Quantitative estimation of 3'5' cyclic AMP phosphodiesterase using anion exchange resin in a batch process.

An improved method for the preparation of adenosine 3′-phosphate 5′-phosphosulfate (PAPS) is described which includes: enzymes from Chlorella for PAPS synthesis; conversion of ATP to AMP after PAPS formation with hexokinase (EC 2.7.1.1) and myokinase (EC 2.7.4.3); and separation of PAPS on DEAE-Sephadex using triethylammonium bicarbonate buffers. Any specific activity can be obtained by using appropriate concentrations of carrier-free ³⁵S and nonradioactive sulfate in the incubations. Between 300 and 2000 μmol of PAPS per batch can be obtained depending on the scale of the preparation. The PAPS is over 95% pure radiochemically and shows only one ultraviolet-absorbing spot on paper electrophoresis at pH 5.8. Adenosine 5′-phosphosulfate (APS) is prepared by incubating PAPS with a 3′-nucleotidase (EC 3.1.3.6) from rye grass. Quantitative conversion of PAPS to APS is obtained, and the APS is purified by column chromatography in the same manner as for PAPS. The APS obtained is better than 95% pure radiochemically and shows only one uv-absorbing spot on paper electrophoresis at pH 5.8.     

The Isolation and Characterization of the Acid-Soluble Nucleotides from the Tubers of Jerusalem Artichoke (Helianthus tuberosus L.)

The acid-soluble nucleotides were extracted from the tubers of Jerusalem artichoke with percbloric acid, and separated and purified by means of adsorption on and elution from active charcoal, repeated chromatography on columns of Dowex I (Cl^-), followed by paper chromatography. The following nucleotides have been characterized and/or identified: 5′-AMP, 3′-AMP, ADP, ATP, 5′-GMP, 2′-GMP, 3′-GMP, 2′,3′-cyclic GMP, GDP, GTP, 5′-UMP, UDP, UTP, NADP, UDP-glucose, UDP-galactose, UDP-fructose, UDP-N-acetylhexosamine and GDP-mannose.^** Neither cytosine ribonucleotides nor deoxyribonucleotides have been detected. The significance of these observations is discussed.     

Choline sulfokinase of Penicillium chrysogenum: partial purification and kinetic mechanism.

Choline sulfokinase (3′-phosphoadenosine 5′-phosphosulfate (PAPS):choline sulfotransferase, EC 2.8.2.6) was purified approximately 30-fold from the mycelium of Penicillium chrysogenum. The K_m for PAPS is 12 μm. The enzyme is remarkably specific for the adenosine 3′,5′ (or 2′-5′)-diphosphate moiety. 3′,5′-ADP (PAP) has a K_i of 2.5 to 14 μm (depending on the choline concentration) whereas the K_i values of 3′-AMP, 5′-AMP, and 5′-ADP are at least 300-fold higher. The enzyme is also highly specific for choline (K_m = 17 μM). Of a number of other amino alcohols tested, none were potent inhibitors and only dimethylaminoethanol served as a reasonably good substrate (K_m = 800 μmV = 35% of V with choline). Triethylaminoethanol was a significantly poorer substrate (K_m = 2800 μM; V = 2% of V with choline). The purified enzyme is relatively stable when stored frozen in the presence of 25% sucrose. In the absence of sucrose, the maximum activity decreases and the K_m for choline increases. (The K_m for PAPS remains constant.) The age-inactivated enzyme can be restored to full activity (original V and K_m for choline) by a 10-min preincubation with 50 mm mercaptoethanol. However, prolonged incubation (24 h) with 50 mm mercaptoethanol results in irreversible denaturation. Initial velocity studies established that the enzyme follows a sequential kinetic mechanism. Product inhibition studies suggest a rapid equilibrium random binding sequence. Choline-O-phosphate (a dead-end inhibitor) is linearly competitive with choline and a linear mixed type inhibitor with respect to PAPS. Choline analogs lacking the alcohol (or ester) group (e.g., trimethylammonium, neurine, chlorocholine) are competitive dead-end inhibitors with respect to choline but are uncompetitive with respect to PAPS. Thiocholine is a linear mixed type inhibitor with respect to PAPS, but the reciprocal plots are almost parallel. These results suggest that the analogs lacking an oxygen atom have a negligible affinity for the free enzyme and bind predominantly to the enzyme-PAPS complex.     

