Fluorescent cytochemical detection of sialidase activity using 5-bromo-4-chloroindol-3-yl-alpha- D- N-acetylneuraminic acid as the substrate.
A novel fluorescent cytochemical method for sialidase activity was developed using 5-bromo-4-chloroindol-3-yl-alpha- D- N-acetylneuraminic acid (X-Neu5Ac) as the substrate. Intact nuclei were isolated from porcine liver and incubated at 37 degrees C for 3 h with 1 mM X-Neu5Ac at pH 4.8. The nuclei were stained with blue color that was derived from the oxidized compound of the reaction product X (5-bromo-4-chloro-3-hydroxyindole). A specific sialidase inhibitor, 2,3-dehydro-2-deoxy- N-acetylneuraminic acid, suppressed the staining in a dose-dependent manner. Despite the specificity of the cytochemical reaction, the staining was too weak to analyze the staining distribution and pattern of individual nuclei. To attain more sensitive detection of sialidase activity, the nuclei were incubated with X-Neu5Ac in the presence of Fast Red Violet LB. Individual nuclei of porcine liver were clearly stained with fluorescence that was produced by the conjugated compound of product X with Fast Red Violet LB. This fluorescent cytochemical method was also employed successfully for detection of sialidase activity of intact GOTO neuroblastoma cells in culture. The present method should provide a useful tool for investigating the localization and stage-specific expression of sialidase activity in tissues and cells.
Saito
Megumi
M
Department of Clinical Pharmacology and Therapeutics, University of Shizuoka School of Pharmaceutical Sciences, Yada 52-1, Japan. saitom@ys2.u-shizuoka-ken.ac.jp
Hagita
Hitoshi
H
Iwabuchi
Yoshiharu
Y
Fujii
Ikuo
I
Ikeda
Kiyoshi
K
Ito
Masaki
M
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology; D004396:Coloring Agents; D005456:Fluorescent Dyes / Q000737:chemistry; D006651:Histocytochemistry / Q000379:methods*; D007211:Indoles / Q000737:chemistry / Q000378:metabolism*; D008099:Liver / Q000201:enzymology; D009438:Neuraminic Acids / Q000737:chemistry / Q000378:metabolism*; D009439:Neuraminidase / Q000378:metabolism*; D009930:Organic Chemicals; D013379:Substrate Specificity; D013552:Swine; D014407:Tumor Cells, Cultured / Q000201:enzymology
C089351:5-bromo-4-chloroindol-3-yl 5-acetamido-3,5-dideoxy-glycero-galacto-2-nonulopyranosidonic acid; D004396:Coloring Agents; D005456:Fluorescent Dyes; D007211:Indoles; D009438:Neuraminic Acids; D009930:Organic Chemicals; C117141:fast red violet; D009439:Neuraminidase