Photoaffinity labeling of glucosyltransferase of the dolichol cycle from rat mammary gland.
UDP-Glc:dolichol phosphate glucosyltransferase from lactating rat mammary gland has been partially purified by a combination of (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography on DEAE-TSK, and affinity chromatography. The partially purified enzyme exhibited several protein bands when examined by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions; among these, a 35-kDa polypeptide was quite prominent and appeared to be enriched during purification. Photoaffinity labeling of the partially purified enzyme preparation with 5-azido-[beta-32P]UDP-Glc identified a 35-kDa polypeptide. Labeling of a solubilized enzyme preparation from crude and stripped microsomes also revealed a 35-kDa band on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Photoinsertion of the probe in this polypeptide is enhanced by the presence of dolichol phosphate and Mg2+. Competition studies with UDP-Glc, UDP-glucuronic acid, other sugar nucleotides, and Glc-1-phosphate provide evidence to validate the specificity of photoaffinity labeling. These studies indicate that this 35-kDa polypeptide is involved in the synthesis of dolichol-P-Glc in rat mammary tissue. The possibility that this polypeptide may represent glucosyltransferase has been discussed.
Shailubhai
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K
Department of Animal Sciences, University of Maryland, College Park 20742.
Illeperuma
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C
Tayal
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M
Vijay
I K
IK
D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D001667:Binding, Competitive; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D008321:Mammary Glands, Animal / Q000201:enzymology*; D051381:Rats; D014532:Uridine Diphosphate Glucose / Q000031:analogs & derivatives / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; C060227:5-azidouridine 5'-diphosphoglucose; D005964:Glucosyltransferases; C033917:UDPglucose dolicholphosphate glucosyltransferase; D014532:Uridine Diphosphate Glucose