Treatment of rat hepatocytes with 0.5 mM concentrations ofThe latter increase did not result in an increased incorporation of uridine and cytidine results in increased cellular concentrations radioactivity into the glycoconjugates. It was estimated that, in of UTP, UDP-sugars and CTP, whereas that of CMP-Nuntreated cells, the ratio of radioactivity incorporated from acetylneuraminate remained unchanged [Pels Rijcken, Overdijk, [3H]glucosamine into glycoconjugate-bound N-acetylhexosamine Van den Eijnden and Ferwerda (1993) Biochem. J. 293,207-213]. and N-acetylneuraminate amounted to 2:3. In pretreated cells The incorporation of radioactivity from 3H-labelled sugars into this ratio changed to approx. 2: 1. Overall, the data show that the cell-associated and secreted glycoconjugate fraction was pretreatment resulted in an increased incorporation of N-acetylinfluenced by these altered cellular concentrations of the nucleohexosamine into cell-associated and secreted glycoconjugates, tides. For [3H]glucosamine, pretreatment with uridine resulted in accompanied by a reduction in sialylation. It was concluded that a reduction of the glycosylation in both fractions. Increases in an increased availability of UDP-N-acetylhexosamine caused the the secreted fractions were observed for fucose with both uridine increased incorporation of N-acetylhexosamine. The elevated and cytidine and for N-acetylglucosamine with uridine only. This study demonstrates that protein glycosylation can be acetylmannosamine, the specific radioactivity of CMP-N-acetylregulated at the level of the availability of the various nucleotideneuraminate showed an almost 2-fold increase on pretreatment.sugars in the Golgi lumen.

Human al-acid glycoprotein (AGP) was separated into a non-bound (AGP-A; 46%), a retarded (AGP-B; 39%) and a bound fraction (AGP-C; 150/,) using concanavalin A (ConA) -Sepharose chromatography. The apparent molecular masses, as determined by SDS-PAGE, of the three fractions were 43.5, 42.3 and 41.2 kDa, respectively.The occurrence of N-linked di-, tri-and tetraantennary glycans on these three molecular forms (AGP-A, -B, and -C) was studied by sequential lectin-affinity chromatography of the 14C-labelled glycopeptides. These were obtained by extensive pronase treatment followed by N-[14C]acetylation of the peptide moieties.The glycopeptides of AGP-A did not bind to ConA-Sepharose whereas for AGP-B and AGP-C 18% and 44%, respectively, of the glycopeptides were bound as diantennary structures. Glycopeptide fractions of all three forms of AGP which were not bound to ConA-Sepharose were shown to contain equal amounts of both tri-and tetraantennary glycans by chromatography with Phaseolus vulgaris leukoagglutinating lectin (L-PHA).With the assumption that each molecule contains five glycosylation sites, it could be shown that AGP-A contains no diantennary structures whereas AGP-B and AGP-C contain one and two diantennary structures, respectively. In addition each of the molecular forms contains equal amounts of tri-and tetraantennary structures on the remaining glycosylation sites. The results of this study, therefore, exclude a uniformity of glycan chains in the three molecular forms of AGP.The degree of sialylation of each of the molecular forms was investigated by chromatography on L-PHAagarose and Ricinus communis agglutinin-I -agarose both before and after desialylation of the glycopeptides. It was shown that about 90% of the biantennary glycans of both AGP-B and AGP-C were disialylated while the remainder were monosialylated. The degree of sialylation of the tri-and tetraantennary glycans was identical for the three molecular forms. In each case, one or more terminal galactose residues occurred on at least 20% of the tri-and 65% of the tetraantennary chains.It is suggested that the decrease in the exposure of galactose residues from AGP-A to AGP-C is related to the concomittant decrease in branching of the glycans of the three molecular forms. The relevance of these findings to studies on the function of AGP during inflammatory and liver diseases is discussed. Abbreviations. AGP, ccl-acid glycoprotein; ConA, concanavalin A; L-PHA, Phaseolus vulgaris leukoagglutinating lectin; RCAI, Ricinus communis agglutinin I; CO, LO, RO, non-retarded or nonbound, and Cn, Ln, Rn (n = 1-4) retarded or bound glycopeptide fractions on ConA-Sepharose, L-PHA-agarose and RCA,-agarose, respectively. The fraction names are sometimes used sequentially, e.g. COLlR3, which indicates the fraction of glycopeptides that was not bound on ConA-Sepharose but retarded sequentially on position L1 of L-PHA-agarose and bound on position R3 of RCAI-agarose. Use of the prefix 'a' denotes that the fraction is desialylated by mild acid hydrolysis. fini...

