Functional molecular mass of a vertebrate hyaluronan synthase as determined by radiation inactivation analysis.
Hyaluronan (HA), a linear polysaccharide composed of N-acetylglucosamine-glucuronic acid repeats, is found in the extracellular matrix of vertebrate tissues as well as the capsule of several pathogenic bacteria. The HA synthases (HASs) are dual-action glycosyltransferases that catalyze the addition of two different sugars from UDP-linked precursors to the growing HA chain. The prototypical vertebrate hyaluronan synthase, xlHAS1 (or DG42) from Xenopus laevis, is a 588-residue membrane protein. Recently, the streptococcal enzyme was found to function as a monomer of protein with approximately 16 lipid molecules. The vertebrate enzymes are larger than the streptococcal enzymes; based on the vertebrate HAS deduced amino acid sequence, two additional membrane-associated regions at the carboxyl terminus are predicted. We have utilized radiation inactivation to measure the target size of yeast-derived recombinant xlHAS1. The target size of HAS activity was confirmed using two internal standards. First, samples were spiked with glucose-6-phosphate dehydrogenase, an enzyme of known molecular weight. Second, parallel samples of native xlHAS1 and a xlHAS1-green fluorescent protein fusion (833 residues) were compared; substantial confidence was gained by using this novel internal standard. Our test also corroborated the basic tenets of radiation inactivation theory. We found that the vertebrate HAS protein functions catalytically as a monomer.
Pummill
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PE
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., Oklahoma City, OK 73104, USA.
Kempner
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DeAngelis
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PL
D000818:Animals; D004307:Dose-Response Relationship, Radiation; D004583:Electrons; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects*; D016695:Glycosyltransferases; D049452:Green Fluorescent Proteins; D000076002:Hyaluronan Synthases; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D008565:Membrane Proteins; D008970:Molecular Weight; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000528:radiation effects; D014166:Transferases; D029867:Xenopus Proteins; D014982:Xenopus laevis
D008164:Luminescent Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D049452:Green Fluorescent Proteins; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases