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1.
Vincenzetti S.; Cambi A.; Maury G.; Bertorelle F.; Gaubert G.; Neuhard J.; Natalini P.; Salvatori D.; De Sanctis G.; Vita A. 《Protein engineering, design & selection : PEDS》2000,13(11):791-799
Site-directed mutagenesis on human cytidine deaminase (CDA)was employed to mutate specifically two highly conserved phenylalanineresidues, F36 and F137, to tryptophan; at the same time, theunique tryptophan residue present in the sequence at position113 was mutated to phenylalanine. These double mutations wereperformed in order to have for each protein a single tryptophansignal for fluorescence studies relative to position 36 or 137.The mutant enzymes thus obtained, W113F, F36W/W113F and F137W/W113F,showed by circular dicroism and thermal stability an overallstructure not greatly affected by the mutations. The titrationof Trp residues by N-bromosuccinimide (NBS) suggested that residueW113 of the wild-type CDA and W36 of mutant F36W/W113F are buriedin the tertiary structure of the enzyme, whereas the residueW137 of mutant F137W/W113F is located near the surface of themolecule. Kinetic experiments and equilibrium experiments withFZEB showed that the residue W113 seems not to be part of theactive site of the enzyme whereas the Phe/Trp substitution inF36W/W113F and F137W/W113F mutant enzymes had a negative effecton substrate binding and catalysis, suggesting that F137 andF36 of the wild-type CDA are involved in a stabilizing interactionbetween ligand and enzyme. 相似文献
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S Vincenzetti A Cambi E Balducci P Natalini R Volpini A Vita 《Canadian Metallurgical Quarterly》1997,42(3):469-476
Cytidine deaminase, a tetrameric enzyme purified from human placenta, was shown to contain a single atom of tightly bound zinc per subunit by Inductively Coupled Plasma Optical Emission Spectrometry analysis. The metal appears to be involved in catalysis, as suggested by the inhibition exerted by 1,10-phenanthroline and dipicolinic acid. This hypothesis is further supported by the finding that the presence of substrate protects the enzymatic activity from dipicolinic acid inhibition. Furthermore the total cysteine residues per subunit were investigated by sulphydryl groups titrating agents. 相似文献
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Cambi A; Vincenzetti S; Neuhard J; Costanzi S; Natalini P; Vita A 《Protein engineering, design & selection : PEDS》1998,11(1):59-63
By site-directed mutagenesis on human cytidine deaminase (CDA), five mutant
proteins were obtained: C65A, C99A, C102A, E67D and E67Q. The three
cysteine mutants were completely inactive, whereas E67D and E67Q showed a
specific activity about 200- and 200000-fold lower, respectively, than the
wild-type CDA. Zinc analysis revealed that only E67D, E67Q and C65A
contained 1 mol Zn2+/mol subunit as in the wild- type CDA. Kinetic
measurements with the specific carboxylic group reagent
N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline performed on wild-type CDA
suggest that Glu67 is essential for the catalytic process. Furthermore,
when both native and denatured CDA was titrated with
5,5'-dithiobis(2-nitrobenzoic acid) six sulfhydryl groups were detected,
whereas in the denatured and reduced enzyme nine such groups were found,
according to the sequence data. When p-hydroxymercuriphenyl sulfonate was
used, nine sulfhydryl groups were detectable and the release of 1 mol of
zinc per mole of CDA subunit was revealed by the metal indicator dye
4-(2-pyridylazo)resorcinol. It seems plausible that the limiting step for
the maintenance of zinc in the active site is the formation of coordination
between Cys99 and Cys102, whereas Cys65 could lead the zinc to the correct
position and orientation within the active site.
