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991.
Catalytic and structural role of a metal-free histidine residue in bovine Cu-Zn superoxide dismutase
Cu-Zn superoxide dismutase (SOD) contains a conserved, metal-free His residue at an opening of the backbone beta-barrel in addition to six Cu- and/or Zn-bound His residues in the active site. We examined the protonation and hydrogen bonding state of the metal-free His residue (His41) in bovine SOD by UV Raman spectroscopy. Analysis of the His Raman intensity at 1406 cm(-1) in a D2O solution has shown that His41 has a pKa of 9.4, consistent with the NMR and X-ray structures at acidic to neutral pH, in which two imidazole nitrogen atoms of cationic His41 are hydrogen bonded to the main chain C=O groups of Thr37 and His118. Upon deprotonation of His41 at pH 9.4, the Thr37-His41-His118 hydrogen bond bridge breaks on the His118 side and SOD loses 70% of its activity. Concomitantly, hydrogen-deuterium exchange is accelerated for amide groups of beta-strands, indicating an increased conformational fluctuation of the beta-barrel. Thr37 and His41 are in direct contact with Leu36, whose hydrophobic side chain closes off the opening of the beta-barrel, while His118 is indirectly connected to Arg141 that assists the docking of superoxide to Cu. These Raman findings strongly suggest that the His41-mediated hydrogen bond bridge plays a crucial role in keeping the protein structure suitable for highly efficient catalytic reactions. The catalytic and structural role of His41 is consistent with the observation that the mutation of His43 in human SOD (equivalent to His41 in bovine SOD) to Arg largely reduces the dismutase activity and the protein structural stability. 相似文献
992.
Dozol H Maechling C Graff R Matsuda A Shuto S Spiess B 《Biochimica et biophysica acta》2004,1671(1-3):1-8
Four adenophostin analogues lacking the adenine moiety were subjected to 31P- and 1H-NMR titrations in order to determine the acid-base behaviour of the individual ionisable groups of the molecules and the complex interplay of intramolecular interactions resulting from the protonation process. For the two trisphosphorylated compounds, the curve pattern of the phosphorus nuclei corresponds to the superimposition of the titration curves of a monophosphorylated polyol and a polyol carrying two vicinal phosphates, suggesting that the two phosphate moieties behave independently. Also, the general shape of 1H-NMR titration curves of the studied compounds is very close to that of adenophostin A, indicating that the adenine moiety does not specifically interact with the phosphorylated sugar moieties. The curves show, however, that both trisphosphorylated compounds adopt slightly different preferential conformations which could contribute to explain the difference in their affinity for Ins(1,4,5)P3 receptor. Their macroscopic as well as the microscopic protonation constants are higher than those of adenophostin A, indicating that the adenine moiety plays a base-weakening effect on the phosphate groups. Further analysis of the microscopic protonation constants confirms that the compound whose conformation is the closest to that of adenophostin A also shows the highest biological activity. The two bisphosphorylated analogues studied behave very similarly, suggesting that the deletion of the hydroxymethyl group on the pentafuranosyl ring only weakly influences the protonation process of the phosphate groups that bear the glucopyranose moiety. 相似文献
993.
Lin H Sugimoto Y Ohsaki Y Ninomiya H Oka A Taniguchi M Ida H Eto Y Ogawa S Matsuzaki Y Sawa M Inoue T Higaki K Nanba E Ohno K Suzuki Y 《Biochimica et biophysica acta》2004,1689(3):219-228
Gaucher disease (GD) is the most common form of sphingolipidosis and is caused by a defect of beta-glucosidase (beta-Glu). A carbohydrate mimic N-octyl-beta-valienamine (NOV) is an inhibitor of beta-Glu. When applied to cultured GD fibroblasts with F213I beta-Glu mutation, NOV increased the protein level of the mutant enzyme and up-regulated cellular enzyme activity. The maximum effect of NOV was observed in F213I homozygous cells in which NOV treatment at 30 microM for 4 days caused a approximately 6-fold increase in the enzyme activity, up to approximately 80% of the activity in control cells. NOV was not effective in cells with other beta-Glu mutations, N370S, L444P, 84CG and RecNciI. Immunofluorescence and cell fractionation showed localization of the F213I mutant enzyme in the lysosomes of NOV-treated cells. Consistent with this, NOV restored clearance of 14C-labeled glucosylceramide in F213I homozygous cells. F213I mutant beta-Glu rapidly lost its activity at neutral pH in vitro and this pH-dependent loss of activity was attenuated by NOV. These results suggest that NOV works as a chemical chaperone to accelerate transport and maturation of F213I mutant beta-Glu and may suggest a therapeutic value of this compound for GD. 相似文献
994.
