Purification and kinetic characterization of a dopamine-sulfating form of phenol sulfotransferase from human brain

The kinetic and biochemical properties of a purified, monoamine-sulfating form of phenol sulfotransferase (M-PST) from human brain are described. M-PST activity was separated and purified from phenol-sulfating activity by anion-exchange chromatography on DEAE-cellulose and subsequently purified on AffiGel Blue and Sephacryl S-200, routinely giving a final purification of over 20 000-fold, with approximately a 3% yield. The molecular weight of the active species, as estimated by gel filtration chromatography, was 250 000. The purified enzyme was inhibited by NaCl (50% at 325 mM) and showed an optimum for dopamine sulfation at pH 7.0. Of the monoamine substrates examined, 4-methoxytyramine was the most extensively sulfated at 20 microM, while at higher substrate concentrations (200 microM), tyramine was the apparent preferred substrate. Kinetic analysis demonstrated that sulfation by M-PST proceeds via an ordered, bisubstrate reaction mechanism, where 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is the leading substrate. True Km values for dopamine and PAPS were 2.9 and 0.35 microM, respectively. The product inhibitor 3'-phosphoadenosine 5'-phosphate possessed a Ki of 0.07 microM, while the dead-end inhibitor ATP exhibited a Ki of 170 microM.     

Human Platelet Phenol Sulfotransferase: Partial Purification and Detection of Two Forms of the Enzyme

To begin to study the usefulness of platelet phenol sulfotransferase (PST) as a possible measure of the enzyme activity in other organs such as the brain, we purified human platelet PST 36-120-fold. Activity toward 3-methoxy-4-hydroxyphenylglycol (MHPG), dopamine, 5-hydroxytryptamine (5-HT), and phenol eluted in the same Sephadex G-100 and Affi-Gel Blue column fractions. Specific activities of the enzyme with MHPG, dopamine, 5-HT, and phenol as substrates were 1198, 1068, 401, and 408 units/mg protein, respectively. Optimal assay conditions were established for each substrate. Apparent Km values were 598 microM, 21 microM, 19 microM, and 500 microM for MHPG, dopamine, phenol, and 5-HT, respectively. Apparent Km values for 3'-phosphoadenosine-5'-phosphosulfate (PAPS) with the same four substrates ranged from 0.11 to 0.25 microM. The pH optima were 6.3 for phenol, 6.8 for dopamine, and 7.0 for MHPG and 5-HT. An additional pH optimum at 8.6 was present for 5-HT. A thermolabile form of the enzyme measured with dopamine and 5-HT, as well as a thermostable form measured with phenol, were present. Dichloronitrophenol (10(-5) M) noncompetitively inhibited the thermostable enzyme activity by 96% but decreased the thermolabile activity by only 36%. These studies provide the basis for a more accurate comparison of human platelet PST with the enzyme in the human brain and in other tissues.     

Purification and characterization of bile salt sulfotransferase from human liver

Bile salt sulfotransferase, the enzyme responsible for the formation of bile salt sulfate esters, was purified extensively from normal human liver. The purification procedure included DEAE-Sephadex chromatography, taurocholate-agarose affinity chromatography, and preparative isoelectrofocusing. The final preparation had a specific activity of 18 nmol min-1 mg protein-1, representing a 760-fold purification from the cytosol fraction with a overall yield of 15%. The human enzyme has a Mr of 67,000 and a pI of 5.2. DEAE-Sephadex chromatography of the cytosol fraction revealed only a single species of activity. The limiting Km for the sulfuryl donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), is 0.7 microM. The limiting Km for the sulfuryl acceptor, glycolithocholate (GLC), is 2 microM. Reciprocal plots were intersecting. Product inhibition studies established that adenosine 3',5'-diphosphate (PAP) was competitive with PAPS (Ki = 0.2 microM) and noncompetitive with respect to GLC. GLC sulfate was competitive with GLC (Ki = 2.2 microM) and noncompetitive with respect to PAPS. Also, 3-ketolithocholate, a dead-end inhibitor, was competitive with GLC (Ki = 0.6 microM) and noncompetitive with respect to PAPS. Iso-PAP (the 2' isomer of PAP) was competitive with PAPS (Ki = 0.3 microM) and noncompetitive with GLC. The cumulative results of the steady-state kinetics experiments point to a random mechanism for the binding of substrates and release of products. The purified enzyme displays no activity toward estrone, testosterone, or phenol. Among the reactive substrates tested, the Vmax/Km values are in the order GLC greater than 3-beta OH-5-cholenic acid greater than glycochenodeoxycholate greater than glycocholate. p-Chloromercuribenzoate inactivated the enzyme. Either PAPS or GLC protected against inactivation, suggesting the presence of a sulfhydryl group at the active site.     

Phenol sulfotransferase in human lung

Human lung phenol sulfotransferase was about 1000-fold purified using the earlier described procedure. 2-Naphthol, p-nitrophenol, phenol, salicylamide, p-methylphenol, o-methoxyphenol, adrenaline, and dopamine were tested as substrates for human lung PST using the barium hydroxide procedure and the ECTEOLA-cellulose method. Km values for sulfate donor (PAPS) and for different sulfate acceptors were determined. 2,6-Dichloro-4-nitrophenol was found to be a competitive inhibitor of human lung PST with Ki = 8.87 +/- 0.08 microM. High salt concentration and Mg2+, Mn2+, and Zn2+ inhibited lung PST. The molecular weight of human lung PST was estimated as 38,000 and 35,000 by gel filtration and SDS-gel electrophoresis, respectively.     

Inhibition and binding studies of coenzyme A and bovine phenol sulfotransferase.

Phenol sulfotransferases (PSTs, EC 2.8.2.1) catalyze sulfonyl group transfer from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the hydroxyl oxygen of aromatic acceptor substrates. The structural overlap between PAPS and coenzyme A (CoA) suggested a possible role of this common acyl carrier in modulating PST activity. To test this hypothesis, purified recombinant bovine PST was examined by kinetic and affinity chromatographic approaches. After demonstrating PST enzyme inhibition by CoA, systematic variation of CoA and PAPS concentrations indicated simple competitive inhibition with K(i) = 1. 3 microM. PST bound to CoA-agarose, attached via the pantetheinyl thiol group, was eluted with PAP but not by 2-naphthol. This observation was consistent with the pattern of inhibition. Additional members of the sulfotransferase superfamily, as well as acylated CoAs, should be further investigated.     

Two Phenol Sulfotransferase Species from One cDNA: Nature of the Differences

A phenol sulfotransferase from rat liver (EC 2.8.2.9), expressed inEscherichia colifrom a single cDNA, was purified as two separable but catalytically active proteins. The proteins appeared to be identical to each other and to the natural liver sulfotransferase by comparison of their amino acid constitution, amino-terminal end group, and interaction with a polyclonal antibody raised against the liver enzyme. Each of the recombinant forms, α and β, catalyzed the sulfuryl group transfer from 4-nitrophenylsulfate to an acceptor phenol, a reaction in which 3′-phospho-adenosine 5′-phosphate (PAP) is a necessary intermediate. Only form β, however, catalyzes the physiological transfer of a sulfuryl group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to the free phenol. Evidence is presented that sulfotransferase α, but not β, has 1 mol of PAP tightly bound per enzyme dimer. The ability to utilize PAPS as a sulfate donor could be altered: form α could be treated and purified as form β to acquire the ability to use PAPS, whereas form β was treated by extended incubation with PAP, lost its ability to use PAPS, and was purified as form α.     

Purification and activation of brain sulfotransferase.

Galactosylceramide sulfotransferase (EC 2.8.2.11) catalyzes the biosynthesis of sulfatide from galactocerebroside and adenosine 3'-phosphate 5'-phosphosulfate (PAPS). This enzyme is developmentally controlled, reaching a maximum activity in the brains of mice corresponding to that of maximum myelination. The product, sulfatide, is an important component of myelin. This transferase from mouse brain has been purified 2600-fold using a combination of pyridoxal 5'-phosphate- and ATP-ligated columns. The purified enzyme yielded a single band following SDS-polyacrylamide gel electrophoresis with an apparent M(r) of 31,000. The entire purification procedure can be completed in 1 day. The pH optimum for the enzyme is 7.0. The Km for PAPS is 1.2 x 10(-6) M, and the Km for cerebroside is 2.6 x 10(-5) M. Cerebroside concentrations > 80 pmol/ml are inhibitory. Enzyme preparations were associated with several lipids. Vitamin K+P(i) activated purified preparations of the sulfotransferase and maintained enzyme activity during storage at -80 degrees C.     