A bioluminescence method for determining adenosine 3'-phosphate 5'-phosphate (PAP) and adenosine 3'-phosphate 5'-sulfatophosphate (PAPS) in biological materials.

A rapid, sensitive, bioluminescence technique for detecting PAPS (adenosine 3′-phosphate 5′-sulfatophosphate) in biological materials is described. PAPS is first hydrolysed in 0.2 n HCl to PAP (adenosine 3′-phosphate 5′-phosphate) and is then assayed by the luciferin-luciferase system of the sea pansy, Renilla reniformis, which is specific for PAP. This bioluminescence system produces light at a rate that is proportional to the amount of PAP present. Light emission is measured in a liquid scintillation spectrometer with the two photomultipliers out of coincidence.Very low amounts of PAPS (10–100 pmoles) have been determined in extracts of yeast and various plant tissues by this method. The production of PAPS in extracts of young wheat leaves is enhanced by including either 5′-AMP or 3′-AMP in the reaction mixture. It is possible that these nucleotides protect PAPS from enzymes that degrade this compound, e.g., a nucleotidase.     

Assay of sulfotransferases.

Two methods are described for the assay of sulfotransferases which are active with sulfate acceptors bearing the hydroxyl functional group. Assays were developed for enzymes which transfer sulfate from 3′-phosphoadenosine–5′-phosphosulfate (PAPS) to sterols, phenols, and simple alcohols thereby forming the corresponding sulfate esters. With a filter binding assay, useful with crude and purified enzyme preparations, a radioactive sterol substrate is used and subsequently separated from labeled product, allowing the determination of between 50 and 400 pmol of product. In a second method, [³⁵S]PAPS is used and the labeled product is separated from PAPS and inorganic sulfate by a thin-layer technique in which product migrates close to the solvent front; the assay is useful with a broad array of substrates and is more sensitive than the filter binding assay.     

Purification of orotate phosphoribosyltransferase and orotidylate decarboxylase by affinity chromatography on Sepharose dye derivatives.

Blue Dextran-Sepharose and Cibacron Blue F3GA-Sepharose (Blue Sepharose) were found to act as affinity adsorbents for orotate phosphoribosyltransferase (PRTase) and orotidine 5′-monophosphate (OMP) decarboxylase from bakers' yeast. Experiments with columns of Blue Dextran-Sepharose and partially purified preparations of the PRTase and decarboxylase revealed that both enzymes were selectively eluted by a low concentration (0.1–2 mm) of their respective substrate or immediate product. On the other hand, a much higher concentration (50–400 mm) of NaCl was required to displace these two enzymes from the above columns. Larger scale experiments showed that OMP decarboxylase in crude extracts was purified about 5700- and 6600-fold on Blue Sepharose using 0.5 mm OMP and 2 mm uridine 5′-monophosphate (UMP) as the eluting ligand, respectively. In contrast, orotate PRTase did not bind to Blue Sepharose unless crude extracts were first subjected to gel filtration. The resulting preparation of orotate PRTase, purified about sixfold with respect to cell-free extracts, was purified an additional 200- and 40-fold when the enzyme was eluted from Blue Sepharose with 0.5 mm OMP and 1 mm 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P), respectively. Blue Dextran-Sepharose, on the other hand, was found to provide a lower degree of enzyme purification and exhibited a lower sample-binding capacity. Samples of the PRTase and decarboxylase that had been purified about 200- and 6000-fold, respectively, on Blue Sepharose displayed a major protein band and one or more minor bands when subjected to polyacrylamide gel electrophoresis. Enzyme activity coincided with the major band in all cases.     

Partial purification and properties of ethanolamine kinase from spinach leaf.

