title: Hydrolysis of a novel lysosomotropic enzyme substrate for beta-galactosidase within intact cells.
abstract: With the goal of improving the detection of lysosomal sphingolipid hydrolases within intact cells, we have recently synthesized a new fluorophor, O-[4-(1-imidazolyl)butyl]-2,3-dicyano-1,4-hydroquinonyl beta-D-galactopyranoside (Im-DCH-beta-Gal). In the present study, we evaluated the interaction of Im-DCH-beta-Gal and its tetraacetate derivative, Im-DCH-beta-Gal(OAc)4, with living human fibroblasts. Im-DCH-beta-Gal was shown to be a specific substrate for human lysosomal beta-galactosidase in cell homogenates. Im-DCH-beta-Gal(OAc)4 was taken up and hydrolyzed by normal fibroblasts under physiological culture conditions. Very little hydrolysis of Im-DCH-beta-Gal(OAc)4 was observed in fibroblasts genetically deficient in lysosomal acid beta-galactosidase or in normal cells pretreated with the lysosomal inhibitors chloroquine and ammonium chloride. Analysis of substrate processing by cells indicated that normal and acid beta-galactosidase-deficient cells showed similar rates of uptake and deacetylation of Im-DCH-beta-Gal(OAc)4, with an 80% decrease in the rate of deglycosylation of substrate by beta-galactosidase-deficient fibroblasts. However, under our conditions, the fluorescent product was not well retained by cells. Our results indicate that this novel class of compounds may be useful in measuring lysosomal enzyme function in intact cells and may have application as a fluorescent marker for genetically altered cells.
authors.affiliation: Developmental and Metabolic Neurology Branch, National Institute of Neurological Disordrs and Stroke, National Institutes of Health, Bethesda, MD 20892.
authors.forename: C R
authors.initials: CR
authors.lastname: Kaneski
authors.forename: S A
authors.initials: SA
authors.lastname: French
authors.forename: M R
authors.initials: MR
authors.lastname: Brescia
authors.forename: M J
authors.initials: MJ
authors.lastname: Harbour
authors.forename: S P
authors.initials: SP
authors.lastname: Miller
mesh_terms: D002460:Cell Line; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000378:metabolism; D005690:Galactose / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D005733:Gangliosidoses / Q000201:enzymology; D006801:Humans; D008247:Lysosomes / Q000201:enzymology*; D008856:Microscopy, Fluorescence; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; C090874:O-(4-(1-imidazolyl)butyl)-2,3-dicyano-1,4-hydroquinonylgalactopyranoside; D001616:beta-Galactosidase; D005690:Galactose