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171.
172.
173.
Protonation of histidine and histidine-tryptophan interaction in the activation of the M2 ion channel from influenza a virus 总被引:3,自引:0,他引:3
The M2 protein of influenza A virus forms a homotetramer ion channel in the lipid membrane. The channel is specific for proton conductance and is activated by low pH with a transition midpoint at pH 5.7. We have studied the structure of the transmembrane domain of the M2 ion channel by using UV resonance Raman spectroscopy, with special attention to the side chains of histidine (His37) and tryptophan (Trp41) residues. The Raman spectra provide direct evidence that the imidazole ring of His37 is protonated upon channel activation at low pH. Concomitantly, the UV resonance Raman scattering from Trp41 shows an unusual intensity change, which is ascribed to a cation-pi interaction between the protonated (cationic) imidazole ring of His37 and the indole ring of Trp41. The protonation of His37 and the Raman intensity change of Trp41 do not occur in the presence of amantadine that blocks the M2 ion channel. These observations clearly show that the protonation of His37 and concomitant cation-pi interaction with Trp41 is a key step in the activation of the M2 ion channel. The His37-Trp41 interaction associated with the channel activation is explained by assuming a conformational transition of His37 induced by electrostatic repulsion among the protonated imidazole rings of four His37 residues in the tetramer channel. Trp41 may play a role in stabilizing the channel open state through cation-pi interaction with His37. A molecular model for the activation of M2 ion channel is proposed on the basis of the gating mechanism. 相似文献
174.
Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups and transports electron equivalents across the vesicle membranes to convert intravesicular monodehydroascorbate radical to ascorbate. We found previously that treatment of oxidized cytochrome b(561) with diethyl pyrocarbonate caused specific N-carbethoxylation of three fully conserved residues (His88, His161, and Lys85) located at the extravesicular side. The modification lead to a selective loss of the electron-accepting ability from ascorbate without affecting the electron donation to monodehydroascorbate radical [Tsubaki, M., Kobayashi, K., Ichise, T., Takeuchi, F., and Tagawa, S. (2000) Biochemistry 39, 3276-3284]. In the present study, we found that these modifications lead to a drastic decrease of the midpoint potential of heme b at the extravesicular side from +60 to -30 mV. We found further that the O-carbethoxylation of one tyrosyl residue (Tyr218) located at the extravesicular side was significantly enhanced under alkaline conditions, leading to a very slow reduction process of the oxidized heme b with ascorbate. On the other hand, the presence of ascorbate during the treatment with diethyl pyrocarbonate was found to suppress the carbethoxylation of His88, His161, and Tyr218, whereas the modification level of Lys85 was not affected. Concomitantly, the final reduction level of heme b with ascorbate was protected, although the fast reduction phase was not fully restored. These results suggest that the two heme-coordinating histidyl residues (His88 and His161) are also a part of the ascorbate binding site. Tyr218 and Lys85 may have a role in the recognition/binding process for ascorbate and are indispensable for the fast electron transfer reaction. 相似文献
175.
Takasaki Y Kogure T Takeuchi K Kaneda K Yano T Hirokawa K Hirose S Shirai T Hashimoto H 《Journal of immunology (Baltimore, Md. : 1950)》2001,166(7):4780-4787
Proliferating cell nuclear Ag (PCNA) occurs as a component of multiprotein complexes during cell proliferation. We found the complexes to react with murine anti-PCNA mAbs, but not with anti-PCNA Abs in lupus sera. The complexes were purified from rabbit thymus extract by affinity chromatography using anti-PCNA mAbs (TOB7, TO17, and TO30) and analyzed by ELISA, immunoprecipitation, immunoblotting, and HPLC gel filtration. That PCNA was complexed with other proteins was demonstrated by its copurification with a group of proteins excluded by an HPLC G3000 SW column. Although immunoblot analysis showed the mAbs to react exclusively with the 34-kDa PCNA polypeptide, they nonetheless immunoprecipitated the same group of proteins, confirming the interaction of the isolated PCNA with other proteins. Anti-PCNA sera, including AK, which reacts with biologically functional sites on PCNA, did not react with complexed PCNA, but did react with it once it was dissociated from the complexes. PCNA complexes in turn reacted with murine anti-DNA mAbs, as well as with Abs against p21, replication protein A, DNA helicase II, cyclin-dependent kinases 4 and 5, and topoisomerase I. These findings suggest that the PCNA complexes purified using anti-PCNA mAbs comprise the "protein machinery" for DNA replication and cell cycle regulation. They also suggest that anti-PCNA mAbs are useful tools with which to characterize the protein-protein interactions within PCNA complexes, as well as the autoimmune responses to proteins interacting with PCNA, which may shed light on the mechanisms of autoantibody production in lupus patients. 相似文献
176.
