Fluorescent cytochemical detection of sialidase activity using 5-bromo-4-chloroindol-3-yl-α-D-N-acetylneuraminic acid as the substrate

Abstract

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.

Sialidase Activity of Influenza A Virus in an Endocytic Pathway Enhances Viral Replication

“…After incubation for 5 or 90 min at 37°C, the cells were fixed and permeabilized with cold methanol for 5 min. To visualize the in situ enzymatic activity of influenza virus NA adsorbed on or after invasion into cells, cells at 5 or 90 min postinfection were incubated with 0.01 mM X-Neu5Ac and Fast Red Violet LB (1 g/ml) (ICN Biomedicals Inc., Aurora, Ohio) in 0.1 M sodium acetate buffer (pH 5.0) for 1 h at 37°C (20). Endosomes were detected by a monoclonal antibody against early endosome antigen 1 (EEA1) (BD Biosciences Clontech, Palo Alto, CA).…”

Distribution of Sialidase Activity and the Role of Sialidase in the Brain

“…An artificial substrate for sialidase, 5-bromo-4-chloroindol-3-yl-α-D-N-acetylneuraminic acid (X-Neu5Ac), has been used for staining sialidase activity in tissue. Since X-Neu5Ac is ionized at neutral pH, it is thought that it is difficult for X-Neu5A to permeate through cell membranes (21). After cleavage of X-Neu5Ac with sialidase on the cell surface, the compound X is easily oxidized to a water-insoluble blue compound that is deposited on tissue (22).…”

“…After cleavage of X-Neu5Ac with sialidase on the cell surface, the compound X is easily oxidized to a water-insoluble blue compound that is deposited on tissue (22). Saito et al used an azo dye, FRV LB, to enhance the detection sensitivity of sialidase activity with X-Neu5Ac (21). After cleavage of X-Neu5Ac with sialidase, compound X reacts with FRV LB, producing a water-insoluble fluorescent compound (Fig.1).…”

“…This compound reached maximum fluorescent intensity at a 696-nm emission wavelength with a 550-nm excitation wavelength. This fluorescent method was employed successfully for detection of sialidase activity on the surfaces of cultured neuroblastoma cells (21).…”

Imaging of Sialidase Activity and Its Clinical Application

“…An improved method, which converts X into a fluorescent derivative by adding Fast Red Violet LB as a sensitizer to X-Neu5Ac, was developed for imaging of sialidase activity. 5) However, this reaction is susceptible to pH conditions and requires two reaction steps. Additionally, the product does not show a sufficient Stokes shift.…”