Comparison of synthetic and natural glucosylceramides as substrate for glucosylceramidase assay.
Commercially available [3H]glucosylceramide is derived from spleen tissue of patients with Gaucher's disease. When such tritiated glucosylceramide was diluted with unlabelled glucosylceramide from different sources and used as the substrate for assays of glucosylceramidase, the apparent activities obtained differed drastically. When diluted with synthetic N-stearoyl- or N-lignoceroyl-glucosyldihydrosphingosine, the release of [3H]glucose was 4-5 times greater than when diluted with unlabelled glucosylceramide from Gaucher spleen, with either brain or fibroblast homogenate as the enzyme source. The Ki values of the synthetic substrates were 15-30 times greater than the Km for the natural mixture, indicating much lower affinity of the synthetic substrates for the enzyme. Although the reason for the reduction in affinity could not be identified, caution is required when the commercial [3H]glucosylceramide is to be diluted with unlabelled glucosylceramide.
D001921:Brain / Q000201:enzymology; D002554:Cerebrosides / Q000378:metabolism*; D005347:Fibroblasts / Q000201:enzymology; D005776:Gaucher Disease / Q000201:enzymology; D005947:Glucose / Q000378:metabolism; D005959:Glucosidases / Q000378:metabolism*; D005962:Glucosylceramidase / Q000378:metabolism*; D005963:Glucosylceramides / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D013154:Spleen / Q000032:analysis
D002554:Cerebrosides; D005963:Glucosylceramides; D005959:Glucosidases; D005962:Glucosylceramidase; D005947:Glucose