Precursors of the pyrimidine moiety of thiamine

1. A method was devised for obtaining the pyrimidine moiety of thiamine in a pure form after its excretion into the medium by de-repressed washed-cell suspensions of mutants of Salmonella typhimurium LT2. 2. By using amino acid-requiring mutants, this excretion of pyrimidine moiety was shown to be dependent on the presence of both methionine and glycine. 3. In the presence of either [Me-¹⁴C]methionine or [G-¹⁴C]methionine, methionine-requiring mutants did not incorporate radioactivity into the pyrimidine moiety. 4. In contrast, both [1-¹⁴C]glycine and [2-¹⁴C]glycine were incorporated into the pyrimidine moiety excreted by glycine-requiring mutants, and this occurred with little or no dilution of specific radioactivity. 5. The possible requirement for methionine as a cofactor and the significance of the incorporation of both carbon atoms of glycine are discussed.     

Synthesis of the spirosuccinimide moiety of Asperparaline A

2,6,6-Trimethyl-2-azaspiro[4.4]nonane-1,3-dione (9), a spirosuccinimide moiety of asperparaline A (1), was synthesized by starting from 2,2-dimethylcyclopentanone (4) via trinitrile 6 in five steps in a moderate yield. This conversion establishes a model study for synthesis of the spirosuccinimide moiety of asperparaline A (1).     

On the oligosaccharide moiety of angiotensin-converting enzyme.

Two glycopeptide fractions in a pronase digest of rabbit pulmonary angiotensin-converting enzyme were resolved by gel filtration. GP-I, the minor component (～1 mole/mol enzyme) contained mannose, galactose, glucose $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and sialic acid in an approximate molar ratio of 1:5:3:4:1:2 and molar equivalents of aspartic acid, threonine and serine. GP-II, the major oligosaccharide unit (～ 12 moles/mol enzyme, ～ 90% of total carbohydrate), contained fucose, mannose, galactose, $\text{N}$-acetylglucosamine, sialic acid and aspartic acid in a molar ratio of 1:4:4:4:1:1. Although accounting for about one-quarter of the weight of the enzyme, GP-II did not compete with the intact glycoprotein for binding to goat antienzyme antibodies. Some structural features of GP-II were deduced by periodate oxidation and digestion with various glycosidases.     

Differential mutagenicity of streptozotocin analogs of the carbohydrate moiety

The mutagenicity of streptozotocin (SZN), 8 of its analogs and N-msthyl-N-nitrosourea (MNU) were compared in Salmonella typhimurium. SZN and its analogs carry MNU attached to the carbohydrate moiety at the C-2 position. The C-1 analogs tested were α- and β-methyl-SZN, α-ethyl-SZN, β-propyl-SZN, α- and β-butyl-SZN; in 2 analogs glucose was replaced by α- or β-inositol. When the ability of these compounds to revert the hisG46 auxotroph was compared, they fell into 4 groups which differed by about 10-fold in mutagenicity from one another. The most mutagenic was (i) SZN, followed by (ii) β-methyl-SZN; (iii) α-methyl-SZN, α-ethyl-SZN, β-propyl-SZN, α- and β-butyl-SZN; (iv) α and β-inositol-MNU. These results suggest that the presence of the glucose moiety is conducive to a high level of mutagenicity of SZN. Alterations of the glucose moiety by addition of larger alkyl groups, especially in the α position lead to decreased mutagenicity. The least mutagenic analogs are those in which the glucose moiety is replaced by inositols.The mutagenicity of SZN, β-methyl-SZN and of β-butyl-SZN was also compared in a mouse tissue-mediated assay. SZN was about 500-fold more mutagenic than its β-methyl analog, while the β-butyl analog was not mutagenic.Depletion of SZN and 4 of its analogs from the medium in presence of bacteria was determined spectrophotometrically. The more mutagenic compounds were depleted more rapidly but the quantitative differences in mutagenicity between these compounds could not be accounted for by depletion alone.     

