Enzymatic sulfation of steroids. V. Partial purification and some properties of sulfotransferase III, the major glucocorticoid sulfotransferase of liver cytosols from male rats

1978 ◽  
Vol 56 (11) ◽  
pp. 1028-1035 ◽  
Author(s):  
Sanford S. Singer ◽  
James Gebhart ◽  
Edward Hess
Keyword(s):  
Molecular Weight ◽  
Sulfhydryl Groups ◽  
Male Rats ◽  
Partial Purification ◽  
Ph Optimum ◽  
Fold Enrichment ◽  
Competitive Inhibitors ◽  
Sequential Mechanism ◽  
Inhibition Studies ◽  
Estradiol 17Β

This manuscript describes purification of sulfotransferase III (STIII), the major hepatic glucocorticoid sulfotransferase of male rats, 77.8 ± 16 fold from cytosol. This represents a probable 250–345 fold enrichment, compared with homogenates. Purified STIII has a molecular weight of 61 500 ± 2500 from Sephadex G-100 chromatography. It is markedly activated by 5 mM divalent Ba, Ca, Co, Cr, Mg, Mn, and Ni salts; inhibited strongly by 5 mM divalent Zn and Cd; and unaffected by 8 mM ADP, ATP, and AMP. Comparison of the ability of purified STIII to sulfate equimolar Cortisol, estradiol-17β, testosterone, and dehydroepiandrosterone suggests that the enzyme may sulfate glucocorticoids preferentially. However, its Cortisol sulfotransferase activity is inhibited by a variety of steroids. Of these, dehydroepiandrosterone, dexamethasone, and progesterone were tested extensively. They were found to be competitive inhibitors. STIII has a sharp pH optimum at pH 6.0 ± 0.1. However, it is routinely assayed at pH 6.8, as explained in the text. It exhibits a sequential mechanism and Km values of 6.82 ± 1.2 and 6.28 ± 0.64 μM for Cortisol and 3′-phosphoadenosine-5′-phosphosulfate, respectively. It also possesses essential sulfhydryl groups, as shown by p-hydroxymercuribenzoate inhibition studies.

Enzymatic sulfation of steroids. XI. The extensive purification and some properties of hepatic sulfotransferase III from female rats

1980 ◽  
Vol 58 (8) ◽  
pp. 660-666 ◽  
Author(s):  
Sanford S. Singer ◽  
Lawrence Bruns
Keyword(s):  
Protein Band ◽  
Female Rats ◽  
Male Rats ◽  
Ph Optimum ◽  
Single Protein ◽  
Sequential Mechanism ◽  
Liver Homogenates ◽  
Inhibition Studies ◽  
Estradiol 17Β ◽  
Ordered Sequential Mechanism

Our earlier studies showed that livers from female rats contained three glucocorticoid sulfating enzymes we named sulfotransferases I, II, and III, (STI, STII, and STIII, respectively). In this report STIII from female Charles River CD rats was purified 1010- to 1300-fold compared with liver homogenates. The most highly purified STIII fraction electrophoresed as a single protein band. The molecular weight of STIII was 68 300 ± 4900. Its pH optimum for cortisol sulfation was pH 6.0 ± 0.1. However, it was routinely assayed at pH 6.8 for reasons enumerated in the text. Cortisol sulfation by STIII proceeded by either an ordered sequential mechanism or by an Iso Theorell-Chance mechanism at pH 6.8. The Km's for cortisol and the reaction coenzyme, 3′-phosphoadenosine-5′-phosphosulfate were 6.48 ± 0.78 and 6.78 ± 1.26 μM, respectively. Comparison of the ability of the enzyme to sulfate 40 μM cortisol, estradiol-17β, testosterone, deoxycorticosterone, and dehydroepiandrosterone, showed that the glucocorticoid was sulfated preferentially. Interestingly, its cortisol sulfotransferase activity was inhibited by a number of steroids. p-Hydroxymercuribenzoate inhibition studies indicated the presence of essential sulfhydryl groups in STIII. The enzyme was activated by divalent Ba, Ca, Co, Cr, Mg, Mn, and Ni salts. It was inactivated by Zn2+ and Cd2+ salts. The text compares STIII from female rats with other steroid sulfotransferases including the major glucocorticoid sulfotransferase from male rats.

