Synthesis of new α-heterocyclic α-aminoesters

alpha-Heterocyclic alpha-aminoesters were obtained in good yields by reaction of a glycine cation equivalent and different heterocyclic nucleophiles; diastereoselectivity using a carbohydrate (galactopyranose) as N-protecting group was modest.     

Specific dissociation of αB subunits from α-crystallin

Exposure of bovine α-crystallin to 0.1 M glycine at pH 7 decreases the average molar mass of the protein from 700 to 420 kDa. When the pH is lowered to 2.5, in the same buffer, the αB chains specifically dissociate from the aggregates, leaving a particle of 290 kDa containing only αA chains. The decrease in the molar mass corresponds to the mass of the αB chains in the original aggregate. The pH-dependent dissociation is fully reversible. Similar changes were observed with rat and kangaroo α-crystallins but the dogfish protein was not affected. Sedimentation velocity analyses and fluorescence spectroscopy yielded a pK, for the dissociation, of 3.7 for α-crystallin and 4.0 for a homopolymer constructed from purified αB₂ polypeptides. An αA₂ homopolymer was virtually unaffected by the lowering of pH. The products from the dissociation were isolated and their properties studied by sedimentation analysis and acrylamide quenching of tryptophan fluorescence. The αB chains were found to be completely denatured, whereas the structure of the αA chains, in the 290 kDa, particle, were only slightly altered. Comparisons of the sequences of the various proteins examined suggested that decreased ionization of aspartic acid 127 in the αB chain was responsible for the specific dissociation of this polypeptide.     

Lysosomal α-D-mannosidase

-mannosidosis in the human is an autosomal recessive lysosomal storage disease caused by a deficiency of lysosomal -D-mannosidasea actvity. Lysosomal -D-mannosidase is involved in the catabolism of N-linked glycoproteins through the sequential degradation of high-mannose, hybrid and complex oligosaccharides. This review is focused on human, mouse, bovine and feline genes coding for lysosomal -D-mannosidase. In particular the exon-intron structure of the genes, their promoters, and the identification of mutations causing the disease have been examined. The construction, by homologous recombination, of a mouse model of -mannosidosis is reported.     

Synthesis of α-mannosylated ergot alkaloids employing α-mannosidase

The ergot alkaloids elymoclavine, ergometrine and chanoclavine were α-mannosylated with α-mannosidase as catalyst. The kinetic reaction with p-nitrophenyl α-mannoside as glycosyl donor gave ca 28 % yield of chanoclavine α-mannoside, whereas the equilibrium reaction with mannose as the glycosyl donor gave ca 11 % yield. However, in the case of elymoclavine and ergometrine, higher yields of α-mannosides were obtained with the equilibrium approach (18 and 13 %).     

Microbial α-mannosidases

Microbial α-mannosidases are used in the analysis of glycopeptides and the developmental regulation of lysosomal enzymes. This survey presents comparison of properties of this high molecular weight, oligomeric protein from a number of microbial sources.     

Thermostable extracellular α-amylase and α-glucosidase ofLipomyces starkeyi

Summary Thermostable, extracellular -amylase and -glucosidase were produced byLipomyces starkeyi CBS 1809 in a medium containing maize starch and soya bean meal. Contrary to published findings which suggested a single cell-bound amylolytic system for another strain ofL. starkeyi, this study revealed the presence of two enzymes — an -amylase and an -glucosidase inL. starkeyi CBS 1809. The enzymes were separated by solvent and salt precipitation and ion-exchange chromatography on DEAE-Biogel-A. The -amylase and -glucosidase had pH optima at 4.0 and 4.5 and temperature optima at 70°C and 60°C, respectively. While the low pH optima are not unique the enzymes are very distinctive in yeasts in having very high temperature optima. The -glucosidase had highest activities on maltose and isomaltose (100) with relative rates of activity on maltotriose, isomaltotriose and p-nitrophenyl--d-glucoside of 59, 48 and 22, respectively. It was inactive towards sucrose. Both the -amylase and -glucosidase ofL. starkeyi were located extracellularly and had molecular weights of 76,000 and 35,000, respectively.     