Structure of the lipid moiety of the Leishmania donovani lipophosphoglycan

The lipid moiety of the lipophosphoglycan of Leishmania donovani had been isolated and characterized as a novel lyso-alkylphosphatidylinositol. Treatment of lipophosphoglycan with either 10% NH4OH or a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus liberated a monoalkylglycerol substituent. Structural characterization of the monoalkylglycerol by gas-liquid chromatography-mass spectrometry indicated the presence of two saturated, unbranched hydrocarbons: a C24 alkyl chain comprising 78% of the lipid with the remaining 22% as a C26 alkyl chain. Periodate sensitivity demonstrated that the alkyl side chain is linked to the C-1 position of the glycerol backbone. Treatment of lipophosphoglycan with nitrous acid released 1-O-alkylglycerophosphorylinositol due to an unacetylated glucosamine residue linked to the inositol of the lyso-alkylphosphatidylinositol. Quantitative analysis of the organic solvent-soluble product of nitrous acid deamination of lipophosphoglycan confirmed the expected ratio of inositol:phosphate:1-O-alkylglycerol as 1:1:1. These results suggest that L. donovani anchors its lipophosphoglycan with a unique lipid component.     

Enzymic nature of the protein moiety of protochlorophyllide holochrome

The enzymic nature of the protein moiety of protochlorophyll(ide) holochrome was studied by following the fate of the [(14)C]protochlorophyll(ide) formed when dark-grown barley (Hordeum vulgare) or bean (Phaseolus vulgaris) leaves are incubated in the dark with 3 mm 4-delta-[(14)C]aminolevulinic acid. It was found that: [List: see text]Since turnover of protochlorophyll(ide) was not observed, these results show that there is a free exchange between the old "endogenous" and the new delta-aminolevulinic-acid-induced protochlorophyll(ide) molecules on the active site of the holochrome protein. These results are consistent with the hypothesis that the holochrome protein acts as an enzyme.     

Diversity of the peptide moiety of human airway mucins

A lambda gt11 cDNA library constructed from human tracheal mucosa was screened with rabbit antibodies raised to chemically deglycosylated pronase glycopeptides from bronchial mucins. This library allowed us to select 20 positive clones. The total or partial nucleotide sequences of 14 of them were classified in three different types of organization or families. Antibodies purified from total immuneserum on each positive clone were specific either of goblet cells or goblet cells and mucous cells, showing a differential cellular expression of the peptide axis of mucins. The use of each type of nucleic probe in Northern blot analysis confirmed the existence of a great heterogeneity of the RNAs contributing to a great peptide heterogeneity.     

Novel interactions of large P3 moiety and small P4 moiety in the binding of the peptide mimetic factor VIIa inhibitor

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is seen as a promising target for developing new anticoagulant drugs. A novel peptide mimetic factor VIIa inhibitor, ethylsulfonamide-d-biphenylalanine-Gln-p-aminobenzamidine, shows 100-fold selectivity against thrombin in spite of its large P3 moiety, unlike previously reported FVIIa/TF selective inhibitors. X-ray crystal structure analysis reveals that the large P3 moiety, d-biphenylalanine, and the small P4 moiety, ethylsulfonamide, make novel interactions with the 170-loop and Lys192 of FVIIa/TF, respectively, accompanying ligand-induced conformational changes of the 170-loop, Gln217, and Lys192. Structural comparisons of FVIIa with thrombin and amino acid sequence comparisons among coagulation serine proteases suggest that these interactions play an important role in achieving selective inhibition for FVIIa/TF.     

Serine incorporation into the selenocysteine moiety of glutathione peroxidase

The selenium in mammalian glutathione peroxidase is present as a selenocysteine ([Se]Cys) moiety incorporated into the peptide backbone 41-47 residues from the N-terminal end. To study the origin of the skeleton of the [Se]Cys moiety, we perfused isolated rat liver with 14C- or 3H-labeled amino acids for 4 h, purified the GSH peroxidase, derivatized the [Se]Cys in GSH peroxidase to carboxymethylselenocysteine ([Se]Cys(Cm)), and determined the amino acid specific activity. Perfusion with [14C]cystine resulted in [14C]cystine incorporation into GSH peroxidase without labeling [Se]Cys(Cm), indicating that cysteine is not a direct precursor for [Se]Cys. [14C]Serine perfusion labeled serine, glycine (the serine hydroxymethyltransferase product), and [Se]Cys(Cm) in purified GSH peroxidase, whereas [3-3H]serine perfusion only labeled serine and [Se]Cys(Cm), thus demonstrating that the [Se]Cys in GSH peroxidase is derived from serine. The similar specific activities of serine and [Se]Cys(Cm) strongly suggest that the precursor pool of serine used for [Se] Cys synthesis is the same or similar to the serine pool used for acylation of seryl-tRNAs.