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Beta-D-Glucuronyltransferase, which transfers D-glucuronic acid (GlcA) from UDP-GlcA to N-acetyl-D-galactosamine (GalNAc) at the nonreducing end of chondro-pentasaccharide-PA (pyridylamino-), GalNAcbeta1-(4GlcAbeta1-3GalNAcbeta1)2-PA, was purified 339-fold with an 11.0% yield from 2-d-old chick corneas by chromatography on DEAE-Sepharose, WGA-agarose, heparin-Sepharose, and 1st and 2nd UDP-GlcA-agarose (in the presence of Gal) columns. The activity was detected by fluorescence of PA residues of the product. The purified enzyme has an optimum pH of 7.0 (Mes buffer), and much higher activity toward chondro-heptasaccharide-PA than toward the chondro-pentasaccharide-PA, but no activity toward p-nitrophenyl-beta-GalNAc. The enzyme activity was almost completely inhibited by GalNAc (20 mm). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme fraction showed one band of 38 kDa with many other bands. The amino acid sequence was determined for the tryptic digests of the 38 kDa band protein. The sequences determined showed no homology to those of several beta-glucuronyltransferases reported previously. It seems that the enzyme is involved in the elongation of chondroitin sulfate chains in vivo.

A morphine UDP-glucuronyltransferase was purified from liver microsomes of untreated Sprague-Dawley rats. A new gel, omega(beta-carboxypropionylamino)octyl Sepharose 4B, was prepared by coupling monomethylsuccinate with omega-aminooctyl Sepharose 4B and this was used as an efficient tool for the separation of microsomal enzymes. Emulgen 911 solubilized microsomes were applied to a column packed with the gel and eluted at pH 7.4 while increasing KCl concentration in a stepwise manner. An isoform was further purified with UDP-hexanolamine Sepharose 4B gel. The purified UDP-glucuronyltransferase (morphine UGT of untreated rat, morphine UGTUT) exhibited a molecular weight of 52000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and was capable of glucuronidating the 3-hydroxyl group of morphine. The isoform catalyzed to a small extent the glucuronidation of 4-hydroxybiphenyl; however, no glucuronidation activity towards androsterone, testosterone, bilirubin, 4-nitrophenol and the 6-hydroxyl group of morphine was observed. The difference in properties, compared with morphine UGT (molecular weight 56000) which was purified previously from phenobarbital-treated rats, is discussed.

The hepatic microsomal UDP-glucuronyltransferase (UDPGT) for p-phenyl benzoic acid (PPBA) in the developing fetus of rat was investigated. The kinetic properties of UDPGT in microsome of fetus changed during developmental period. The value of the Vmax for PPBA and uridine 5'-phosphoglucuronic acid (UDPGA) per mg protein increased with the day of gestation, remarkably between the 19th and 21st day of gestation. The developmental profile of the Vmax per g liver was similar to the Vmax per mg protein. Whereas the Vmax in whole liver showed rapid increase during development comparing with the Vmax per g liver. The Km for PPBA and UDPGA showed slower increase with the day of gestation than the corresponding Vmax.

Uridine diphosphate (UDP)-glucuronosyltransferase (GT) active on androsterone (AD) and 4-nitrophenol (NP) was solubilized from male rat liver microsomes of the Wistar strain. The precipitate obtained in the 60%-satd. ammonium sulfate was purified by diethylaminoethyl (DEAE)-cellulose chromatography and affinity chromatography on UDP-hexanolamine Sepharose 4B. DEAE-cellulose chromatography showed the existence of two peaks of GT active on AD and NP. Peak I was found in rats with the high-activity and low-activity phenotypes in terms of AD glucuronidation and had high NP-GT and low AD-GT activities. In contrast, peak II was found only in rats with the high-activity phenotype, corresponded to high AD-GT activity and had comparatively low NP-GT activities. The corresponding peak in rats with the low-activity phenotype had only NP-GT activity. Comparison of Km values for AD obtained from microsomes and purified enzymes provides evidence that AD-GT isoenzyme should be deficient in Wistar rats with the low-activity phenotype and that AD glucuronidation should be catalyzed poorly by other GT isoenzyme in these rats.

The protective effect of SKF 525-A on the suppression of cytochrome P-450 content and monooxygenase activities by treatment with CoCl2 and polyriboinosinic acid-polyribocytidylic acid [poly(I.C] was compared as a part of studies of suppression of drug metabolizing enzymes by interferon inducers. Induction of heme oxygenase activity by CoCl2 and poly (I.C) was not altered by simultaneous treatment with SKF 525-A. Depression of cytochrome P-450 content and benzphetamine N-demethylase activity by treatment with CoCl2 was prevented by co-treatment with SKF 525-A. This effect was explained by the prevention of release of heme from cytochrome P-450 by forming metabolic intermediate complexes with metabolites of SKF 525-A. On the other hand, poly(I.C) significantly suppressed P-450 content and benzphetamine N-demethylase and benzo [a] pyrene hydroxylase activities, even under simultaneous treatment with SKF 525-A. This inhibition by poly (I.C) was accompanied by weak staining of proteins corresponding to cytochrome P-450 in SDS gel electrophoresis. In addition, the activity of non-heme enzyme, 4-hydroxybiphenyl glucuronyltransferase, was suppressed by treatment with poly (I.C) but not by CoCl2-treatment. These findings strongly suggested that, unlike CoCl2, poly (I.C) suppressed cytochrome P-450 content and monooxygenase activities due to decreased synthesis or increased degradation of the apoprotein of cytochrome P-450 with slight contribution of the induced heme oxygenase.