Adult male rats were injected intraventricularly with N-[3H]acetylmannosamine. After different time intervals the rats were killed and free sialic acid, CMP-sialic acid, lipid- and protein-bound sialic acid were isolated from brain and the specific radioactivities determined. Maximal specific radioactivity was reached after approximately 4 h for CMP-sialic acid, after 10-12 h for free sialic acid and after approximately 42 h for lipid- and protein-bound sialic acid. After some days the specific radioactivities of all four pools were the same and decreased equally, with a calculated turnover rate of approximately 3.5 weeks. The conclusion was that this phenomenon was the result of reutilisation of sialic acid and/or precursors. Therefore, the calculated turnover is not the turnover of bound sialic acid, but merely the rate of leakage of sialic acid and/or precursors out of the brain, so that no real turnover can be measured by this method. The first few hours after injection the specific radioactivity of CMP-sialic acid rose above that of free sialic acid. It is supposed that a compartmentalization exists of free sialic acid. The newly synthesised sialic acid molecules are not secreted into the cytoplasmic pool but are preferentially used for the synthesis of CMP-sialic acid. The results and conclusions are discussed in view of the general problems concerning turnover measurements of glycoconjugates.

The enzymes UDP-N-acetylglucosamine pyrophosphorylase, UDP-N-acetylglucosamine 2-epimerase, N-acetylmannosamine kinase, N-acetylglucosamine kinase and N-acetylglucosamine 2-epimerase, which are involved in the metabolism of N-acetylneuraminic acid, were studied in rat with regard to their subcellular localization and tissue distribution. The subcellular distribution studies in liver indicated that the enzymes are localized in the soluble cell fraction. In other tissues the comparison of enzyme activities in homogenates with that in high-speed supernatants led to a similar conclusion. UDP-N-acetylglucosamine pyrophosphorylase, N-acetylglucosamine kinase and N-acetylglucosamine 2-epimerase were detected in almost all tissues studied. UDP-N-acetylglucosamine 2-epimerase and N-acetylmannosamine kinase, two enzymes considered to be key enzymes in the N-acetylneuraminic acid biosynthesis, were detected only in sialoglycoprotein-secreting tissues, i.e. liver, salivary gland and intestinal mucosa. The low activity of the key enzymes in other tissues suggests that the biosynthetic pathway of N-acetylneuraminic acid is not the same in various tissues.

Pyrimidine nucleotide metabolism in rat hepatocytes was studied by measurement of the labelling kinetics of the various intermediates after double labelling with [14C]orotic acid and [3H]cytidine, the precursors for the de novo and the salvage pathways respectively. For the uridine nucleotides, differences were found for the 14C/3H ratios in the UDP-sugars, in UMP (of RNA) and in their precursor UTP, suggesting the existence of separated flows of the radioactive precursors through the de novo and the salvage pathways. Higher ratios in the UDP-sugars, which are synthesized in the cytoplasm, and a lower ratio in UMP (of RNA) relative to the 14C/3H ratio in UTP indicated that UTP derived from orotic acid is preferentially used for the cytoplasmic biosynthesis of the UDP-sugars. Uridine, derived from cytidine, is preferentially used for the nuclear-localized synthesis of RNA. In contrast to these findings, the 14C/3H ratios in the cytidine derivatives CMP-NeuAc and CMP (of RNA), and in the liponucleotides CDP-choline and CDP-ethanolamine, were