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11.
Uteroferrin, an acid phosphatase with a spin-coupled and redox-active binuclear iron center, is paramagnetic in its pink, enzymatically active, mixed-valence (S = 1/2) state. Phosphate, a product and inhibitor of the enzymatic activity of uteroferrin, converts the pink, EPR-active form of the protein to a purple, EPR-silent species. In contrast, molybdate, a tetrahedral oxyanion analog of phosphate, transforms the EPR spectrum of uteroferrin from a rhombic to an axial form. With both electron spin echo envelope modulation (ESEEM) and electron nuclear double resonance (ENDOR) spectroscopies, we observe a hyperfine interaction of [95Mo]molybdate with the S = 1/2, Fe(II)-Fe(III) center of the protein. A pair of 95Mo resonances centered at the 95Mo Larmor frequency at the applied magnetic field and separated by a hyperfine coupling constant of 1.2 MHz is evident. Therefore, a single monomeric species of molybdate is close to, and likely a ligand of, the binuclear cluster. 1H ENDOR studies on uteroferrin reveal at least six sets of lines mirrored about the 1H Larmor frequency. Two pairs of these lines become reduced in intensity when the protein is exchanged against D2O. Moreover, ESEEM and 2H ENDOR spectra display resonances at the 2H Larmor frequency. Therefore, the metal-binding region of the protein is accessible to solvent. Additional deuterium lines observable by ESEEM spectroscopy provide evidence for a population of strongly coupled, readily exchangeable protons associated with the binuclear center. The measured hyperfine coupling constants for these deuterons are orientation-dependent with splittings of nearly 4 MHz at g3 = 1.59 and less than 1 MHz at g1 = 1.94. In the presence of molybdate, ESEEM spectra of D2O-exchanged samples reveal a resonance at the 2H Larmor frequency, with no evidence of spectral components due to strongly coupled deuterons. 1H ENDOR studies of the uteroferrin-molybdate complex show at least seven pairs of lines, mirrored about the 1H Larmor frequency, of which one pair becomes attenuated in amplitude upon deuteration. The active site thus remains accessible to solvent in the presence of molybdate. 相似文献
12.
Participation of glutamic acid 23 of T4 endonuclease V in the beta-elimination reaction of an abasic site in a synthetic duplex DNA.
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T4 endonuclease V catalyzes the hydrolysis of the glycosyl bond of a thymine dimer in a DNA duplex and the cleavage of the 3'-phosphate by beta-elimination. We have previously identified a catalytic site for the first reaction (pyrimidine dimer-glycosylase activity) by systematic mutagenesis (Doi et al. Proc. Natl. Acad. Sci. USA 1992 in press) and by x-ray crystallography (Morikawa et al. Science, 256: 523-526, 1992). The results showed that replacement of Glu23 with either glutamine or aspartic acid completely abolished the glycosylase activity. We describe the investigation of the second reaction (apurinic/apyrimidinic endonuclease activity), using twenty two mutants of T4 endonuclease V plus a DNA mini duplex containing an abasic site. Replacement of Glu23 by glutamine abolished the second reaction, but replacement with aspartic acid did not. The pH optima of the mutant (23 Asp) and the wild type were found to be 5.0 and 5.5, respectively. We conclude that the carboxylate anion in position 23 may act as a general base in the beta-elimination reaction of the endonuclease. 相似文献
13.
Conformational dynamics of DNA affected by intercalation and minor groove binding: direct observation of large DNA. 总被引:1,自引:0,他引:1
Y Matsuzawa K Minagawa K Yoshikawa M Matsumoto M Doi 《Nucleic acids symposium series》1991,(25):131-132
Using fluorescence microscopy, we have observed moving DNA molecules in solution and analyzed the "higher-order" structure in a quantitative manner. It was found that EB (ethidium bromide), an intercalator, has the effect to increase the persistent length. In other words, EB expands DNA. Whereas, DAPI (4',6-diamidino-2-phenylindole), a minor groove binding drug, decreases the persistent length. It is demonstrated that the direct observation of DNA molecules with fluorescence microscopy is quite useful to study the interaction of various chemical compounds with DNA molecules. 相似文献
14.
Analyses of intramolecular disulfide bonds in proteins by polyacrylamide gel electrophoresis following two-step alkylation 总被引:1,自引:0,他引:1
A method that makes use of polyacrylamide gel electrophoresis was developed for the analysis of intramolecular disulfide bonds in proteins. Proteins with different numbers of cleaved disulfide bonds are alkylated with iodoacetic acid or iodoacetamide as the first step. The disulfide bonds remaining were reduced by excess dithiothreitol, and the newly generated free sulfhydryl groups were alkylated with the reagent not yet used (iodoacetamide, iodoacetic acid, or vinyl-pyridine) as the second step. This treatment made it possible for lysozyme (Mr, 14,000; 4 disulfides), the N-terminal half-molecule of conalbumin (Mr, 36,000; 6 disulfides), the C-terminal half-molecule of conalbumin (Mr, 40,000; 9 disulfides), and whole conalbumin (Mr, 78,000; 15 disulfides) to be separated by acid-urea polyacrylamide gel electrophoresis into distinct bands depending on the number of disulfide bonds cleaved. The method allowed us to determine the total number of disulfide bonds in native proteins and to assess the cleaved levels of disulfide bonds in partially reduced proteins. Two-step alkylation used in combination with radioautography was especially useful for the analysis of disulfide bonds in proteins synthesized in complex biological systems. 相似文献
15.
Several physicochemical parameters of ovalbumin were examined at acid pH. The intrinsic viscosity and far UV-CD spectrum at pH 2 did not differ from those at pH 7. But the near UV-CD spectrum, difference absorption spectrum around 250-320 nm, and fluorescence spectrum showed micro-environmental changes around the aromatic amino acid residues in acid solution. The reactivity of one of the four sulfhydryl groups with 2,2'-dithiodipyridine increased at pH below 5. The rate of denaturation by urea and that of surface tension decay were high in the low pH range. We concluded that at low pH (around 2), ovalbumin molecules kept their native globular conformation, but that their chain flexibility increased and they were very susceptible to denaturation. This state might be equivalent to the molten-globule state observed with some globular proteins in acidic region. 相似文献
16.
T Ishida K Kamiichi A Kuwahara M Doi M Inoue 《Biochemical and biophysical research communications》1986,136(1):294-299
The crystal structure of title complex has been analyzed by X-ray diffraction method as a model for elucidating the possible interaction between the phenylalanyl residue of proteins and the N7-protonated or methylated guanine base of nucleic acids. The guanine base is associated with the benzene ring of phenylalanine by stacking interaction, and further connected with the carboxyl group by the formation of a pair of hydrogen bonds. These two interaction modes are suggested to be responsible for the specific recognition of base sequence by protein. 相似文献
17.
Translational coupling in Escherichia coli of a heterologous Bacillus subtilis-Escherichia coli gene fusion. 总被引:1,自引:0,他引:1
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The efficient expression in Escherichia coli of the Tn9-derived chloramphenicol acetyltransferase (EC 2.3.1.28) gene fused distal to the promoter and N terminus of the Bacillus subtilis aprA gene was dependent on the initiation of translation from the ribosome-binding site in the aprA gene. 相似文献
18.
Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to beta-lactam antibiotics in Staphylococcus aureus. 总被引:44,自引:10,他引:34
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M Matsuhashi M D Song F Ishino M Wachi M Doi M Inoue K Ubukata N Yamashita M Konno 《Journal of bacteriology》1986,167(3):975-980
A novel penicillin-binding protein, PBP-2' (Mr about 75,000), is known to be induced in excessively large amount by most beta-lactam compounds in cells of a clinically isolated strain of Staphylococcus aureus, TK784, that is highly resistant to beta-lactams and also most other antibiotics. This protein has very low affinities to most beta-lactam compounds and has been supposed to be the cause of the resistance of the cells to beta-lactams. A 14-kilobase DNA fragment was isolated from the cells that carried the gene encoding this penicillin-binding protein and also a genetically linked marker that is responsible for the resistance to tobramycin. This DNA was cloned on plasmid pACYC184 and was shown to cause both production of PBP-2' and resistance to tobramycin in Escherichia coli cells. However, the formation of PBP-2' in E. coli was only moderate and was independent of normal inducer beta-lactams. The PBP-2' formed in the E. coli cells showed slow kinetics of binding to beta-lactams similar to that of PBP-2' formed in the original S. aureus cells and gave a similar pattern of peptides to the latter when digested with the proteolytic V8 enzyme of S. aureus. 相似文献
19.
The single-stranded region of the amino acid acceptor stem corresponding to the 3'-end of E. coli tRNAMetf was replaced by ligation of chemically synthesized ribooligonucleotides, in order to change the length of the single-stranded CCA terminus. The chemically synthesized ribooligomers, CCA, ACCA, AACCA and CAACCA, were ligated to nuclease-treated E. coli tRNAMetf, which lacked the ACCA sequence at the 3'-end. The methionine acceptor activities of these modified tRNAs were examined using E. coli methionyl-tRNA synthetase. Ligation of the chemically synthesized pentamer (AACCA) to the acceptor terminus restored the methionine acceptor activity, whereas ligation of the hexamer (CAACCA) or trimer (CCA) to the acceptor terminus did not Modification of the acceptor terminus had no effect on the formylation of accepted methionine. 相似文献
20.
Native and non-native conformation-specific antibodies directed to the loop region of hen egg-white lysozyme 总被引:1,自引:0,他引:1
Two types of antibodies were differentiated in conventional guinea pig anti-hen egg-white lysozyme (HEL) antisera. The specificities of both antibodies were directed to the loop I region (mainly directed to Cys64--Cys80 loop) but the antibodies were distinct in respect of reactivities with native HEL. One type of antibody reacted with HEL and loop-peptides of HEL but not with the completely reduced and carboxymethylated form of loop-peptides (native conformation specific antibody: NC-Ab). On the other hand, the other type of antibody did not react with HEL but reacted with loop-peptides and also with the completely reduced and carboxymethylated form of loop-peptides (non-native conformation specific antibody: NNC-Ab). The percentage of NNC-Ab in loop I reactive antibody fraction from pooled guinea pig anti-HEL antisera obtained by two different immunization methods was about 25%. Since the affinities of the NNC-Ab to loop-related peptides were higher by one order of magnitude than those of the NC-Ab to the same peptides, care is necessary in evaluating antigenic determinants in native protein. The immunization of guinea pigs with Ploop I . II [sequence 57-107 (Cys64-Cys80, Cys76-Cys94)] evoked an antibody population having specificity similar to but not identical with that of the NNC-Ab type anti-loop I antibody in conventional anti-HEL antisera. 相似文献