Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae.

TLDR

The authors used an affinity‑purified rabbit antibody against human milk galactosyltransferase to localize the enzyme in HeLa cells via immunofluorescence and protein A‑gold electron microscopy. Immunolocalization revealed galactosyltransferase confined to two to three trans cisternae of the Golgi apparatus, co‑distributed with thiamine pyrophosphatase but absent from the acid phosphatase‑positive trans‑most cisterna, indicating a concerted role of both enzymes in glycosylation.

Abstract

An affinity-purified, monospecific rabbit antibody against soluble human milk galactosyltransferase was used to localize the enzyme in HeLa cells by immunofluorescence and by the protein A-gold technique at the electron microscope level. Specific immunofluorescence was observed in a juxtanuclear cytoplasmic region which was identified, on immunostained thin sections of low-temperature Lowicryl K4M-embedded HeLa cells, as Golgi apparatus. Label by gold particles was limited to two to three trans cisternae of the Golgi apparatus, indicating a compartmentalization of galactosyltransferase in the cisternal stack. Combination of preembedding thiamine pyrophosphatase cytochemistry, with postembedding immunostaining for galactosyltransferase proved codistribution of the two enzymes. However, the acid phosphatase-positive, trans-most cisterna was negative for galactosyltransferase. The close topological association of both galactosyltransferase and thiamine pyrophosphatase (or nucleoside diphosphatase) suggests a concerted action of both enzymes in glycosylation.