巻号一覧
119 巻, 6 号
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Hiroyuki Motoshima, Tadashi Ueda, Taiji Imoto1996 年 119 巻 6 号 p. 1019-1023
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーWe developed a sensitive method for analyzing the conformation of the transition state in the unfolding of hen lysozyme. The activation free energy changes of mutant lysozymes with Gly-Pro and Pro-Gly sequences at the same sites (Gly47Pro47', Pro47Gly47', Gly101Pro102, Prol01Gly102, Gly117Pro118, Pro117Gly118, Gly121Pro122, and Pro121G1y122 lysozymes) were obtained for the unfolding in aqueous solution at pH 5.5 and 35°C. Since we had shown that the difference of energies of the unfolded state in lysozymes having an introduced Gly-Pro or Pro-Gly sequence at the same site was much smaller than the difference of energies of the folded states [Motoshima, H., Ueda, T., Hashimoto, Y., Tsutsumi, M., and Imoto, T. (1995) J. Biochem. 118, 1138-1144], we could estimate the difference of energies of the folded and the transition states unequivocally. We defined the °-value as the ratio of the difference in the free energy change in the transition state to that in the free energy change in the folded state between lysozymes with Gly-Pro and Pro-Gly sequences at the same site. The °-values gave information on how much the mutated sites retained the folded structure in the transition state. These values were 0.45 around position 47, which is located in the β-sheet structure, 0.12 at position 101-102, which is located in the loop at the upper part of the active site, 0.17 at position 117-118, which is located in the β-turn and 0.64 at position 121-122, which is located in the 310-helix. Therefore, in the transition state in the unfolding of lysozyme, it was found that the 310-helical region had a similar structure to the intact region, while both the β-turn and the loop at the upper part of the active site were considerably unfolded. The β-sheet structure was also moderately disrupted in the transition state.抄録全体を表示PDF形式でダウンロード (514K) -
Yuki Kunioka, Toshio Ando1996 年 119 巻 6 号 p. 1024-1032
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーWe have studied transglutaminase-catalyzed incorporation of monodansylcadaverine and monobiotincadaverine into rabbit skeletal muscle heavy meromyosin (HMM). The incorporation of dansylcadaverine reached saturation at 4 mol per 1 mol of HMM. An electrophoretogram of the chymotryptic digest of the dansyl-labeled HMM revealed that the labeling took place primarily in the S-2 region of HMM. Atomic force microscopic images and electron micrographs of the complexes of the biotinylated HMM and UltraAvidin-coated fluorescent polyacrylamide nanoparticles revealed that the biotinylated site on S-2 was very close to the C-terminus (near the S-2/light meromyosin junction). In keeping with this result, together with HMM's key sites being localized on the S-1 region, the enzymatic conjugation of biotincadaverine had no influence upon the actin-activated ATPase activity of HMM or upon the ability of HMM to actuate sliding of actin filaments in in vitro motility assay. Attachment of an UltraAvidin-coated fluorescent nanobead to the biotinylated HMM also did not alter the motile activity of HMM. Thus, we can optically pinpoint individual HMM molecules in a sample, which will facilitate handling and manipulation of single HMM molecules and observation of their functional behavior.抄録全体を表示PDF形式でダウンロード (3237K) -
Shin Murai, Toshiaki Arata, Akio Inoue1996 年 119 巻 6 号 p. 1033-1037
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーWe studied the difference in the structure of head B (Pi-burst head) and head A of myosin by limited tryptic digestion of myosin subfragment-1 (S-1), and using antibodies (anti-A and anti-B) which bind specifically with each head. The antibodies were prepared using peptides with sequences identical to those around the reactive lysine residue of heads A and B. When myosin subfragment-1 (S-1) was cleaved limitedly by trypsin, S-1 heavy chain (100 kDa) was digested into fragments of 25, 50, and 20 kDa. Two fragments with molecular masses of 75 and 27 kDa were transiently produced in the initial phase of digestion. Anti-A and anti-B antibodies bound only with peptides that contained the reactive lysine residue [S-1 heavy chain (100 kDa), 75-, 27-, and 25-kDa peptides], thus showing specific binding with antigen peptide. However, the 27-kDa fragment bound more strongly with anti-B antibody than with anti-A antibody. When S-1 was separated into fractions rich in S-1A and S-1B using insoluble anti-A or anti-B antibody, each antibody bound more strongly with the S-1 heavy chain (100 kDa) of its corresponding fraction by Western immunoblotting. These results suggest that the antibodies react specifically with peptides even after SDS-PAGE and membrane-blotting, and that the structure of the 25 kDa-50 kDa junction differs between heads A and B of myosin.抄録全体を表示PDF形式でダウンロード (2998K) -
Shunji Izuta, Mizue Kohsaka-Ichikawa, Toyofumi Yamaguchi, Mineo Saneyo ...1996 年 119 巻 6 号 p. 1038-1044
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーIn order to elucidate the biological activities of cordycepin (3'-deoxyadenosine) and related 3'-deoxyribonucleosides on eukaryotic DNA replication, the inhibitory effects of triphosphate derivatives of 3'-deoxyadenosine (3'-dATP), 8-azido-3'-deoxyadenosine (8-N3-3'-dATP), 3'-deoxyguanosine (3'-dGTP), 3'-deoxyuridine (3'-dUTP), 5-fluoro-3'-deoxyuridine (5-F-3'-dUTP), 3'-deoxycytidine (3'-dCTP), and 5-fluoro-3'-deoxycytidine (5-F-3'-dCTP) on DNA primase and replicative DNA polymerases α, δ, and ε purified from cherry salmon (Oncorhynchus masou) testes or calf thymus were examined. All analogs, except 8-N3-3'-dATP, showed strong inhibitory effects on DNA primase, but none of them inhibited DNA polymerases α, δ, or ε. Kinetic analysis revealed that the inhibition modes of them were competitive with respect to the incorporation of natural substrate that had the corresponding base moiety and non-competitive with respect to other substrates. Based on the kinetic data, we compared the affinities of 3'-dNTPs between DNA primase and RNA polymerases I and II, since 3'-dNTPs also inhibit eukaryotic RNA polymerases. Although the Ki values for DNA primase were much larger than those for RNA polymerases, the Ki/Km values, which indicate the affinity of the analog to the enzyme, were very similar.抄録全体を表示PDF形式でダウンロード (1956K) -
Shin-ya Ohki, Kazunori Miura, Moyoko Saito, Ken-ichi Nakashima, Hirono ...1996 年 119 巻 6 号 p. 1045-1055
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーUsing two- and three-dimensional NMR techniques, 1H and main-chain 15N resonances of the N-terminal half domain of yeast calmodulin (YCM0-N) in the presence of Mg2+ and Ca2+ (Mg2+- and Ca2+-forms) were assigned. The secondary structures of YCM0-N in both forms were determined. The NOESY and 15N-edited NOESY spectra of YCM0-N in each form indicate that there is a hydrophobic core and that two Ca2+-binding loops are connected by a short antiparallel β-sheet. There are four helices (A, B, C, and D named from the N-terminus) for YCM0-N in the Mg2+-form. The B-helix is, however, not formed in the Ca2+-form. The Ca2+-binding of YCM0-N was monitored by (1H, 15N)-HSQC at various Ca2+ concentrations. The observed spectral changes as a function of Ca2+-concentration can not readily be grouped into a small number of classes; each residue shows individual spectral change. There is no apparent relationship between the spectral change and the type or location of the amino acid concerned.抄録全体を表示PDF形式でダウンロード (1190K) -
Itaru Yamamoto, Tetsuhiko Matsunaga, Kei Sakata, Yuko Nakamura, Seiich ...1996 年 119 巻 6 号 p. 1056-1061
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーWe previously reported the augmentation by sodium butyrate (NaBu) of IL-4-induced IgE production in LPS-stimulated murine B-lymphocytes. Histone deacetylase inhibition may be involved in the molecular mode of action of NaBu. Thus, this study was accomplished to examine the involvement of histone hyperacetylation in IL-4-induced class switching promotion by NaBu with a specific histone deacetylase inhibitor, trichostatin A (TSA). TSA was found to enhance IL-4-dependent IgE production in a concentration-dependent manner (0.3-30nM) as did NaBu, although neither compound affected the IgE production in the absence of IL-4. The effect of combined addition of suboptimal concentrations of NaBu and TSA to the culture appeared to be additive, not synergistic. Moreover, the intense of enhancement of IgE production observed by addition of both compounds at optimal concentrations appeared to be equivalent to that obtained with each maximal concentration. Furthermore, enhancement of IgE production by TSA or NaBu was confirmed to be absolutely IL-4 dependent and was not due to the shift of kinetics. On the other hand, various cell cycle inhibitors such as caffeine and theophylline (G1 or G2-arrest), hydroxyurea (S-arrest), and colchicine (M-arrest), none of which have the ability to inhibit histone deacetylase, were shown to have no effect. These findings suggest that histone hyperacetylation plays an important regulatory role in the modification of IL-4-dependent class switching to Cε in LPS-stimulated murine B-cells by NaBu.抄録全体を表示PDF形式でダウンロード (763K) -
Ken-Ichi Kodaira, Masaya Oki, Makiko Kakikawa, Hisashi Kimoto, Akira T ...1996 年 119 巻 6 号 p. 1062-1069
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe microvirid phage φK, specific for Escherichia coli K12, contains a circular singlestranded (SS) DNA in the icosahedral virion, which comprises four phage gene products, F (capsid), G (major spike), H (minor spike), and J (core). φKhT, a host-range mutant of φK, can grow on E. coli C and B, besides K12, and is more thermosensitive than the parental phage φK. Sequencing analysis revealed that the genome of φK and φKhT consists of 6, 089 nucleotides (nt), and codes for eleven genes, whose sequences are similar to those of α3, φX174, and G4 infective to strain C. In φKhT, two nt had changed: one is in the gene G, resulting in replacement of the 75th codon Ala with Ser, and the other is at 67th codon of the gene H: Val to Ala. Chemically synthesized gene J protein composed of 23 amino acids (aa) binds to φK SS DNA more tightly than and preferentially over the host E. coli SS-DNA-binding protein (SSB). These results indicate that the two spike proteins G and H are involved in the determination of φK host-range, and support a model in which the gene J protein functions in packaging the viral SS DNA into the virion vesicle.抄録全体を表示PDF形式でダウンロード (1802K) -
Yukio Shirasawa, Sumie Sato, Toshio Osawa, Akikazu Hirashima1996 年 119 巻 6 号 p. 1070-1075
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーTo investigate the dominant-negative inhibition of Moloney murine leukemia virus (MoMuLV) proliferation by polymerization-defective mutants of reverse transcriptase (RT), we constructed several plasmids harboring the Mo-MuLV RT gene in which the YVDD sequence, one of the conserved sequences in RNA-dependent polymerases, was altered, and then transformed mouse NIH3T3 cells and Escherichia coli with the mutant plasmids. Mouse NIH3T3 cells expressing these mutant RT genes were highly resistant to Mo-MuLV proliferation. The mutant RT expressed in E. coli exhibited no polymerization activity, but it retained its binding activity to the template RNA and inhibited in vitro poly (dG) synthesis occurring with the wild-type RT. These results suggest that the competition for binding of the two types of enzymes to the template is responsible for the resistance to Mo-MuLV proliferation and that the YVDD sequence of Mo-MuLV may be a good target for dominant-negative inhibition of retroviral proliferation.抄録全体を表示PDF形式でダウンロード (2076K) -
Takahiko Nojima, Itaru Nitta, Takuya Ueda, Kimitsuna Watanabe1996 年 119 巻 6 号 p. 1076-1079
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーWe demonstrate here that ribosomes from not only Escherichia coli and Thermus thermophilus [Nitta et al. (1994) J. Biochem. 115, 803-807; J. Biochem., (1995) 118, 841-849] but also yeast and bovine mitochondria catalyze peptide synthesis promoted by a high concentration of pyridine in the absence of soluble protein factors and chemical energy sources, and compare some characteristic features of the reactions among these organisms. Sensitivities against antibiotics, chloramphenicol and cycloheximide, showed the same tendency to those in the in vitro aqueous translation systems of these organisms, suggesting that the basic mechanism for peptide synthesis is the same among these organisms. The optimal concentration of pyridine was centered at 50% for all systems, although the dependencies on the pyridine concentrations and the yields of the products were different from one another. All these systems required Mg2+, and only mitochondrial system showed the extra Mn2+-requirement, which enhanced the yield by several fold. The optimum reaction temperatures coincided closely with the growing temperatures of the organisms except for the mitochondrial system, which showed the highest activity above 80°C. The rationale for these observations remains to be solved.抄録全体を表示PDF形式でダウンロード (1849K) -
Thiên-Ngoc Pham, Kaeko Hayashi, Ryo Takano, Hiroshi Nakazawa, Ha ...1996 年 119 巻 6 号 p. 1080-1085
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーFungal protease inhibitor F (FPI-F) from silkworm hemolymph is a novel serine protease inhibitor of the Bombyx family. The cDNA of FPI-F was introduced into a baculovirus vector and a recombinant virus was isolated and plaque-purified. The protease inhibitory activities increased in the culture medium of insect cells and in the hemolymph of silkworms infected with the recombinant virus. Judged from the behavior on ion-exchange and reversed-phase chromatographies, amino acid compositions, amino-terminal sequences, and CD spectra, the recombinant FPI-F was identical with native FPI-F. Infection with the recombinant virus caused inhibition of larval development of the silkworm. However, the degree of the effect was different in two strains, Shinryukaku and Taiheichoan, indicating that the selection of the strain of silkworm is important in using the baculovirus expression system.抄録全体を表示PDF形式でダウンロード (700K) -
