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NICOLAY GENOV BRUNO FILIPPI PAVLINA DOLASHKA KEITH S. WILSON CHRISTIAN BETZEL 《Chemical biology & drug design》1995,45(4):391-400
The stability towards thermal and chemical (guanidine hydrochloride, GnHCl) denaturation of six inhibited subtilases (mesentericopeptidase, subtilisins BPN′, Carlsberg and DY, proteinase K and thermitase) has been investigated by kinetic and equilibrium studies. The unfolding processes were monitored by circular dichroic and fluorescence spectroscopy. Experiments in the absence and presence of extraneous calcium in the concentration range 2×10?3-10?1 M were performed. The presence of calcium in the weak calcium binding site changes the denaturation drastically. The heat- (or GnHCl-) induced unfolding curves obtained using CD spectroscopy show two independent transitions which seem not to have been resolved before. The presence of Ca2+ in the second (third in the case of thermitase) binding site increases the Tm, values by 11-21 °C and the δGD(H2O) values obtained from denaturation experiments in GnHCl by 6.7-7.2 kcal/mol when an extraneous Ca2+ concentration of 2 × 10?2 M was used. One interpretation is that the initial step of denaturation in the presence of added calcium is the formation of a partially unfolded intermediate form, retaining a highly ordered structure with 60-85% of the a-helix structure of the native enzyme. This intermediate then unfolds at a temperature considerably higher than that of the same proteinases in the absence of added Ca2+. The free energy of stabilization of the intermediates is increased by 1.8-2.8 times in comparison with that for the unfolding reactions of the subtilases with empty Ca2/Ca3 binding sites. A second interpretation is that the two steps in the unfolding curves correspond to enzyme without and with calcium in the weak binding site. Fluorescence experiments confirm the mechanism involving the formation of intermediate states. The results are discussed in relation to the X-ray models of the six subtilases. 相似文献
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GENERALIZED FAMILIAL BRONCHOMALACIA 总被引:1,自引:0,他引:1
E. AGOSTI G. De FILIPPI R. FIOR F. CHIUSSI 《Acta paediatrica (Oslo, Norway : 1992)》1974,63(4):616-618
ABSTRACT. Agosti, E., De Filippi, G., Fior, R. and Chiussi, F. (Paediatric Clinic, University of Trieste, Departments of Radiology and Otorhinolaryngology, Istituto per I'Infanzia and Department of Paediatrics, Ospedale Civile Palmanova, Trieste, Italy). Generalized familial bronchomalacia. Acta Paediatr Scand, 63: 616, 1974.—Generalized bronchomalacia was found in a 4-month-old infant with chronic respiratory distress since the first day of life. The diagnosis was confirmed by endoscopy and radiography. The patient differs in several respects from other cases reported. The disorder seems to be familial. The clinical symptoms were unusually severe and no evidence of bronchiectasis could be demonstrated. 相似文献
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PAOLO RUZZA ANDREA CALDERAN BRUNO FILIPPI BARBARA BIONDI ARIANNA DONELLA DEANA LUCA CESARO LORENZO A. PINNA GIANFRANCO BORIN 《Chemical biology & drug design》1995,45(6):529-539
Tyrosine protein kinases (TPKs) of the src family contain two major phosphoacceptor sites which are homologous to the Tyr 416 and Tyr 527 of pp60c-src. The former represents the main autophosphorylation sites of these enzymes, and its phosphorylation correlates with increased kinase activity. It has previously been demonstrated that the Src-like tyrosine kinase expressed by the oncogene lyn displays a high affinity toward the heptapeptide H-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-OH, which reproduces the main autophosphorylation site of the Src family enzymes [Donella-Deana, A., Marin, O., Brunati, A.M. & Pinna, L.A. (1992) Eur. J. Biochem. 204 , 1159–1163]. Our study was addressed to the synthesis of some derivatives of this sequence in order to obtain both peptide substrates suitable for the detection of the Src-like tyrosine kinase activity and active site-directed inhibitors specific for this class of enzymes. For this purpose we synthesized by classical solution methods the heptapeptide and its dimeric form. Moreover, in order to improve the proteolytic resistance of these peptides we also synthesized their cyclic derivatives and their N-terminal acetylated and C-terminal amidated analogs. The correlation between the different structural properties induced by the modifications of the native sequence and the propensity of the peptides to act as Lyn substrates was examined. The kinetic data obtained indicate that the extent of the peptide phosphorylation varies considerably depending on the flexibility and length of the analogs. While the cyclization and the C-terminal amidation of the heptapeptide are detrimental for the Lyn activity, dimeric derivatives display very favourable kinetic constants. In particular the cyclic dimer is an especially suitable substrate for the tyrosine kinase and a powerful inhibitor of both the phosphorylation activity of Lyn and the enzyme autophosphorylation. © Munksgaard 1995. 相似文献