An immunocytochemical technique for the demonstration of intracellular antigens (secretory proteins) on thin sections is reported. Staphylococcal protein A which reacts with the Fc fragment of IgG molecules was labeled with colloidal gold as a marker. The antigenic sites were visualized on aldehyde-fixed and Epon-embedded tissue in a two step procedure. The specific antisera were applied to thin sections for binding to the antigens and then visualized by the protein A-gold complex. By using this technique different secretory proteins of the exocrine and endocrine pancreas were localized. The protein A-gold technique is proposed as a general method for visualization of antigenic sites on thin sections.

In Saccharomyces cerevisiae, transfer of N-linked oligosaccharides is immediately followed by trimming of ER-localized glycosidases. We analyzed the influence of specific oligosaccharide structures for degradation of misfolded carboxypeptidase Y (CPY). By studying the trimming reactions in vivo, we found that removal of the terminal α1,2 glucose and the first α1,3 glucose by glucosidase I and glucosidase II respectively, occurred rapidly, whereas mannose cleavage by mannosidase I was slow. Transport and maturation of correctly folded CPY was not dependent on oligosaccharide structure. However, degradation of misfolded CPY was dependent on specific trimming steps. Degradation of misfolded CPY with N-linked oligosaccharides containing glucose residues was less efficient compared with misfolded CPY bearing the correctly trimmed Man8GlcNAc2 oligosaccharide. Reduced rate of degradation was mainly observed for mis- folded CPY bearing Man6GlcNAc2, Man7GlcNAc2 and Man9GlcNAc2 oligosaccharides, whereas Man8GlcNAc2 and, to a lesser extent, Man5GlcNAc2 oligosaccharides supported degradation. These results suggest a role for the Man8GlcNAc2 oligosaccharide in the degradation process. They may indicate the presence of a Man8GlcNAc2-binding lectin involved in targeting of misfolded glycoproteins to degradation in S. cerevisiae.

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 lowtemperature 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.Biosynthesis of complex type heteroglycans proceeds in two distinct stages in different cellular organelles: first, N-glycosylation involving the en bloc transfer of a lipid-linked oligosaccharide takes place in the rough endoplasmic reticulum (for recent review, see reference 27) where part of it is processed by glucosidases (6); thereafter, the glycoproteins move to the Golgi apparatus where N-linked glycans are further processed (10) before being elongated by sequential action of terminal glycosyltransferases which include N-acetyl-D-glucosaminyl-, galactosyl-, fucosyl-, and sialyltransferases (for review, see reference 26). On the basis of cell fractionation studies (3,5, 11,15) and autoradiographic (1, 19) evidence, chain elongation is assumed to occur in the Golgi apparatus. Accordingly, galactosyltransferase activity has frequently been selected as marker for Golgi fractions (see 3,5,8, 11,14,15,26 for selected references). In order to obtain conclusive in situ evidence of the Golgi-association of galactosyltransferase (UDP-galactose: ,ß-D-N-acetylglucosaminyl-protein 8 (1-4) transferase, EC 2.4 . (23,24). We report here the first ultrastructural localization of a glycosyltransferase, namely galactosyltransferase, and demonstrate its presence in a distinct Golgi subcompartment which is composed of two to three thiamine pyrophosphatase-positive transGolgi cisternae. MATERIALS AND METHODSGalactosyltransferase was purified from pooled human milk (9). The immunization schedule included six subcutaneous injections of 0.2 mg of galactosyltransferase emulsified in complete Freund's adjuvant into rabbits. Antibody production was monitored by an enzyme-linked immunosorbent assay (ELISA) (2). Monospecific antibodies were obtained by affinity purification on immobilized galactosyltransferase. Their specificity towards purified galactosyltransferase was checked by ELISA (2) and the immunoreplica technique essentially according to Towbin et al. (29), except th...

No abstract

The recently developed low temperature embedding procedure with the resin Lowicryl

No abstract

No abstract

No abstract