Bovine pepsinogen A: Isolation and parial characterization of isoforms with high activity

The goal of this study was to purify bovine pepsinogen by a simple method allowing the preparation of large amount of pure protein. The purified protein and antisera are needed to develop diagnostic methods for further investigations in animals susceptible of gastric disorders or helminthosis. Pepsinogen isoforms were separated from extracts of bovine fundic mucosa by ammonium sulfate precipitations and chromatography on DEAE and hydroxyapatite. The isoforms showed a high activity in indirect proteolytic assay. Sequence analysis gave the following amino acid sequence SVVKIPLVKK for fraction 1, 2 and SVVKIPLVKKKSLRQNLIENGKLKE for fraction 3. The Mass spectrometry revealed isoforms with different masses from 39,864 to 40,181 Da. The estimated organic phosphate content ranged from 0.98 to 3.9 moles of phosphate per molecule. The protocol, with few steps, gave consequent quantities of pure and active protein available for further studies including the development of RIA and ELISA as diagnostic tools in gastrointestinal diseases

Stability domains, substrate-induced conformational changes and hinge-bending motions in a psychrophilic phosphoglycerate kinase : a microcalorimetric study

peer reviewedThe cold-active phosphoglycerate kinase from the Antarctic bacterium Pseudomonas sp. TACII18 exhibits two distinct stability domains in the free, open conformation. It is shown that these stability domains do not match the structural N- and C-domains as the heat-stable domain corresponds to about 80 residues of the C-domain, including the nucleotide binding site, whereas the remaining of the protein contributes to the main heat-labile domain. This was demonstrated by spectroscopic and microcalorimetric analyses of the native enzyme, of its mutants, and of the isolated recombinant structural domains. It is proposed that the heat-stable domain provides a compact structure improving the binding affinity of the nucleotide, therefore increasing the catalytic efficiency at low temperatures. Upon substrate binding, the enzyme adopts a uniformly more stable closed conformation. Substrate-induced stability changes suggest that the free energy of ligand binding is converted into an increased conformational stability used to drive the hinge-bending motions and domain closure