caa a22 4500 477101968 CHVBK 20180405111544.0 cr unu---uuuuu 170330e19960601xx s 000 0 eng 10.1007/BF00196565 doi (NATIONALLICENCE)springer-10.1007/BF00196565 Partial purification and characterization of UDP-glucose: betanidin 5- O - and 6- O -glucosyltransferases from cell suspension cultures of Dorotheanthus bellidiformis (Burm. f.) N.E.Br [Elektronische Daten] [Susanne Heuer, Thomas Vogt, Hartmut Böhm, Dieter Strack] Uridine 5′-diphosphoglucose-dependent glucosyl-transferases (UDP-glucose:betanidin 5-O- and 6-O-glucosyltransferases; 5-GT and 6-GT; EC 2.4.1) catalyze the regiospecific transfer of glucose to the 5- and 6-hydroxy group of betanidin in the formation of betanin and gomphrenin I, respectively. Both GT activities were partially purified from cell suspension cultures of Dorotheanthus bellidiformis (Burm. f.) N.E. Br. Isoelectric focusing of crude protein extracts indicated the presence of three 5-GT isoforms and a single 6-GT form. The 5-GT isoforms were partially separated from each other and completely from the 6-GT. Studies of the glucosyltransferase activities were focused on the major isoform of the 5-GTs and the 6-GT, which displayed the same pH optimum near 7.5 in K-phosphate buffer. A 3- and 2.5-fold enrichment and 11% and 10% recovery of the 5-GT and 6-GT, respectively, were routinely achieved; however, a 3300-fold enrichment of the major 5-GT isoform and a 6-fold enrichment of the 6-GT were also achieved. Both enzymes are monomers and displayed apparent native Mrs near 55 000. The maxima of the reaction temperature were at 50 °C for the 5-GT and at 37°C for the 6-GT with respective apparent energies of activation of 51 and 53 kJ · mol−1. Kinetic studies indicated that the apparent Michaelis constants (apparent K m) of the GTs for one substrate were dependent on the concentration of the second substrate. However, the relationship between the apparent K m values and the dissociation constants (K i) were different; m > K i applies for the 5-GT and K m < K i for the 6-GT activity. Consequently, this results in a predominant formation of betanin at low substrate concentrations, but a predominant formation of gomphrenin I at high substrate concentrations, assuming that both enzymes may compete freely for their substrates. This might explain why we could not observe a correlation between extractable 5-GT and 6-GT activities and the in-vivo accumulation of the respective products from cell-suspension cultures of D. bellidiformis. Springer-Verlag, 1996 Betacyanin nationallicence Betalain nationallicence Betanin nationallicence Dorotheanthus (cell culture) nationallicence Glucosyltransferase nationallicence Gomphrenin nationallicence BSA : bovine serum albumin nationallicence DOPA : l-(3,4-dihydroxyphenyl)-alanine nationallicence 5-GT : UDP-glucose:betanidin 5-O-glucosyltransferase nationallicence 6-GT : UDP-glucose:betanidin 6-O-glucosyl-transferase nationallicence Mr : molecular mass nationallicence Heuer Susanne Institut für Pharmazeutische Biologie der Technischen Universität Braunschweig, Mendelssohnstrasse 1, D-38106, Braunschweig, Germany aut Vogt Thomas Institut für Pflanzenbiochemie (IPB), Abteilung Sekundärstoffwechsel, Weinberg 3, D-06120, Halle (Saale), Germany aut Böhm Hartmut Deutsches Institut für Ernährungsforschung, Arthur-Scheunert-Allee 114-116, D-14558, Bergholz-Rehbrücke, Germany aut Strack Dieter Institut für Pflanzenbiochemie (IPB), Abteilung Sekundärstoffwechsel, Weinberg 3, D-06120, Halle (Saale), Germany aut Planta Springer-Verlag 199/2(1996-06-01), 244-250 0032-0935 199:2<244 1996 199 425 https://doi.org/10.1007/BF00196565 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00196565 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Heuer Susanne Institut für Pharmazeutische Biologie der Technischen Universität Braunschweig, Mendelssohnstrasse 1, D-38106, Braunschweig, Germany aut NATIONALLICENCE 700 1- Vogt Thomas Institut für Pflanzenbiochemie (IPB), Abteilung Sekundärstoffwechsel, Weinberg 3, D-06120, Halle (Saale), Germany aut NATIONALLICENCE 700 1- Böhm Hartmut Deutsches Institut für Ernährungsforschung, Arthur-Scheunert-Allee 114-116, D-14558, Bergholz-Rehbrücke, Germany aut NATIONALLICENCE 700 1- Strack Dieter Institut für Pflanzenbiochemie (IPB), Abteilung Sekundärstoffwechsel, Weinberg 3, D-06120, Halle (Saale), Germany aut NATIONALLICENCE 773 0- Planta Springer-Verlag 199/2(1996-06-01), 244-250 0032-0935 199:2<244 1996 199 425 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer