Reductive cleavage of Xaa-proline peptide bonds by mild alkaline borohydride treatment employed to release O-glycosidically linked carbohydrate units of glycoproteins

During the deglycosylation reaction of fish egg polysialoglycoproteins under the conditions of 1 M NaBH4 in 0.1 M NaOH at 37 degrees C for 48 h, a marked loss of the glycine content has been encountered, besides the serine and threonine residues to which the carbohydrate units are linked. The chemical basis behind this phenomenon has been elucidated by amino acid analysis first of the major glycopeptides (carbohydrate-(O)Thr-Gly-Pro-Ser) derived from desialylated polysialoglycoproteins and subsequently six proline-containing peptides before and after treatment under similar conditions. It has thus been established that -Xaa-Pro- sequences are remarkably susceptible to reductive cleavage under such mild aqueous conditions. In view of the finding that the reductive cleavage of insulin B-chain, which contains a single proline residue adjacent and C-terminal to a threonine residue, led to about 80% loss of the threonine residue, deglycosylation with alkaline borohydride reagents warrants a special comment. The decreased amounts of serine or threonine residues cannot be related simply to the degree of glycosylation of these residues. The above results are therefore discussed in the relation to other work.     

Effects of exogenous soluble dextrans on insoluble glucan synthesis by Streptococcus mutans glucosyltransferase

Production of water-insoluble glucan (ISG) from sucrose by cell-free Streptococcus mutans AHT glucosyltransferase (GTF) first rapidly increased, and then sharply declined, as the amounts of water-soluble Dextrans T20 approximately T500 present, were increased. The decline of ISG synthesis was accompanied by an increased synthesis of the water-soluble fraction (SG). Prolonged incubation, however, induced enhanced synthesis of ISG even at higher dextran concentrations. The concentration of dextran required to stimulate or suppress ISG synthesis depended on the amounts of GTF used, but the extent of the stimulation was almost identical for the same GTF/dextran ratio. Thus, ISG synthesis is stimulated by the presence of dextrans at relatively low concentrations, but retarded at higher concentrations by being shifted to SG synthesis. ISG produced in the presence of dextrans contained higher proportions of alpha-1,6 glucosidic linkage and lower molecular size fractions, and possessed lower viscosity. These ISG products did not exhibit the coalescence of two component fibrils as observed with control ISG. These changes combined may contribute to the reduction of ISG-dependent adherence to glass of S. mutans cells by the presence of soluble dextrans, irrespective of their molecular size and concentration.     

An X-ray study on the interaction between indole ring and pyridine coenzymes: Crystal structure of 1-methyl-3-carbamoylpyridinium: Indole-3-acetic acid (1:1) monohydrate charge-transfer complex

The crystal and molecular structures of the title complex has been determined by X-ray diffraction methods, as a model for tryptophan residues in protein-pyridine coenzyme interactions. The structure was solved by direct methods and was refined by standard methods (final R = 0.073). The light-green crystals consist of alternate layers of indole-3-acetic acid and 1-methyl-3-carbamoylpyridinium molecules piled up to the c-direction, and are stabilized by the crystal water participating in hydrogen bonds in the a- and b-directions. The parallel stackings and interplanar spacing distances between indole and pyridinium rings strongly suggest a II–II¹ charge transfer from the indole ring to the lowest unoccupied orbital of the pyridinium ring in the ground state. Furthermore, this crystal structure provides evidence that quaternization of the N1 position enhances electron-acceptor properties of pyridine. On the other hand, the proton magnetic resonance spectra suggest that the stacking mode between both rings in solution is very similar to the one observed in the crystal structure.     

In vitro microwave effects on human neutrophil precursor cells (CFU-C)

Human marrow cells were irradiated with 2450-MHz CW microwaves in a fluid-filled waveguide irradiation system. Cell exposure was conducted by placing a marrow cell suspension in 20-μl glass microcapillary tubes that were positioned in the exposure chamber, and irradiated at power densities from 31 to 1,000 mW/cm² (with corresponding specific absorption rates of 62 to 2,000 mW/g) for 15 minutes. The temperature of the sample was maintained at a fixed point. Sham-irradiated (SI) and microwave-irradiated (MWI) cells were cultured in a methylcellulose culture system for neutrophil colony proliferation. There was no reduction in neutrophil colony number on days 6–7 or 12–14 in cells exposed at 31 or 62 mW/cm², but as the power density was increased to 1,000 mW/cm², there was a reduction in colony number of MWI cells compared with SI cells. The microwave interaction with the human neutrophil colony-forming cells was apparently not related to temperature rise, or to the state of cell cycle, and was irreversible.     

13C and 1H NMR studies of helix-coil transition of poly(β-benzyl-L-aspartate) and poly(γ-benzyl-L-glutamate): Behavior in nonprotonating solvent mixtures,and origin of solvent-induced chemical shift

From the results of ¹³C-nmr measurement of poly(β-benzyl-L -aspartate) and its model compounds in dimethyl sulphoxide/deuterated chloroform mixtures, it was found that the side chain of poly(β-benzyl-L -aspartate) is solvated by dimethyl sulphoxide in the region more than dimethyl sulphoxide 20% (v/v), where the backbone maintains the α-helix. The chemical shift differences in the benzyl group carbons of poly(γ-benzyl-L -glutamate) (trifluoroacetic acid/deuterated chloroform) accompanied by the helix-coil transition, originate from the interaction between the ester group of the side chain and trifluoroacetic acid. The chemical shift difference in the ester carbon is similar. On the other hand, the chemical shift differences of the side-chain carbons in the alkyl portion (C^β, C^γ) originate not only from the interaction between the ester group of the side chain and trifluoroacetic acid, but also from some other unknown factors. The chemical shift differences of the side-chain carbons of poly(β-benzyl-L -aspartate) originate from the interaction between the ester group of the side chain and trifluoroacetic acid.     

Fast atom bombardment tandem mass spectrometric analysis of N-carbamoylamino acids.

Using fast atom bombardment (FAB) and tandem mass spectrometry (MS/MS), we examined 12 synthetic N-carbamoylamino acids (CAA) as tert-butyldimethylsilyl (TBDMS) derivatives. In FAB mass spectrometry and FAB MS/MS, spectra of protonated molecules for CAA provide specific cleavages involving the TBDMS carbamoyl moiety. The daughter scan spectrum of the parent ion indicated that it was useful for structural elucidation and differentiation of structural isomers of CAA. We have also identified each CAA separately in a mixture using a neutral loss scan for characteristic ions. In addition, we demonstrated that CAA in urine samples from patients with ornithine carbamoyl transferase deficiency gave collision-induced dissociation (CID) spectra which correspond well with CID spectra obtained using synthetically prepared CAA.