Ethanolamine kinase was purified 60-fold by fractionation with ammonium sulfate, freeze-thawing, and gel filtration from a 100,000g supernatant from spinach leaf. The 100,00g supernatant preparation was stable for weeks, but the partially purified preparation lost half of the ethanolamine kinase activity in 10–14 days at 0–4 °C or ?20 °C. A molecular weight of 110,000 was estimated by gel filtration on Sephadex G-200. The reaction required ethanolamine (K_m, 42 μm), MgATP (K_m, 63 μm), and free magnesium ions. The enzyme was inhibited by MgATP, with an apparent K_i of 6.7 mm. Ethanolamine kinase was inhibited by calcium (in the presence of magnesium) and o-phenanthroline. EDTA above 0.9 mm inhibited the formation of phosphorylethanolamine and EGTA stimulated at low concentrations (0.4-0.9 mm) and inhibited at 1.8 mm. Ethanolamine kinase was inhibited by monomethylethanolamine and dimethylethanolamine, but not by choline (5 mm). The ethanolamine kinase and choline kinase activities of the 100,000g supernatant preparation could be separated by gel electrophoresis     

Isolation and identification of glycosaminoglycans associated with purified nuclei from rat liver

Nuclei isolated from rat liver were purified extensively and then subjected to extraction of glycosaminoglycans by the conventional method with a slight modification including the treatments with amylase nucleases, (DNAase and RNAase), and sialidase in addition to the pronase treatment. The nuclear glycosaminoglycan fraction thus prepared was subjected to chromatography on Dowex 1-X2 (Cl^?) and electrophoresis before or after digestion with specific enzymes such as Streptomyces hyaluronidase, chondroitinase ABC and AC. These results together with the results of chemical analyses have revealed that the purified nuclei from rat liver contain glycosaminoglycans equivalent to 0.2–0.3 μg hexuronic acid per mg DNA. A major component of the nuclear glycosaminoglycans has been identified as hyaluronic acid, while a minor component as chondroitin sulfate A (or C). Preliminary investigations have shown that most of the nuclear glycosaminoglycans are associated with the chromatin fraction.     

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.     

Trehalase from the cellular slime mold Dictyostelium discoideum: Purification and characterization of the homogeneous enzyme from myxamoebae

Trehalase (α-α′-trehalose 1-d-glucohydrolase, EC 3.2.1.28) was solubilized from myxamoebae of the cellular slime mold Dictyostelium discoideum by a freeze-thaw cycle and was subsequently purified to homogeneity using the techniques of ethanol fractionation, molecular sieve chromatography, DEAE-cellulose ion-exchange chromatography, chromatofocusing, and preparative polyacrylamide disc gel electrophoresis. The 1000-fold purified enzyme had a specific activity of about 104 units/mg, which was accompanied by a net recovery of 5 to 7% of the original activity. The purified enzyme was maximally active at pH 5.5, showed high specificity for trehalose, and exhibited a typical hyperbolic response as a function of trehalose concentration with a K_m of 1.2 mm. The enzyme was maximally active at 50 °C and had an energy of activation of 12–13 kcal/mol. Thermal stability studies demonstrated that full enzymatic activity was recovered following a 5-min incubation of trehalase at temperatures up to 45–50 °C. Analysis of various compounds for inhibitory effects indicated that Tris and urea were slightly effective, reducing enzymatic activity by 28 and 6% at concentrations of 100 and 10 mm, respectively. Of five heavy metals tested, HgCl₂ was the most inhibitory, reducing activity by 58% when present at a final concentration of 1.0 mm. Enzymatic activity was not affected by any adenine derivative examined (e.g., ATP, ADP, AMP, cAMP, adenosine, and adenine). The molecular weight of the native enzyme was determined by molecular sieve chromatography, pore gradient electrophoresis, and electrophoresis as a function of acrylamide concentration. All three methods yielded a value of about 10⁵ ± 5 × 10³. Estimation of the subunit or monomer molecular weight by sodium dodecyl sulfate-gel electrophoresis indicated a value of 95–100 × 10³. The isoelectric point as determined in 7.5% polyacrylamide gels with pH 3–10 ampholytes was 7.2–7.3. The purified enzyme adsorbed to concanavalin A-Sepharose in the presence of KCl (0.1 m) and was eluted with α-methylmannoside, thereby suggesting an association between trehalase and carbohydrate. In agreement with this conclusion was the observation that trehalase could be specifically stained for carbohydrate with the Alcian blue and periodic acid-Schiff's reagents following polyacrylamide disc gel electrophoresis.     

