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Domain characteristics of the cyanogen bromide fragment 121-316 of thermolysin.
No full textThe molecule of thermolysin was shown by X-ray crystallography to be composed of two structural domains of equal size comprising residues 1-157 and 158-316. In order to explore the possibility that these domains correspond to globular fragments able to refold autonomously, we have investigated the conformational and stability properties of fragment 121-316, which was obtained by limited chemical cleavage of thermolysin with cyanogen bromide. As judged by far-ultraviolet circular dichroism measurements, in aqueous solution under neutral conditions the fragment maintains a relative amount of helical structure which is comparable to that exhibited by the corresponding region in native thermolysin. The secondary structure attained by the fragment appears remarkably stable to the denaturing action of heat. By measuring the temperature dependence of the dichroic signal at 220 nm a Tm near 74 degrees was obtained. Immunodiffusion analyses indicated that the fragment recognizes and precipitates antibodies raised in rabbits using native thermolysin as immunogen. The overall conformational and immunochemical data indicate that fragment 121-316 of thermolysin is able to refold into a stable structure of native-like characteristics independently of the rest of the molecule. The results of this study complement those previously reported for fragment 206-316 (Vita, C., Fontana, A., Seeman, J.R. & Chaiken, I.M. (1979) Biochemistry 18
Nuovo metodo HPLC per la determinazione del contenuto degli amminoacidi liberi presenti negli idrolizzati proteici ottenuti dal carniccio.Vicenza 26.2.1999
No full textDomain Characterization of the Carboxyl-Terminal Fragment 206-316 of Thermolysin: pH and Ionic Strength Dependence of Conformation
No full textProperties of Sepharose-Bound beta-Lactamase from Enterobacter Cloacae
No full textbeta-Lactamase from Enterobacter cloacae P99 was immobilized onto Sepharose by the cyanogen bromide activation method and the properties of the Sepharose-bound enzyme were compared with those of soluble and cell-bound enzyme. The immobilized beta-lactamase showed enhanced stability to storage at 4 degrees C (approximately 1 year) in respect to the free enzyme in solution (few days). The optimum pH for activity is similar for both Sepharose- and cell-bound beta-lactamase and extends over a broader pH range (pH 6-9) than the soluble enzyme (pH 8-9). Immobilization leads also to significant enhancement of thermal stability. Effective enzyme inhibition by flucloxacillin occurs with both soluble and Sepharose-bound beta-lactamase, whereas the cell-bound enzyme is much less (10(-5) times) inhibited. These results indicate that immobilized beta-lactamase could be usefully employed as a tool for investigating the properties of newly designed beta-lactamase inhibitors
Fluphenazine phototoxicity: a mechanism involving the trifluoromethyl group
No full textFluphenazine is a neuroleptic drug used for the long-term treatment of mental disorders, in particular in the therapy of various psychoses, including schizophrenia and mania.
Occurrence of skin phototoxic and photoallergic reactions is observed when patients expose themselves to sunlight during treatment. In order to identify the mechanism of fluphenazine phototoxicity, the drug was irradiated with UVA in different solvents and in presence of nucleophilic substrates of biological relevance, like serine, lysine and a serine-containing oligopeptide. The photoproducts were characterized by mass spectroscopy and, for three of them, by NMR spectroscopy. A minor product in which N-oxidation occurred was found in all conditions. The major product which formed in water was a carboxylic acid derived from the hydrolytic defluorination of the CF3 group. In methanol and ethanol the main product was the ester of the carboxylic acid. Serine also bound fluphenazine through the same mechanism. Although with low yield, a similar product was identified in a 14-peptide irardiated in presence of the drug. Lysine did not react in the same conditions. These results suggest that photobinding of fluphenazine to serine residues of proteins may occur in vivo, thus initiating the processes leading to photoallergy
Folding of thermolysin fragments. Identification of the minimum size of a carboxyl-terminal fragment that can fold into a stable native-like structure.
No full textThe COOH-terminal cyanogen bromide fragment 206-316 of thermolysin has been shown to possess protein domain characteristics that are able to refold into a stable native-like structure (Fontana et al., 1982). We now report the results of limited proteolysis of this fragment with the aim of identifying the minimum size of a COOH-terminal fragment of thermolysin that is able to fold by itself. Proteolysis with subtilisin, chymotrypsin, thermolysin and trypsin allowed us to isolate to homogeneity eight different subfragments, which can be grouped in two sets of peptides, i.e. (218-222)-316 and (252-255)-316. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. In addition, even the smallest fragment isolated (sequence 255-316) shows co-operative and reversible unfolding transitions mediated by heat (tm 65 degrees C) and guanidine hydrochloride (midpoint transition at 2.5 M denaturant), as often observed with globular proteins. From the kinetics of the proteolytic digestion and analysis of the isolated subfragments, it is concluded that proteases lead to a stepwise degradation of fragment 206-316 from its NH2-terminal region, leading to the highly helical fragment (252-255)-316, quite resistant to further proteolytic digestion. The results of this study provide evidence that it is possible to isolate stable supersecondary structures of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain of thermolysin
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