A quantitative cytochemical assay of beta-galactosidase in single cultured human skin fibroblasts.
A quantitative cytochemical method for the measurement of beta-galactosidase activity in cultured human skin fibroblasts has been developed using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside as the indigogenic substrate. The method relies upon the oxidation of the primary reaction product by ferro/ferricyanide during which an insoluble indigo dye is generated as the final reaction product. The reaction was linear with time up to 60 min using the final cytochemical standard procedure. The enzyme showed maximum activity at pH 4.0 to 4.1. The concentration optima of indigogenic substrate and potassium ferro/ferricyanide were 3.67 mM and 3.13 mM respectively. The presence of sodium chloride activated beta-galactosidase up to 100 mM, but was inhibitory above that concentration. The enzyme was inhibited by N-ethylmaleimide, N-acetyl-D-galactosamine and heparin. The enzyme molecules were shown to diffuse out of the cells using media without a suitable inert colloid stabilizer. However, diffusion was completely prevented by using polyvinyl alcohol (PVA) grade G18/140. Air-drying of cells was essential to make the cell membrane permeabel to the substrate and, thereby, to avoid a pronounced lag phase. However, in a biochemical analysis, air-drying itself caused a decrease in enzyme activity to 43% of the control. Even after air-drying lysosomal latency could still be demonstrated by using PVA grade G04/140. Control persons, one carrier of and two patients with beta-galactosidase deficiency were easily identified as belonging to three separate groups by using the cytochemical assay. It is proposed that the quantitative cytochemical approach may also be applied to cultured human amniotic fluid cells or chorion biopsies giving a rapid prenatal diagnosis of beta-galactosidase deficiency due to the small number of cells needed in the analysis.
D002478:Cells, Cultured; D005260:Female; D005347:Fibroblasts / Q000201:enzymology; D005696:Galactosidases / Q000032:analysis*; D006580:Genetic Carrier Screening; D006651:Histocytochemistry / Q000379:methods*; D006801:Humans; D007211:Indoles; D007787:Lactose Intolerance / Q000175:diagnosis; D011247:Pregnancy; D011296:Prenatal Diagnosis; D012867:Skin / Q000201:enzymology*; D013661:Tay-Sachs Disease / Q000175:diagnosis / Q000235:genetics; D001616:beta-Galactosidase / Q000032:analysis*
D007211:Indoles; C035455:5-bromo-4-chloro-3-indoxyl phosphate; D005696:Galactosidases; D001616:beta-Galactosidase