A kinetic investigation of the aps-kinase from Chlamydomonas reinhardii CW 15

The reaction kinetics of APS-kinase from Chlamydomonas reinhardii showed that the enzyme formed PAPS from APS upon the addition of ATP. Evidence for a ³⁵S-labelled protein intermediate between APS and PAPS has been obtained. The APS-kinase activity could only be measured in the presence of low concentrations of APS (20 ± 10 μM) and of ATP (0.2 ± 0.05 mM) due to substrate inhibition. The inhibition was partially overcome by low concentrations of 3′,5′-PAP (10,μM). The rates of PAPS formation obtained with cell extracts from the alga varied from 2 to 6 nM PAPS/mg protein/min (33–100 × 10^?12 kat/mg).     

Simultaneous determination of thiabendazole and its major metabolite, 5-hydroxythiabendazole,in bovine tissues using gradient liquid chromatography with thermospray and atmospheric pressure chemical ionisation mass spectrometry

A novel method is presented for the determination of thiabendazole and 5-hydroxythiabendazole in animal tissues. Samples are homogenised in buffer at pH=7.0, extracted with ethyl acetate and cleaned up using CN solid-phase extraction columns. Thiabendazole and 5-hydroxythiabendazole are separated chromatographically using gradient elution and analysed by liquid chromatography–mass spectrometry. Deuterated thiabendazole is employed as an internal standard for thiabendazole determination; 5-hydroxythiabendazole is quantified via external standards. Samples are screened by monitoring the protonated molecular ions at m/z=202 for thiabendazole, 206 for deuterated thiabendazole and 218 for 5-hydroxythiabendazole using thermospray LC–MS. Positives are confirmed by multiple ion monitoring using APCI LC–MS. Validation of the method was carried out at 50, 100 and 200 μg kg⁻¹. Recoveries for thiabendazole in bovine muscle, liver and kidney ranged from 96–103% with C.V.s between 0.7 and 4.8% and for 5-hydroxythiabendazole recoveries ranged from 70–85% with C.V.s between 3.1 and 11.5%.     

Simple,rapid and sensitive method for the determination of indomethacin in plasma by high-performance liquid chromatography with ultraviolet detection

A micro method for determination of indomethacin in plasma was developed. Following deproteinization of plasma with acetonitrile containing internal standard (mefenamic acid), the separation of indomethacin and internal standard was achieved by high-performance liquid chromatography using a 7 μm LiChrosorb-RP18 column (250×4 mm I.D.) at 50°C. The mobile phase was 6 mM phosphoric acid–acetonitrile (50:50). The flow-rate was kept at 2.0 ml/min and the column effluent was monitored at 205 nm. The coefficients of variation of the method estimated at 0.2 and 1.0 μg/ml were 4.2 and 2.3%, and the detection limit of the drug was about 0.05 μg/ml (S/N=5). The method requires minimum pretreatment of the plasma with a small sample volume (25 μl), and is very suitable for therapeutic drug monitoring of indomethacin in premature infants with symptomatic patent ductus arteriosus.     

