Phosphosulfate Sulfotransferase Activity Research Articles

Effect of chilling on the diurnal rhythm of enzymes involved in protection against oxidative stress in a chilling‐tolerant and a chilling‐sensitive maize genotype

The effect of chilling on diurnal changes in activity of adenosine 5′‐phosphosulfate sulfotransferase, glutathione reductase (EC 1.6.4.2) and glutathione transferase (EC 2.5.1.18) was analysed in the second leaf of Z 7, a chilling‐tolerant, and Penjalinan, a chilling‐sensitive maize (Zea mays L.) genotype. Nitrate reductase (EC 1.6.6.1) was measured for comparison. All enzyme activities examined changed with a typical diurnal rhythm in both genotypes cultivated at 25°C. Adenosine 5′‐phosphosulfate sulfotransferase and nitrate reductase activity peaked during the light period, then decreased and reached lowest levels at the end of the dark period. Glutathione reductase activity increased in the dark and decreased during the light period. Maximum glutathione transferase activities were measured in the middle of the light period, minimal ones in the middle of the dark period. At 12°C these diurnal changes were eliminated in all enzymes examined of both genotypes.The average adenosine 5′‐phosphosulfate sulfotransferase and glutathione reductase activity were higher in the chilling‐tolerant Z 7 than in the sensitive Penjanilan at 12°C in the light. Increased levels of both enzymes may contribute in establishing increased levels of cysteine and reduced glutathione in the chilling‐tolerant Z 7. Indeed it has been shown before that the chilling‐tolerant maize genotypes contain higher levels of both compounds at low temperatures than chilling‐sensitive ones.

Glutathione synthesis in maize genotypes with different sensitivities to chilling

The effect of chilling on enzymes, substrates and products of sulfate reduction, gultathione synthesis and metabolism was studied in shoots and roots of maize (Zea mays L.) genotypes with different chilling sensitivity. At full expansion of the second leaf, chilling at 12 °C inhibited dry weight increase in shoots and roots compared to controls at 25 °C and induced an increase in adenosine 5′-phosphosulfate sulfotransferase and γ-glutamylcysteine synthetase (EC 6.3.2.2) activity in the second leaf of all genotypes tested. Glutathione synthetase (EC 6.3.2.3) activity was about one order of magnitude higher than γ-glutamylcysteine synthetase activity, but remained unchanged during chilling except for one genotype. During chilling, cysteine and glutathione content of second leaves increased to significantly higher levels in the two most chilling-tolerant genotypes. Comparing the most tolerant and most sensitive genotype showed that chilling induced a greater incorporation of35S from [35S]sulfate into cysteine and glutathione in the chilling-tolerant than in the sensitive genotype. Chilling decreased the amount of35S-label incorporated into proteins in shoots of both genotypes, but had no effect on this incorporation in the roots. Glutathione reductase (EC 1.6.4.2) and nitrate reductase (EC 1.6.6.1) activity were constitutively higher in the chilling-tolerant genotypes, but showed no changes in most examined genotypes during 3 d at 12 °C. Our results indicate that in maize glutathione is involved in protection against chilling damage.

Effect of Chilling on Assimilatory Sulfate Reduction and Glutathione Synthesis in Maize

The contents of cysteine, γ-glutamylcysteine and glutathione, as well as the activity of enzymes of assimilatory sulfate reduction and glutathione synthesis in second leaves and root tips of maize seedlings ( Zea mays L. cv. LG 11) cultivated at low temperatures, were compared with controls grown at 25 °C. In vitro nitrate reductase (EC 1.6.6.1) activity was measured for comparison. Compared with the controls, adenosine 5'-phosphosulfate sulf otransf erase activity was 3.5- and 5.5-fold higher at 12 °C in second leaves and root tips, respectively. After 3 days of growth, the activity of y,-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3) in the second leaves was also significantly higher at 12 °C compared with 25 °C. Consistent with these enzyme activities, there was an increase in cysteine and glutathione contents both in second leaves and root tips after 3 days of growth at 12 °C. Nitrate reductase activity of second leaves and root tips was not affected by growth at 12 °C, indicating that the increase in adenosine 5'-phosphosulfate sulfotransferase activity and of the enzymes of glutathione synthesis was specific. Our results demonstrate that the increased activity of a key enzyme of assimilatory sulfate reduction and of the enzymes of glutathione synthesis contribute to the increased glutathione levels measured at 12 °C.

