Detection of metabolic conversions of ascorbate-2-monophosphate and ascorbate-2-sulfate to ascorbic acid in tiger prawn (Penaeus monodon) using high-performance liquid chromatography and colorimetry

Biochemical conversions of ascorbate-2-monophosphate and ascorbate-2-sulfate to ascorbic acid by acid phosphatase and ascorbate-2-sulfate sulfohydrolase, respectively, were found in extracts of a hepatopancreas of Penaeus monodon, bovine liver and tilapia liver. Both enzymes were assayed with high-performance liquid chromatography (HPLC) and colorimetry. Colorimetry was based on the reduction of a color of 2,6-dichlorophenolindophenol (DCIP) when ascorbic acid was released from enzymatic activity. Assay of acid phosphatase either with HPLC or with colorimetry was found to be equally reliable. However, sensitivity of the HPLC assay was slightly higher than that of colorimetry; HPLC was able to detect activity as little as 1 nmol ascorbic acid released per min, whereas colorimetry was limited at 6–7 nmol/min. Assay of ascorbate-2-sulfate sulfohydrolase in crude extracts with the HPLC technique was found to be more specific than that with the colorimetric assay. The excess reduction of DCIP color not related to the sulfohydrolase activity was observed in the colorimetric technique. An accumulation of ascorbic acid in a hepatopancreas of P. monodon fed with feeds supplemented with phosphorylated or sulfated ascorbic acid was higher than that of the prawn fed with feed without ascorbic acid. The accumulated ascorbic acid was possibly from the activity of acid phosphatase or the sulfohydrolase that hydrolyzed phosphorylated or sulfated derivatives in vivo, respectively. Metabolism of the ascorbate derivatives in the prawn is discussed.     

Copurification of L-ascorbate-2-sulfate sulfohydrolase and arylsulfatase activities from the liver of a marine gastropod, Charonia lampas.

Ascorbate-2-sulfate sulfohydrolase was purified 184-fold from a crude extract of the liver of Charonia lampas. In all purification steps including phosphocellulose, first and second Sephadex G-150 column chromatographies, the enzyme activity eluted together with arylsulfatase [ED 3.1.6.1] activity, and was separated from glycosulfatase ]EC 3.1.6.3] activity. The nonidentity of ascorbate-2-sulfate sulfohydrolase and glycosulfatase was further confirmed by an isoelectric focussing study. Ascorbate-2-sulfate sulfohydrolase had an isoelectric point, pI, of 4.9, and had maximum activity at pH 4.0. Its molecular weight was estimated to be about 154.000.     

Ascorbate-2-phosphate inhibition of ascorbate-2-sulfate sulfohydrolase from bovine liver.

The hydrolysis of ascorbate-2-sulfate by the enzyme, ascorbate-2-sulfate sulfohydrolase, purified from bovine liver has been shown to be powerfully inhibited by ascorbate-2-phosphate. The inhibition by ascorbate phosphate is competitive with a K_I of 0.3 μM. Na₂HPO₄ also inhibits by an apparent non-competitive process. The Na₂HPO₄ concentration at 50% inhibition is 7.7 μM. A possible control role for ascorbate phosphate in ascorbate biochemistry is suggested.     

Effects of various compounds on the ascorbate-2-sulfate sulfohydrolase and arylsulfatase activities copurified from the liver of Charonia lampas.

The effects of various compounds on ascorbate-2-sulfate sulfohydrolase and arylsulfatase (EC 3.1.6.1) activities in the copurified preparation from the liver of Charonia lampas were investigated. The former activity was competively inhibited by inorganic phosphate and sulfate. The latter was not affected by sulfate. Higher concentrations of sodium chloride inhibited the former and activated the latter. Neither of the activities was inhibited by sulfhydryl reagents. Both activities were competively inhibited by the other substrate.     

Uridine diphospho-N-acetylgalactosamine-4-sulfate sulfohydrolase activity of human arylsulfatase B and its deficiency in the Maroteaux-Lamy syndrome.