Purine catabolism in man: inhibition of 5′-phosphomonoesterase activities from placental microsomes

1976 ◽  
Vol 54 (12) ◽  
pp. 1055-1060 ◽  
Author(s):  
Irving H. Fox ◽  
Pamela J. Marchant
Keyword(s):  
Alkaline Phosphatase ◽  
Product Inhibition ◽  
Maximum Activity ◽  
Ph Optimum ◽  
Phosphomonoesterase Activity ◽  
Competitive Inhibitors ◽  
Sequential Mechanism ◽  
Mechanism Of Interaction ◽  
Noncompetitive Inhibitor ◽  
Inhibition Studies

The 5′-phosphomonoesterase activity of 5′-nucleotidase (EC 3.1.3.5) and alkaline phosphatase (EC 3.1.3.1) participates in the catabolism of purine ribonucleotides to uric acid in humans.Initial velocity studies of 5′-nucleotidase suggest a sequential mechanism of interaction between AMP and MgCl2, with a Km of 14 and 3 μM, respectively. With product inhibition studies the apparent Ki's for adenosine, inosine, cytidine, and inorganic phosphate were 0.4, 3.0, 5.0, and 42 mM, respectively. A large number of nucleoside mono-, di-, and tri-phosphate compounds were inhibitors of the enzyme. Allopurinol ribonucleotide, ADP, or ATP were competitive inhibitors when AMP was the substrate, with a Ki slope of 10, 20, or 54 μM, respectively. GTP was a noncompetitive inhibitor, with a Ki slope of 120 μM.The phosphomonoesterase activity of human placental microsomal alkaline phosphatase had a pH optimum of 10.0 and had only 18% of maximum activity at pH 7.4. Substrates and inhibitors included almost any phosphorylated compound. The Km for AMP was 0.4 mM and the apparent Ki for Pi was 0.6 mM. Activity was increased only 19% by 5 mM MgCl2.These observations suggest that 5′-nucleotidase and alkaline phosphatase may be inhibited by ATP and Pi, respectively, under normal intracellular conditions, and that AMP may be preferentially hydrolyzed by 5′-nucleotidase.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

The β-glucosidases of porcine kidney

1977 ◽  
Vol 55 (2) ◽  
pp. 140-145 ◽  
Author(s):  
Julian N. Kanfer ◽  
Richard A. Mumford ◽  
Srinivasa S. Raghavan
Keyword(s):  
Sodium Taurocholate ◽  
Hydrolytic Activity ◽  
Acidic Ph ◽  
Porcine Kidney ◽  
Ph Optimum ◽  
Pig Kidney ◽  
Competitive Inhibitors ◽  
Triton X ◽  
Hydrolysis Of ◽  
Estradiol 17Β

Some of the properties of a partially purified particle bound and soluble β-glucosidase (EC 3.2.1.21) from pig kidney were compared. The soluble β-glucosidase (1) hydrolyzed 4-methylumbelliferyl-β-D-glucoside (4-MU-β-D-glucoside) 17α-estradiol 3β-glucoside, 17α-estradiol 17β-glucoside, and salicin, but not glucosylceramide, (2) possessed a broad pH optimum (5.5–7.0), (3) had an isoelectric point of 4.9, and (4) was inhibited by Triton X-100. Several compounds were found to be competitive inhibitors of its hydrolytic activity, gluconolactam and estrone β-glucoside being the most effective. In contrast, a particulate β-glucosidase purified from the same tissue (1) had an acidic pH optimum (5.0), (2) was stimulated by sodium taurocholate and 'Gaucher's factor' for the hydrolysis of both 4-MU-β-glucoside and glucosylceramide, and (3) was capable of catalyzing a transglucosylation reaction employing 4-MU-β-D-glucoside or glucosylceramide as the glucosyl donor, and [l4C]ceramide as acceptor.

Partial purification and properties of an L-arabinose dehydrogenase from Azospirillum brasiliense

1983 ◽  
Vol 29 (2) ◽  
pp. 242-246 ◽  
Author(s):  
Norman J. Novick ◽  
Max E. Tyler
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Gel Filtration ◽  
Reducing Agent ◽  
Partial Purification ◽  
Ph Optimum ◽  
Glycine Buffer ◽  
Purification And Properties ◽  
Filtration Technique

An L-arabino-aldose dehydrogenase responsible for the oxidation of L-arabinose to L-arabino-γ-lactone has been purified 59-fold from L-arabinose grown cells of Azospirillum brasiliense. The dehydrogenase was found to be specific for substrates with the L-arabino-configuration at carbons 2, 3, and 4. Km values for L-arabinose of 75 and 140 μM were found with NADP and NAD as coenzymes, respectively. The enzyme had a pH optimum of 9.5 in glycine buffer and was stable when heated to 55 °C for 5 min. No enhancement of activity in the presence of any divalent cation or reducing agent tested was found. L-Arabinose dehydrogenase had a molecular weight of 175 000 as measured by the gel filtration technique.

scholarly journals The partial purification and characterization of cytosol alcohol dehydrogenase from Astasia