Adenosine 5‘‐Phosphosulfate Sulfotransferase from Norway Spruce: Biochemical and Physiological Properties*

AbstractBiochemical and physiological properties of adenosine 5′‐phosphosulfate sulfotransferase, a key enzyme of assimilatory sulfate reduction, from spruce trees growing under field conditions were studied. The apparent Km for adenosine 5′‐phosphosulfate (APS) was 29 ± 5.5μM, its apparent Mr was 115,000. 5′‐AMP inhibited the enzyme competitively with a Ki of 1 mM, but also stabilized it. MgS04 at 800 mM increased adenosine 5′‐phosphosulfate sulfotransferase activity by a factor of 3, concentrations higher than lOOOmM were inhibitory. Treatment of isolated shoots with nutrient solution containing 1 or 2 mM sulfate, and 3 or 10 mM glutathione, respectively, induced a significant decrease in extractable adenosine 5′‐phosphosulfate sulfotransferase activity over 24h, whereas GSH as well as S2‐ up to 5mM cysteine and up to 200 mM SO32‐ had no effect on the in vitro activity of the enzyme. As with other enzymes involved in assimilatory sulfate reduction, namely ATP sulfurylase (EC 2.7.7.4), sulfite reductase (EC 1.8.7.1) and O‐acetyl‐L.‐serine sulfhydrylase (EC 4.2.99.8), adenosine 5′‐phosphosulfate sulfotransferase was still detected at appreciable activities in 2‐ and 3‐year‐old needles. Adenosine 5′‐phosphosulfate sulfotransferase activity was low in buds and increased during shoot development, parallel to the chlorophyll content. The enzyme activity was characterized by an annual cycle of seasonal changes with an increase during February and March.

Regulation of Sulfate Assimilation by Light and O-Acetyl-l-Serine in Lemna minor L.

The effect of 0.5 millimolar O-acetyl-l-serine added to the nutrient solution on sulfate assimilation of Lemna minor L., cultivated in the light or in the dark, or transferred from light to the dark, was examined. During 24 hours after transfer from light to the dark the extractable activity of adenosine 5'-phosphosulfate sulfotransferase, a key enzyme of sulfate assimilation, decreased to 10% of the light control. Nitrate reductase (EC 1.7.7.1.) activity, measured for comparison, decreased to 40%. Adenosine 5'-triphosphate (ATP) sulfurylase (EC 2.7.7.4.) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8.) activities were not affected by the transfer. When O-acetyl-l-serine was added to the nutrient solution at the time of transfer to the dark, adenosine 5'-phosphosulfate sulfotransferase activity was still at 50% of the light control after 24 hours, ATP sulfurylase and O-acetyl-l-serine sulfhydrylase activity were again not affected, and nitrate reductase activity decreased as before. Addition of O-acetyl-l-serine at the time of the transfer caused a 100% increase in acid-soluble SH compounds after 24 hours in the dark. In continuous light the corresponding increase was 200%. During 24 hours after transfer to the dark the assimilation of (35)SO(4) (2-) into organic compounds decreased by 80% without O-acetyl-l-serine but was comparable to light controls in its presence. The addition of O-acetyl-l-serine to Lemna minor precultivated in the dark for 24 hours induced an increase in adenosine 5'-phosphosulfate sulfotransferase activity so that a constant level of 50% of the light control was reached after an additional 9 hours. Cycloheximide as well as 6-methyl-purine inhibited this effect. In the same type of experiment O-acetyl-l-serine induced a 100-fold increase in the incorporation of label from (35)SO(4) (2-) into cysteine after additional 24 hours in the dark. Taken together, these results show that exogenous O-acetyl-l-serine has a regulatory effect on assimilatory sulfate reduction of L. minor in light and darkness. They are in agreement with the idea that this compound is a limiting factor for sulfate assimilation and seem to be in contrast to the proposed strict light control of sulfate assimilation.

