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A 13C-n.m.r. study of intermediates in the L-type pentose phosphate cycle.

The structures in aqueous solution of all major contributing forms of D-altro-heptulose 1,7-diphosphate, and D-glycero-D-altro and D-glycero-D-ido-octulose 1,8-diphosphates have been established by 13C-n.m.r. spectroscopy. Assignments to individual carbon atoms were made with the aid of isotopically enriched analogues and by comparison with related sugars.

Heptoses↗

Isolation and characterization of the 36-kDa D-mannose 6-phosphate receptor from porcine testis.

A D-mannose 6-phosphate receptor was isolated from total membranes of porcine testis, and its interaction with ligands was examined. The receptor was a glycoprotein comprised of several 36-kDa sub-units with an isoelectric point (pI) of 6.1. The binding of the receptor to the insoluble phosphomannan core occurred in the absence of divalent cations, but was selectively stimulated by MnCl2 and effectively inhibited by D-mannose 6-phosphate, D-fructose 1-phosphate, and pentamannosyl monophosphate. The phosphate group and HO-2 of D-mannose 6-phosphate are important in the receptor-ligand interaction, HO-4 probably contributes to a lesser extent, and HO-1 seems to have no interaction.

Animals↗

Preparation and application of a pentamannosyl monophosphate-bovine serum albumin conjugate.

Pentamannosyl monophosphate, derived from Hansenula holstii O-phosphomannan, was conjugated to bovine serum albumin by reductive amination. The conjugate inhibited the binding of the porcine testis mannose 6-phosphate receptor to the insoluble phosphomannan core. A mannose 6-phosphate receptor with a molecular weight of 200,000 was purified from porcine liver membranes, using an affinity matrix of the conjugate attached to Sepharose 4B. Rabbits were immunised with the conjugate, and the antisera were purified on a phosphomannan core-Sepharose 4B column in order to give an antibody which was specific for the 6-phosphate group and the equatorial HO-4 of D-mannose 6-phosphate. On Western blot analysis using the purified antibodies, ovalbumin, which contained a typical high-mannose type of oligosaccharide, was not recognised. However, a testicular glycoprotein fraction formed an immunostaining band. These results indicate the effectiveness of the conjugate as a ligand for mannose 6-phosphate receptors. The antibodies highly specific for mannose 6-phosphate may be used to detect or purify lysosomal enzymes.

Animals↗

Large-scale synthesis of beta-L-fucopyranosyl phosphate and the preparation of GDP-beta-L-fucose.

A practical 15-mmol large-scale synthesis of beta-L-fucopyranosyl dicyclohexylammonium phosphate from L-fucose in 63% overall yield was developed. The synthesis took advantage of a neighboring Bz-2 group participating in a Koenigs-Knorr-like glycosylation. The sugar phosphate was transformed into the activated sugar nucleoside, guanosine diphosphate beta-L-fucopyranose, on a gram scale.

Carbohydrate Conformation↗

Inhibition of polymorphonuclear leukocyte oxidative metabolism by exogenous phospholipase C.

We studied the effects of exogenous, purified phospholipase C (PLC) on neutrophil oxidative metabolism, lysosomal enzyme release and aggregation. We found that PLC inhibited O2- and H2O2 generation and oxygen consumption, but did not alter glucose oxidation via the hexose monophosphate shunt. In contrast, we found a striking stimulation of aggregation and release of the lysosomal enzymes lysozyme and beta-glucuronidase. In experiments designed to further characterize the mechanism of the PLC effect on membrane activation we studied the effect of PLC on intracellular calcium concentration [Ca2+]i and found that PLC did not interfere with the fMLP-mediated rise in [Ca2+]i, suggesting that its inhibitory effect on the respiratory burst does not involve inhibition of early signal transduction events. In addition, we found that PLC alone results in mobilization of intracellular Ca2+ stores, consistent with its stimulatory effect on aggregation and lysosomal enzyme release.

Calcium↗