Imidazolethyl-Phosphoramidate α-Oligonucleotides

Abstract

α-ODNs conjugated to imidazole groups via phosphoramidate internucleosidic linkages were synthesized. The presence of the imidazolethyl-phosphoramidate linkage improved the affinity of α-ODNs for their nucleic acid targets.     

Asymmetric acetalation of α,α-trehalose: Synthesis of α-d-galactopyranosyl α-d-glucopyranoside and 6-deoxy-6-fluoro-α-d-glucopyranosyl α-d-glucopyranoside

Selective acetalation of α,α-trehalose with ethyl or methyl isopropenyl ether and toluene-p-sulphonic acid in N,N-dimethylformamide gave the 4,6-isopropylidene acetal as the major product, isolated as its hexa-acetate 1 (38%). The gluco-galacto analogue 6 of α,α-trehalose was synthesized from 1 by the sequence: hydrolysis of the isopropylidene group with trifluoroacetic acid, mesylation of the resulting diol, benzoate displacement, and saponification of the product. Deacetylation of 1 followed by benzylation and hydrolysis of the acetal group furnished a hexa-O-benzyl derivative 9. Tosylation of the primary hydroxyl group in 9, treatment of the product with tetrabutylammonium fluoride in acetonitrile, and subsequent catalytic hydrogenolysis of the benzyl groups gave 6-deoxy-6-fluoro-α,α-trehalose (12). Compounds 6 and 12 and 6-deoxy-6-iodo-α,α-trehalose are substrates for cockchafer trehalase, but have very low V_max values.     

Anomer Selective Formation of l-Menthyl α-D-Glucopyranoside by α-Glucosidase-catalyzed Reaction

l-Menthol was glucosylated by the α-glucosidase (EC 3.2.1.20) of Saccharomyces cerevisiae using maltose as glucosyl donor. When 50 mg of l-menthol and 1M maltose in 10 mM citrate–phosphate buffer (pH 7.0) were incubated for 24 h at 30°C, a menthylglucoside was selectively obtained as a product. The molar conversion yield based on supplied menthol was 4.5%. The product was identified as l-menthyl α-D-glucopyranoside (α-MenG) by ¹³C-NMR analysis.     

Human α2macroglobulin

Summary Human ₂macroglobulin combines two unique features: the non-active site directed inhibition of virtually all endoproteases and the selective clearance of ₂M-endoprotease complexes by receptor-mediated endocytosis. To study the molecular details of the mechanisms involved, primary amines were found to be worthwhile probes at three specific levels: the inactivation of native ₂M, the derivatization by factor XIII and the cellular process of receptor-recycling. In this paper published data are supplemented with recently obtained evidence to discuss and speculate on the possible action or involvement of transglutaminase activities, indicated by the effects of the primary amines.     

The α-ketoglutarate-dehydrogenase complex

Damage from oxidative stress and mitochondrial dysfunction occur together in many common neurodegenerative diseases. The enzymes that form the mitochondrial alpha-ketoglutarate- dehydrogenase complex (KGDHC), a key and arguably rate-limiting enzyme system of the tricarboxylic acid cycle, might mediate the interaction of these processes. KGDHC activity is reduced in numerous age-related neurodegenerative diseases and is diminished by oxidative stress. In Alzheimer's disease (AD), the reduction correlates highly to diminished mental performance. Thus, research has focused on the mechanisms by which select oxidants reduce KGDHC and the consequences of such a reduction. Diminished KGDHC in cells is associated with apoptosis without changes in the mitochondrial membrane potential. Studies of isolated mitochondria and of animal models suggest that a reduction in KGDHC can predispose to damage by other toxins that promote neurodegeneration. Diminished oxidative metabolism can be plausibly linked to pathological features of neurodegenerative diseases (e.g., reduced mental function, the plaques and tangles in AD). Thus, reductions in KGDHC might be central to the pathophysiology of these diseases. Studies of proteins, cells, animal models, and humans suggest that treatments to diminish, or bypass, the reduction in KGDHC might be beneficial in age-related neurodegenerative disorders.     