Regulation of Assimilatory Sulfate Reduction by Herbicide Safeners in Zea mays L.

Effects of the herbicide safeners N,N-diallyl-2,2-dichloroacetamide and 4-dichloroacetyl-3,4-dihydro-3-methyl-2H-1,4-benzooxazin (CGA 154281) on the contents in cysteine and glutathione, on the assimilation of (35)SO(4) (2-), and on the enzymes of assimilatory sulfate reduction were analyzed in roots and primary leaves of maize (Zea mays) seedlings. Both safeners induced an increase in cysteine and glutathione. In labeling experiments using (35)SO(4) (2-), roots of plants cultivated in the presence of safeners contained an increased level of radioactivity in glutathione and cysteine as compared with controls. A significant increase in uptake of sulfate was only detected in the presence of CGA 154281. One millimolar N,N-diallyl-2,2-dichloroacetamide applied to the roots for 6 days increased the activity of adenosine 5'-phosphosulfate sulfotransferase about 20- and threefold in the roots and leaves, respectively, compared with controls. CGA 154281 at 10 micromolar caused a sevenfold increase of this enzyme activity in the roots, but did not affect it significantly in the leaves. A significant increase in ATP-sulfurylase (EC 2.7.7.4) activity was only detected in the roots cultivated in the presence of 10 micromolar CGA 154281. Both safeners had no effect on the activity of sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8). The herbicide metolachlor alone or combined with the safeners induced levels of adenosine 5'-phosphosulfate sulfotransferase, which were higher than those of the appropriate controls. Taken together these results show that the herbicide safeners increased both the level of adenosine 5'-phosphosulfate sulfotransferase activity and of the thiols cysteine and glutathione. This indicates that these safeners may be involved in eliminating the previously proposed regulatory mechanism, in which increased concentrations of thiols regulate assimilatory sulfate reduction by decreasing the activities of the enzymes involved.

Effect of high and low sulfate concentrations on adenosine 5′‐phosphosulfate sulfotransferase activity from Lemna minor

The effect of Na2SO4 concentrations from 0 to 17.6 mM in the nutrient solution of Lemna minor L. strain 6580 on adenosine 5′‐phosphosulfate sulfotransferase activity was examined. Routinely, the plants were cultivated on 0.88 mA SO42−. The enzyme activity was increased by 50 to 100% after transfer to 0 or 0.0088 mM SO42−. Transfer back to 0.88 mM rapidly decreased the enzyme activity to the initial level. Cultivation on 17.6 mM Na2SO4 redueed extractable adenosine 5′‐phosphosulfate sulfotransferase by 50%. The original level was rapidly re‐established on 0,88 mM. In control experiments, a decrease in adenosine 5′‐phosphosulfate sulfotransferase activity was also induced by K2 SO4, whereas NaCl caused a small increase. This indicates that the observed effects are dependent on the sulfate ion. ATP‐sulfurylase activity measured for comparison was only significantly affected by the omission of sulfate, which induced a 20% increase, indicating that this enzyme activity from Lemna minor is less suseeptible to changes in medium sulfate than adenosine 5′‐phosphosulfate sulfotransferase. A close relationship between adenosine 5′‐phosphosulfate sulfotransferase activity and the content of asparagine, glutamine, non‐protein thiols and sulfate in the tissue was detected, indicating a positive control mechanism induced by amides and a negative mechanism induced by thiols and sulfate.

Effect of SO2, on the activity of adenosine 5′‐phosphosulfate sulfotransferase from spruce trees (Picea abies) in fumigation chambers and under field conditions

The effect of SO2 on adenosine 5′‐phosphosulfate sulfotransferase activity and various other parameters of needles from spruce (Picea abies L.) was studied using potted grafts in outdoor fumigation chambers and trees growing near a factory. In summer and autumn fumigation of grafted spruce, SO2, decreased the extractable activity of adenosine 5′‐phosphosulfate sulfotransferase to 12–50% of the controls, and reduced the amount of 35S from sulphate incorporated into protein by excised branches to a comparable degree. SO2 treatment in January and February inhibited the increase in adenosine 5′phosphosulfate sulfotransferase activity measured in the controls during this time. ATP‐sulfurylase activity was less affected by SO2. fumigation. In trees growing near a factory with high SO2. emission, the activity of adenosine 5′‐phosphosulfate sulfotransferase was about 35% of that of trees from a control area. The low enzyme activity was correlated with a high content of sulfate and compounds containing thiol groups.

Intercellular Localization of Assimilatory Sulfate Reduction in Leaves of Zea mays and Triticum aestivum

The intercellular distribution of assimilatory sulfate reduction enzymes between mesophyll and bundle sheath cells was analyzed in maize (Zea mays L.) and wheat (Triticum aestivum L.) leaves. In maize, a C(4) plant, 96 to 100% of adenosine 5'-phosphosulfate sulfotransferase and 92 to 100% of ATP sulfurylase activity (EC 2.7.7.4) was detected in the bundle sheath cells. Sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8) were found in both bundle sheath and mesophyll cell types. In wheat, a C(3) species, ATP sulfurylase and adenosine 5'-phosphosulfate sulfotransferase were found at equivalent activities in both mesophyll and bundle sheath cells. Leaves of etiolated maize plants contained appreciable ATP sulfurylase activity but only trace adenosine 5'-phosphosulfate sulfotransferase activity. Both enzyme activities increased in the bundle sheath cells during greening but remained at negligible levels in mesophyll cells. In leaves of maize grown without addition of a sulfur source for 12 d, the specific activity of adenosine 5'-phosphosulfate sulfotransferase and ATP sulfurylase in the bundle sheath cells was higher than in the controls. In the mesophyll cells, however, both enzyme activities remained undetectable. The intercellular distribution of enzymes would indicate that the first two steps of sulfur assimilation are restricted to the bundle sheath cells of C(4) plants, and this restriction is independent of ontogeny and the sulfur nutritional status of the plants.

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.

SO2 and assimilatory sulfate reduction in beech leaves

The effect of SO2 on the extractable activity of ATP sulfurylase (EC 2.7.7.4.). adenosine 5′‐phosphosulfate sulfotransferase, ribulosebisphosphate carboxylase, chlorophyll, protein, sulfate, and amino acids was examined in leaves of potted grafts of beech (Fagus sylvatica L.) treated in outdoor fumigation chambers. Addition of 0.025 and 0.075 μl SO2 1−1 to unfiltered ambient air caused a decrease in the extractable activity of adenosine 5′‐phosphosulfate sulfotransferase to about 20 to 30% of the controls. Neither the extractable activity of ATP sulfurylase and ribulosebisphosphate carboxylase nor the content in chlorophyll, total amino acids and protein were significantly affected by SO2, but there was an increase in the sulfate content. Leaves treated with 0.075 μl SO2 1−1 contained more alanine and cysteine and less serine than the controls. After transfer of the SO2‐treated beech trees to control chambers there was an increase in adenosine 5′‐phosphosulfate sulfotransferase activity, but no significant decrease in SO2−4‐sulfur.