995.
Selection and Characterization of Nickel-Tolerant Tobacco Cells 总被引:3,自引:2,他引:1
R. Nakazawa Y. Kameda T. Ito Y. Ogita R. Michihata H. Takenaga 《Biologia Plantarum》2004,48(4):497-502
Tobacco (Nicotiana tabacum L. cv. BY-2) cell lines tolerant to 700 M Ni in which unselected cells can not grow, were selected. The Ni-tolerant cells were also more tolerant to Co, but not to Cd than unselected cells. Ni concentrations in Ni-tolerant cells were always higher than those in medium. Since buthionine sulfoximine did not affect their Ni-tolerance, it is suggested that phytochelatins are not involved in Ni-tolerance of Ni-tolerant cells. On the other hand, histidine contents in Ni-tolerant and unselected cells, which were treated with Ni, were higher that those treated without Ni, and the degree of the elevation of histidine contents by Ni-treatment was higher in Ni-tolerant cells than in unselected cells. Additionally, exogenous histidine reduced the inhibitory effect of Ni on the growth of unselected cells. In addition, the cells that were tolerant to histidine-analogue, had higher contents of histidine and Ni-tolerance. These results suggest that histidine is involved in Ni-tolerance and the detoxification of Ni in symplast in Ni-tolerant cells. 相似文献
996.
Furutani Y Kato A Kawai R Fibriani A Kojima S Hirose S 《Biology of reproduction》2004,71(5):1583-1590
We determined the cDNA and gene structures of guinea pig caltrin II, a unique member of the calcium transporter inhibitors containing a whey acidic protein (WAP) motif, and we established that it is a secretory protein with a potential 21-amino acid signal peptide in its N-terminus. Northern blot analysis and in situ hybridization histochemistry indicated that the expression of caltrin II is restricted to luminal epithelial cells in the seminal vesicles. Its message levels markedly decreased either after castration (and were restored by simultaneous administration of testosterone) or after treatment of the animals with estradiol, suggesting that the expression of caltrin II is androgen-dependent. Recombinant caltrin II had an elastase-inhibitor activity. Comparison of sequence between the caltrin II and related genes and their molecular evolutionary analyses revealed that caltrin II and seminal vesicle secretory proteins (SVPs) appear to be evolved from a common ancestor gene that is made by the fusion of semenogelin and trappin genes. Caltrin II and SVPs lost the transglutaminase substrate domain and the WAP motif, respectively, within a single exon, resulting in the exertion of different functions. 相似文献
997.
Dynamic structure of vesicle-bound melittin in a variety of lipid chain lengths by solid-state NMR
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Solid-state 31P- and 13C-NMR spectra were recorded in melittin-lecithin vesicles composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Highly ordered magnetic alignments were achieved with the membrane surface parallel to the magnetic field above the gel-to-liquid crystalline phase transition temperature (Tc). Using these magnetically oriented vesicle systems, dynamic structures of melittin bound to the vesicles were investigated by analyzing the 13C anisotropic and isotropic chemical shifts of selectively 13C-labeled carbonyl carbons of melittin under the static and magic-angle spinning conditions. These results indicate that melittin molecules adopt an alpha-helical structure and laterally diffuse to rotate rapidly around the membrane normal with tilt angles of the N-terminal helix being -33 degrees and -36 degrees and those of the C-terminal helix being 21 degrees and 25 degrees for DLPC and DPPC vesicles, respectively. The rotational-echo double-resonance method was used to measure the interatomic distance between [1-13C]Val8 and [15N]Leu13 to further identify the bending alpha-helical structure of melittin to possess the interhelical angles of 126 degrees and 119 degrees in DLPC and DPPC membranes, respectively. These analyses further lead to the conclusion that the alpha-helices of melittin molecules penetrate the hydrophobic cores of the bilayers incompletely as a pseudo-trans-membrane structure and induce fusion and disruption of vesicles. 相似文献
998.
999.
Higashi K Yoshida K Nishimura K Momiyama E Kashiwagi K Matsufuji S Shirahata A Igarashi K 《Journal of biochemistry》2004,136(4):533-539
Following the report that agmatine has an anti-proliferative effect on cell growth through induction of antizyme [Satriano et al. (1998) J. Biol. Chem. 273, 15313-15316], we examined the effects of 16 different diamines on cell growth. Many diamines had little or no effect on cell growth, but agmatine and 1,6-hexanediamine had anti-proliferative effects, with agmatine having the strongest effect. Inhibition of cell growth occurred after 2 days, and inhibitory effects paralleled the degree of antizyme induction. Decreased spermine levels indicated that induction of spermidine/spermine N(1)-acetyltransferase was also involved in the inhibition of cell growth by agmatine and 1,6-hexanediamine. The frameshift efficiency (ratio of antizyme synthesis with or without frameshift) measured in a rabbit reticulocyte cell-free system was also increased by 1,3-propanediamine and cis-1,4-cyclohexanediamine in addition to agmatine and 1,6-hexanediamine. However, the intracellular levels of 1,3-propanediamine and cis-1,4-cyclohexanediamine were low when these compounds were added to the cell-culture medium. Other diamines had no effect on cell growth or frameshift efficiency. The results suggest that the presence of two amino-groups separated by an appropriate distance is important for the enhancement of frameshifting by diamines. 相似文献
1000.
Mukhopadhyay D Asari A Rugg MS Day AJ Fülöp C 《The Journal of biological chemistry》2004,279(12):11119-11128
The formation of the hyaluronan-rich cumulus extracellular matrix is crucial for female fertility and accompanied by a transesterification reaction in which the heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI)-related proteins are covalently transferred to hyaluronan. Tumor necrosis factor-induced protein-6 (TNFIP6) is essential for this transfer reaction. Female mice deficient in TNFIP6 are infertile due to the lack of a correctly formed cumulus matrix. In this report, we characterize the specificity of TNFIP6-mediated HC transfer from IalphaI to hyaluronan. Hyaluronan oligosaccharides with eight or more monosaccharide units are potent acceptors in the HC transfer, with longer oligosaccharides being somewhat more efficient. Epimerization of the N-acetyl-glucosamine residues to N-acetyl-galactosamines (i.e. in chondroitin) still allows the HC transfer although at a significantly lower efficiency. Sulfation of the N-acetyl-galactosamines in dermatan-4-sulfate or chondroitin-6-sulfate prevents the HC transfer. Hyaluronan oligosaccharides disperse cumulus cells from expanding cumulus cell-oocyte complexes with the same size specificity as their HC acceptor specificity. This process is accompanied by the loss of hyaluronan-linked HCs from the cumulus matrix and the appearance of oligosaccharide-linked HCs in the culture medium. Chondroitin interferes with the expansion of cumulus cell-oocyte complexes only when added with exogenous TNFIP6 before endogenous hyaluronan synthesis starts, supporting that chondroitin is a weaker HC acceptor than hyaluronan. Our data indicate that TNFIP6-mediated HC transfer to hyaluronan is a prerequisite for the correct cumulus matrix assembly and hyaluronan oligosaccharides and chondroitin interfere with this assembly by capturing the HCs of the IalphaI-related proteins. 相似文献