Enzymic transfer of glucose and xylose from uridine diphosphate glucose and uridine diphosphate xylose to bilirubin by untreated and digitonin-activated preparations from rat liver.
1. Digitonin-treated and untreated homogenates, cell extracts and washed microsomal preparations from liver of Wistar R rats are capable of transferring sugar from UDP-glucose or UDP-xylose to bilirubin. No formation of bilirubin glycosides occurred with UDP-galactose or d-glucose, d-xylose or d-glucuronic acid as the sources of sugar. 2. Procedures to assay digitonin-activated and unactivated bilirubin UDP-glucosyltransferase and bilirubin UDP-xylosyltransferase were developed. 3. In digitonin-activated microsomal preparations the transferring enzymes had the following properties. Both enzyme activities were increased 2.5-fold by pretreatment with digitonin. They were optimum at pH6.6-7.2. Michaelis-Menten kinetics were followed with respect to UDP-glucose. In contrast, double-reciprocal plots of enzyme activity against the concentration of UDP-xylose showed two intersecting straight-line sections corresponding to concentration ranges where either bilirubin monoxyloside was formed (at low UDP-xylose concentrations) or where mixtures of both the mono- and di-xyloside were synthesized (at high UDP-xylose concentrations). Both enzyme activities were stimulated by Mg(2+); Ca(2+) was slightly less, and Mn(2+) slightly more, stimulatory than Mg(2+). Of the activities found in standard assay systems containing Mg(2+), 58-78% (substrate UDP-glucose) and 0-38% (substrate UDP-xylose) were independent of added bivalent metal ion. Double-reciprocal plots of the Mg(2+)-dependent activities against the concentration of added Mg(2+) were linear. 4. In comparative experiments the relative activities of liver homogenates obtained with UDP-glucuronic acid, UDP-glucose and UDP-xylose were 1:1.5:2.7 for untreated preparations and 1:0.29:0.44 after activation with digitonin. 5. Bilirubin UDP-glucuronyltransferase was protected against denaturation by human serum albumin, whereas bilirubin UDP-xylosyltransferase was not. 6. Digitonin-treated and untreated liver homogenates from Gunn rats were inactive in transferring sugar to bilirubin from UDP-glucuronic acid (in agreement with the work of others), UDP-glucose or UDP-xylose.
D000818:Animals; D001663:Bilirubin / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D004789:Enzyme Activation; D005947:Glucose / Q000378:metabolism*; D005964:Glucosyltransferases / Q000378:metabolism; D005965:Glucuronates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000494:pharmacology; D008862:Microsomes, Liver / Q000201:enzymology; D011489:Protein Denaturation; D051381:Rats; D012709:Serum Albumin; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*; D014994:Xylose / Q000378:metabolism*
D005965:Glucuronates; D012709:Serum Albumin; D014539:Uridine Diphosphate Sugars; D008345:Manganese; D014994:Xylose; D005964:Glucosyltransferases; D008274:Magnesium; D005947:Glucose; D004072:Digitonin; D001663:Bilirubin; D002118:Calcium