The hydrolysis of UDP-N-acetylgalactosamine-4-sulfate by human arylsulfatase B has been demonstrated with an enzyme preparation purified 200-fold from placenta. No hydrolysis was observed with arylsulfatase A. UDP-N-acetylgalactosamine-4-sulfate is the first fully characterized physiological compound shown to be a substrate for arylsulfatase B, confirming that arylsulfatase B is an N-acetylgalactosamine-4-sulfate sulfohydrolase. Cultured fibroblasts derived from patients with Maroteaux-Lamy syndrome were deficient in UDP-N-acetylgalactosamine-4-sulfate sulfohydrolase to the same extent that they were deficient in arylsulfatase B.     

Ascorbic acid-2-sulfate sulfhohydrolase activity of human arylsulfatase A.

Pure human arylsulfatase A (EC 3.1.6.1) was found to hydrolyze ascorbic acid 2-sulfate to ascorbic acid and inorganic sulfate at rates from 200 to 2000 mumol/mg per h depending on the method of assay. This rate was lower than that observed with the synthetic substrate 4-nitrocatechol sulfate, but higher than that seen with the physiological substrate cerebroside sulfate. Extracts of cultured fibroblasts from normal subjects were also shown to hydrolyze ascorbic acid 2-sulfate; extracts of fibroblasts from patients with metachromatic leukodystrophy, known to be deficient in arylsulfatase A, did not. Similarly, hydrolysis of ascorbic acid 2-sulfate was not observed when a partially purified preparation of human arylsulfatase B was tested under a variety of conditions. Thus, in the human, arylsulfatase A appears to be the major, if not the only, ascorbic acid-2-sulfate sulfohydrolase.     

Partial characterization of steroid sulfohydrolase and steroid sulfotransferase activities in purified porcine Leydig cells

Subcellular fractions of purified pig Leydig cells from 7 different animals have been investigated with respect to their abilities to catalyze the sulfation of several steroids and the hydrolysis of the sulfated forms of these same steroids. Considerable estrone sulfate sulfohydrolase of pH optimum 7.5 and high apparent Km was found to be concentrated in the 105,000 g pellet but no evidence was obtained, in any subcellular fraction, for the presence of any activity toward the 3-sulfate of pregnenolone, dehydroepiandrosterone (DHA) or delta 5-androstene-3 beta,17 beta-diol (androstenediol). Cytosolic sulfotransferase activity toward estrone, pregnenolone, DHA and androstenediol was present in each animal. The activity toward these 4 substrates was eluted from a gel filtration column as a single peak of apparent molecular weight 43 KDa. Upon chromatofocusing, a sharp estrogen sulfotransferase peak of apparent pI 6.1 and pH optimum 9.5, was clearly separated from the neutral steroid sulfotransferase which eluted over a more acidic pH range in a manner suggestive of the presence of several isozymes. This latter, which exhibited a wide pH optimum range between 6 and 8.5, was most active toward androstenediol, and least active toward pregnenolone. The estrogen sulfotransferase exhibited Michaelis-Menten kinetics (apparent Km = 4 microM). The neutral steroid sulfotransferase activity increased in velocity with increasing androstenediol or DHA concentration up to 1 microM beyond which considerable substrate inhibition occurred. It appears from these data that neutral steroid sulfates synthesized in the pig Leydig cell are not subject to enzymic desulfation in the same cells.     

Inhibition of Helicobacter pylori glycosulfatase activity toward gastric sulfomucin by nitecapone.

