Synthesis and characterization of 5-azido-UDP-glucuronic acid. A new photoaffinity probe for UDP-glucuronic acid-utilizing proteins.
A new active site-directed photoaffinity analogue, [beta-32P]5-azido-UDP-glucuronic acid (UDP-GlcA), was enzymatically synthesized from [beta-32P]5-N3UDP-Glc using UDP-glucose dehydrogenase. The product was characterized by its mobility on ion exchange and two thin-layer chromatographic systems, by its UV absorbance at 288 nm, and the loss of this absorbance after UV irradiation of the compound. Photoincorporation of [beta-32P]5-N3UDP-GlcA into bovine liver UDP-Glc dehydrogenase (EC 1.1.1.22) was saturable with an apparent Kd of 12.5 microM, and was inhibited by the known active-site effectors UDP-GlcA, UDP-Glc, and UDP-xylose. When human liver microsomes with known UDP-glucuronosyltransferase (EC 2.4.1.17) activities were photolabeled with [beta-32P]5-N3UDP-GlcA, major photolabeled bands of 35-37 and 50-54 kDa were detected. When rat liver microsomes from phenobarbital-injected rats were photolabeled with [beta-32P]5-N3UDP-GlcA, there was a marked increase in photoincorporation of a 51-kDa protein as compared with control animals. Evidence is presented which suggests that the photolabeled 51-54-kDa proteins in the liver microsomes from both tissues are UDP-glucuronosyltransferase and that [beta-32P]5-N3UDP-GlcA represents a new alternative approach in the study of UDP-glucuronosyltransferase and other UDP-GlcA-utilizing enzymes.
Drake
R R
RR
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205.
Zimniak
P
P
Haley
B E
BE
Lester
R
R
Elbein
A D
AD
Radominska
A
A
D000345:Affinity Labels / Q000138:chemical synthesis* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D014453:Glucuronosyltransferase / Q000378:metabolism; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology; D015394:Molecular Structure; D010777:Photochemistry; D051381:Rats; D011916:Rats, Inbred F344; D014533:Uridine Diphosphate Glucose Dehydrogenase / Q000378:metabolism; D014535:Uridine Diphosphate Glucuronic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism
D000345:Affinity Labels; C071932:5-azido-UDP-glucuronic acid; D014535:Uridine Diphosphate Glucuronic Acid; D014533:Uridine Diphosphate Glucose Dehydrogenase; D014453:Glucuronosyltransferase