A rapid and simple assay method for UDP-glucose:ceramide glucosyltransferase.
We have developed a simple rapid method for measuring UDP-glucose:ceramide glucosyltransferase; the method utilizes ceramide immobilized on the surface of silica gel and [14C]UDP-glucose as substrate. The reaction product, [14C]glucosylceramide, formed on the surface of the silica gel was easily separated from free [14C]UDP-glucose, either by centrifugation or by filtration. The reliability of this solid phase method was evaluated by using rat brain membrane fraction as an enzyme source. This enzyme had an optimal pH of 6.4-6.5 and required Mn2+, Mg2+ in the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Apparent Km values of 8.7 microM for UDP-glucose and 292 microM for ceramide were determined using the new method. Under the optimal conditions, the solid phase method yielded 2-5-times more product than did the method using micellar system. Moreover, the reaction was highly quantitative in its enzyme dose-activity relationship.
Matsuo
N
N
Biological Science Laboratories, Kao Corporation, Tochigi, Japan.
Nomura
T
T
Imokawa
G
G
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002250:Carbon Radioisotopes; D002413:Cations, Divalent / Q000494:pharmacology; D002518:Ceramides / Q000302:isolation & purification*; D002793:Cholic Acids / Q000494:pharmacology; D002855:Chromatography, Thin Layer / Q000379:methods; D003902:Detergents / Q000494:pharmacology; D005964:Glucosyltransferases / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics; D008297:Male; D011865:Radioisotope Dilution Technique; D051381:Rats; D011919:Rats, Inbred Strains; D014532:Uridine Diphosphate Glucose / Q000378:metabolism*
D002250:Carbon Radioisotopes; D002413:Cations, Divalent; D002518:Ceramides; D002793:Cholic Acids; D003902:Detergents; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D014532:Uridine Diphosphate Glucose