-terminal regulatory domain

ATP sulfurylase from Penicillium chrysogenum is a homohexameric enzyme that is subject to allosteric inhibition by 3'-phosphoadenosine 5'-phosphosulfate. In contrast to the wild type enzyme, recombinant ATP sulfurylase lacking the C-terminal allosteric domain was monomeric and noncooperative. All kcat values were decreased (the adenosine 5'-phosphosulfate (adenylylsulfate) (APS) synthesis reaction to 17% of the wild type value). Additionally, the Michaelis constants for MgATP and sulfate (or molybdate), the dissociation constant of E.APS, and the monovalent oxyanion dissociation constants of dead end E.MgATP.oxyanion complexes were all increased. APS release (the k6 step) was rate-limiting in the wild type enzyme. Without the C-terminal domain, the composite k5 step (isomerization of the central complex and MgPPi release) became rate-limiting. The cumulative results indicate that besides (a) serving as a receptor for the allosteric inhibitor, the C-terminal domain (b) stabilizes the hexameric structure and indirectly, individual subunits. Additionally, (c) the domain interacts with and perfects the catalytic site such that one or more steps following the formation of the binary E.MgATP and E.SO4(2-) complexes and preceding the release of MgPPi are optimized. The more negative entropy of activation of the truncated enzyme for APS synthesis is consistent with a role of the C-terminal domain in promoting the effective orientation of MgATP and sulfate at the active site.     

Crystal structure of ATP sulfurylase from Saccharomyces cerevisiae, a key enzyme in sulfate activation

ATP sulfurylases (ATPSs) are ubiquitous enzymes that catalyse the primary step of intracellular sulfate activation: the reaction of inorganic sulfate with ATP to form adenosine-5'-phosphosulfate (APS) and pyrophosphate (PPi). With the crystal structure of ATPS from the yeast Saccharomyces cerevisiae, we have solved the first structure of a member of the ATP sulfurylase family. We have analysed the crystal structure of the native enzyme at 1.95 Angstroms resolution using multiple isomorphous replacement (MIR) and, subsequently, the ternary enzyme product complex with APS and PPi bound to the active site. The enzyme consists of six identical subunits arranged in two stacked rings in a D:3 symmetric assembly. Nucleotide binding causes significant conformational changes, which lead to a rigid body structural displacement of domains III and IV of the ATPS monomer. Despite having similar folds and active site design, examination of the active site of ATPS and comparison with known structures of related nucleotidylyl transferases reveal a novel ATP binding mode that is peculiar to ATP sulfurylases.     

ATP sulfurylase from higher plants : purification and preliminary kinetics studies on the cabbage leaf enzyme

ATP sulfurylase was purified extensively from green cabbage (Brassica capitata L.) leaf. The enzyme appears to be an asymmetric dimer composed of 57,000 dalton subunits. Initial velocity and product inhibition studies of the forward and reverse reactions point to an obligately ordered kinetic mechanism with MgATP adding before MoO₄²⁻ (or SO₄²⁻). and MgPPi leaving before AMP + MoO₄²⁻ (or adenosine-5′-phosphosulfate [APS]). The addition of excess purified fungal APS kinase to assay mixtures increased the rate of ³⁵SO₄²⁻ incorporation and MgPPi formation and extended the linearity of the forward reaction. This effect can be ascribed to the continual removal of APS, a potent product inhibitor of ATP sulfurylase. The specific activities of the enzyme in the APS synthesis, molybdolysis, MgATP synthesis, and sulfate-dependent [³²P]-MgPPi-MgATP exchange assays were 3.3, 38, 38, and 4.3 micromole product formed per minute per milligram protein, respectively.     

Asparagine metabolism and asparaginase activity in a euryhaline Chlamydomonas species.

A Chlamydomonas species isolated from a marine environment possesses an L-asparaginase, an enzyme not yet reported in the microalgae. This enzyme enabled the organism to grow as well with asparagine as sole nitrogen source as with inorganic nitrogen sources (NO3-, NH4+). Only the amide nitrogen was used for growth since growth did not occur on aspartate and aspartate accumulated in the media when cells were either grown on asparagine or during short-term incubations with L-[U-14C]asparagine. Cells grown on NO3-, NH4+, or L-asparagine in batch culture possessed equivalent asparaginase activities. However, nitrogen-limited cells possessed four times the activity of cells grown with sufficient nitrogen for normal growth, regardless of the possessed the lowest activity per cell, while lag phase and stationary phase cells possessed greater activity. The enzyme behaved like a periplasmic space enzyme since (1) breaking the cells did not release into solution more activity than was shown by whole cells and (2) whole cells converted L-[U-14C]asparagine to [14C]aspartate with little intracellular accumulation of radioactivity. Cell-free preparations of the enzyme possessed a Km value for asparagine of 1.1 x 10-4 M, with no glutaminase activity.