The inhibition of pepsin-catalysed reactions by products and product analogues. Kinetic evidence for ordered release of products

1. The inhibition of pepsin-catalysed hydrolysis of N-acetyl-l-phenylalanyl-l-phenylalanylglycine by products and product analogues was studied. 2. The non-competitive nature of the inhibition by the product N-acetyl-l-phenylalanine confirms an ordered release of products, and points to a common mechanism (involving an amino-enzyme) for pepsin-catalysed transpeptidation and hydrolysis reactions. 3. N-Acetyl-l-phenylalanine ethyl ester is also a non-competitive inhibitor, but here the inhibition is of the ;dead-end' type. No ethanol is detectable in reaction mixtures, indicating that this ester cannot act as an amino group acceptor in a transpeptidation process. 4. The same is true for N-methanesulphonyl-l-phenylalanine methyl and methyl thiol esters. No methanethiol is liberated when the methyl thiol ester is present as an inhibitor of the hydrolytic reaction, and the hope that such a thiol ester would effectively trap the amino-enzyme was not fulfilled.     

Biophysical characterization of recombinant proteins expressing the leucine zipper-like domain of the human immunodeficiency virus type 1 transmembrane protein gp41.

Envelope oligomerization is thought to serve several crucial functions during the life cycle of human immunodeficiency virus type 1 (HIV-1). We recently reported that virus entry requires coiled-coil formation of the leucine zipper-like domain of the HIV-1 transmembrane envelope glycoprotein gp41 (C. Wild, T. Oas, C. McDanal, D. Bolognesi, and T. Matthews, Proc. Natl. Acad. Sci. USA 89:10537-10541, 1992; C. Wild, J. W. Dubay, T. Greenwell, T. Baird, Jr., T. G. Oas, C. McDanal, E. Hunter, and T. Matthews, Proc. Natl. Acad. Sci. USA 91:12676-12680, 1994). To determine the oligomeric state mediated by this region of the envelope, we have expressed the zipper motif as a fusion partner with the monomeric maltose-binding protein of Escherichia coli. The biophysical properties of this protein were characterized by velocity and equilibrium sedimentation, size exclusion chromatography, light scattering, and chemical cross-linking analyses. Results indicate that the leucine zipper sequence from HIV-1 is capable of multimerizing much larger and otherwise monomeric proteins into extremely stable tetramers. Recombinant proteins containing an alanine or a serine substitution at a critical isoleucine residue within the zipper region were also generated and similarly analyzed. The alanine- and serine-substituted proteins behaved as tetrameric and monomeric species, respectively, consistent with the influence of these same substitutions on the helical coiled-coil structure of synthetic peptide models. On the basis of these findings, we propose that the fusogenic gp4l structure involves tetramerization of the leucine zipper domain which is situated approximately 30 residues from the N-terminal fusion peptide sequence.     

Structural studies of the capsular polysaccharide and lipopolysaccharide O-antigen of Aeromonas salmonicida strain 80204-1 produced under in vitro and in vivo growth conditions.

Aeromonas salmonicida is a pathogenic aquatic bacterium and the causal agent of furunculosis in salmon. In the course of this study, it was found that when grown in vitro on tryptic soy agar, A. salmonicida strain 80204-1 produced a capsular polysaccharide with the identical structure to that of the lipopolysaccharide O-chain polysaccharide. A combination of 1D and 2D NMR methods, including a series of 1D analogues of 3D experiments, together with capillary electrophoresis-electrospray MS (CE-ES-MS), compositional and methylation analyses and specific modifications was used to determine the structure of these polysaccharides. Both polymers were shown to be composed of linear trisaccharide repeating units consisting of 2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA), 3-[(N-acetyl-L-alanyl)amido]-3,6-dideoxy-D-glucose[3-[(N-acetyl-L-alanyl)amido]-3-deoxy-D-quinovose, Qui3NAlaNAc] and 2-acetamido-2,6-dideoxy-D-glucose (2-acetamido-2-deoxy-D-quinovose, QuiNAc) and having the following structure: [-->3)-alpha-D-GalpNAcA-(1-->3)-beta-D-QuipNAc-(1-->4)-beta-D-Quip3NAlaNAc-(1-]n, where GalNAcA is partly presented as an amide and AlaNAc represents N-acetyl-L-alanyl group. CE-ES-MS analysis of CPS and O-chain polysaccharide confirmed that 40% of GalNAcA was present in the amide form. Direct CE-ES-MS/MS analysis of in vivo cultured cells confirmed the formation of a novel polysaccharide, a structure also formed in vitro, which was previously undetectable in bacterial cells grown within implants in fish, and in which GalNAcA was fully amidated.     

Stabilization of Microsatellite Sequences by Variant Repeats in the Yeast Saccharomyces Cerevisiae

We examined the effect of a single variant repeat on the stability of a 51-base pair (bp) microsatellite (poly GT). We found that the insertion stabilizes the microsatellite about fivefold in wild-type strains. The stabilizing effect of the variant base was also observed in strains with mutations in the DNA mismatch repair genes pms1, msh2 and msh3, indicating that this effect does not require a functional DNA mismatch repair system. Most of the microsatellite alterations in the pms1, msh2 and msh3 strains were additions or deletions of single GT repeats, but about half of the alterations in the wild-type and msh6 strains were large (>8 bp) deletions or additions.     

Protein synthesis by hybrid ribosomes reconstructed from rabbit reticulocyte ribosomal core-particles and amphibian or fungal split-proteins.

It was shown that high-salt (2.75 M-NH4Cl/69mM-MgCl2) shock treatment at 0 degrees C of the larger subparticles (L-subparticles) of rabbit, Xenopus laevis and Neurospora crassa cytoplasmic ribosomes yielded split-protein fractions that were not only functionally equivalent but also interchangeable. Thus, although the remaining core-particles were inactive in both the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis, activity was restored when they were combined with either homologous or heterologous split-protein fractions. This technique was used to prepare active hybrid L-subparticles, e.g. rabbit cores/N. crassa split-proteins, and also active hybrid ribosomes, e.g. rabbit smaller subparticle/X. laevis core-particle/rabbit split-proteins. Rabbit and X. laevis split-protein fractions labelled with 14C by reductive methylation with [14C]formaldehyde and sodium cyanoborohydride were both shown to bind to rabbit core-particles in approximate correlation with the degree of re-activation. The split-protein fractions of rabbit and X. laevis L-subparticles were analysed by two-dimensional and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The molecular weights (measured in sodium dodecyl sulphate gels) of the split-proteins of rabbit and X. laevis L-subparticles were found to be similar. These results demonstrate that the peptidyltransferase active centre of cytoplasmic ribosomes of eukaryotes has components that are interchangeable over a wide evolutionary range. Evidently the essential molecular architecture of the active centre is highly conserved.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.