A glycosulfatase activity toward gastric sulfomucin was identified in the extracellular material elaborated by H. pylori. The enzyme exhibited maximum activity at pH 5.7 in the presence of Triton X-100 and CaCl2, and displayed on SDS-PAGE an apparent molecular weight of 30kDa. The H. pylori glycosulfatase effectively caused desulfation of N-acetylglucosamine-6-sulfate and galactose-6-sulfate of the carbohydrate chains of mucins, as well as that of glucose-6-sulfate of glyceroglucolipids, but was ineffective towards galactosyl- and lactosylceramide sulfates which contain galactose-3-sulfate. The glycosulfatase activity towards human gastric sulfomucin was affected by an antiulcer agent, nitecapone, which at its optimal concentration (100 micrograms/ml) caused a 61% inhibition. The results show that H. pylori through its glycosulfatase activity causes desulfation of sulfated mucins and glyceroglucolipids of the protective mucus layer, and that nitecapone is able to interfere with this detrimental action.     

Steroid sulfatase activity in a Peptococcus niger strain from the human intestinal microflora.

A strictly anaerobic gram-positive coccus, identified as Peptococcus niger, that developed sulfatase activity towards steroid-3-sulfate esters was isolated from human fecal material. This strain desulfated the arylsulfate esters estrone-3-sulfate (100%) and beta-estradiol-3-sulfate (50%); only trace amounts of desulfated estriol-3-sulfate were found. In addition, alkylsulfatase activity was found towards the 3 alpha-sulfates of 5 alpha-androstane-17-one and 5 beta-androstane-17-one and towards the 3 beta-sulfates of 5 alpha-androstane-17-one, delta 5-androstene-17-one, 5 alpha-pregnane-20-one, and delta 5-pregnene-20-one, all of which were 100% desulfated. No sulfatase activity was found towards the 17-sulfate esters of beta-estradiol or delta 4-androstene-3-one-17 alpha-ol. The nonsteroid arylsulfate esters paranitrophenyl sulfate, paranitrocatechol sulfate, and phenolphthalein disulfate were desulfated 70, 40, and 40%, respectively. In addition to its sulfatase activity, this strain also developed C-17 oxidoreductase activity towards the estrogens and androsta(e)nes and C-3 oxidoreductase activity towards androsta(e)nes and pregna(e)nes.     

Steroid sulfatase activity in a Peptococcus niger strain from the human intestinal microflora

A strictly anaerobic gram-positive coccus, identified as Peptococcus niger, that developed sulfatase activity towards steroid-3-sulfate esters was isolated from human fecal material. This strain desulfated the arylsulfate esters estrone-3-sulfate (100%) and beta-estradiol-3-sulfate (50%); only trace amounts of desulfated estriol-3-sulfate were found. In addition, alkylsulfatase activity was found towards the 3 alpha-sulfates of 5 alpha-androstane-17-one and 5 beta-androstane-17-one and towards the 3 beta-sulfates of 5 alpha-androstane-17-one, delta 5-androstene-17-one, 5 alpha-pregnane-20-one, and delta 5-pregnene-20-one, all of which were 100% desulfated. No sulfatase activity was found towards the 17-sulfate esters of beta-estradiol or delta 4-androstene-3-one-17 alpha-ol. The nonsteroid arylsulfate esters paranitrophenyl sulfate, paranitrocatechol sulfate, and phenolphthalein disulfate were desulfated 70, 40, and 40%, respectively. In addition to its sulfatase activity, this strain also developed C-17 oxidoreductase activity towards the estrogens and androsta(e)nes and C-3 oxidoreductase activity towards androsta(e)nes and pregna(e)nes.     

The deposition of calcium pyrophosphate by NTP pyrophosphohydrolase of matrix vesicles from fetal bovine epiphyseal cartilage

About 5 mumol CaPPi/mg protein was deposited within 3 h in the presence of the reaction mixtures containing 1 mM ATP, 2 mM Ca2+, 1 mM Pi, and 17 micrograms of purified NTP pyrophosphohydrolase. At 1 mM ATP, 50% of the deposition was inhibited by 0.5-1 mM of various substrate and product analogues including AMP, ADP, and ethylene hydroxyl diphosphonate. The magnitude of inhibition on NTP pyrophosphohydrolase activity was in the order of AMP = CMP = ADP greater than adenosine greater than adenine greater than NAD = NADP. AMP, CMP, ADP, and adenosine are competitive inhibitors. The modes of inhibition by adenine, NAD, and NADP differ from the competitive inhibition. Ribose, 3'-AMP, 2'-AMP, and cAMP did not inhibit the enzyme activity.     