1974 ◽  
Vol 141 (2) ◽  
pp. 469-475 ◽  
Author(s):  
Rolf Morosoli ◽  
Nicole Bégin-Heick
Keyword(s):  
Molecular Weight ◽  
Alcohol Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Total Activity ◽  
Growth Conditions ◽  
Partial Purification ◽  
Heat Inactivation ◽  
Kinetic Properties ◽  
Ph Optimum

1. The cytosol alcohol dehydrogenase (alcohol–NAD oxidoreductase, EC 1.1.1.1) of Astasia longa was partially purified and characterized from cells grown in the presence of air+CO2 (95:5) or of O2+CO2 (95:5). 2. Under both these growth conditions, the cells contained a fraction, ADHII, which was characterized by its electrophoretic properties, by a high degree of resistance to heat inactivation, by a sharp pH optimum at 8.2 and by its kinetic properties. The estimated molecular weight of this fraction was approx. 150000, which is similar to that of yeast alcohol dehydrogenase. 3. Cells grown in air+CO2 (95:5) contain another fraction, ADHI, which can be further separated into two subfractions by polyacrylamide-gel electrophoresis and by DEAE-cellulose chromatography. This was termed fraction ‘ADHI-air’. 4. In addition to fraction ADHII, cells grown in the presence of O2 have a twofold increase in fraction ADHI-air activity as well as two new fractions that could not be demonstrated in air-grown cells. These new fractions which we have called fraction ‘ADHI-O2’, account for about 10% of the total activity. 5. The ADHI fractions (air) and (O2) have similar broad pH–activity curves and similar kinetic properties, both having a lower Km for ethanol and NAD than fraction ADHII. However, they differ from each other with respect to their activity with various substrates. The estimated molecular weight of these two ADHI fractions and their chromatographic behaviour on hydroxyapatite and on DEAE-cellulose also distinguish them.

UDP-D-Xylose: Flavonol 3-O-Xylosyltransferase from Young Leaves of Euonymus alatus f. ciliato-dentatus

1991 ◽  
Vol 46 (11-12) ◽  
pp. 1003-1010 ◽  
Author(s):  
Nariyuki Ishikura ◽  
Zhi-qing Yang
Keyword(s):  
Molecular Weight ◽  
Ammonium Sulfate ◽  
Isoelectric Point ◽  
Deae Cellulose ◽  
Sulfhydryl Groups ◽  
Hydroxyl Group ◽  
Soluble Enzyme ◽  
Ph Optimum ◽  
Euonymus Alatus ◽  
Young Leaves

From the young leaves of Euonymus alatus f. ciliato-dentatus, a novel enzyme, UDP-D-xylose: flavonol 3-O-xylosyltransferase (F3XT), catalyzing the transfer of D-xylose from UDP-D-xylose to the 3 position of 3,5,7,4′-tetrahydroxyflavone (kaempferol), was detected and purified about 16-fold by precipitation with ammonium sulfate and DEAE-cellulose CC, by which F3XT was separated from two coexisting flavonol O-glucosyltransferases (FGT). Thus, F3XT was isolated as a soluble enzyme with a pH optimum of 7.0 in Tris-HCl buffer. The molecular weight of F3XT , which had an isoelectric point at pH 6.1, was estimated by elution from a column of Sephadex G-100 to be about 48 kDa. The activity of F3XT was stimulated by 14 mM 2-ME and strongly inhibited by 1 mAbstractм Cu2+, 1 mм Zn2+, and various re­ agents that react with sulfhydryl groups. Among the substrates tested for F3XT , kaempferol was the best. The Km values for kaempferol and UDP-xylose were determined to be 0.83 jim and 25 μM, respectively. F3XT mediated the transfer of xylose exclusively to the 3-hydroxyl group of kaempferol. Isorhamnetin, quercetin and fisetin also can function as xylosyl acceptor though less efficiently, but neither the 7-O-glucosides nor the 3-O-glucosides of kaempferol and quercetin were able to accept D-xylose. Dihydroflavonols were not xylosylated.

scholarly journals Partial purification and properties of an acid nucleotidase from the postmicrosomal supernatant of rat spleen

1978 ◽  
Vol 169 (3) ◽  
pp. 597-605 ◽  
Author(s):  
Hans Tjernshaugen
Keyword(s):  
Molecular Weight ◽  
Catalytic Properties ◽  
Partial Purification ◽  
Ph Optimum ◽  
Phosphatase Inhibitors ◽  
Purification And Properties ◽  
Optimum Ph ◽  
Liver Lysosomes ◽  
Rat Liver Lysosomes ◽  
Single Enzyme