Takanori So, Tadashi Ueda, Yoshito Abe, Tomoko Nakamata, Taiji Imoto1996 年 119 巻 6 号 p. 1086-1093
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーWe selectively introduced monomethoxypolyethylene glycol (mPEG) 5000, 2000, and 550 into Asp119 in lysozyme. To examine how the mPEGs were present on the surface of the modified lysozyme, the activities, binding abilities to the Fab fragment of anti-lysozyme monoclonal antibody, net charges and nuclear magnetic resonance (NMR) spectra of mPEG lysozymes were examined. With the increase in molecular weight of mPEG, the activities and binding abilities to the Fab of mPEG lysozyme decreased. However, introduced mPEG5000 did not cause complete inhibition of the activities and binding abilities to the Fab, while the maximum length of mPEG5000 was so great that it largely covered the surface of the lysozyme molecule. Analyses of the net charges and NMR suggested that the introduced mPEG preferentially assumed a folded conformation on the surface rather than spread all over the surface. Based on the structure of mPEG lysozyme, the mechanism of the reduced immunogenicity of mPEG lysozyme was discussed.抄録全体を表示PDF形式でダウンロード (1803K) -
Shimpei Morita, Masami Fukase, Masami Yamaguchi, Yuhei Morita1996 年 119 巻 6 号 p. 1094-1099
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe substrate specificity of a novel serine protease isolated from soybean seeds, cultivar Keburi, was investigated using various peptide-MCAs and several neuropeptides involving single and paired basic amino acid sequences. The protease was quite specific for arginine residue at the P1 site of the active center, and it recognized paired Arg-Arg and cleaved at the linkage between Arg-Arg or after Arg-Arg in peptide and protein molecules. This is the first protease in plant tissues which resembles in substrate specificity the arginine-specific serine proteinases from porcine gastric and intestinal mucosa, recognizing paired basic amino acid sequences.抄録全体を表示PDF形式でダウンロード (3439K) -
Yoshiaki Nagata, Jun Nakamura, Taibo Yamamoto1996 年 119 巻 6 号 p. 1100-1105
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーSareoplasmic reticulum (SR) vesicles were isolated from scallop muscle by the method of Abe et at. (J. Biochem. 112, 822-827, 1992) and their thermolability was examined in the presence and absence of Ca2+. When SR was preincubated at 38°C in the presence of 0.1mM Ca2+, Ca2+ transport activity decreased as a function of time with a half-inhibition time of about 5 min. Activities of the Ca2+-dependent ATPase, phosphoenzyme (EP) formation and E2 to E1 transition were decreased by the heat treatment in parallel with the Ca2+ transport activity. In contrast, when SR was preincubated at 38°C in the presence of 2-5 mM EGTA, all of these activities, except for the Ca2+ transport, were markedly protected from the heat inactivation. The uncoupling between Ca2+ transport and the ATPase reaction did not lead to a rise in the Ca2+ permeability of SR membrane. Plots of the ATPase activity or steadystate level of EP against pCa in the thermal incubation medium revealed a typical sigmoidal curve with a half-inhibition concentration and Hill number of about 0.5μM and 1.80, respectively. These results suggest that 2 mol of Ca2+ must be removed from the highaffinity Ca2+ binding sites on the ATPase to stabilize the Ca2+-ATPase against heat inactivation. The protection from heat inactivation disappeared if SR was preincubated at 38°C after having been solubilized with a nonionic detergent, but returned when the detergent was removed to reconstitute the SR membrane. These results suggest that the protection of ATPase from thermal inactivation in EGTA may require a membrane structure in which the ATPase molecules exist in an appropriate arrangement.抄録全体を表示PDF形式でダウンロード (682K) -
Yoshiaki Kouzuma, Keiko Kawano, Makoto Kimura, Nobuyuki Yamasaki, Tomo ...1996 年 119 巻 6 号 p. 1106-1113
