Analysis of the streptococcal hyaluronic acid synthase complex using the photoaffinity probe 5-azido-UDP-glucuronic acid.
The mucopolysaccharide, hyaluronic acid, is an important component of both mammals and pathogenic streptococci. This high molecular weight polymer is synthesized by a membrane-associated, multisubunit hyaluronate synthase which utilizes UDP-glucuronic acid and UDP-N-acetylglucosamine as substrates. Using the photoaffinity probe, [beta-32P]5-azido-UDP-glucuronic acid, three streptococcal membrane proteins (42, 33, and 27 kDa) specifically photoincorporated this probe. Labeling of these proteins was enhanced in the presence of UDP-N-acetylglucosamine, whereas UDP-galactose or UDP-glucose had no effect on incorporation. UDP-glucuronic acid inhibited the labeling of the three proteins in a dose-dependent manner. Detergent-solubilized membrane proteins from transposon-inactivated hyaluronic acid capsule mutants no longer incorporated the probe. This was also the case when membranes from stationary phase organisms were tested. Finally, glucuronic acid no longer was incorporated into high molecular weight hyaluronic acid with either the mutant or stationary phase preparations. Further biochemical analysis will be required to demonstrate the exact role each of the proteins play in hyaluronic acid biosynthesis.
van de Rijn
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Wake Forest University Medical Center, Winston-Salem, North Carolina 27157.
Drake
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D000345:Affinity Labels / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D014453:Glucuronosyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D016695:Glycosyltransferases; D000076002:Hyaluronan Synthases; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins; D008970:Molecular Weight; D013045:Species Specificity; D013297:Streptococcus pyogenes / Q000201:enzymology*; D014166:Transferases; D014535:Uridine Diphosphate Glucuronic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D029867:Xenopus Proteins
D000345:Affinity Labels; D046911:Macromolecular Substances; D008565:Membrane Proteins; D029867:Xenopus Proteins; C071932:5-azido-UDP-glucuronic acid; D014535:Uridine Diphosphate Glucuronic Acid; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases