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Biosynthesis of heparin. Solubilization and partial characterization of N- and O-sulphotransferases.

ABSTRACT: Assay methods were developed enabling separate determination of N- and O-sulphotransferase activities in an enzyme preparation from mouse mastocytoma. N-Desulphoheparin and chemically N-acetylated heparan sulphate were used as specific exogenous sulphate acceptors in the transfer of [35S]sulphate residues from adenosine 3'-phosphate 5'-[35S]sulphatophosphate to amino and hydroxyl groups respectively. The resulting 35S-labelled polysaccharides were isolated as their cetylpyridinium complexes on filter paper. Sulphotransferases were solubilized from a mastocytoma microsomal fraction by treatment with detergent-alkali. The pH optimum for both enzymes was about 7.5 Km with regard to adenosine 3'-phosphate 5'-sulphatophosphate was estimated to be 2 X 10(-5) M for the N-sulphotransferase and 1 X 10(-4) M for the O-sulphotransferase(s). The enzymes required bivalent cations for maximum activity, Mn2+ stimulating both the N- and O-sulphotransferase four- to five-fold, whereas Ca2+ increased the N- but not the O-sulphotransferase activity. The O-sulphotransferase was found to be more sensitive to heat-inactivation, 60% of the activity being lost after 1 min at 50 degrees C, whereas only 15% of the N-sulphotransferase activity was lost. In contrast, the N-sulphotransferase was selectively inhibited (or inactivated) by NaCl; at 0.125 M-NaCl concentration the O-sulphotransferase activity was essentially unaffected, whereas the N-sulphotransferase activity was depressed by 80%. These results strongly indicate that N- and O-sulphate-transfer reactions should be ascribed to different enzymes, or, alternatively, to separate and independent active sites on the same enzyme molecule.

SUBMITTER: Jansson L

PROVIDER: S-EPMC1165591 | biostudies-other | 1975 Jul

REPOSITORIES: biostudies-other

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Project description:Lipophosphoglycan (LPG), is the major cell surface molecule of promastigotes of all Leishmania species. It is comprised of three domains: a conserved glycosylphosphatidylinositol anchor linked to a repeating phosphorylated disaccharide (P2; PO4-6Gal beta 1-4Man alpha 1-) backbone and capped with a neutral oligosaccharide. In Leishmania major the backbone is substituted at the C(O)3 of the Galp residue with side chains containing Galp, Glcp and Arap residues whereas in Leishmania donovani the backbone is unsubstituted. We report the solubilization of a (beta 1-3) galactosyltransferase [(beta 1-3)GalT] from a L. major microsomal preparation using Triton X-100. Solubilization occurs with a 10-fold stimulation of enzyme activity. This (beta 1-3)GalT specifically transfers Gal residues from UDP-Gal to exogenously added L. donovani LPG acceptor. Depolymerization of the [14C]Gal-labelled LPG product with mild acid and analysis by high-performance anion-exchange chromatography detected only the phosphotrisaccharide. (P3; PO4-6([14C]Gal beta 1-3-4Man alpha 1-) found in L. major LPG. This contrasts with the activity of the membrane-bound enzyme which also synthesizes the larger phosphosaccharide units[Ng, Handman and Bacic (1994) Glycobiology 4, 845-853]. This suggests that more than one (beta 1-3)GalT is involved in the addition of these Gal units and that the solubilized activity is the (beta 1-3)GalT that adds the first beta Gal residue to the acceptor. The (beta 1-3)GalT was partially purified by lectin-affinity chromatography and used to establish the K(m) values for UDP-Gal (445 microM) and L. donovani acceptor (280 microM as P2 molar equivalent) in kinetic assays. Inhibition studies with various glycosides and mono- and di-saccharides established the P2 repeating unit as the minimum acceptor structure recognized by (beta 1-3)GalT. The detergent-solubilized (beta 1-3)GalT was reversibly inactivated by millimolar concentrations of univalent anionic salts. The (beta 1-3)GalT had an absolute requirement for Mn2+ and also required Mg2+ for optimum activity; Mg2+ cannot substitute for Mn2+, which is loosely bound to beta (1-3)GalT and is probably involved in the correct folding of the enzyme. The (beta 1-3)GalT was unaffected by Ca2+ ions, but were irreversibly inactivated by micromolar levels of transition metal ions (Cu2+ > Zn2+ > Ni2 > Co2+). The (beta 1-3)GalT activity was also inhibited by diethyl pyrocarbonate, but not by N-ethylmaleimide or iodoacetamide, suggesting that active-site histidine residues, rather than cysteine residue(s), are important for enzyme activity.

Project description:Epididymal delta 4-steroid 5 alpha-reductase (cholestenone 5 alpha-reductase), the enzyme that catalyses the conversion of testosterone into the biologically active metabolite dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one), is a membrane-bound enzyme found in both nuclear and microsomal subcellular fractions. In order to characterize epididymal delta 4-steroid 5 alpha-reductase, it was first necessary to solubilize the enzymic activity. Of the various treatments tested, a combination of 0.5% (w/v) Lubrol WX, 0.1 M-sodium citrate and 0.1 M-KCl maintained enzymic activity at control values and solubilized 66% of total epididymal delta 4-steroid 5 alpha-reductase activity in an active and stable form. The sedimentation coefficient of solubilized delta 4-steroid 5 alpha-reductase, as determined in continuous sucrose density gradients, was greater for the microsomal than for the nuclear enzyme (11.6S compared with 10.1S). Although the apparent Km values of the enzyme for testosterone were similar in nuclear and microsomal subcellular fractions (range 1.75 x 10(-7) - 4.52 x 10(-7)M), the apparent Km of the enzyme for NADPH was about 30-fold greater for the microsomal enzyme than for the nuclear enzyme. The apparent Km of the enzyme for either substrate was not significantly altered after solubilization. The relative capacity of steroids to inhibit the enzymic activity, the pH optima and the effects of Ca2+ and Mg2+ were similar for membrane-bound and solubilized delta 4-steroid 5 alpha-reductase in both the nuclear and the microsomal fractions. The results reported demonstrate that epididymal delta 4-steroid 5 alpha-reductase can be solubilized in an active and stable form with no significant changes in the kinetic characteristics of the enzyme after solubilization; furthermore, kinetic and molecular-size differences observed for the nuclear and the microsomal forms of the enzyme suggest that there may exist at least two forms of epididymal delta 4-steroid 5 alpha-reductase.

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Biosynthesis of heparin. Solubilization and partial characterization of N- and O-sulphotransferases.

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