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーTwo proteinaceous cysteine proteinase inhibitors (cystatins) referred to as Sca and Scb were purified to homogeneity from the seeds of sunflower (Heliantas annuus) by gel filtration on Sephadex G-75 followed by a series of ion-exchange column chromatographies and reverse-phase HPLC (RP-HPLC). The isoelectric points (pI) of Sea and Scb were estimated to be 5.6 and 9.6, respectively. The inhibitory potencies of these two cystatins were examined with cysteine proteinases from various sources, such as plants, mammals, and bacteria. Papain was strongly inhibited by both Sca and Scb with K1 values of 5.6×10-9 and 1.7×10-10M, respectively. Sca and Scb were also found to be potent inhibitors of ficin (K1 values of 1.9×10-6 and 2.8×10-9M, respectively). Rat cathepsin H was inhibited strongly by Scb and slightly by Sea. Although rat cathepsins B and L were significantly inhibited by Seb, they were scarcely affected by Sea. Neither Sca nor Scb inhibited Arg-gingipain, an arginine-specific cysteine proteinase of Porphyromonas gingivalis. The complete amino acid sequences of the two inhibitors were determined by protein chemical methods. The proteins Sea and Scb consist of 83 and 101 amino acid residues with Mr of 9, 330 and 11, 187, respectively, and there are identical residues at 34 positions in the two sequences, that is at 42% of the residues compared. Comparison of their sequences with those of other cystatins revealed that Sea shares 59-73% identical residues with other phytocystatins, while Scb shows less identity to other phytocystatins, sharing only 28-38% identical residues. Furthermore, only 20-27% of the residues of both cystatins, Sca and Scb, are identical to those of the animal cystatins.抄録全体を表示PDF形式でダウンロード (1744K) -
Chiaki Setoyama, Retsu Miura, Yasuzo Nishina, Kiyoshi Shiga, Hisashi M ...1996 年 119 巻 6 号 p. 1114-1117
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe cDNA for porcine kidney D-amino acid oxidase (DAO) was cloned by means of the reverse transcription-polymerase chain reaction system from porcine kidney RNA and over-expressed in Escherichia coli which had been transformed with a vector containing the DAO cDNA. The expressed DAO was purified to homogeneity by a three-step procedure, i.e., heat-treatment, DEAE Sepharose column chromatography, and hydroxyapatite column chromatography. The purified DAO preparation, rDAO (recombinant DAO), showed an identical UV-visible absorption spectrum and catalytic activity with those of the wild-type enzyme purified from porcine kidney. Crystallization of rDAO was performed by the hanging-drop method and crystals of suitable quality for X-ray crystallography were obtained. The crystals so obtained diffracted to 2.5 Å with a conventional X-ray source, and to 2.0 Å with synchrotron radiation. The crystals belong to the orthorhombic space group P212121 with unit cell dimensions of a=110.3, b=92.9, c=71.6 Å. A Vm value of 2.35 Å3/Da indicates that there are two subunits related by a twofold non-crystallographic axis in the asymmetric unit. Two heavy atom derivatives have been identified.抄録全体を表示PDF形式でダウンロード (1528K) -
Hwei-Ling Peng, Wen-Ling Deng, Yin-Hsiu Yang, Hwan-You Chang1996 年 119 巻 6 号 p. 1118-1123
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe acoD gene, which encodes a dihydrolipoamide dehydrogenase component of the acetoin dehydrogenase enzyme system of Klebsiella pneumoniae was isolated and the nucleotide sequence determined. The gene is capable of encoding a protein of 465 amino acid residues with conserved binding domains for NAD and FAD, and two redox-active cysteine residues. The acoD gene product exhibited a Michaelis constant of 170μM for NAD, while NADP can not be used as a substrate. The purified enzyme appeared to be a dimer of the acoD gene product. It did not associate tightly with the El and E2 components of either acetoin dehydrogenase or 2-oxoglutarate dehydrogenase to form an active multi-enzyme complex.抄録全体を表示PDF形式でダウンロード (1794K) -
Vesa P. Kontinen, Miyuki Yamanaka, Ken-ichi Nishiyama, Hajime Tokuda1996 年 119 巻 6 号 p. 1124-1130
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーSecE, an essential membrane component of the Escherichia coli protein translocase, consists of 127 amino acid residues. Only a part of the second putative cytoplasmic region comprising some 13 residues is essential for the SecE function as long as the proper topological arrangement is retained. The Trp84 and Pro85 residues of this region are conserved in all eubacterial SecE homologues. The conservation of positively charged residues corresponding to Arg8O and Lys8l is also substantial. We deleted or replaced these residues to assess their roles in the SecE function. Deletion of the Arg80-Lys81 dipeptide did not abolish the SecE function whereas that of Trp84 or Pro85 caused a loss of the function. Strikingly, however, replacement of Pro85 with either