相似文献
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Cambi Alessandra; Vincenzetti Silvia; De Sanctis Giampiero; Neuhard Jan; Natalini Paolo; Vita Alberto 《Protein engineering, design & selection : PEDS》2001,14(10):807-813
We cloned, purified and characterized two extremophilic cytidinedeaminases: CDABcald and CDABpsy, isolated from Bacillus caldolyticus(growth at 72°C) and Bacillus psychrophilus (growth at 10°C),respectively. We compared their thermostability also with themesophilic counterpart, CDABsubt, isolated from Bacillus subtilis(growth at 37°C). The DNA fragments encoding CDABcald andCDABpsy were sequenced and the deduced amino acid sequencesshowed 70% identity. High sequence similarity was also foundwith the mesophilic CDABsubt. Both enzymes were found to behomotetramers of approximately 58 kDa. CDABcald was found tobe highly thermostable, as expected, up to 65°C, whereasCDABpsy showed higher specific activity at lower temperaturesand was considerably less thermostable than CDABcald. Afterpartial denaturation at 72°C for 30 min, followed by renaturationon ice, CDABcald recovered 100% of its enzymatic activity, whereasCDABpsy as well as CDABsubt were irreversibly inactivated. Circulardichroism (CD) spectra of CDABcald and CDABpsy at temperaturesranging from 10 to 95°C showed a markedly different thermostabilityof their secondary structures: at 10 and 25°C the CD spectrawere indistinguishable, suggesting a similar overall structure,but as temperature increases up to 5070°C, the -helicesof CDABpsy unfolded almost completely, whereas its ß-structureand the aromatic amino acids core remained pretty stable. Nosignificant differences were seen in the secondary structuresof CDABcald with increase in temperature. 相似文献
6.
B Bussolati F Mariano A Cignetti A Guarini V Cambi R Foà G Piccoli G Camussi 《Canadian Metallurgical Quarterly》1998,161(3):1493-1500
IL-12 is chemotactic for NK cells and polymorphonuclear neutrophils (PMN), but not for monocytes. In the present study, we evaluated whether the chemotactic effect of IL-12 is a direct phenomenon or is dependent on the generation of secondary mediators. The results obtained indicate that IL-12 induces a dose- and time-dependent synthesis of platelet-activating factor (PAF) from PMN and NK cells and of reactive oxygen radicals (ROS) from PMN. Monocytes and CD56-negative PBMC cells did not synthesize PAF or ROS after challenge with IL-12. The production of ROS by PMN was significantly inhibited by two chemically different PAF receptor antagonists (WEB 2170 and CV 3988), suggesting an autocrine stimulation of PMN by PAF newly synthesized after the challenge with IL-12. Moreover, the IL-12-induced chemotaxis of PMN and NK cells was significantly reduced by both WEB 2170 and CV 3988, suggesting that synthesized PAF mediates the chemotactic effect of IL-12. Preincubation with superoxide dismutase, which blocks the formation of superoxide anions, also reduced the chemotactic effect of IL-12 on PMN, but not on NK cells, suggesting that superoxide anion generation is relevant only for the IL-12-induced chemotaxis of PMN. In conclusion, the results of the present study indicate that IL-12-induced PAF synthesis plays a critical role in triggering the events involved in the motogenic response of PMN and NK to IL-12. 相似文献
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M Shafiee JF Griffon G Gosselin A Cambi S Vincenzetti A Vita S Eriksson JL Imbach G Maury 《Canadian Metallurgical Quarterly》1998,56(9):1237-1242
The stereoselectivities of recombinant human deoxycytidine kinase (EC 2.7.1.74) (dCK) and of recombinant human cytidine deaminase (EC 3.5.4.5) (CDA) were investigated with respect to a series of cytidine analogs, most of them having the unnatural L-stereochemistry. The enantioselectivity of dCK was always low and generally favored the L-enantiomers in the case of beta-2',3'-dideoxycytidine (beta-ddC), 5-fluoro-beta-2',3'-dideoxycytidine (beta-FddC) and beta-cytidine (beta-riboC). Concerning beta-2'-deoxycytidine, dCK showed a preference for the D-enantiomer. All other examined beta-L-cytidine analogs, [1-beta-L-lyxofuranosyl cytosine (beta-L-lyxoC), l-beta-L-xylofuranosyl cytosine (beta-L-xyloC), and 5-fluoro-1-beta-L-xylofuranosyl cytosine (beta-L-Fxylo C)], were substrates of dCK regardless of the nature of the pentose. None of the studied alpha-L-anomers (alpha-L-riboC, alpha-L-araC, alpha-L-lyxoC, or alpha-L-xyloC) was a substrate of dCK. Contrasting with the relaxed enantioselectivity of dCK, CDA had a strict requirement for D-cytidine analogs since none of the already listed beta-L- or alpha-L analogs was a substrate or an inhibitor of the enzyme. The conjunction of the preceding stereochemical properties of dCK and CDA confers to L-cytidine analogs important potentialities in antiviral and anticancer therapies. 相似文献
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