ABSTRACT
A strain producing 5-del-proinsulin was designed on the basis of an industrial strain producing human recombinant proinsulin. 5-Del-proinsulin is an analog of human recombinant proinsulin. The new strain contains a deletion of 5 amino acid residues in the C-end region of the B-chain and a residue of tyrosine at B25. A method of oligonucleotide-directed mutagenesis was applied. Comparison of the electrophoregrams of the inclusion bodies of the initial and resulting strains made it possible to conclude that the pattern of the proteins was the same and the electrophoretic mobility of the recombinant proteins was practically identical.
Subject(s)
Gene Deletion , Proinsulin/genetics , Receptor, Insulin/metabolism , Tyrosine/genetics , Amino Acid Sequence , Base Sequence , Binding Sites/genetics , Escherichia coli , Genetic Code , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligonucleotides/genetics , Recombinant Proteins/geneticsABSTRACT
The effect of proteinases of plant and microbial origin on polymyxin M was studied. It was shown that this antibiotic was absolutely stable to the effect of papain and ficin. On hydrolysis with subtilisin there formed polymyxin decyclized analogs not described earlier. Their isolation, purification and biological activity are described. The structure of these compounds was assessed by one- and two-dimensional 1H NMR spectroscopy. The role of various functional groups, their space orientation and impact on antimicrobial activity of the compounds are discussed.
Subject(s)
Polymyxins/analysis , Ficain/pharmacology , Papain/pharmacology , Peptide Hydrolases/pharmacology , Structure-Activity RelationshipABSTRACT
Activity of polymyxin B nonapeptide alone and in combination with other antibiotics against clinical strains of Pseudomonas and enteric bacteria was studied. It was shown that nonapeptide was highly active against Pseudomonas and moderately active against enteric bacteria. In combination with rifampicin, fusidic acid or erythromycin the nonapeptide had a potentiating effect on the tested strains.
Subject(s)
Anti-Bacterial Agents/pharmacology , Gram-Negative Bacteria/drug effects , Polymyxin B/pharmacology , Polymyxins/pharmacology , Drug Resistance, Microbial , Drug Synergism , Microbial Sensitivity Tests , Time FactorsABSTRACT
LPCC and HPLC revealed that polymyxin M was a mixture of five components of the polymyxin nature: PM1, PM2, PMx, PMy and PMz. The individual compounds PM1, PM2 and PMz were isolated. Their physicochemical properties and data on antimicrobial activity are presented.
Subject(s)
Polymyxins/isolation & purification , Bacteria/drug effects , Chromatography, High Pressure Liquid , Polymyxins/analysis , Polymyxins/pharmacologyABSTRACT
Spatial structure of polypeptide antibiotic polymyxin M in water was studied by one-and two-dimensional (COSY, COSY-45, RELAY) H NMR spectroscopy. Analysis of the signal spectral parameters revealed two intramolecular hydrogen bonds in the cyclic part of the molecule which was analogous to the structure of polymyxin B. However, configuration of both the beta-turns in the polymyxin M structure differed from that of the detected earlier beta-turns in the structure of polymyxin B.
Subject(s)
Polymyxins/analysis , Hydrogen , Magnetic Resonance Spectroscopy , Molecular Conformation , Solutions , Structure-Activity Relationship , WaterABSTRACT
Polymyxin B and its shortened analog were studied comparatively by 1H-NMR spectroscopy. Analysis of the signal chemical shifts, constants of spin-spin interaction of 3J HN-C alpha H and temperature coefficients of the NH signal chemical shifts revealed absolute structural identity of both molecules cyclic parts. This proved that there was no conformative interaction between the cyclic and linear parts of the polymyxin B molecule. Comparison of the results with the data on the biological activity showed that the hydrophobic N-end moiety of the polymyxin B molecule played a specific role in its antibacterial effect and toxicity.