Gly, Ser, or Ala, and that of Trp84 with Lys did not abolish the SecE function. These results indicate that the strong conservation of these residues does not reflect their obligatory requirement for the SecE function. A chimeric SecE possessing the cytoplasmic region of the E. coli SecE and the following region of the Bacillus subtilis SecE was able to form the translocation machinery together with SecA, SecY, and SecG. Although a Len to Arg mutation at position 108 has been thought to cause a loss of signal recognition fidelity and thereby suppress a signal sequence defect, the same mutation at position 111 caused a complete loss of the function. The levels of SecY and SecG in the secEcsE501 mutant, which expresses SecE at a decreased level and is sensitive to low temperature, increased upon the expression of functional SecE derivatives, irrespective of the site of mutation, suggesting that the levels of SecY and SecG are co-operatively determined by the level of functional, but not non-functional, SecE. Based on these results, the SecE function in the translocase is discussed.抄録全体を表示PDF形式でダウンロード (3368K) -
Pierre Gans, Jean-Pierre Simorre, Michael Caffrey, Dominique Marion, P ...1996 年 119 巻 6 号 p. 1131-1142
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーSequence-specific 1H and 15N assignments have been made for the amino acids of the ferrocytochrome c2 from Rhodobacter sphaeroides. Initial assignments were made by analysis of a series of homonuclear 2D COSY, TOCSY, and NOESY spectra obtained with the unlabeled protein. 2D and 3D1H-15N correlated spectra obtained for a uniformly 15N-labeled ferrocytochrome c2 were used to confirm and extend the assignments. Partial 13C assignments have also been made by means of HSQC experiments on 13C at natural abundance, in particular for about two-thirds of the 13Cα. Medium-range NOE connectivities, together with 3J(HCαNH) coupling constants, indicated the presence of five helices at positions 6-16, 60-68, 74-82, 84-91, and 109-120. No other regular secondary structure was observed. This folding is similar to that previously observed for the ferrocytochrome c2 of Rhodobacter capsulatus in solution, which exhibits approximately 50% sequence identity. Moreover, the rotation rates of the aromatic rings of phenylalanine or tyrosine, when conserved, were similar to those observed for R. capsulatus. Furthermore, CαH chemical shifts, which are sensitive to the secondary structure and ring current effects of the heme, appear to be very similar for the two proteins. Consequently, the solution structure of R. sphaeroides ferrocytochrome c2 appears to be very similar to that of R. capsulatus ferrocytochrome c2. These results are compared with the X-ray crystal structure of the R. sphaeroides ferrocytochrome c2.抄録全体を表示PDF形式でダウンロード (4833K) -
Yasuo Sugiyama, Yasuo Mukohata1996 年 119 巻 6 号 p. 1143-1149
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe bacterial opsins can be refolded to regenerate the chromophore by transfer from SDS to DMPC/CHAPS/SDS mixed micelles in the presence of retinal. A sequential refolding model has been proposed for bacterioopsin [Booth et al. (1995) Nature Struct. Biol. 2, 139-143]. However, the roles of DMPC and CHAPS in the refolding process are not clear. In this study we measured the effects of DMPC and CHAPS on the refolding of bacterial opsins in vitro by CD and fluorescence spectroscopy. In contrast to in experiments in the presence of large amounts of DMPC, the process of retinal binding pocket formation was a rate-determining step in overall chromophore regeneration with relatively low concentrations of DMPC. CHAPS triggered α-helix formation and long-range interactions between the helices within 1s by providing a suitable hydrophobic environment for bacterial opsins. This CHAPS-induced transient molten globule-like structure would be identical to I1 postulated by Booth et al., to which DMPC bound and induced the proper packing of the side chains to form a retinal binding pocket. If DMPC was not present, CHAPS induced another conformation change in bacterial opsins, which led to denaturation. DMPC dependence of chromophore regeneration and the maintenance of the retinal binding pocket suggested that retinal binding pocket formation was part of the large structure changes during stable apoprotein formation.抄録全体を表示PDF形式でダウンロード (870K) -
Norio Kaneda, Amjad H. Talukder, Hiroko Nishiyama, Shinichi Koizumi, T ...1996 年 119 巻 6 号 p. 1150-1156