NAD-specific isocitrate dehydrogenase from bovine heart. Interaction with Ca2+ chelators.

The activity of NAD-specific isocitrate dehydrogenase was inhibited by EDTA, EGTA and other nitrogen-containing polycarboxylate Ca2+ chelators in the absence and in the presence of ADP by a mechanism that could not be attributed solely to the removal of free Ca2+. Carboxymethyltartronate (2-oxapropane-1,1,3-tricarboxylate), an oxygen ether polycarboxylate chelator, did not inhibit when ADP was absent. The activation by ADP, a positive effector of the enzyme, decreased with increasing concentration of carboxymethyltartronate, paralleling the removal of free Ca2+ by this chelator. The following were found when free Ca2+ was decreased to negligible concentrations (5-50 nM) with carboxymethyltartronate. (1) Free Ca2+ enhanced, but was not absolutely required for, activation by ADP. (2) Activation of enzyme activity by magnesium citrate neither required nor was increased by Ca2+ when ADP was absent. However, the potentiation of citrate activation by ADP was facilitated by free Ca2+. (3) The reversal of NADPH inhibition of enzyme activity by ADP did not absolutely require Ca2+, but it was enhanced by free Ca2+. (4) The inhibition of enzyme activity by NADH was not reversed by ADP either with or without Ca2+.     

Nucleoside pyrophosphatase activity associated with pig kidney alkaline phosphatase

1. A study was made of the hydrolysis, at pH9.0, of ATP and ADP catalysed by pig kidney alkaline phosphatase. Both of these nucleoside pyrophosphates are substrates for the enzyme; K(m) values are 4x10(-5)m for ATP and 6.3x10(-5)m for ADP. V(max.) for ADP is approximately double that of ATP. 2. Above 0.1mm approximately, both ATP and ADP are inhibitory, but the inhibition is reversible by the addition of Mg(2+) ions to form MgATP(2-) or MgADP(-) complexes. The complexes, besides being non-inhibitory, are also substrates for the enzyme with K(m) values identical with those of the respective free nucleotides. 3. Mg(2+) ions are inhibitory when present in excess of ATP or ADP. The degree of inhibition is greater with ATP as substrate, but with both ATP and ADP a mixed competitive-non-competitive type of inhibition is observed. 4. It is suggested that under normal conditions the enzyme is inhibited by cellular concentrations of ATP plus ADP but that an increase in the concentration of Mg(2+) ions stimulates activity by relieving nucleoside pyrophosphate inhibition. The properties may be of importance in the regulation of the transport of bivalent cations.     

Properties of enzyme fraction A from Chlorella and copurification of 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase, adenosine 5'phosphosulfate sulfohydrolase and adenosine-5'-phosphosulfate cyclase activities.

Enzyme fraction A from Chlorella which catalyzes the formation of adenosine 5'-phosphosulfate from adenosine 3'-phosphate 5'-phosphosulfate is further characterized. Fraction A is found to contain an Mg2+ -activated and Ca2+ -inhibited 3' (2')-nucleotidase specific for 3' (2'), 5'-biphosphonucleosides. This activity has been named 3' (2), 5'-biphosphonucleoside 3' (2')-phosphohydrolase. The A fraction is also found to contain an activity which catalyzes the formation of adenosine 3':5'-monophosphate (cyclic AMP) from adenosine 5'-phosphosulfate (adenosine 5'-phosphosulfate cyclase). Under the same conditions of assay, 5'-ATP and 5'-ADP are not substrated for cyclic AMP formation. Unlike the 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase activity, the adenosine 5'-phosphosulfate cyclase activity does not require Mg2+, requires NH+4 or Na+, and is not inhibited by Ca2+. The A fraction also contains an adenosine 5'-phospho sulfate sulfohydrolase activity which forms 5'-AMP and sulfate. The three activities remain together during purification and acrylamide gel electrophoresis of the purified preparation yields a pattern where only one protein band has all three activities. The phosphohydrolase can be separated from the other two activities by affinity chromatography on agarose-hexyl-adenosine 3'n5'-bisphosphate yielding a phosphohydrolase preparation showing a single band on gel electrophoresis. The adenosine 5'-phosphosulfate cyclase may provide an alternate route of cyclic AMP formation from sulfate via ATP sulfurylase, but its regulatory significance in Chlorella, if any, remains to be demonstrated. In sulfate reduction, the phosphohydrolase may serve to provide a readily utilized pool of adenosine 5'-phosphosulfate as needed by the adenosine 5'-phosphosulfate sulfotransferase. The cyclase and sulfohydrolase activities would be regarded as side reactions incidental to this pathway, but may be of importance in other metabolic and regulatory reactions.     

Pyruvate dehydrogenase kinase isoform 2 activity limited and further inhibited by slowing down the rate of dissociation of ADP

Pyruvate dehydrogenase kinase 2 (PDK2) activity is enhanced by the dihydrolipoyl acetyltransferase core (E2 60mer) that binds PDK2 and a large number of its pyruvate dehydrogenase (E1) substrate. With E2-activated PDK2, K(+) at approximately 90 mM and Cl(-) at approximately 60 mM decreased the K(m) of PDK2 for ATP and competitive K(i) for ADP by approximately 3-fold and enhanced pyruvate inhibition. Comparing PDK2 catalysis +/- E2, E2 increased the K(m) of PDK2 for ATP by nearly 8-fold (from 5 to 39 microM), increased k(cat) by approximately 4-fold, and decreased the requirement for E1 by at least 400-fold. ATP binding, measured by a cold-trapping technique, occurred at two active sites with a K(d) of 5 microM, which equals the K(m) and K(d) of PDK2 for ATP measured in the absence of E2. During E2-aided catalysis, PDK2 had approximately 3 times more ADP than ATP bound at its active site, and the pyruvate analogue, dichloroacetate, led to 16-fold more ADP than ATP being bound (no added ADP). Pyruvate functioned as an uncompetitive inhibitor versus ATP, and inclusion of ADP transformed pyruvate inhibition to noncompetitive. At high pyruvate levels, pyruvate was a partial inhibitor but also induced substrate inhibition at high ATP levels. Our results indicate that, at physiological salt levels, ADP dissociation is a limiting step in E2-activated PDK2 catalysis, that PDK2.[ADP or ATP].pyruvate complexes form, and that PDK2.ATP.pyruvate.E1 reacts with PDK2.ADP.pyruvate accumulating.     

Selective inhibition of glutathione S-transferases by 17 beta-estradiol disulfate.

Enzyme activity of homogeneous glutathione S-transferases A, B, and C with reduced glutathione and 1-chloro-2,4-dinitrobenzene was inhibited in varying degrees by 50 μm concentrations of monosulfate and disulfate derivatives of several steroids. In contrast, transferase AA activity was not affected. Of the inhibitors tested, estradiol-3,17-disulfate and estradiol-3-sulfate were the most inhibitory, followed by pregnenolone sulfate, estradiol-17-sulfate, dehydroisoandrosterone sulfate, and cortisol sulfate. Transferases A and C were most affected, especially by estradiol disulfate and estradiol-3-sulfate, which exhibited essentially complete inhibition at a concentration of μm. Double reciprocal plots of estradiol disulfate inhibition with respect to 1-chloro-2,4-dinitrobenzene concentration showed uncompetitive inhibition with transferases A and C and noncompetitive inhibition with transferase B (ligandin). With reduced glutathione as the variable substrate, transferases A and C exhibited noncompetitive inhibition kinetics, while transferase B showed partial noncompetitive kinetics.     