Purification and properties of mitochondrial uracil-DNA glycosylase from rat liver

Uracil-DNA glycosylase from rat liver mitochondria, an inner membrane protein, has been purified approximately 575,000-fold to apparent homogeneity. During purification two distinct activity peaks, designated form I and form II, were resolved by phosphocellulose chromatography. Form I constituted approximately 85% while form II was approximately 15% of the total activity; no interconversion between the forms was observed. The major form was purified as a basic protein with an isoelectric point of 10.3. This enzyme consists of a single polypeptide with an apparent Mr of 24,000 as determined by recovering glycosylase activity from a sodium dodecyl sulfate-polyacrylamide gel. A native Mr of 29,000 was determined by glycerol gradient sedimentation. The purified enzyme had no detectable exonuclease, apurinic/apyrimidinic endonuclease, DNA polymerase, or hydroxymethyluracil-DNA glycosylase activity. A 2-fold preference for single-stranded uracil-DNA over a duplex substrate was observed. The apparent Km for uracil residues in DNA was 1.1 microM, and the turnover number is about 1000 uracil residues released per minute. Both free uracil and apyrimidinic sites inhibited glycosylase activity with Ki values of approximately 600 microM and 1.2 microM, respectively. Other uracil analogues including 5-(hydroxymethyl)uracil, 5-fluorouracil, 5-aminouracil, 6-azauracil, and 2-thiouracil or analogues of apyrimidinic sites such as deoxyribose and deoxyribose 5'-phosphate did not inhibit activity. Both form I and form II had virtually identical kinetic properties, and the catalytic fingerprints (specificity for uracil residues located in a defined nucleotide sequence) obtained on a 152-nucleotide restriction fragment of M13mp2 uracil-DNA were almost identical. These properties differentiated the mitochondrial enzyme from that of the uracil-DNA glycosylase purified from nuclei of the same source.     

Purification and characterization of rat liver minoxidil sulphotransferase.

Minoxidil (Mx), a pyrimidine N-oxide, is used therapeutically as an antihypertensive agent and to induce hair growth in patients with male pattern baldness. Mx NO-sulphate has been implicated as the agent active in producing these effects. This paper describes the purification of a unique sulphotransferase (ST) from rat liver cytosol that is capable of catalysing the sulphation of Mx. By using DEAE-Sepharose CL-6B chromatography, hydroxyapatite chromatography and ATP-agarose affinity chromatography, Mx-ST activity was purified 240-fold compared with the activity in cytosol. The purified enzyme was also capable of sulphating p-nitrophenol (PNP) at low concentrations (less than 10 microM). Mx-ST was purified to homogeneity, as evaluated by SDS/PAGE and reverse-phase h.p.l.c. The active form of the enzyme had a molecular mass of 66,000-68,000 Da as estimated by gel exclusion chromatography and a subunit molecular mass of 35,000 Da. The apparent Km values for Mx, 3'-phosphoadenosine 5'-phosphosulphate and PNP were 625 microM, 5.0 microM and 0.5 microM respectively. However, PNP displayed potent substrate inhibition at concentrations above 1.2 microM. Antibodies raised in rabbits to the pure enzyme detected a single band in rat liver cytosol with a subunit molecular mass of 35,000 Da, as determined by immunoblotting. The anti-(rat Mx-ST) antibodies also reacted with the phenol-sulphating form of human liver phenol sulphotransferase, suggesting some structural similarity between these proteins.     

Endocrine steroid sulfotransferases: porcine endometrial estrogen sulfotransferase

Porcine endometrial estrogen sulfotransferase has been isolated and its properties examined. This enzyme only appeared in uteri from ovariectomized gilts which had been primed with estrogen and treated with progesterone. The most stable form of the enzyme was obtained via chromatofocusing of the 100,000 g supernatant from secretory endometrium. A molecular weight of 31 KDa was determined for this sulfotransferase by molecular sieve (Sephadex G-200 Superfine) and disk-gel electrophoresis. The active protein displayed a pI of 6.1, pH optimum of 7.6-7.8 and a requirement of 10 mM Mg2+ for maximum transfer of sulfate from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to estrone (E1). Km of the reaction was 24 +/- 4.7 microM for PAPS and 24 +/- 9.8 nM for E1 as substrate. Porcine endometrial sulfotransferase thus displayed a much greater affinity for E1 than a similar enzyme previously isolated from bovine adrenals. As has been observed of sulfotransferases from other tissues, an endogenous substrate (presumed to be E1) accompanies the enzyme throughout its purification.     