Recombinant Thymosin α1

An artificial gene encoding thymosin ₁ was obtained by chemoenzymatic synthesis and cloned into Escherichia coli. An expressing recombinant plasmid containing the hybrid protein gene, which encodes amino acid sequences of thymosin ₁ and the Saccharomyces cerevisiae intein Sce VMA, was constructed. The expression of the hybrid protein from the resulting hybrid gene in E. coli, the properties of the resulting hybrid protein, and the conditions for its nonenzymatic cleavage to thymosin ₁ were studied.     

Photo-oxygenation of α-Ionone

Photo-oxygenation of α-ionone was studied to clarify the relationship between the maturity of aroma and photo-oxygenative change of α-ionone. α-Ionone was converted to oxygenated derivatives which were identified as 2,3-epoxy-β-ionone, 3,4-epoxy-α-ionone, 4-keto-β-ionone (trans- and cis-form), 5-keto-α-ionone and 3,4-dihydroxy-α-ionone.     

Helix induction potential of N-terminal α-methyl, α-amino acids

Summary A series of longer analogues of the C-peptide of RNAse A has been synthesized with the aim of assessing the helix induction potential in water of α-methyl, α-amino acids at the N-terminus of the chain. The circular dichroism data indicate that one isovaline residue is effective in increasing the helix content of the 13-residue peptide by about 7%.     

Evolutionary History of Eukaryotic α-Glucosidases from the α-Amylase Family

Although some α-glucosidases from the α-amylase family (glycoside hydrolase family GH13) have been studied extensively, their exact number, organization on the chromosome, and orthology/paralogy relationship were unknown. This was true even for important disease vectors where gut α-glucosidase is known to be receptor for the Bin toxin used to control the population of some mosquito species. In some cases orthologs from related species were studied intensively, while potentially important paralogs were omitted. We have, therefore, used a bioinformatics approach to identify all family GH13 α-glucosidases from the selected species from Metazoa (including three mosquito species: Aedes aegypti, Anopheles gambiae, and Culex quinquefasciatus) as well as from Fungi in an effort to characterize their arrangement on the chromosome and evolutionary relationships among orthologs and among paralogs. We also searched for pseudogenes and genes coding for enzymatically inactive proteins with a possible new function. We have found GH13 α-glucosidases mostly in Arthropoda and Fungi where they form gene families, as a result of multiple lineage-specific gene duplications. In mosquito species we have identified 14 α-glucosidase (Aglu) genes of which only five have been biochemically characterized so far, two are putative pseudogenes and the rest remains uncharacterized. We also revealed quite a complex evolutionary history of the eukaryotic α-glucosidases probably involving multiple losses of genes or horizontal gene transfer from bacteria.     

β-Galactosidase α-complementation

Summary Studies on -galactosidase -complementation are reviewed. The isolation and structure of two -galactosidase fragments that form an enzymically active complex are described. One of these is a cyanogen bromide peptide from whole -galactosidase; the other is a dimeric-protein from a lacZ deletion mutant of Escherichia coli. The mechanism most likely involves an initial binding of two cyanogen bromide peptides to the dimer, followed by formation of a tetramer, and finally a slow conformational change of the complex to a native-like enzyme. The overall reaction is essentially irreversible. A region of the polypeptide chain involved in dimer-dimer contact must be supplied by the cyanogen bromide peptide. -Complemented enzyme contains overlapping sequences. Proteolytic experiments were carried out to determine the origin of the funtionally important segment. The effect on a-complementation of amino acid substitutions at four positions in the polypeptide chain was investigated. The implications of these results for -galactosidase structure and for proteins in general are discussed.