Soluble and membrane-bound low-Km 5'-nucleotidase was isolated from high-speed supernatants and membrane fractions derived from the electric organ of the electric ray (Torpedo marmorata) or from bovine brain cerebral cortex. Purification of both enzymes included chromatography on concanavalin A-Sepharose and AMP-Sepharose. The contribution to the total of soluble enzyme activity was lower in electric organ (1.6 %) than in bovine cerebral cortex (27.9 %). Membranebound and soluble forms have very similar Km values for AMP and are inhibited by micromolar concentrations of ATP. Both forms cross-react with, and are inhibited by, an antibody against the membrane-bound surface-located (ecto-) 5'-nucleotidase from electric organ. The HNK-1 carbohydrate epitope is present on both forms of the Torpedo enzyme, but is entirely absent from bovine cerebral-cortex 5'-nucleotidase. An antibody specific for the inositol 1,2-(cyclic)monophosphate that is formed on phospholipase C cleavage of an intact glycosyl-phosphatidylinositol (GPI) anchor binds to the soluble, but not to the membrane-bound, form of the enzyme from both sources. Our results suggest that soluble low-Km 5'-nucleotidase in both electric organ and bovine brain is derived from the membrane-bound GPIanchored form of the enzyme by the action of a phospholipase C and is not a soluble cytoplasmic enzyme.

A cDNA encoding a 5'-nucleotidase was identified by screening a lgtlO cDNA library from the electric lobe of Discopyge ommata using a cDNA probe containing the complete open reading frame coding for the rat liver enzyme. Nucleotide sequence analysis defines an open reading frame of 577 amino acids, corresponding to a calculated molecular mass of 63833 Da. The N-terminus of the mature protein, as determined by direct protein sequencing, is preceded by 29 amino acid residues comprising a signal peptide. The C-terminus contains a stretch of hydrophobic amino acids, considered to be cleaved on post-translational modification and exchanged for glycosylphosphatidylinositol as a membrane anchor. The predicted protein contains four potential N-linked glycosylation sites. Electric ray 5'-nucleotidase shares 61 % amino acid identity with the enzymes from rat liver and human placenta, and about 23 YO with bacterial proteins possessing 5'-nucleotidase activity and also additional enzyme activities like UDP-glucose hydrolase. Polyclonal antibodies raised against 5'-nucleotidase from mammalian sources or the electric ray electric organ reveal mutual crossreactivity. Interestingly, there are 5 -7 domains highly conserved in procaryotes and vertebrates in enzymes exhibiting 5'-nucleotidase, 3'-nucleotidase or phosphodiesterase activity. 5'-nucleotidase isolated from Torpedo electric organ hydrolyzes UDP-glucose at 8 % of the rate of AMP hydrolysis. The possible phylogenetic origin of vertebrate 5'-nucleotidase from multifunctional nucleotide hydrolases is discussed.

Carbohydrates Carbohydrates U 0500Regioselective Synthesis of Galactosylated Tri-and Tetrasaccharides by Use of β-Galactosidase from Bacillus circulans. -(FARKAS, E.; SCHMIDT, U.; THIEM*, J.; KOWALCZYK, J.; KUNZ, M.; VOGEL, M.; Synthesis 2003, 5, 699-706; Inst. Org. Chem., Univ. Hamburg, D-20146 Hamburg, Germany; Eng.) -M. Paetzel

The present review addresses the role of food constituents in the aetiology of metabolic conditions and chronic diseases, mostly related to energy metabolism and substrate regulation, such as obesity and non-insulin-dependent diabetes mellitus. Second, attention is paid to malnutrition, a major cause of mortality and morbidity in developing countries, which may be a cause of concern in Europe because of the increasing number of elderly people in the population. Finally, the role of diet during exercise, a condition of enormous substrate demands, is evaluated. Based on a critical evaluation of the existing knowledge in the literature, implications for future research in relation to functional foods are discussed.

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