Human Brain Phenol Sulfotransferase: Biochemical Properties and Regional Localization

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of catecholamines and phenol and catechol drugs. The human blood platelet contains a thermolabile (TL) form of PST that catalyzes the sulfate conjugation of dopamine and other monoamines and a thermostable (TS) form that catalyzes the sulfate conjugation of micromolar concentrations of phenol and p-nitrophenol. Experiments were performed to determine whether the brain contains forms of PST analogous to the TL and TS forms found in the human platelet, and to determine whether there are regional variations in human brain PST activity. We found that the human brain contains at least two forms of PST, forms that are similar to the platelet TS and TL forms of the enzyme with respect to substrate specificity, apparent Km constants, thermal stability, and sensitivity to inhibitors. Optimal conditions were determined for the measurement of these two activities in brain homogenates. The stability of PST activities in the human brain after death was determined in five samples of cerebral cortex that were obtained during clinically indicated neurosurgical procedures. An average of 76 +/- 8% and 80 +/- 9% (mean +/- SEM) of the basal TL and TS PST activities, respectively, remained in these five samples of cerebral cortex after 8 h of storage under simulated post-mortem conditions. Six human brains were then obtained less that 8 h after death from patients who had no neurological disease prior to death. The mean activities of the TL and TS forms of PST were measured in 17 different regions of the six brains. If the pituitary was excluded from consideration, TL and TS PST activities both varied approximately fivefold among these regions, and both activities were highest in cerebral cortex. However, the average TS activity in the anterior pituitary, a tissue of non-neural origin embryologically, was 6.5-fold greater than the highest average TS PST activity found in cerebral cortex.     

Activation of purified guanylate cyclase by nitric oxide requires heme. Comparison of heme-deficient, heme-reconstituted and heme-containing forms of soluble enzyme from bovine lung

Bovine lung soluble guanylate cyclase was purified to apparent homogeneity in a form that was deficient in heme. Heme-deficient guanylate cyclase was rapidly and easily reconstituted with heme by reacting enzyme with hematin in the presence of excess dithiothreitol, followed by removal of unbound heme by gel filtration. Bound heme was verified spectrally and NO shifted the absorbance maximum in a manner characteristic of other hemoproteins. Heme-deficient and heme-reconstituted guanylate cyclase were compared with enzyme that had completely retained heme during purification. NO and S-nitroso-N-acetylpenicillamine only marginally activated heme-deficient guanylate cyclase but markedly activated both heme-reconstituted and heme-containing forms of the enzyme. Restoration of marked activation of heme-deficient guanylate cyclase was accomplished by including 1 microM hematin in enzyme reaction mixtures containing dithiothreitol. Preformed NO-heme activated all forms of guanylate cyclase in the absence of additional heme. Guanylate cyclase activation was observed in the presence of either MgGTP or MnGTP, although the magnitude of enzyme activation was consistently greater with MgGTP. The apparent Km for GTP in the presence of excess Mn2+ or Mg2+ was 10 microM and 85-120 microM, respectively, for unactivated guanylate cyclase. The apparent Km for GTP in the presence of Mn2+ was not altered but the Km in the presence of Mg2+ was lowered to 58 microM with activated enzyme. Maximal velocities were increased by enzyme activators in the presence of either Mg2+ or Mn2+. The data reported in this study indicate that purified guanylate cyclase binds heme and the latter is required for enzyme activation by NO and nitroso compounds.     

Adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum. Purification and kinetic characterization

Adenosine 5'-phosphosulfate (APS) kinase, the second enzyme in the pathway of inorganic sulfate assimilation, was purified to near homogeneity from mycelium of the filamentous fungus, Penicillium chrysogenum. The enzyme has a native molecular weight of 59,000-60,000 and is composed of two 30,000-dalton subunits. At 30 degrees C, pH 8.0 (0.1 M Tris-chloride buffer), 5.5 microM APS, 5 mM MgATP, 5 mM excess MgCl2, and "high" salt (70-150 mM (NH4)2SO4), the most highly purified preparation has a specific activity of 24.7 units X mg of protein-1 in the physiological direction of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) formation. This activity is nearly 100-fold higher than that of any previously purified preparation of APS kinase. APS kinase is subject to potent substrate inhibition by APS. In the absence of added salt, the initial velocity at 5 mM MgATP plus 5 mM Mg2+ is maximal at about 1 microM APS and half-maximal at 0.2 and 4.4 microM APS. In the presence of 200 mM NaCl or 70-150 mM (NH4)2SO4, the optimum APS concentration shifts to 4-6 microM APS; the half-maximal values shift to 1-1.3 and 21-27 microM APS. The steady state kinetics of the reaction were investigated using a continuous spectrophotometric assay. The families of reciprocal plots in the range 0.25-5 mM MgATP and 0.8-5.1 microM APS are linear and intersect on the horizontal axis. Appropriate replots yield KmMgATP = 1.5 mM, KmAPS = 1.4 microM, and Vmax, = 38.7 units X mg of protein-1. Excess APS is an uncompetitive inhibitor with respect to MgATP (K1APS = 23 microM). PAPS, the product of the forward reaction, is also uncompetitive with MgATP. PAPS is not competitive with APS. In the reverse direction, the plots have the characteristics of a rapid equilibrium ordered sequence with MgADP adding before PAPS. The kinetic constants are KmPAPS = 8 microM, KiMgADP = 560 microM, and Vmaxr = 0.16 units X mg of protein-1. Iso-PAPS (the 2'-phosphate isomer of PAPS) is competitive with PAPS and uncompetitive with respect to MgADP (Ki = 6 microM). APS kinase is inactivated by phenylglyoxal, suggesting the involvement of an essential argininyl residue. MgATP or MgADP at 10 Ki protect against inactivation. APS or PAPS at 600 and 80 Km, respectively, are ineffective alone, but provide nearly complete protection in the presence of 0.1 Ki of MgADP or MgATP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sulfating-activity and stability of cDNA-expressed allozymes of human phenol sulfotransferase, ST1A3*1 ((213)Arg) and ST1A3*2 ((213)His), both of which exist in Japanese as well as Caucasians.

We recently found single amino acid substitutions ((213)Arg/His and (223)Met/Val) in polymorphic human phenol-sulfating phenol sulfotransferase (SULT: cDNAs encoding ST1A3, P PST or HAST1/2) among Caucasians and African-Americans. In a Japanese population (n = 143), allele frequencies of (213)Arg and (213)His were 83.2 and 16. 8%, respectively, but the (223)Val allele was not found. (213)His homozygosity was reportedly associated with both very low (>7-fold) sulfating activities of p-nitrophenol (at 4 microM) and low thermostability in platelets. Sulfating-activity determinations using recombinant (213)Arg- and (213)His-forms (ST1A3*1 and ST1A3*2, respectively) did not, however, reveal appreciable deficiency in [(35)S]3'-phosphoadenosine 5'-phosphosulfate (PAPS)-dependent sulfation of p-nitrophenol (4 microM) by ST1A3*2 (7.5 vs. 10.2 nmol/min/nmol SULT for ST1A3). Kinetic parameters for p-nitrophenol for p-nitrophenol sulfation supported the slight decrease in sulfating activities at 4 microM (K(m), 0.82 vs. 1.75 microM; V(max), 13.2 vs. 13.1 nmol/min/nmol SULT, respectively, for ST1A3*1 and *2). p-Nitrophenyl sulfate-dependent 2-naphthol sulfation by ST1A3*2 was 69% of that by ST1A3*1 (p<0.05). However, ST1A3*2 was remarkably unstable at 45 and 37 degrees C as compared to ST1A3*1. The lower p-nitrophenol sulfating activity of ST1A3*2 may explain the lower platelet p-nitrophenol sulfation in ST1A3*2 homozygotes. Protein instability and ST1A3 gene regulation may be both involved in the polymorphism of p-nitrophenol sulfation in human tissues.     

