N-glycosylation of purified rat and rabbit hepatic UDP-glucuronosyltransferases.
Five UDP-glucuronosyltransferases (UDPGTs) have been isolated to apparent homogeneity from rat and rabbit liver and have been characterized for their glycoprotein nature by reacting these proteins with commercially available endo- and exoglycosidases. The enzymes studied were rat hepatic p-nitrophenol, 17 beta-hydroxysteroid, and 3 alpha-hydroxysteroid UDPGTs and rabbit hepatic p-nitrophenol and estrone UDPGTs. Hydrolysis of oligosaccharide moieties was evidenced by an increase in the mobility (decreased apparent molecular weight) of the protein subunits after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified rabbit hepatic estrone and p-nitrophenol UDPGTs were hydrolyzed by almond glycopeptidase A and endo-beta-N-acetylglucosaminidase H from Streptomyces plicatus (endo H), but not by endo-beta-N-acetylglucosaminidase D from Diplococus pneumoniae (endo D) suggesting that these transferases are glycoproteins of the high mannose type and not of the complex type. Likewise, purified rat hepatic 3 alpha-hydroxysteroid and p-nitrophenol UDPGTs were substrates for glycopeptidase A and endo H but not for endo D. One enzyme, 17 beta-hydroxysteroid UDPGT, was not glycosylated since it was not hydrolyzed by any of the three endoglycosidases. All four glycosylated UDPGTs could serve as substrates for jack bean alpha-mannosidase, confirming the high mannose nature of the oligosaccharide. Deglycosylation of the purified UDPGTs by endo H did not have an effect on the catalytic activities of these proteins.
Green
M D
MD
Department of Pharmacology, University of Iowa, Iowa City 52242.
Tephly
T R
TR
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D014453:Glucuronosyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D008862:Microsomes, Liver / Q000201:enzymology*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains
D007527:Isoenzymes; D014453:Glucuronosyltransferase; D006026:Glycoside Hydrolases