Detection and characterization of UDP-GalNAc: chondroitin N-acetylgalactosaminyltransferase in bovine serum using a simple assay method.
We report the occurrence, in bovine serum of a soluble form, of N-acetylgalactosaminyl-transferase activity, which is involved in chondroitin sulfate biosynthesis, and we describe a simple assay method for the enzyme. Chondroitin with the general structure [GlcA-GalNAc], was found to serve as acceptor substrates for the enzyme. To develop a routine assay, bovine serum was incubated with polymeric chondroitin and UDP-[3H]GalNAc. After the removal of labeled endogenous acceptors derived from serum by trichloroacetic acid treatment, the labeled chondroitin sulfate chains were separated from residual unincorporated label by centrifugation using syringe columns packed with Sephadex G-25, and the effluent fractions were monitored by scintillation counting. This simple method allowed us to perform multiple assays and to establish assay conditions for the serum N-acetylgalactosaminyltransferase involved in chondroitin polymerization.
Kitagawa
H
H
Department of Biochemistry, Kobe Pharmaceutical University.
Tsuchida
K
K
Ujikawa
M
M
Sugahara
K
K
D000818:Animals; D002021:Buffers; D002417:Cattle; D002807:Chondroitin / Q000378:metabolism*; D002845:Chromatography / Q000379:methods*; D003911:Dextrans; D006863:Hydrogen-Ion Concentration; D007477:Ions; D008670:Metals; D017350:N-Acetylgalactosaminyltransferases / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D013594:Syringes
D002021:Buffers; D003911:Dextrans; D007477:Ions; D008670:Metals; D002807:Chondroitin; C025614:sephadex; D017350:N-Acetylgalactosaminyltransferases