Peptide-Induced Morphogenesis in the Nematode-Trapping Fungus Arthrobotrys oligospora

The present investigation shows the ability of peptides to induce capture organ formation in Arthrobotrys oligospora when applied in a synthetic low nutrient medium. Under certain conditions casitone was shown to induce capture organ formation. The active principle in casitone was concentrated and purified by alternating procedures of ion exchange chromatography and gel chromatography in pyridine-acetic acid buffers. Crude casitone solutions were applied to columns of Dowex 50 W-X2 and eluted stepwise with 0.1–1.0 M pyridine-acetic acid pH 3.2–5.1. Active portions, free from most acid and neutral amino acids, were further purified on columns of Sephadex G-10 in 0.1 M pyridine-acetic acid pH 4.6. Aromatic amino acids and large molecules in the void volume could be separated from an active peptide mixture which was subjected to renewed ion exchange chromatography on Bio-Rad AG 50 W-X2. By stepwise and/or gradient elution in 0.1–0.5 M pyridine-acetic acid pH 3.2 fairly purified peptides were obtained. The composition of the test medium is an important factor in spontaneous capture organ formation. The peptides isolated from casitone induced capture organ formation, when given to the fungus in a synthetic mineral salt medium supplied with thiamin and biotin. Similar effects were obtained with small synthetic peptides in the same concentration (0.1 mg/ml). A large variety of peptides seem to be active when applied in a suitable medium. This was especially true for peptides with Rf > Rf_leu on thin layers of cellulose developed with butanol-acetic acid-water (4: 1: 1). Of the peptides investigated valyl-peptides exerted the most drastic effect.     

Purification and structural analysis of the modulator of the glucocorticoid-receptor complex. Evidence that modulator is a novel phosphoglyceride

Modulator is the low molecular weight heat-stable inhibitor of glucocorticoid-receptor complex activation. We have purified modulator to apparent homogeneity from heated rat liver cytosol. This was accomplished using Sephadex G-15 gel filtration, Dowex 1 anion-exchange chromatography, and preparative silica high-performance liquid chromatography. The modulator preparation was judged to be homogeneous by analytical silica high-performance liquid chromatography, two-dimensional silica thin-layer chromatography, and proton nuclear magnetic resonance spectroscopy. The apparent concentration of modulator in rat liver cytosol is 6.5 microM. The purified modulator inhibits heat activation of the rat liver glucocorticoid-receptor complex and stabilizes the steroid binding ability of the unoccupied rat liver glucocorticoid receptor in a dose-dependent manner. At a concentration of 5-6.5 microM, modulator inhibits receptor activation and stabilizes the unoccupied receptor by 50%. At a concentration of 500-630 microM, sodium molybdate also inhibits receptor activation and stabilizes the unoccupied receptor by 50%. Thus, modulator appears to be the endogenous factor that exogenous sodium molybdate mimics in vitro. Chemical analysis of the purified modulator following two-dimensional silica thin-layer chromatography indicates that modulator is an aminophospholipid. Physical analysis of the purified modulator by infrared and nuclear magnetic resonance spectroscopy, as well as mass spectrometry, demonstrates that modulator is an ether aminophosphoglyceride.     

Purification and characterization of acyl-CoA carboxylase from the goose uropygial gland which produces multimethyl-branched acids and evidence for its identity with avian acetyl-CoA carboxylase