The kinetic properties of citrate synthase from rat liver mitochondria

1. Citrate synthase (EC 4.1.3.7) was purified 750-fold from rat liver. 2. Measurements of the Michaelis constants for the substrates of citrate synthase gave values of 16mum for acetyl-CoA and 2mum for oxaloacetate. Each value is independent of the concentration of the other substrate. 3. The inhibition of citrate synthase by ATP, ADP and AMP is competitive with respect to acetyl-CoA. With respect to oxaloacetate the inhibition by AMP is competitive, but the inhibition by ADP and ATP is mixed, being partially competitive. 4. At low concentrations of both substrates the inhibition by ATP is sigmoidal and a Hill plot exhibits a slope of 2.5. 5. The pH optimum of the enzyme is 8.7, and is not significantly affected by ATP. 6. Mg(2+) inhibits citrate synthase slightly, but relieves the inhibition caused by ATP in a complex manner. 7. At constant total adenine nucleotide concentration made up of various proportions of ATP, ADP and AMP, the activity of citrate synthase is governed by the concentration of the sum of the energy-rich phosphate bonds of ADP and ATP. 8. The sedimentation coefficient of the enzyme, as measured by activity sedimentation, is 6.3s, equivalent to molecular weight 95000.     

Effect of nucleoside 5′-di- and 5′-tri-phosphates on pancreatic ribonuclease activity

1. ADP, ATP and GDP inhibited the phosphotransferase activity, the release of cyclic nucleotides from RNA, of ribonuclease. No significant inhibition was elicited by pyrimidine 5'-nucleoside diphosphates, CDP and UDP. 2. Inhibition by ADP, AMP, adenosine, adenine, NAD and NADP was insignificant at the concentrations tested. Small inhibition was observed with high concentrations of AMP and only when soluble RNA was the substrate. 3. Inhibition by ADP was found to be ;uncompetitive'. 4. Results seem to indicate that at least for optimum inhibition the polyphosphate of the purine nucleoside is essential. They further suggest that the inhibitor acts by combining with the enzyme only when the enzyme is bound to the substrate.     

Caspase activity during cell stasis: avoidance of apoptosis in an invertebrate extremophile, Artemia franciscana

Evaluation of apoptotic processes downstream of the mitochondrion reveals caspase-9- and low levels of caspase-3-like activities in partly purified extracts of Artemia franciscana embryos. However, in contrast to experiments with extracts of human hepatoma cells, cytochrome c fails to activate caspase-3 or -9 in extracts from A. franciscana. Furthermore, caspase-9 activity is sensitive to exogenous calcium. The addition of 5 mM calcium leads to a 4.86 +/- 0.19 fold (SD) (n = 3) increase in activity, which is fully prevented with 150 mM KCl. As with mammalian systems, high ATP (>1.25 mM) suppresses caspase activity in A. franciscana extracts. A strong inhibition of caspase-9 activity was also found by GTP. Comparison of GTP-induced inhibition of caspase-9 at 0 and 2.5 mM MgCl(2) indicates that free (nonchelated) GTP is likely to be the inhibitory form. The strongest inhibition among all nucleotides tested was with ADP. Inhibition by ADP in the presence of Mg(2+) is 60-fold greater in diapause embryos than in postdiapause embryos. Because ADP does not change appreciably in concentration between the two physiological states, it is likely that this differential sensitivity to Mg(2+)-ADP is important in avoiding caspase activation during diapause. Finally, mixtures of nucleotides that mimic physiological concentrations in postdiapause and diapause states underscore the depressive action of these regulators on caspase-9 during diapause. Our biochemical characterization of caspase-like activity in A. franciscana extracts reveals that multiple mechanisms are in place to reduce the probability of apoptosis under conditions of energy limitation in this embryo.