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Plant Physiology 97:396-401 (1991)
© 1991 American Society of Plant Biologists

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Metabolism and Enzymology

Partial Purification, Photoaffinity Labeling, and Properties of Mung Bean UDP-Glucose:Dolicholphosphate Glucosyltransferase 1

Richard R. Drake, Jr.2, Gur P. Kaushal2, Irena Pastuszak3 and Alan D. Elbein2

Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78284

UDP-glucose:dolichylphosphate glucosyltransferase has been purified 734-fold from Triton X-100 solubilized mung bean (Phaseolus aureus) microsomes. The partially purified enzyme has broad pH optima of activity from 6.0 to 7.0 and is maximally stimulated with 10 millimolar MgCl2. The Km for UDP-glucose was determined as 27 micromolar, and the Km for dolichol-P was 2 micromolar. Using the UDP-glucose photoaffinity analog, 5-azido-UDP-glucose, a polypeptide of 39 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels was identified as the catalytic subunit of the enzyme. Photoinsertion into this 39-kilodalton polypeptide with [32P]5-azido-UDP-glucose was saturable, and was maximally protected with the native substrate UDP-glucose. 5-Azido-UDP-glucose behaves competitively with UDP-glucose in enzyme assays, and upon photolysis inhibits activity in proportion to its concentration. This study represents the first subunit identification of a plant glycosyltransferase involved in the biosynthesis of the lipid-linked oligosaccharides that are precursors of N-linked glycoproteins.

2 Present address: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205.

3 Present address: Department of Biochemistry, Medical School, Lublin, Lubartowska 85, Poland.

1 This research was supported by grant DK 21800 from the National Institutes of Health.






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Copyright © 1991 by the American Society of Plant Biologists