1. The dephosphorylation of 3′-AMP, 3′-dAMP, 3′-CMP and 3′-dCMP was studied in the postmicrosomal supernatant of rat spleen and liver. In both organs 3′-AMP and 3′-dAMP were dephosphorylated at an appreciable rate, in both the presence and the absence of Mg2+. The pH optimum for this dephosphorylation was in the range 4.5–5.0. 3′-CMP and 3′-dCMP were very slowly degraded, though the activity towards 3′-dCMP increased somewhat in the presence of Mg2+. The optimum pH for this Mg2+-dependent dephosphorylation was 5.5–6.0. 2. The rate of dephosphorylation of 3′-AMP and 3′-dAMP per mg of protein was about 5 times as high in spleen as in liver. 3. The dephosphorylation of 3′-AMP could be ascribed to a single enzyme with pH optimum about 4.5. The activity towards 3′-dAMP could be resolved into one component coinciding with the 3′-dAMP-degrading enzyme, and one Mg2+-requiring component probably identical with the soluble deoxyinosine-activated nucleotidase. The dephosphorylation of 3′-dCMP seemed to be performed only by the latter enzyme. 4. The enzyme dephosphorylating 3′-AMP was purified 200-fold from the postmicrosomal supernatant and its physical and catalytic properties were compared with those of acid nucleotidase (EC 3.1.3.31) purified from rat liver lysosomes. The two enzymes were identical in all properties tested (substrate specificity, Km, molecular weight, response to phosphatase inhibitors), but some of the data differed from earlier reports on the acid nucleotidase. 5. The subcellular localization of the acid nucleotidase, its relationship to the acid phosphatase(s) and its role in the breakdown of nucleic acid constituents are discussed.

Characterization and substrate specificity of fumarylacetoacetate fumarylhydrolase

1977 ◽  
Vol 55 (1) ◽  
pp. 1-8 ◽  
Author(s):  
D. J. Mahuran ◽  
Ronald H. Angus ◽  
Carl V. Braun ◽  
S. S. Sim ◽  
Donald E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Sulfhydryl Groups ◽  
Ph Optimum ◽  
Amino Terminal ◽  
Diketo Acids ◽  
Hydrolysis Of

The molecular weight of fumarylacetoacetate fumarylhydrolase (EC 3.7.1.2) is 86 000 ± 10 000, as determined by gel filtration. The enzyme appears to be a dimer with a monomer molecular weight of 38 000 – 43 000, as determined by gel electrophoresis, gel filtration in guanidine–hydrochloride, and ultracentrifugation. The subunits appear to be identical, as only one band is seen in gel electrophoresis, only one protein peak is detected in gel filtration in guanidine–hydrochloride, and only one amino-terminal amino acid (proline) is detected. Three free sulfhydryl groups per denatured monomer are detected by reaction with 5,5′-dithiobis(2-nitrobenzoic acid), while for the active enzyme only two sulfhydryl groups react with this reagent. The extinction coefficients at 260 and 280 nm, the amino acid composition, and the isoelectric point (6.7) of the enzyme are also reported.The enzyme catalyzes the hydrolysis of six 2,4-diketo acids and three 3,5-diketo acids tested. The Km of the substrates is similar but V varies by a factor of 120. The pH optimum is 7.3. The enzyme did not catalyze the hydrolysis of a number of esters tested.

Characterization and partial purification of β-hydroxybutyrate dehydrogenase from sporulating cells of Bacillus cereus T

1973 ◽  
Vol 19 (6) ◽  
pp. 673-677 ◽  
Author(s):  
E. D. Thompson ◽  
H. M. Nakata
Keyword(s):  
Molecular Weight ◽  
Bacillus Cereus ◽  
Divalent Cation ◽  
Phosphate Buffer ◽  
Partial Purification ◽  
Ph Optimum ◽  
Michaelis Constants

A soluble NAD+-dependent β-hydroxybutyrate (βHB) dehydrogenase was shown to appear 3 to 4 h after the onset of sporulation of Bacillus cereus T. The enzyme was stable in Tris-chloride buffer when frozen, but required 0.05 to 0.1 M of MgCl2 or other divalent cation such as Mn2+, Ba2+, or Ca2+ for stability at 4C. In the presence of phosphate buffer or EDTA, the enzyme lost all activity within 2 min. βHB dehydrogenase was partially purified and shown to have a molecular weight of about 93 000, pH optimum of 8.0 in 0.1 M Tris-chloride buffer, Michaelis constants, Km, of 2.3 × 10−3 M for β-hydroxybutyrate and 9.5 × 10−4 M for NAD+, and was inhibited 40% by 1 × 10−3 M p-hydroxymercuribenzoate. The enzyme from B. cereus T was compared in these respects with βHB dehydrogenases isolated from several non-sporeforming bacteria.

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