Adenosine-5′-phosphosulfate kinase from Thermobifida fusca

Sulfur, as a macronutrient, is essential for all kinds of organisms. Sulfate, the primary available source of sulfur, is firstly activated by adenylation catalyzed by ATP sulfurylase (ATPS) to form adenosine 5′-phosphosulfate (APS), which will be further phosphorylated into 3′-phosphoadenosine 5′-phosphosulfate (PAPS) by APS kinase (APSK). In some organisms, sulfate activating related enzymes are assembled to form sulfate-activating complex (SAC) to facilitate APS synthesis, the thermodynamically unfavorable reaction. In genome of a moderate thermophilic bacterium, Thermobifida fusca, there are presumably GTPasecoupled ATPS and one putative bifunctional ATPS/APSK type SAC. In this study, this putative SAC of T. fusca was prokaryotically expressed, purified and characterized. Activity assays showed that it contained APSK activity, while lacked ATPS activity. SAC of T. fusca was further used as a coupling enzyme to assay APS formation catalyzed by yeast ATPS. Based on the sequence alignment and modeled structure, we infer that the divergences of two conserved motifs and the missing of a loop and a helix-turn-helix motifs may contribute to the deficiency of ATPS activity.     

A factor-dependent sulfotransferase specific for 3′-phosphoadenosine-5′-phosphosulfate (PAPS) in the Cyanobacterium Synechococcus 6301

A sulfotransferase isolated from the Cyanobacterium Synechococcus 6301 was found to be specific for 3-phosphoadenosine-5-phosphosulfate (PAPS). The molecular weight of this transferase has been estimated on a Sephadex-G-100 column to be about 58,000. The K _m for PAPS was determined to be 20 M. The pH optimum was 8.0. The thiol dithioerythritol was needed for activity; other thiols such as glutathione, cysteine, or mercaptoethanol did not catalyze this reaction. The transferase, however, could not react directly with the thiol. A heat-stable factor was needed in this reaction. This factor was purified by conventional techniques and its molecular weight was determined on a Sephadex-G-50 column to be about 11,500. The factor showed normal Michaelis-Menten behavior toward the PAPS-sulfotransferase. It has been identified as thioredoxin. The tranferase was inhibited by 3-5-ADP and 2–5-ADP; all other adenine-containing nucleotides such as 2-AMP, 3-AMP, 5-AMP, ADP, and c-AMP did not influence this reaction.Abbreviation PAPS 3-phosphoadenosine-5-phosphosulfate     

Effect of Diquat (1,1′-Ethylene-2,2′-Dipyridylium Dibromide) on the Photosynthetic Growth of Rhodospirillum rubrum

Diquat (2 x 10(-4)m) inhibited both aerobic and anaerobic growth of Rhodospirillum rubrum. With photosynthetic cultures, diquat affected the synthesis of bacteriochlorophyll more readily than cell mass (turbidity). Diquat retarded the synthesis of bacteriochlorophyll and some protein more readily than that of other cellular constituents such as ribonucleic acid, deoxyribonucleic acid, and cell mass. With cells deficient in phosphate, diquat inhibited the uptake-conversion of inorganic phosphate completely only when 3-(3,4-dichlorophenyl)-1,1'-dimethyl urea and ascorbate were also present.     

The Chelating Ability of Adenosine-5′-Triphosphate-3′-Diphosphate (pppApp)

We have developed a new series of R4L1 Gateway binary vectors (R4L1pGWB), which carry the bialaphos resistance gene (bar) or the UDP-N-acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) gene as selection markers that confer BASTA^® and tunicamycin resistance on plants respectively. R4L1pGWBs have an attR4-attL1-reporter and can accept an attL4-promoter-attR1 entry clone for easy construction of an attB4-promoter-attB1-reporter clone. The new R4L1pGWBs facilitate promoter:reporter analysis in pre-existing transgenic plants that are resistant to kanamycin or hygromycin.     

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.Abbreviations APS Adenosine 5-phosphosulfate - APSSTase Adenosine 5-phosphosulfate sulfotransferase - BSA Bovine serum albumin - BRIJ58 Polyethylene glycolmonostearylether - DTE Dithioerythritol - DTT Dithiothreitol - EDTA Ethylenediaminetetraacetic acid - ME 2-Mercaptoethanol - NADP-GPD NADP-linked glyceraldehyde-3-phosphate dehydrogenase - PAPS Adenosine 3-phosphate 5-phosphate 5-phosphosulfate - POPOP 1,4 Di [2-(5-phenyloxazolyl)]-benzene - PPO 2,5-Diphenyloxazol The results presented in this paper are taken from the Ph. D. thesis of H.F.     

Studies on degradation of adenosine-5′-phosphosulphate (APS) and adenosine-3′-phosphate-5′-phosphosulphate (PAPS) in extracts of Anabaena cylindrica

[³⁵S]Adenosine-5′-phosphosulphate ([³⁵S]APS) and [³⁵S]adenosine-3′-phosphate-5′-phosphasulphate ([³⁵S]PAPS) were rapidly degraded by extracts of Anabaena cylindrica. The loss of radioactivity from these sulphur nucleotides resulted in a corresponding increase of free ³⁵SO₄^ in the incubation mixture. The soluble fraction of the broken cells (S₇₅) hydrolysed both PAPS and APS, whereas the pellet fractions (P₂₀ and P₇₅) hydrolysed PAPS only. The degradation of [³⁵S]PAPS was almost completely suppressed by various 5′-adenine nucleotides, 3′-AMP, nucleotide triphosphates or pyrophosphate, while glucose-6-phosphate, phosphate ions and sodium sulphite were less effective. The hydrolysis of [³⁵S]APS was prevented by sodium fluoride and 5′-AMP, but 3′-AMP was ineffective.     

A new assay for ATP sulfurylase based on differential solubility of the sodium salts of adenosine 5′-phosphosulfate and sulfate

A new assay for ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) has been devised, the key feature of which is the quantitative precipitation of the unreacted substrate as Na₂SO₄, in ethanol:water (5:1), leaving the product adenosine 5′-phosphosulfate (APS) in solution with an 80% yield. This separation procedure, coupled with the use of $\text{[}{}^{35}\text{S]SO}{}_4{}^{\mathrm{2?}}$, makes it possible to assay the synthesis of picomole amounts of [³⁵S]APS and thereby determine accurately the initial velocity of the forward reaction catalyzed by ATP sulfurylase. Extracts from tobacco cells synthesized [³⁵S]APS and a negligible amount of one unknown sulfur compond, but they did not synthesize 3′-phosphoadenosine 5′-[³⁵S]phosphosulfate (PAPS) under the assay conditions adopted, so that the ³⁵S remaining in solution after ethanol precipitation of $\text{[}{}^{35}\text{S]SO}{}_4{}^{\mathrm{2?}}$ is a valid assay of the ATP sulfurylase reaction in these extracts. This assay is simple, rapid, and suitable for assaying multiple samples. Extracts of four additional species of plants were incubated under the same conditions, and each catalyzed the synthesis of [³⁵S]APS according to this assay. The assay is suitable for use with the enzyme from any organism, provided that the product APS is not acted upon by another enzyme.     

Occurrence of Guanosine-5′-diphosphate-3′-diphosphate and Adenosine-5′-triphosphate-3′-diphosphate in Streptomyces galilaeus

Synthetic activity and existence of ppGpp and pppApp in an anthracycline-producing strain Streptomyces galilaeus were determined by radioimmunoassay and ³²P-labeling method during cultivation under both the antibiotic productive and non-productive conditions. The cellular ppGpp(pppGpp)-synthesizing activity was highest at the end of exponential growth, and 3-fold higher in the antibiotic-productive cultivation than in non-productive cultivation. The intracellular level of ppGpp determined by radioimmunoassay was high at the end of exponential growth, and afterwards its level decreased by one fifth. The low level of cellular ppGpp during the period of intense antibiotic production suggests that ppGpp consumption may play an important role in antibiotic production of S. galilaeus. The concentration of pppApp was not determined intracellularly by radioimmunoassay.     

Uptake of Liposomally Entrapped Adenosine-3′-5′-Cyclic Monophosphate in Mouse Brain

[3H] Adenosine-3',5'-cyclic monophosphate (cAMP) could be entrapped efficiently into small unilamellar vesicles when bound to cAMP-dependent protein kinase. The leakage of [3H]cAMP protein kinase complex from liposomes was reduced by more than 60% as compared to free [3H]cAMP. Hyperosmolar mannitol increased the delivery of liposomally entrapped [3H]cAMP protein kinase to the brain with maximum uptake occurring at 10 min after mannitol administration. Optimal delivery to the brain was observed when vesicles composed of total brain lipids or phosphatidylcholine:cholesterol:sulfatides (7:2:1) were used. A slower clearance of liposomally entrapped material from brain tissue was seen under hyperosmolar conditions.     