Activity of Sulfate-assimilating Enzymes in Primary Leaves of Phaseolus vulgaris L. During Dark-induced Senescence

Summary The activity of three enzymes of assimilatory sulfate reduction, ATP sulfurylase, adenosine 5′-phosphosulfat sulfotransferase, and O-acetyl-L-serine sulfhydrylase, was measured in extracts from primary leaves of Phaseolus vulgaris L. seedlings during dark-induced senescence. When 7-day-old seedlings were transferred from continuous light to darkness there was an increase in total activity of ATP sulfurylase (E.C. 2.7.7.4) and adenosine 5′-phosphosulfate sulfotransferase during the first 24 h. This was followed by a rapid decrease. After 6 days in the dark both enzyme activities were no longer detectable. Ribulosebisphosphate carboxylase activity, measured for comparison, increased during the first 3 days in the dark and remained constant during the following 3 days. When the identical experiment was performed with 10-day-old seedlings, ATP sulfurylase and adenosine 5′-phosphosulfate sulfotransferase activity started decreasing already during the first 24 h in the dark with a halflife of 40 and 20 h, respectively. The half-life of ATP sulfurylase and adenosine 5′-phosphosulfate sulfotransferase activity in control seedlings kept in continuous light was 52 and 50 h, respectively. Ribulosebisphosphate carboxylase activity and chlorophyll content decreased at about the same rate in seedlings kept in the light or in the dark with half-lives of more than 100 h. In excised primary leaves from 10-day-old seedlings, the dark-induced decrease in activity of ATP sulfurylase, adenosine 5'-phosphosulfate sulfotransferase, and ribulosebisphosphate carboxylase and the content of chlorophyll was similar to that in leaves of intact plants . Total O-acetyl-L-serine sulfhydrylase activity only decreased by about 30% in leaves of 10-day-old seedlings transferred to the dark for 7 days. The O-acetyl-L-serine sulfhydrylase activity associated with the chloroplasts decreased more rapidly than the extrachloroplastic activity.

Regulation of Sulfate Assimilation in Plants

The correlation between the extractable activities of three key enzymes of assimilatory sulfate reduction and the in vivo incorporation of (35)SO(4) (2-) into amino acids, proteins, and sulfolipids was investigated from greening to senescence in primary leaves of beans (Phaseolus vulgaris L.). The total extractable activity of ATP sulfurylase (EC 2.7.7.4) and of adenosine 5'-phosphosulfate sulfotransferase reached a maximum in the leaves of approximately 7- and 11-day-old seedlings, respectively. During senescence, there was a decrease in both enzyme activities. After approximately 17 days, no appreciable activities remained. In contrast, total O-acetyl-l-serine sulfhydrylase (EC 4.3.99.8) activity decreased to only approximately 50% of the maximal value during the same period. The in vivo incorporation of (35)SO(4) (2-) into amino acid and protein fractions showed a time-course similar to that of the total extractable adenosine 5'-phosphosulfate sulfotransferase activity. Both cysteine and sulfate markedly decreased during senescence. The total extractable activity of ribulosebisphosphate carboxylase (EC 4.1.1.39) was maximal in the primary leaves of 13-day-old seedlings, and approximately 40% of this value was still detectable after 17 days. Taken together with results from the literature, these results show that assimilatory sulfate reduction in primary leaves of P. vulgaris L. stops before CO(2) and nitrate assimilation.

Regulation of adenosine 5′‐phosphosulfate sulfotransferase by sulfur dioxide in primary leaves of beans (Phaseolus vulgaris)

AbstractSO2 inhibited the light‐induced increase of extractable adenosine 5′‐phosphosulfate sulfotransferase in greening primary leaves of bean seedlings (Phaseolus vulgaris L. cv. Saxa (Radio) Stamm Vatter). In green primary leaves containing appreciable extractable adenosine 5′‐phosphosulfate sulfotransferase activity, SO2 treatment for 20 h decreased the activity of the enzyme to between 10 and 20% of the initial level. After removal of SO2 from the air, the extractable adenosine 5′‐phosphosulfate sulfotransferase activity increased after a lag, both in green and greening primary leaves, and was back to the control level after about 48 h. The sulfate concentration was increased about fourfold during SO2 treatment. An increase in sulfate sulfur accompanied by a decrease in adenosine 5′‐phosphosulfate sulfotransferase was also observed when bean seedlings, after excision of the roots, were transferred to nutrient solutions containing high sulfate concentrations, suggesting that sulfate is involved in the regulation of the enzyme.

