digitonin and bilirubin-diglucuronide

The molecular masses of the UDP-glucuronyltransferases were determined by means of radiation-inactivation analysis of sodium dodecyl sulfate-treated lyophilized rat liver microsomal preparations using a calibrated 60Co source. Bilirubin glucuronidation with formation of bilirubin monoglucuronide is catalyzed by a 41,500-Da enzyme; conversion of bilirubin mono- to diglucuronide is catalyzed by a 175,000-Da enzyme. The glucuronidation of estrone and testosterone is mediated by enzymes with molecular masses of 73,500 and 142,000 Da, respectively, and the glucuronidation of p-nitrophenol and phenolphthalein is mediated by enzymes with molecular masses of 109,000 and 159,000 Da, respectively. Our results show that UDP-glucuronyltransferase consists of a heterogenous group of enzymes with strikingly different molecular masses. Our data furthermore suggest that these enzymes may be oligomers composed of one to four subunits with similar molecular masses. Based on these findings, a molecular model of bilirubin UDP-glucuronyltransferase is proposed, consisting of four subunits. For bilirubin diglucuronide formation, the complete tetrameric enzyme is required, whereas formation of monoglucuronide can be mediated by a single subunit. The monomeric monoglucuronide-forming enzyme is resistant to sodium dodecyl sulfate, treatment whereas the tetrameric diglucuronide-forming enzyme is labile, but once inactivated, the diglucuronide-forming enzyme can be reconstituted by decreasing the sodium dodecyl sulfate concentration by means of dialysis.

Topics: Animals; Bilirubin; Detergents; Digitonin; Glucuronosyltransferase; Hexosyltransferases; Male; Microsomes, Liver; Models, Molecular; Molecular Weight; Quaternary Ammonium Compounds; Rats; Rats, Inbred Strains; Sodium Dodecyl Sulfate

The activity of bilirubin-UDP-glucuronyl transferase (UDPGT) in rat liver microsomes was assayed in standardized incubation mixtures employing two different procedures to quantitate formation of conjugated bilirubin: solvent extraction and diazotization with sulfanilic acid, or selective coupling with diazotized ethyl anthranilate at pH 2.7. Both of these procedures yielded equivalent UDPGT activities with several different microsomal preparations, averaging, respectively, 385 and 372 microU protein with untreated microsomes, 646 and 608 microU protein following activation with UDP-N-acetylglucosamine (3.07 mmol/l), and 1370 and 1478 microU protein after treatment with digitonin (0.65%). In rats pretreated with phenobarbital (75 mg/kg per 6 days), a 70-90% increase in UDPGT activity was observed with either diazotization procedure, irrespective of whether 'native', UDP-N-acetylglucosamine-or digitonin-activated enzyme was employed. These studies indicate that, when other conditions are standardized, equivalent information about UDPGT can be obtained with either of two widely employed diazotization reactions and a variety of enzyme activation procedures.

Topics: Animals; Benzenesulfonates; Bilirubin; Diazonium Compounds; Digitonin; Enzyme Activation; Glucuronosyltransferase; Male; Microsomes, Liver; Phenobarbital; Rats; Rats, Inbred Strains; Spectrophotometry; Sulfanilic Acids; Uridine Diphosphate N-Acetylglucosamine

Although the formation of bilirubin monoglucuronide by hepatic microsomes has been easily demonstrable, that of bilirubin diglucuronide, the principal conjugate of bile, has been more difficult. Therefore, an examination of the uridine diphosphate glucuronate-dependent microsomal formation of these two conjugates has been made utilizing a high performance liquid chromatographic method which quantitates the isomeric forms of the products. Initial studies indicated that at high starting bilirubin concentrations, only bilirubin monoglucuronide was formed; whereas at lower concentrations (approximately 15 microM), bilirubin diglucuronide could be formed efficiently, but only under very specific conditions. Untreated microsomes and microsomes treated with Triton X-100 did not differ; each formed monoglucuronide efficiently, yet formed diglucuronide poorly. Digitonin or UDP-N-acetylglucosamine pretreatment, in contrast, was found to facilitate bilirubin diglucuronide formation, the former much more than the latter. The activity of mannose 6-phosphatase, an enzyme located on the inner surface of the microsomal vesicles, did not correlate well with the bilirubin diglucuronide formation. Time course studies with digitonin and UDP-N-acetylglucosamine indicated a precursor-product relation between bilirubin monoglucuronide and bilirubin diglucuronide, and product isomer composition studies indicated that the bilirubin tetrapyrroles were stable (no random dipyrrolic exchange had occurred). Temperature studies with the digitonin-treated preparation demonstrated an increase in monoglucuronide-forming activity over the 0-25 degrees C range, whereas diglucuronide formation increased dramatically over the range from 25 to 35 degrees C. The results indicate that microsomal diglucuronide-forming activity differs characteristically from monoglucuronide-forming activity, and that it is intensely sensitive to the manipulation of its microsomal membrane environment.

Topics: Animals; Bilirubin; Digitonin; Glucuronosyltransferase; Hexosyltransferases; Male; Microsomes, Liver; Rats; Rats, Inbred Strains; Temperature; Time Factors; Uridine Diphosphate N-Acetylglucosamine