Properties of the 3′-phosphoadenosine-5′-phosphosulfate (PAPS) synthesizing systems of brain and liver

Chromatography of brain and liver 100,000g supernatants over HPLC molecular sieve columns revealed striking differences in the molecular weight distribution of ATP-sulfurylase and APS-kinase of the two tissues, pointing to different enzymic species for both enzymes in brain and liver. This was further substantiated by kinetic characterization of the two enzymes of both tissues. APS-kinase of liver is allosterically activated by ATP, while the brain enzyme is not. ATP-sulfurylase of brain is activated at high, but still physiological concentrations of ATP. Brain ATP-sulfurylase is inhibited by phenylalanine.     

The role of glutamine synthetase in the regulation of nitrogenase activity (“switch off” effect) in Rhodospirillum rubrum

We have studied the changes in the activities of both nitrogenase (switch off) and glutamine synthetase in Rhodospirillum rubrum upon addition of ammonium ions or glutamine to nitrogen fixing cultures. Both activities decrease drastically and return in a parallel manner when added ammonia is metabolized. The decrease in glutamine synthetase activity does not seem to be primarily due to adenylylation of the enzyme. Addition of glutamine to cells starved for nitrogen results in inactivation of glutamine synthetase but nitrogenase is only partially switched off.Abbreviations CeMe₃NBr Cetyltrimethylammonium bromide - Hepes N-2-hydroxyethyl-piperazine-N-2 sulfonic acid - MSO methionine-D,L-sulfoximine - Tea-Dmg triethanol amine-3,3-dimethylglutaric acid     

Substrate Properties of Adenosine- and Uridine- 3′, 5′-bisphenylphosphonates for 3′-Nucleotidase/Nucleases

Abstract

3′, 5′-Bisphenylphosphonate and 5′-phenylphosphonate esters of adenosine and uridine were synthesized to investigate the substrate properties of the 3′, 5′-bisphenylphosphonates for 3′-nucleotidase/nucleases. The V _max/apparent K _m, values of the enzymes for them were found to be 9 to 21-fold higher than those for the corresponding nucleoside 3′-phenylphosphonates.     

Defect in 3′-phosphoadenosine 5′-phosphosulfate synthesis in brachymorphic mice: I. Characterization of the defect

Biosynthesis of the undersulfated proteoglycan found in brachymorphic mouse (bm/ bm) cartilage has been investigated. Similar amounts of cartilage proteoglycan core protein, as measured by radioimmune inhibition assay, and comparable activity levels of four of the glycosyltransferases requisite for synthesis of chondroitin sulfate chains were found in cartilage homogenates from neonatal bm/bm and normal mice, suggesting normal production of glycosylated core protein acceptor for sulfation. When incubated with ³⁵S-labeled 3′-phosphoadenosine 5′-phosphosulfate (PAPS), bm/bm cartilage extracts showed a higher than control level of sulfotransferase activity. In contrast, when synthesis was initiated from ATP and ³⁵SO₄^2?, mutant cartilage extracts showed lower incorporation of ³⁵SO₄^2? into endogenous chondroitin sulfate proteoglycan (19% of control level) and greatly reduced formation of PAPS (10% of control level). Results from coincubations of normal and mutant cartilage extracts exhibited intermediate levels of sulfate incorporation into PAPS and endogenous acceptors, suggesting the absence of an inhibitor for sulfate-activating enzymes or sulfotransferases. Degradation rates of ³⁵S]PAPS and of ³⁵S-labeled adenosine 5′-phosphosulfate (APS) were comparable in bm/bm and normal cartilage extracts. Specific assays for both ATP sulfurylase (sulfate adenylyltransferase; ATP:sulfate adenylyltransferase, EC 2.7.7.4) and APS kinase (adenylylsulfate kinase; ATP:adenylylsulfate 3′-phosphotransferase, EC 2.7.1.25) showed decreases in the former (50% of control) and the latter (10–15% of control) enzyme activities in bm/bm cartilage extracts. Both enzyme activities were reduced to intermediate levels in extracts of cartilage from heterozygous brachymorphic mice (ATP-sulfurylase, 80% of control; APS kinase, 40–70% of control). Furthermore, the moderate reduction in ATP sulfurylase activity in bm/bm cartilage extracts was accompanied by increased lability to freezing and thawing of the residual activity of this enzyme. These results indicate that under-sulfation of chondroitin sulfate proteoglycan in bm/bm cartilage is due to a defect in synthesis of the sulfate donor (PAPS), resulting from diminished activities of both ATP sulfurylase and APS kinase, although the reduced activity of the latter enzyme seems to be primarily responsible for the defect in PAPS synthesis.     