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーBy means of a baculovirus expression system, a large amount of mouse midkine (MK) was produced. The protein was purified to homogeneity by heparin-Sepharose column chromatography. The purified protein was of a mature type; the signal peptide was cleaved at the expected site. To examine the neurite-guiding activity of MK, rat embryonic brain cells (embryonic days 17-18) were cultured on plates coated with purified MK in a grid pattern. The cells attached to and extended their neurites along the substrate pattern. This interaction was strongly inhibited by heparin, but not by other glycosaminoglycans. Treatment of the cells with heparitinase was effective for inhibiting their adhesion to the substrate. These data suggest that the heparin-like domain on cell surface heparan sulfate proteoglycan is the primary site for MK binding upon interaction with nerve cells.抄録全体を表示PDF形式でダウンロード (5125K) -
Nam-Ho Choi-Miura, Takashi Tobe, Jun-ichi Sumiya, Yasuko Nakano, Yoshi ...1996 年 119 巻 6 号 p. 1157-1165
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーA novel hyaluronan-binding protein (PHBP) was purified from human plasma by affinity chromatography on hyaluronan-conjugated Sepharose. The contaminating IgM and albumin in the partially purified preparation were removed with anti-IgG antibody-conjugated Sepharose and anti-albumin antibody-conjugated Sepharose, respectively, and no other contaminant was observed. Finally, 800μg of PHBP was isolated from 500ml of human plasma. PHBP gave a single 70-kDa band on SDS-PAGE under non-reducing conditions, and 50-kDa and 17-kDa bands under reducing conditions. Thus, PHBP was a heterodimer composed of 50-kDa and 17-kDa subunits, bridged by a disulfide linkage. Both subunits had novel N-terminal amino acid sequences, indicating that PHBP was a novel hyaluronanbinding protein in human plasma. The amino acid sequence deduced from the nucleotide sequence of the cloned PEEP cDNA exhibited significant homology to that of hepatocyte growth factor activator (HGFA). The results of Northern blot analysis indicated that liver, kidney, and pancreas expressed PHBP mRNA. The predicted structure of PHBP showed three epidermal growth factor (EGF) domains, a kringle domain and a serine protease domain, from its N-terminus, although HGFA has a fibronectin type II domain, an EGF domain, a fibronectin type I domain, an EGF domain, a kringle domain, and a serine protease domain, from its N-terminus.抄録全体を表示PDF形式でダウンロード (4756K) -
Yoshitaka Ikeda, Junichi Fujii, Naoyuki Taniguchi1996 年 119 巻 6 号 p. 1166-1170
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーγ-Glutamyl transpeptidase possesses two histidine residues at positions 383 and 505 which are conserved in all mammalian and bacterial species. In order to elucidate the functions of these residues, we prepared mutants in which these residues were replaced by Ala. Kinetic analysis of the hydrolysis of L-γ-glutamyl-p-nitroanilide indicated that substitution at His-383 decreased the Vmax value to 14% of that of the wild type, but had no effect on Vmax/Km. In reactions involving glycylglycine as the acceptor substrate, the Vmax value of this mutant decreased to 38% with little alteration of Vmax/Km for L-γ-glutamyl-p-nitroanilide as a γ-glutamyl donor, but with a significant reduction of Vmax/Km for the acceptor. These results show that this substitution causes impairment of the step in which the free enzyme is regenerated from the γ-glutamyl enzyme by water or an acceptor substrate. On the other hand, replacement of His-505 resulted in a decrease of the Vmax value for transpeptidation to about 10% of that of the wild type despite no substantial effect on the Vmax value for the hydrolysis reaction. However, this substitution did not affect Vmax/Km for the acceptor on transpeptidation. Thus, the formation of a non-productive enzyme-substrate complex with the acceptor substrate would decrease the Vmax value on transpeptidation. These results suggest that His-383 plays an important catalytic role in facilitating the degradation of the γ-glutamyl-enzyme through hydrolysis or transfer of the γ-glutamyl moiety to an acceptor. It was also shown that His-505 is important in the formation of a complex of the γma;-glutamyl enzyme with the acceptor substrate even though it plays no critical role in the catalysis. Although the pH-dependence profile and the van't Hoff plot for the ionic group responsible for enzyme activity were consistent with the requirement of a histidine residue, neither of the conserved histidines could be assigned as such an ionic group. This suggests that another histidine residue (s) might play an essential role in the enzyme function.抄録全体を表示PDF形式でダウンロード (654K) -
Hisako Muramatsu, Xiao-jun Song, Norio Koide, Hajime Hada, Takao Tsuji ...1996 年 119 巻 6 号 p. 1171-1175
