Identification of the UDP-glucose-binding polypeptide of callose synthase from Beta vulgaris L. by photoaffinity labeling with 5-azido-UDP-glucose.
The photoaffinity probe 5-azidouridine 5'-[beta-32P]diphosphate glucose (5N3[32P]UDP-Glc) was used to identify a 57-kDa polypeptide as a strong candidate for the UDP-Glc-binding polypeptide of UDP-glucose: (1,3)-beta-glucan (callose) synthase from red beet (Beta vulgaris L.) storage tissue. Unlabeled 5N3UDP-Glc was a competitive inhibitor of callose synthase with a Ki of 310 microM. Callose synthase was purified from plasma membranes by a two-step solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, followed by product entrapment, and photoincorporation of radioactivity from 5N3[32P]UDP-Glc was used to identify UDP-Glc-binding polypeptides that copurified with callose synthase activity. Photoinsertion into the 57-kDa band was closely correlated with all catalytic properties examined. Photolabeling of the 57-kDa polypeptide was enriched upon purification of callose synthase by product entrapment, was abolished with increasing levels of unlabeled UDP-Glc, was dependent upon the presence of divalent cations, and the pH dependence of photolabeling correlated with the pH activity profile of callose synthase. In addition, photolabeling of the 57-kDa band did not occur after phospholipase treatment, which destroys enzyme activity. The extent of labeling of this polypeptide thus correlates closely with the activity of callose synthase under a wide variety of conditions. These results imply that the polypeptide at 57 kDa represents the substrate-binding and cation-regulated component of the callose synthase complex of higher plants.
Frost
D J
DJ
Department of Food Science, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, New Brunswick 08903.
Read
S M
SM
Drake
R R
RR
Haley
B E
BE
Wasserman
B P
BP
D000345:Affinity Labels / Q000378:metabolism*; D001386:Azides / Q000378:metabolism*; D001667:Binding, Competitive; D002462:Cell Membrane / Q000201:enzymology; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D010777:Photochemistry; D010944:Plants / Q000201:enzymology*; D029702:Schizosaccharomyces pombe Proteins; D014532:Uridine Diphosphate Glucose / Q000031:analogs & derivatives / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D046911:Macromolecular Substances; D008565:Membrane Proteins; D029702:Schizosaccharomyces pombe Proteins; D014539:Uridine Diphosphate Sugars; C060227:5-azidouridine 5'-diphosphoglucose; D005964:Glucosyltransferases; C020570:1,3-beta-glucan synthase; D014532:Uridine Diphosphate Glucose