caa a22 4500 465734626 CHVBK 20180323111751.0 cr unu---uuuuu 170327e19900301xx s 000 0 eng 10.1007/BF01050379 doi (NATIONALLICENCE)springer-10.1007/BF01050379 Comparative effects of sulfhydryl reagents on membrane-bound and solubilized UDP-glucose:ceramide glucosyltransferase from Golgi membranes. Evidence for partial involvement of a thiol group in the nucleotide sugar binding site of the solubilized enzyme [Elektronische Daten] [I Durieux, M Martel, R Got] We have studied the inactivation of membrane-bound and solubilized UDP-glucose:ceramide glucosyltransferase from Golgi membranes by various types of sulfhydryl reagents. The strong inhibition of the membrane-bound form by the non-penetrant mercurial-type reagents clearly corroborated the fact that in sealed and "right-side-out” Golgi vesicles the ceramide glucosyltransferase is located on the cytoplasmic face. No significant differences in the susceptibility to the various sulfhydryl reagents were noted when solubilized enzyme was assayed, showing that solubilization does not reveal other critical SH groups. The different results obtained must be interpreted with regard to several thiol groups, essential for enzyme activity. No protection by the substrate UDP-glucose against mercurial-type reagents was obtained indicating that these thiol groups were not located in the nucleotide sugar binding domain. A more thorough investigation of the thiol inactivation mechanism was undertaken with NEM (N-ethylmaleimide), an irreversible reagent. The time dependent inactivation followed first order kinetics and provided evidence for the binding of 1 mol NEM per mol of enzyme. UDP-Glucose protected partially against NEM inactivation, indicating that the thiol groups may be situated in or near the substrate binding domain. Inactivation experiments with disulfide reagents showed that increased hydrophobicity led to more internal essential SH groups which are not obviously protected by the substrate UDP-glucose, thus not implicated in the substrate binding domain, but rather related to conformational changes of the enzyme during the catalytic process. Glycoconjugate Journal, 1990 ceramide glucosyltransferase nationallicence sulfhydryl reagents nationallicence solubilized and membrane-bound form nationallicence Chaps : 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate nationallicence Mops : 4-morpholinepropanesulfonic acid nationallicence PC : phosphatidylcholine nationallicence NEM : N-ethylmaleimide nationallicence CPDS : carboxypyridine disulfide (dithio-6,6′-dinicotinic acid) nationallicence DTNB : 5,5′-dithiobis-(2-nitrobenzoic acid) nationallicence DTP : dithiodipyridine nationallicence p -HMB : para-hydroxymercuribenzoate nationallicence DTT : dithiothreitol nationallicence BAL : British anti-Lewisite (dimercaptopropanol) nationallicence Zw 3-14 : Zwittergent 3-14 nationallicence Durieux I. Laboratoire de Biochimie des Membranes (LBTM-CNRS UM 380024), Université Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622, Villeurbanne Cedex, France aut Martel M. Laboratoire de Biochimie des Membranes (LBTM-CNRS UM 380024), Université Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622, Villeurbanne Cedex, France aut Got R. Laboratoire de Biochimie des Membranes (LBTM-CNRS UM 380024), Université Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622, Villeurbanne Cedex, France aut Glycoconjugate Journal Kluwer Academic Publishers 7/2(1990-03-01), 183-192 0282-0080 7:2<183 1990 7 10719 https://doi.org/10.1007/BF01050379 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF01050379 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Durieux I. Laboratoire de Biochimie des Membranes (LBTM-CNRS UM 380024), Université Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622, Villeurbanne Cedex, France aut NATIONALLICENCE 700 1- Martel M. Laboratoire de Biochimie des Membranes (LBTM-CNRS UM 380024), Université Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622, Villeurbanne Cedex, France aut NATIONALLICENCE 700 1- Got R. Laboratoire de Biochimie des Membranes (LBTM-CNRS UM 380024), Université Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622, Villeurbanne Cedex, France aut NATIONALLICENCE 773 0- Glycoconjugate Journal Kluwer Academic Publishers 7/2(1990-03-01), 183-192 0282-0080 7:2<183 1990 7 10719 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer