Methods for analysis of acid alpha-1,4-glucosidase activity in single hybrid cells.
Two methods are described which allow the quantitative assay of the lysosomal enzyme alpha-1,4-glucosidase in single fibroblasts. In the first procedure the substrate was maltose, and liberated glucose was measured with an enzymatic cycling procedure for reduced nicotinamide adenine dinucleotide phosphate. Single cultured fibroblasts were found to have enzyme activities in the range of 0.5-10 X 10(-13) moles glucose/hr. In the second procedure the artificial substrate 4-methylumbelliferyl-alpha-D-glucopyransodie was used. It is hydrolyzed in a single step reaction to the fluorescent product 4-methylumbelliferone (MU). By reducing the incubation volume and by measuring the fluorescence in microdroplets with a microscope fluorometer, a sensitivity of 10(-14) moles MU could be obtained. Activities were found ranging from 0.5-10 X 10(-14) moles MU/hr/cell. Both procedures for single cell analysis proved to be reliable when compared with conventional assays on cell homogenates. Cocultivation and cell fusion studies were performed to demonstrate that these methods can be used to study the metabolic and genetic interaction between normal and enzyme-deficient fibroblasts derived from patients with glycogenosis II.
D005347:Fibroblasts / Q000201:enzymology; D005959:Glucosidases / Q000378:metabolism*; D006008:Glycogen Storage Disease / Q000201:enzymology*; D006009:Glycogen Storage Disease Type II / Q000201:enzymology*; D006801:Humans; D006822:Hybrid Cells; D013050:Spectrometry, Fluorescence / Q000379:methods
D005959:Glucosidases