Analysis of the regulation of adenosine 5′-phosphosulfate sulfotransferase activity inLemna minor L. using15N-density labeling

The role of de novo synthesis in the regulation of adenosine 5'-phosphosulfate sulfotransferase activity by H2S inLemna minor L. was investigate using density labeling with(15)N applied as(15)NO 3 (-) in the culture medium. While adenosine 5'-phosphosulfate sulfotransferase activity was rapidly reduced by H2S and rapidly recovered upon removal of H2S, O-acetyl-L-serine sulfhydrylase (EC 4.2.99.8) did not show changes in extractable activity in response to H2S and could therefore be used as an internal marker enzyme for density labeling. The incorporation of(15)N into adenosine 5'-phosphosulfate sulfotransferase was strongly reduced upon transfer of plants into a H2S-containing atmosphere. Half-maximal labeling was reached only after 70-80 h compared to 40-50 h in the control. After removal of H2S, adenosine 5'-phosphosulfate sulfotransferase activity increased to the initial level within 20 h, and the enzyme reached halfmaximal labeling after only 15 h. The time course of the density increase of O-acetyl-L-serine sulfhydrylase was not affected very significantly by H2S. These results provide evidence that de novo synthesis of enzyme protein is involved in the regulation of adenosine 5'-phosphosulfate sulfotransferase activity by H2S.

Properties and regulation of adenosine 5?-phosphosulfate sulfotransferase from suspension cultures ofNicotiana sylvestris

The properties and the regulation of adenosine 5'-phosphosulfate sulfotransferase extracted from cell suspension cultures ofNicotiana sylvestris was investigated. Optimal adenosine 5'-phosphosulfate sulfotransferase activity was obtained from the cells by extraction with 0.1 M tris-HCl, pH8.0, containing 2 M MgSO4 and 10 mM dithioerythritol. The K m for adenosine 5'-phosphosulfate in the sulfotransferase reaction was about 11 μM. Adenosine 5'-phosphosulfate in concentrations above 50 μM were inhibitory. The extratable adenosine 5'-phosphosulfate sulfotransferase activity decreased during cultivation with sulfate as the sole sulfur source, but after about 3 days it reached a constant level (50 to 100 nmol activated sulfate transferred h(-1) mg(-1) protein) which was maintained for at least 24 h. Addition of 0.5 mM cysteine to the culture medium decreased the extractable adenosine 5'-phosphosulfate sulfotransferase activity and blocked growth completely. With 0.1 mM cysteine an enzyme level of about 10% of the initial value was reached within 6 to 12 h without significant inhibition of growth. The added cysteine was absorbed rapidly and after 24 h cysteine could no longer be detected in the medium. Before the cysteine was completely depleted, the activity of adenosine 5'-phosphosulfate sulfotransferase started to increase, reaching ultimately a level which was comparable to the initial value.

Enzymatic sulfation of glycochenodeoxycholic acid by tissue fractions from adult hamsters.

Using a radiometric assay with glycochenodeoxycholic acid as substrate, bile acid:3'-phosphoadenosine-5'-phosphosulfate sulfotransferase activity was found in 105,000 g supernatant fractions of liver, proximal intestine, and adrenal gland homogenates from adult hamsters. Optimum conditions for measurement of the hepatic enzyme were determined. In both male and female animals sulfation only occurred at the 7 alpha-position. Saturation analysis with glycohenodeoxycholic acid revealed that the higher activity observed in fractions from female compared to male hamsters was due to a 4-fold lower apparent Km (79 muM vs. 317 muM) for this bile acid in the females. The sulfation of glycohenodeoxycholic acid was competitively inhibited by glycolithocholic acid, chenodeoxycholic acid, and ursodeoxycholic acid. The data are consistent with the concept that sulfation of many, if not all, bile acids can occur in vivo.

Regulation of adenosine 5?-phosphosulfate sulfotransferase activity by H2S and cyst(e)ine in primary leaves of Phaseolus vulgaris L.

During chloroplast development in the primary leaves of Phaseolus vulgaris, the extractable activity of adenosine 5'-phosphosulfate sulfotransferase increased ten-fold. When chloroplast development took place in air enriched with 3.5 μl H2S·l(-1) there was a decrease in adenosine 5'-phosphosulfate sulfotransferase activity. Cyst(e)ine in concentrations up to 1 mM (in the external medium) did not affect the increase in adenosine 5'-phosphosulfate sulfotransferase activity in intact plants. In plants with excised roots, 0.75 mM cyst(e)ine inhibited this increase. In green primary leaves, H2S or cyst(e)ine treatment resulted in a decrease of extractable adenosine 5'-phosphosulfate sulfotransferase activity. In intact plants, this effect of cyst(e)ine was observed at a concentration of 1 mM, and in plants with excised roots, 0.25 mM had a comparable effect.In developing plants, the extractable activities of O-acetyl-L-serine sulfhydrylase (EC 4.2.99.9) and ribulosebisphosphate carboxylase (EC 4.1.1.39.) were not affected by H2S or cyst(e)ine.

Studies of sulfate utilization by algae 18. identification of glutathione as a physiological carrier in assimilatory sulfate reduction by Chlorella

Adenosine 5′-phosphosulfate-sulfotransferase is the first enzyme in the pathway of assimilatory sulfate reduction in Chlorella, and transfers the sulfo group from APS (adenosine 5′-phosphosulfate) to a thiol acceptor forming an organic thiosulfate. In vitro, adenosine 5′-phosphosulfate-sulfotransferase transfers to a variety of thiol acceptors, the best among the monothiols is glutathione, the only one to show a regulatory interaction with adenosine 5′-phosphosulfate-sulfotransferase. To identify the physiological acceptor, adenosine 5′-phosphosulfate-sulfotransferase is assayed without thiols; the cell fraction which stimulates adenosine 5′-phosphosulfate-sulfotransferase activity is expected to contain the physiological acceptor. Boiled Chlorella extract contains physiological acceptor when assayed in the presence of adenosine 5′-phosphosulfate-sulfotransferase and NADPH. Physiological acceptor is dialyzable but is retained by filters of 1000 daltons cut off. After passage through DEAE—Sephadex A-25 and Sephadex G-25, physiological acceptor is found to be ninhydrin-positive and shows co-electrophoresis with oxidized glutathione. Upon reduction with dithiothreitol, physiological acceptor moves with reduced glutathione and on acid hydrolysis yields amino acids identical with those from authentic oxidized glutathione. Physiological acceptor, with adenosine 5′-phosphosulfate-sulfotransferase and AP 35 S, yields labeled glutathione—S—SO 3 −. Thiosulfonate reductase from Chlorella reduces glutathione—S—SO 3 − to bound sulfide. Only one active accepting component is found in the boiled extracts.

Localization of adenosine 5?-phosphosulfate sulfotransferase in spinach leaves

Roots of spinach (Spinacia oleracea L.) seedlings contained only a very low activity of adenosine 5'-phosphosulfate sulfotransferase compared to the cotyledons. Adenosine 5'-phosphosulfate sulfotransferase activity increased about tenfold in cotyledons during greening. Preparation of organelle fractions from spinach leaves by a combination of differential and isopycnic density gradient centrifugation showed that adenosine 5'-phosphosulfate sulfotransferase banded with NADP-glyceraldehyde-3-phosphate dehydrogenase, a marker enzyme for intact chloroplasts. In the fractions of peroxisomes, mitochondria and broken chloroplasts virtually no adenosine 5'-phosphosulfate sulfotransferase activity was measured. Comparison with the chloroplast enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase indicates that in spinach, adenosine 5'-phosphosulfate sulfotransferase is localized almost exclusively in the chloroplasts.