Acyl-CoA carboxylase was purified from the 140,000g supernatant of the goose uropygial gland extract by means of Sepharose 4B-CL gel filtration, ammonium sulfate precipitation, and affinity chromatography with monomeric avidin-Sepharose 4B-CL. The purified enzyme showed a pH optimum of 8 and had a specific activity ranging from 2–8 μmol/min/mg protein for acetyl-CoA. Sodium dodecyl sulfate-electrophoresis showed a single band corresponding to a molecular weight of 238,000. Carboxylase activity was stimulated threefold by 20 mm citrate. Maximal activity was observed with 25 mm bicarbonate, 10 mm Mg²⁺, 3 mm ATP, and 1 to 2 mm acyl-CoA. The enzyme carboxylated acetyl-CoA, propionyl-CoA, butyryl-CoA, pentanoyl-CoA, and hexanoyl-CoA, with a V of 8.8, 5.7, 0.9, 0.04, and 0.03 μmol/min/mg, respectively; K_m values for the five CoA esters were quite similar. The carboxylated products from these substrates were analyzed by high-performance liquid chromatography. This carboxylase was inhibited by sodium and chloride ions. Chemical modification of the enzyme with pyridoxal-5′-phosphate showed inhibition of activity that was time and concentration dependent. The inhibition was reversed by dilution except when treated with sodium borohydride before dilution. Acetyl-CoA partially (40%) protected the enzyme from inhibition, whereas 3′-dephosphoacetyl-CoA, which showed a K_m 3.5 times that of acetyl-CoA, was much less efficient in protecting the enzyme against inactivation by pyridoxal phosphate. These results suggest that the ?-amino group of a lysine residue is involved in binding acetyl-CoA via interaction with the 3′-phosphate. Chemical modification of the enzyme with phenylglyoxal showed inhibition of activity that was time and concentration dependent. However, none of the substrates protected the enzyme from inactivation; citrate partially protected the enzyme, possibly by changing the configuration of the enzyme. Amino acid analysis of the protein showed striking similarities with carboxylases purified from other animals. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the gland enzyme showed fusion of precipitation lines with the enzymes from goose liver and chicken liver. These results strongly support the conclusion that the uropygial gland, which synthesizes multimethyl-branched acids, employs the same carboxylase as that present in other tissues.     

Glucagon-induced phosphorylation of pyruvate kinase (type L) in rat liver slices.

The effect of glucagon on the phosphorylation of pyruvate kinase in ³²P-labelled slices from rat liver was investigated. Pyruvate kinase was isolated by immunoadsorbent chromatography. The enzyme was partially phosphorylated in the absence of added hormone (0.2 mol of phosphate/mol of enzyme subunit). Upon incubation with 10^?7 M glucagon, the incorporation of [³²P]phosphate was 0.6–0.7 mol/mol of enzyme subunit. Concomitantly, the concentration of intracellular cyclic 3′,5′-AMP increased from 0.3 to 3.2 μM. The phosphorylation inhibited the enzyme activity at low concentrations of phosphoenolpyruvate (60% at 0.5 mM). Almost maximal phosphorylation of the enzyme was reached within 2 min after the addition of glucagon. The concentration of hormone giving half maximal effect on the pyruvate kinase phosphorylation was about 7×10^?9M. The inactivation of the enzyme paralleled the increase in phosphorylation. It is concluded that pyruvate kinase is phosphorylated in the intact liver cell.     

Purification and Characterization of S-adenosylmethionine Synthetase from Wheat Embryos: Subunit Structure and Molecular Properties

S-adenosylmethionine synthetase from wheat embryos was purified to electrophoretic homogeneity. The mol wt of the enzyme was 174,000 as determined by molecular sieve chromatography on Sephacryl S-200. A single subunit of purified AdoMet synthetase was observed on SOS-PAGE with a mol wt of 84,000 suggesting that the enzyme is a homodimer. The apparent Km of purified enzyme with ATP and methionine is 80 μM and 100 μM, respectively. The pH optimum of the enzyme is 7.75. The enzyme requires MgCb, KCI and reduced glutathione for optimum activity. The ³H-labelled putative S-adenosylmethionine reaction product was converted into ³H-labelled 5′-methyl-thioadenosine by heat treatment (100°C, 10 min, pH 7.0). This proved the authenticity of the reaction product of the AdoMet synthetase in wheat embryos.     

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.     

The effect of thyroxine on transparency and PAPS synthesis in the avian cornea

The normal onset of developing corneal transparency, which begins on Day 14 of chick embryogenesis, can be accelerated by the in vivo application of exogenous thyroxine, as originally demonstrated by Coulombre and Coulombre (1964, Exp. Eye Res.3, 105–114). When thyroxine (5 μg) is injected at Day 6, measurements of ³⁵SO^2?₄ incorporation by corneal homogenates indicate that synthesis of 3′-adenosine phosphosulfate (APS) in the cornea is increased at Day 8, but not that of 3′-phosphoadenosine-5′-phosphosulfate (PAPS). Injection of hormone at Day 9 results in a precocious increase in corneal transparency at Day 12 and a corresponding increase in the synthesis of APS and PAPS.