Biosynthesis of N-acetylneuraminic acid (Neu5Ac), a prominent component of glycoconjugates, is initiated by the action of UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase, EC 5.1.3.14) and N-acetylmannosamine kinase (ManNAc kinase, EC 2.7.1.60). We demonstrate for the first time that the two activities are parts of one bifunctional enzyme in rat liver. The enzyme was purified to homogeneity from rat liver cytosol using salmine sulfate precipitation and chromatography on phenyl-Sepharose, ATP-agarose, and Mono Q. The purification resulted in one polypeptide with an apparent molecular mass of 75 kDa. Immunoprecipitation with a polyclonal antibody against the polypeptide reduced both enzyme activities in equal amounts.Gel filtration analysis of purified UDP-GlcNAc 2-epimerase/ManNAc kinase showed that the polypeptide self-associates as a dimer and as a hexamer with apparent molecular masses of 150 and 450 kDa, respectively. The hexamer was fully active for both enzyme activities, whereas the dimer catalyzed only the phosphorylation of N-acetylmannosamine (ManNAc). Incubation of the dimer with UDP-N-acetylglucosamine led to reassembly of the fully active hexamer; maximal quantities of the hexamer were produced after incubation for 3 h.Kinetic analysis of purified hexameric and dimeric enzyme revealed significantly lower Michaelis constants (93 ؎ 3 to 121 ؎ 15 M for ManNAc and 1.18 ؎ 0.13 to 1.67 ؎ 0.20 mM for ATP) and higher cooperativity (Hill coefficients of 1.42 ؎ 0.16 to 1.17 ؎ 0.06 for ManNAc and 1.30 ؎ 0.09 to 1.05 ؎ 0.14 for ATP) for the hexamer for both substrates of ManNAc kinase. The Michaelis constant of UDP-GlcNAc 2-epimerase for its substrate was 11 ؎ 2 M. The Hill coefficient of 0.45 ؎ 0.07 represents strongly negative cooperativity in substrate binding. UDP-GlcNAc 2-epimerase was feedback inhibited by CMP-Neu5Ac. Complete inhibition was achieved with 60 M CMP-Neu5Ac, and highly positive cooperativity (Hill coefficient of 4.1) was found for inhibitor binding.

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Biosynthesis of procaryotic glycoproteins has been studied in some detail only in the extreme halophile Halobacterium halobium. To extend these studies for a moderate halophile, dolichol phosphate-linked oligosaccharides were isolated and characterized from Haloferax volcanii. Mannosyl (beta 1-->4)galactosyl phosphodolichol could be characterized as a main component by GC/MS permethylation analysis, mass spectrometry and 1H-NMR-spectroscopy. Furthermore, two additional components, present in lower quantities, were partially characterized and identified as a tetrasaccharyl phosphodolichol containing mannose, galactose, and rhamnose in the ratio of 2:1:1 as well as another dihexosyl phosphodolichol. Both the latter compounds contain an additional charged group. All these oligosaccharides were found to be linked to a dolichol consisting of 11 or 12 isoprene units including a saturated omega-terminal isoprene residue.

The synthesis of IV-acyl-2-amino-2-deoxy-hexoses, their metabolism and their incorporation into glycosphingolipids of rat pheochromocytoma cell line PC I2 were investigated. The dala indicate that in PC 12 cells the N-acyl-2.amino-2-dcoxy-hexoses, N-propanoyl-r+glucosaminc and Nbutanoyl-o-glucosamincare metabolized to thecorresponding phosphatcs.and that N-propanoyl-D-glucosamine isalsometabolized to Nmpropanoyl neuraminic acid. Using variously radiolabelled N-acyl-2-amino-2-deoxy-hexoses, their incorporation into glycosphingolipids was shown.

Cadherins are calcium-dependent, transmembrane intercellular adhesion proteins with morphoregulatory functions in the development and maintenance of tissues. In the development of striated muscle, the expression and function of mainly M-, N-, and R-cadherin has been studied so far. While these three cadherins are expressed in skeletal muscle cells, of these only N-cadherin is expressed in cardiac muscle. In this review, M-, N-, and R-cadherin are discussed as important players in the terminal differentiation and possibly also in the commitment of skeletal muscle cells. Furthermore, reports are described which evaluate the essential role of N-cadherin in the formation of heart tissue.