Proteins of the Golgi apparatus. Purification to homogeneity, N-terminal sequence, and unusually large Stokes radius of the membrane-bound form of UDP-galactose:N-acetylglucosamine beta 1-4galactosyltransferase from rat liver.

The Golgi marker enzyme, UDP-galactose:N-acetylglucosamine beta 1-4galactosyltransferase (beta 1-4GalT) was purified 44300-fold in its intact, membrane-bound form from rat liver membranes. The protein was isolated from detergent extracts as a high-M(r) form, having a Stokes radius approximating a globular protein of M(r) 440,000. It is comprised of a single protein component as observed on SDS/polyacrylamide gels, having an M(r) near 51,000, and does not have intermolecular disulfide cross-links. N-terminal sequencing of the enzyme demonstrated that it contains an N-terminal hydrophobic stretch deduced previously from cDNA encoding for the enzyme. Previous studies have indicated that the protein may be translated at either of two AUG sites near the 5' end of the mRNA [Russo, R. N., Shaper, N. L. & Shaper, J. H. (1990) J. Biol. Chem. 265, 3324-3331], giving rise to two polypeptides, one appended with 13 amino acids. In the work described here, evidence was only found for the sequence of the short form, missing a single methionine at the N-terminus. Mild proteolytic treatment cleaved the enzyme, giving rise to low-M(r) forms which were fully catalytically active and which, upon sequencing, were missing a 66-amino-acid stretch from the N-terminus (as compared to the mouse cDNA). Proteolytic treatment was accompanied by conversion of the form having a large Stokes radius to one approximating a globular protein with M(r) near 50,000. The N-terminal stretch appears to contribute to maintenance of the form having a large Stokes radius. This may be the result of interaction with a detergent micelle, dimerization or oligomerization, or interaction with some other large, non-protein molecule, although a detergent exchange still resulted in a form having a large Stokes radius.

A spacer-modified disaccharide as a photoaffinity reagent for the acceptor-binding area of bovine (1----4)-beta-D-galactosyltransferase: comparison of its acceptor properties with those of other 2-acetamido-2-deoxy-beta-D-glucopyranosides.

The spacer-modified disaccharide 1,10-di-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2-azi-1,10- decanediol (10) that mimics the biantennary core heptasaccharide of N-glycoproteins has been synthesised. Compound 10 is an excellent acceptor in galactosyltransferase-catalysed galactosylation by UDP-galactose, is superior (7-8-fold) to analogues that have only one GlcNAc unit, and is an efficient photoaffinity reagent for galactosyltransferase. In the presence of UDP-Gal, no photoaffinity labelling by 10 takes place, which agrees with the mechanism of galactosyltransferase action.

Spacer-modified trisaccharide glycosides that mimic the biantennary Asn-linked oligosaccharide acceptor of (1----4)-beta-D-galactosyltransferase and can be used as competitive inhibitors and for irreversible deactivation.

The biantennary spacer-modified trisaccharide glycoside methyl 3,6-di-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyloxyethyl)-alpha -D- mannopyranoside (5) was synthesised and used together with several 2-acylamino-2-deoxy-D-glucose derivatives in competition experiments with beta-D-galactosyltransferase. Compound 5 was an acceptor substrate (KM 0.18 mM) comparable to the biantennary core heptasaccharide of glycoproteins (KM 0.13 mM). Replacing the N-acetyl group by other N-acyl groups did not alter the kinetic parameters significantly. When the N-acyl group was iodoacetyl, the compound was an irreversible inhibitor.

Properties of IgA-binding receptors on murine T cells: relative importance of Fc alpha R, beta-galactosyltransferase and anti-secretory component reactive proteins (ASCP).

Murine T cells and T-cell lines express receptors for the Fc of IgA (Fc alpha R); however, their molecular properties remain to be elucidated. In the present study, we examined three candidate molecules for IgA-binding receptors including Fc alpha R, beta-galactosyltransferase (beta-GT) and anti-secretory component (SC) reactive proteins (ASCP) expressed on T cells which might participate in the binding of different molecular forms of IgA. T-cell lines derived from CD4+ T cells of mouse Peyer's patches (PP) (designated PPT 4-6 and PPT 4-16) and from cloned PP T helper (Th) cell lines (ThHA1 #9 and #10) bound both monomeric and dimeric IgA (mIgA and dIgA), while the fusion partners (BW 5147 and R1.1) did not. In contrast, both Fc alpha R+ and Fc alpha R- cell lines bound to high molecular weight polymeric or aggregated IgA (pIgA). All cell lines reacted with a monoclonal anti-beta-GT (MoAb) and beta-GT enzyme activity was associated with the cell lysates and membrane fractions of all cells tested. The anti-beta-GT MoAb stained a 47-kDa band on immunoblots which was identical to that seen with native enzyme. mRNA analysis with beta-GT cDNA showed that all cell lines constitutively produced enzyme-specific mRNA. Both Fc alpha R+ T cells and Fc alpha R- control cell lines showed cell surface specific beta-GT activity. This is the first study which shows that mouse T cells produce beta-GT. However, Fc alpha R and beta-GT appear to be separate receptors, because Fc alpha R+ T cells bound mIgA and dIgA, and this treatment did not affect staining with biotinylated anti-beta-GT MoAb. Further, preincubation of the Fc alpha R+ cells with anti-beta-GT MoAb did not block mIgA binding. However, the anti-beta-GT MoAb partially blocked binding of pIgA to both Fc alpha R+ and Fc alpha R- T cells, suggesting that beta-GT may be a receptor for pIgA. Others have shown that T cells may bind IgA through a receptor serologically related to SC. We found that antibodies both to human SC and to rat SC specifically bound to both Fc alpha R+ and Fc alpha R- T cells. Further, a 72-kDa band was detected when cell membrane fractions were analysed with these antisera (ASCP) by solid phase immunoisolation technique and immunoblot analysis. The ASCP is not an IgA-binding receptor, since anti-SC did not block either mIgA or pIgA binding.(ABSTRACT TRUNCATED AT 400 WORDS)