Acycloadenosine Derivatives as Inhibitors of 5′-Deoxy-5′-Methylthioadenosine Phosphorylase (MesADo PASE)

Abstract

Four classes of acyclo amlogs of 5′-methylthioadenosine were synthesized and tested as inhibitors of mammalian methylthicadenosine phosphorylase. Halogenated dihydroxypropyl acycloadenosires were most potent, i.e. K_i = 0.2 - 0.7 uM.     

Synthesis and General Biological Activity of a Small Adenosine-5′-(Carboxamide and Sulfanilamide) Library

A small library of fifty-five adenosine peptide analogs was synthesized, under the Pilot Scale Library (PSL) Program of the NIH Roadmap initiative, from 2′,3′-O-isopropylideneadenosine-5′-carboxylic acid 2. The coupling of amine or sulfanilamide reactants to the free 5′-carboxylic acid moiety of 2, in automated solution-phase fashion, led after acid-mediated hydrolysis to target compounds 3–57 in good yields and high purity. No marked anticancer or antimalarial activity was noted on preliminary cellular testing. Initial screening through the MLPCN program, however, indicates that these analogs may show diverse and interesting biological activities.     

Evidence for the regulation of pyridoxal 5′-phosphate formation in liver by pyridoxamine (pyridoxine) 5′-phosphate oxidase

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) purified from rabbit liver is competitively inhibited by the reaction product, pyridoxal 5′-phosphate. The K_i, 3 μM, is considerably lower than the K_m for either natural substrate (18 and 24 μM for pyridoxamine 5′-phosphate and 25 and 16 μM for pyridoxine 5′-phosphate in 0.2 M potassium phosphate at pH 8 and 7, respectively). The K_i determined using a 10% rabbit liver homogenate is the same as that for the pure enzyme; hence, product inhibition $\text{in}\text{vivo}$ is probably not diminished significantly by other cellular components. Similar determinations for a 10% rat liver homogenate also show strong inhibition by pyridoxal 5′-phosphate. Since the reported liver content of free or loosely bound pyridoxal 5′-phosphate is greater than K_i, the oxidase in liver is probably associated with pyridoxal 5′-phosphate. These results also suggest that product inhibition of pyridoxamine-P oxidase may regulate the $\text{in}\text{vivo}$ rate of pyridoxal 5′-phosphate formation.     

Equilibration of [3H]glucosamine and [35S]sulfate with intracellular pools of UDP-N-acetylhexosamine and 3′-phosphoadenosine-5′-phosphosulfate (PAPS) in cultured fibroblasts

Nucleotides and sugar nucleotides were extracted from cultures of human fibroblasts with perchloric acid, separated by isotachophoresis, and quantified by uv absorption analysis at 254 nm. ATP (936 pmol/μg DNA) was, as expected, the dominating nucleotide pool. The energy charge was estimated to 0.9. The UDP-N-acetylhexosamine pool was also a very prominent compound (596 pmol/μg DNA). After incubation of fibroblasts with [³H]glucosamine, more than 95% of the acid-soluble radioactivity was found in the UDP-N-acetylhexosamine pool. Incubation with [³⁵S]sulfate resulted in the incorporation of [³⁵S]sulfate into 3′-phosphoadenosine-5′-phosphosulfate (PAPS). The latter could, however, only be measured as radioactivity, as the amount was too small to be quantified as total mass. Pulse-labeling of fibroblasts with [³⁵S]sulfate and [³H]glucosamine from 5 min to 16 h showed that [³⁵S]PAPS was equilibrated in less than 10 min, while [³H]glucosamine required a longer time, 2–4 h, to attain a steady state with UDP-N-acetylhexosamine. [¹⁴C]Glucose required approximately the same time as [³H]glucosamine to reach steady state with UDP-acetylhexosamine, which suggests that the reason for the long equilibration time is the slow turnover of this pool.