The human platelet as an independent unit for sulfate conjugation

The human platelets possess a full complement of enzymes capable of synthesizing N-acetyldopamine (NADA) 35sulfate from ATP, Mg++ and sodium 35sulfate. The pH optimum for this three-step overall sulfate conjugation (comprising of the ATP sulfurylase, APS kinase and phenolsulfotransferase reactions) is 8.6 and the reactions proceeded progressively for several hours. Both ATP and Mg++ ions, above their respective optimal concentrations of 5 and 7 mM, inhibited the sulfate conjugation of NADA. The apparent Km values for NADA as determined by the phenolsulfotransferase (PST) and overall reactions were similar in magnitude being 2.6 and 4.8 microM, respectively, while that for sodium 35sulfate was 202 microM. A comparison of these two activities in 62 platelet preparations of normal subjects showed that the rate of the PST reaction was generally higher than the overall reaction even though the PST assay was carried out at suboptimal concentration of PAPS. There was a positive correlation (r = 0.82) between the two sets of data, suggesting that the PST reaction probably has some control over the rate of overall sulfate conjugation.     

An acetylcholinesterase purified from the greenbug (Schizaphis graminum) with some unique enzymological and pharmacological characteristics

An acetylcholinesterase (AChE, EC 3.1.1.7) was purified from the greenbug, Schizaphis graminum (Rondani). The maximum velocities (Vmax) for hydrolyzing acetylthiocholine (ATC), acetyl-(beta-methyl) thiocholine (AbetaMTC), propionylthiocholine, and S-butyrylthiocholine were 78.0, 67.0, 37.4, and 2.3 micromol/min/mg, and the Michaelis constants (Km) were 57.6, 60.6, 31.3, and 33.4 microM, respectively. More than 98% of AChE activity was inhibited by 10 microM eserine or BW284C51, but only 7% of the activity was inhibited by ethopropazine at the same concentration. Based on the substrate and inhibitor specificities, the purified enzyme appeared to be a true AChE. Nondenaturing polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing of the purified AChE revealed three molecular forms. The isoelectric points were 7.3 for the major form and 6.3 and 7.1 for two minor forms. The major form of purified AChE showed molecular masses of 129 kDa for its native protein and 72 kDa for its subunits on SDS-PAGE. However, the purified AChE exhibited some distinctive characteristics including: (1) lack of affinity to the affinity ligand 3-(carboxyphenyl) ethyldimethyl ammonium, which has been used widely in purification of AChE from various insect species; and (2) 20-200-fold higher substrate-inhibition thresholds for ATC and AbetaMTC than AChE from other insect species. These biochemical properties may reflect structural differences of AChE purified from the greenbug compared with that from other insect species.     

3-Hydroxy-3-methylglutaryl-coenzyme A synthase from ox liver. Purification, molecular and catalytic properties.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) was purified to homogeneity from ox liver and obtained essentially free from acetoacetyl-CoA thiolase activity. The purification procedure included substrate elution from cellulose phosphate and chromatofocusing. The relative molecular mas was about 100 000 and S20,w0 was 6.36S. The enzyme appears to be a dimer of identical subunits (Mr 47 900). The Km for acetoacetyl-CoA is extremely low (less than 0.5 microM), and acetoacetyl-CoA (Acac-CoA) gives marked substrate inhibition (KiAcac-CoA = 3.5 microM) that is competitive with respect to acetyl-CoA. Both CoA and DL-3-hydroxy-3-methylglutaryl-CoA give mixed product inhibition with respect to acetyl-CoA, which is compatible with a Ping Pong mechanism in which both products can form kinetically significant complexes with two forms of the enzyme. The two forms are most likely to be free enzyme and an acetyl-enzyme intermediate.     

Direct photoaffinity labeling of proteins with adenosine 3'-[32P]phosphate 5'-phosphosulfate. Atractyloside inhibits labeling of a Mr = 34,000 protein in an adrenal medullary Golgi fraction

Direct photoaffinity labeling with radioactively labeled adenosine 3'-phosphate 5'-phosphosulfate (PAPS) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography was used to identify PAPS binding proteins in a Golgi membrane preparation of bovine adrenal medulla. [3'-32P]PAPS was synthesized from adenosine 5'-phosphosulfate (APS) and [gamma-32P]ATP using APS kinase prepared from yeast and was purified by reverse-phase ion pair high performance liquid chromatography. Upon irradiation with UV light, [3'-32P]PAPS, as well as [35S]PAPS under conditions which minimized sulfotransferase-catalyzed incorporation of 35SO4 from [35S]PAPS into proteins, bound selectively to a 34-kDa protein of the Golgi membrane preparation. PAPS binding to the 34-kDa protein was strongly inhibited by the presence of 50 microM atractyloside. The 34-kDa PAPS binding protein therefore appears to be similar to the mitochondrial ATP/ADP translocator with regard to both molecular weight and inhibition by atractyloside of adenine nucleotide binding. Photoaffinity labeling will be useful in the purification and functional identification of the 34-kDa protein.     

