title: Stimulation of the dithiol-dependent reductases in the vitamin K cycle by the thioredoxin system. Strong synergistic effects with protein disulphide-isomerase.
abstract: It has been shown previously that the thioredoxin system (thioredoxin + thioredoxin reductase + NADPH) may replace dithiothreitol (DTT) as a cofactor for vitamin KO and K reductase in salt-washed detergent-solubilized bovine liver microsomes. Here we demonstrate that the system can be improved further by adding protein disulphide-isomerase (PDI) to the components mentioned above. Moreover, NADPH may be replaced by reduced RNAase as a hydrogen donor. In our in vitro system the various protein cofactors were required at concentrations 2-5 orders of magnitude lower than that of DDT, whereas the maximal reaction rate was about 3-fold higher. PDI stimulated the thioredoxin-driven reaction about 10-fold, with an apparent Km value of 8 microM. These data suggest that in the vitro system the formation of disulphide bonds is somehow linked to the vitamin K-dependent carboxylation of glutamate residues. In vivo, both disulphide formation and vitamin K-dependent carboxylation are post-translational modifications taking place at the luminal side of the endoplasmic reticulum of mammalian secretory cells. The possibility that the reactions are also coupled in vivo is discussed.
authors.affiliation: Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
authors.forename: B A
authors.initials: BA
authors.lastname: Soute
authors.forename: M M
authors.initials: MM
authors.lastname: Groenen-van Dooren
authors.forename: A
authors.initials: A
authors.lastname: Holmgren
authors.forename: J
authors.initials: J
authors.lastname: Lundström
authors.forename: C
authors.initials: C
authors.lastname: Vermeer
mesh_terms: D000818:Animals; D019735:Carbon-Carbon Ligases; D002417:Cattle; D003067:Coenzymes / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism; D054477:Glutaredoxins; D005980:Glutathione Reductase / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008954:Models, Biological; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases; D019704:Protein Disulfide-Isomerases; D011506:Proteins / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D013552:Swine; D013879:Thioredoxins / Q000378:metabolism*; D014812:Vitamin K / Q000378:metabolism*
chemical_list: D003067:Coenzymes; D004220:Disulfides; D054477:Glutaredoxins; D007328:Insulin; D011506:Proteins; D014812:Vitamin K; D013879:Thioredoxins; D009249:NADP; D010088:Oxidoreductases; D005980:Glutathione Reductase; D012260:Ribonucleases; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D008025:Ligases; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase; D004229:Dithiothreitol