Metabolism of conjugated sterols in eggplant. Part 1. UDP-glucose : sterol glucosyltransferase.
A membrane-bound UDP-glucose : sterol glucosyltransferase from Solanum melongena (eggplant) leaves was partially purified and its specificity as well as molecular and kinetic properties were defined. Among a wide spectrum of 3-OH steroids (i.e. typical plant sterols, androstane, pregnane and cholestane derivatives, steroidal alkaloids and sapogenins) and triterpenic alcohols, the highest activity was found with 22-oxycholesterol. UDP-glucose appeared to be the best sugar donor. The enzyme preparation was also able to utilize UDP-galactose, TDP-glucose and CDP-glucose as a sugar source for sterol glucosylation, however, at distinctly lower rates. The investigated glucosyltrasferase was stimulated by 2-mercaptoethanol, Triton X-100 and negatively charged phospholipids, and inhibited in the presence of UDP, mono-, di- and triacylglycerols, divalent cations such as Zn(2+), Co(2+), high ionic strength, cholesteryl glucoside, galactoside and xyloside and some amino acid-modifying reagents (SITS, DIDS, PLP, DEPC, pCMBS, NEM, WRK and HNB). Our results suggest that unmodified residues of lysine, tryptophan, cysteine, histidine and dicarboxylic amino acids are essential for full enzymatic activity and indicate that a glutamic (or aspartic) acid residue is necessary for the binding of sugar donor, i.e. UDP-glucose in the active site of the GT-ase while histidine and cysteine residues are both important for the binding of the nucleotide-sugar as well as of the steroidal aglycone.
Potocka
Anna
A
Institute of Biochemistry, Warsaw University, Warszawa, Poland.
Zimowski
Jan
J
D001671:Biochemistry / Q000379:methods; D002241:Carbohydrates / Q000737:chemistry; D002784:Cholesterol / Q000031:analogs & derivatives / Q000737:chemistry; D003035:Cobalt / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology; D005964:Glucosyltransferases / Q000378:metabolism*; D007477:Ions; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D017830:Octoxynol / Q000494:pharmacology; D018515:Plant Leaves / Q000378:metabolism; D032323:Solanum melongena / Q000201:enzymology*; D013261:Sterols / Q000378:metabolism*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry
D002241:Carbohydrates; D003902:Detergents; D007477:Ions; D013261:Sterols; C027578:cholesteryl glucoside; D003035:Cobalt; D008623:Mercaptoethanol; D017830:Octoxynol; D002784:Cholesterol; D005964:Glucosyltransferases; C023256:UDPglucose-sterol glucosyltransferase; D015032:Zinc; D014532:Uridine Diphosphate Glucose