A method for determination of galactosyltransferase I activity synthesizing the proteoglycan linkage region.
An assay method was devised for measuring the activity of galactosyltransferase I (UDP-D-galactose:D-xylose galactosyltransferase), which is one of the enzymes synthesizing the linkage region between the core protein and glycosaminoglycan chains of proteoglycan. For this method, the reaction mixture contained a fluorescent substrate, 4-methylumbelliferyl-beta-D-xyloside as an acceptor, UDP-galactose as a donor and D-galactal as a competitive inhibitor of endogenous beta-galactosidase in the enzyme solution. The reaction mixture was incubated at 37 degrees C with enzyme solution prepared from an extract of cultured cells, and galactosyl-xylosyl-4-methylumbelliferone was produced as a reaction product. Measurement of galactosyltransferase I activity was performed by separation and quantitative analysis of this reaction product using high-performance liquid chromatography. Utilizing this method, easier and more sensitive detection of galactosyltransferase I activity in a cell-free system became possible. Application of the method revealed that cultured human skin fibroblasts contained galactosyltransferase I activity.
Higuchi
T
T
Department of Biochemistry, Hirosaki University School of Medicine, Japan.
Tamura
S
S
Takagaki
K
K
Nakamura
T
T
Morikawa
A
A
Tanaka
K
K
Tanaka
A
A
Saito
Y
Y
Endo
M
M
D001667:Binding, Competitive; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005347:Fibroblasts / Q000201:enzymology; D005690:Galactose / Q000031:analogs & derivatives / Q000494:pharmacology; D005700:Galactosyltransferases / Q000032:analysis / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry; D011509:Proteoglycans / Q000096:biosynthesis*; D012867:Skin / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D014531:Uridine Diphosphate Galactose / Q000378:metabolism
D006025:Glycosaminoglycans; D007202:Indicators and Reagents; D009844:Oligosaccharides; D011509:Proteoglycans; C002038:galactal; D014531:Uridine Diphosphate Galactose; D006923:Hymecromone; C014111:methylumbelliferyl-beta-D-xyloside; D005700:Galactosyltransferases; C063053:xylosylprotein 4-beta-galactosyltransferase; D005690:Galactose