Seki E Tsutsui H Nakano H Tsuji N Hoshino K Adachi O Adachi K Futatsugi S Kuida K Takeuchi O Okamura H Fujimoto J Akira S Nakanishi K 《Journal of immunology (Baltimore, Md. : 1950)》2001,166(4):2651-2657
IL-18, produced as biologically inactive precursor, is secreted from LPS-stimulated macrophages after cleavage by caspase-1. In this study, we investigated the mechanism underlying caspase-1-mediated IL-18 secretion. Kupffer cells constantly stored IL-18 and constitutively expressed caspase-1. Inhibition of new protein synthesis only slightly reduced IL-18 secretion, while it decreased and abrogated their IL-1beta and IL-12 secretion, respectively. Kupffer cells deficient in Toll-like receptor (TLR) 4, an LPS-signaling receptor, did not secrete IL-18, IL-1beta, and IL-12 upon LPS stimulation. In contrast, Kupffer cells lacking myeloid differentiation factor 88 (MyD88), an adaptor molecule for TLR-mediated-signaling, secreted IL-18 without IL-1beta and IL-12 production in a caspase-1-dependent and de novo synthesis-independent manner. These results indicate that MyD88 is essential for IL-12 and IL-1beta production from Kupffer cells while their IL-18 secretion is mediated via activation of endogenous caspase-1 without de novo protein synthesis in a MyD88-independent fashion after stimulation with LPS. In addition, infection with Listeria monocytogenes, products of which have the capacity to activate TLR, increased serum levels of IL-18 in wild-type and MyD88-deficient mice but not in caspase-1-deficient mice, whereas it induced elevation of serum levels of IL-12 in both wild-type and caspase-1-deficient mice but not in MyD88-deficient mice. Taken together, these results suggested caspase-1-dependent, MyD88-independent IL-18 release in bacterial infection. 相似文献
177.
The effect of jasplakinolide. an actin-polymerizing and filament-stabilizing drug, on the growth, encystation, and actin cytoskeleton of Entamoeba histolytica and Entamoeba invadens was examined. Jasplakinolide inhibited the growth of E. histolytica strain HM-1:IMSS and E. invadens strain IP-1 in a concentration-dependent manner, the latter being more resistant to the drug. The inhibitory effect of jasplakinolide on the growth of E. histolytica trophozoites was reversed by removal of the drug after exposure to 1 microM for 1 day. Encystation of E. invadens as induced in vitro was also inhibited by jasplakinolide. Trophozoites exposed to jasplakinolide in encystation medium for 1 day did not encyst after removal of the drug, whereas those exposed to the drug in growth medium for 7 days did encyst without the drug. The process of cyst maturation was unaffected by jasplakinolide. Large round structures were formed in trophozoites of both amoebae grown with jasplakinolide; these were identified as F-actin aggregates by staining with fluorescent phalloidin. Accumulation in trophozoites of both amoebae of actin aggregates was observed after culture in jasplakinolide. Also, E. invadens cysts formed from trophozoites treated with jasplakinolide contained the actin aggregate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis revealed that the jasplakinolide treatment led to an increase in the proportion of F-actin associated with formation of the aggregate. The results suggest that aggregates are formed from the cortical flow of F-actin filaments, and that these filaments would normally be depolymerized but are artificially stabilized by jasplakinolide binding. 相似文献
178.
Cytostatin, which is isolated from a microbial cultured broth as a low molecular weight inhibitor of cell adhesion to extracellular matrix (ECM), has anti-metastatic activity against B16 melanoma cells in vivo. In this study, we examined a target of cytostatin inhibiting cell adhesion to ECM. Cytostatin inhibited tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin upon B16 cell adhesion to fibronectin. While the amount of FAK was not affected by cytostatin, electrophoretically slow-migrating paxillin appeared. Alkaline phosphatase treatment diminished cytostatin-induced slow-migrating paxillin. Furthermore, cytostatin increased intracellular serine/threonine-phosphorylated proteins and was found to be a selective inhibitor of protein phosphatase 2A (PP2A). Cytostatin inhibited PP2A with an IC(50) of 0.09 microgram/ml in a non-competitive manner against a substrate, p-nitrophenyl phosphate, but it had no apparent effect on other protein phosphatases including PP1, PP2B and alkaline phosphatase even at 100 microgram/ml. On the contrary, dephosphocytostatin, a cytostatin analogue, without inhibitory effect on PP2A did not affect B16 cell adhesion including FAK and paxillin. These results indicate that cytostatin inhibits cell adhesion through modification of focal contact proteins such as paxillin by inhibiting a PP2A type protein serine/threonine phosphatase. This is the first report that describes a drug with anti-metastatic ability that inhibits PP2A selectively. 相似文献
179.
180.
Takami H Nakasone K Ogasawara N Hirama C Nakamura Y Masui N Fuji F Takaki Y Inoue A Horikoshi K 《Extremophiles : life under extreme conditions》1999,3(1):29-34
The nucleotide sequences of three independent fragments (designated no. 3, 4, and 9; each 15–20 kb in size) of the genome
of alkaliphilic Bacillus sp. C-125 cloned in a λ phage vector have been determined. Thirteen putative open reading frames (ORFs) were identified in
sequenced fragment no. 3 and 11 ORFs were identified in no. 4. Twenty ORFs were also identified in fragment no. 9. All putative
ORFs were analyzed in comparison with the BSORF database and non-redundant protein databases. The functions of 5 ORFs in fragment
no. 3 and 3 ORFs in fragment no. 4 were suggested by their significant similarities to known proteins in the database. Among
the 20 ORFs in fragment no. 9, the functions of 11 ORFs were similarly suggested. Most of the annotated ORFs in the DNA fragments
of the genome of alkaliphilic Bacillus sp. C-125 were conserved in the Bacillus subtilis genome. The organization of ORFs in the genome of strain C-125 was found to differ from the order of genes in the chromosome
of B. subtilis, although some gene clusters (ydh, yqi, yer, and yts) were conserved as operon units the same as in B. subtilis.
Received: April 17, 1998 / Accepted: June 23, 1998 相似文献