Purification and characterization of phosphomannose isomerase-guanosine diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa

We report here the purification and characterization of phosphomannose isomerase-guanosine 5'-diphospho-D-mannose pyrophosphorylase, a bifunctional enzyme (PMI-GMP) which catalyzes both the phosphomannose isomerase (PMI) and guanosine 5'-diphospho-D-mannose pyrophosphorylase (GMP) reactions of the Pseudomonas aeruginosa alginate biosynthetic pathway. The PMI and GMP activities co-eluted in the same protein peak through successive fractionation on hydrophobic interaction, ion exchange, and gel filtration chromatography. The purified enzyme migrated as a 56,000 molecular weight protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the native protein migrated as a monomer of 54,000 molecular weight upon gel filtration chromatography. The apparent Km for D-mannose 6-phosphate was 3.03 mM, and the Vmax was 830 nmol/min/mg of enzyme. For the GMP forward reaction, apparent Km values of 20.5 microM and 29.5 microM for D-mannose 1-phosphate and GTP, respectively, were obtained from double reciprocal plots. The GMP forward reaction Vmax (5,680 nmol/min/mg of enzyme) was comparable to the reverse reaction Vmax (5,170 nmol/min/mg of enzyme), and the apparent Km for GDP-D-mannose was determined to be 14.2 microM. Both reactions required Mg2+ activation, but the PMI reaction rate was 4-fold higher with Co2+ as the activator. PMI (but not GMP) activity was sensitive to dithiothreitol, indicating the involvement of disulfide bonds to form a protein structure capable of PMI activity. DNA sequencing of a cloned mutant algA gene from P. aeruginosa revealed that a point mutation at nucleotide 961 greatly decreased the levels of both PMI and GMP in a crude extract.     

Partial Purification and Characterization of Detergent-Solubilized N-Sulfotransferase Activity Associated with Calf Brain Microsomes

Calf brain 3'-phosphoadenosine 5'-phosphosulfate (PAPS):proteoheparan sulfate (PHS) N-sulfotransferase activity is solubilized by extracting salt-washed microsomes with 1% Cutscum. A protocol is described for the partial purification of the sulfotransferase activity utilizing: (1) diethylaminoethyl (DEAE)-Sephacel, (2) heparin-Sepharose CL-6B, and (3) 3',5'-ADP-agarose as chromatographic supports. Sulfotransferase activity was followed by using 3'-phosphoadenosine 5'-phospho[35S]sulfate and endogenous acceptors in heat-inactivated microsomes as exogenous substrates. Two chromatographically distinct fractions (ST1 and ST2) of sulfotransferase activity are resolved on DEAE-Sephacel. Both sulfotransferase activities have been partially purified and characterized. An apparent purification of the two N-sulfotransferase fractions of 22- to 29-fold, relative to the microsomal activity, is achieved by this procedure. Since ST1 appears to represent approximately 24% of the total microsomal activity, a purification of 89-fold has been estimated for this fraction. Neither sulfotransferase activity was stimulated by MnCl2, MgCl2, or CaCl2 added at 10 mM, nor inhibited by the presence of 10 mM EDTA. ST1 and ST2 are optimally active at pH 7.5-8. Apparent Km values for PAPS of 2.3 microM and 0.9 microM have been determined for ST1 and ST2, respectively. ST1 exhibits N-sulfotransferase activity primarily and is inhibited by phosphatidylserine whereas the ST2 fraction contains a mixture of N- and O-sulfotransferase activity and is stimulated by phosphatidylserine, phosphatidylcholine, and lysophosphatidylcholine. The detection of two chromatographically distinct sulfotransferase activities raises the possibility that N-sulfation of proteoheparan sulfates could be catalyzed by more than one enzyme, and that N-sulfation and O-sulfation of proteoglycans are catalyzed by separate enzymes in nervous tissue.