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーMidkine (MK) is a growth factor that promotes neurite outgrowth and survival of neurons, and enhances the plasminogen activator in endothelial cells. A highly sensitive enzymelinked immunoassay for MK was developed, involving affinity-purified anti-MK antibodies, their biotinylated form, and avidin-β-galactosidase. The amount of bound avidin-β-galactosidase was determined using a fluorogenic substrate, 4-methylumbelliferyl-β-D-galactoside. This method allowed the detection of human and mouse MK in the range of 50pg-10ng. Pleiotrophin, which is related to MK in its amino acid sequence, did not show any cross reactivity. Employing this method, the MK levels in the developing mouse brain were determined. The MK level was 2μg/g of wet tissue on the 12th day of gestation, and then steadily decreased during embryogenesis and postnatal development to 30ng/g two months after birth. The assay method can also be applied to serum samples. Although the MK levels in the sera of normal human subjects were low or undetectable, 0.6-8ng/ml of MK was detected in samples in the majority of cases of hepatocellular carcinomas.抄録全体を表示PDF形式でダウンロード (525K) -
Sachiko Okuno, Takako Kitani, Hitoshi Fujisawa1996 年 119 巻 6 号 p. 1176-1181
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーCalmodulin-dependent protein kinase IV (CaM-kinase IV), which plays crucial roles in the functioning of Ca2+ in the central nervous system and immune system, is markedly activated upon phosphorylation by the action of CaM-kinase IV kinase. Northern and Western blot analyses of CaM-kinase IV kinase showed relatively weak reactions in the rat cerebellum, where the activity of CaM-kinase IV kinase has been demonstrated to exist, indicating that CaM-kinase IV kinase isoforms distinct from the enzyme cloned from the cerebral cortex may exist in the cerebellum. When the crude extracts of rat cerebral cortex, brain stem, and cerebellum were immunotitrated with antibody against the cloned enzyme, only approximately 46, 56, and 25% of the enzyme activity of the respective extracts were immunoprecipitated. Thus, at least two distinct isoforms of CaM-kinase IV kinase appear to exist in the brain.抄録全体を表示PDF形式でダウンロード (3316K) -
Shiao-Shek Tang, Gu-Gang Chang1996 年 119 巻 6 号 p. 1182-1188
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe kinetic mechanism of the endogenous glutathione transferase (GST) activity of octopus S-crystallin was investigated by steady-state kinetics. Biphasic double-reciprocal plots were obtained for both glutathione and the hydrophobic substrate 1-chloro-2, 4-dinitrobenzene (CDNB). Substrate inhibition was observed only for CDNB with Ksi value of 29.7±0.01mM. The catalytic constant for S-crystallin was three orders of magnitude smaller than that for the digestive gland GST of the same species. The initial-velocity studies indicated that the enzyme reaction might conform to a steady-state random Bi-Bi kinetic mechanism, being similar to the reaction of GST from other sources. The pH-rate profiles also suggest that the same chemical mechanism for the nucleophilic aromatic substitution between GSH and CDNB was employed for S-crystallin. The interaction of Tyr7 with the bound GSH lowered the pKa value of the sulfhydryl group of GSH to 6.82-6.85, which is 2.32-2.35 pH unit smaller than that found in aqueous solution. This lowering of pKa value produces the thiolate anion of GSH, a better nucleophile to attack the ipso carbon of CDNB, resulting in formation of Meisenheimer complex intermediate. Removing the chloride ion from this intermediate complex produces the conjugate product. Using the method devised by Wang and Srivastava (Anal. Biochem. 216, 15-26, 1994), the functional unit of the dimeric S-crystallin was estimated to be a monomer. The possible biological implications of the endogenous detoxification ability of cephalopods S-crystallin are discussed.抄録全体を表示PDF形式でダウンロード (880K) -
Kanako Momoeda, Keiko Hirota, Tadahito Utsuki, Yoshiaki Tsuchida, Kazu ...1996 年 119 巻 6 号 p. 1189-1195
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーA dramatic change in the glycosphingolipid composition in murine lung occurred between 1 day and 1 week after birth. GlcCer and LacCer were the predominant neutral glycosphingolipids prenatally and 1 day after birth, and the concentrations of globo- and ganglioseries glycosphingolipids increased abruptly from 1 week after birth, reaching maxima at 2-3 weeks. To explore the functional significance of the change, we examined the role of neutral glycosphingolipids as receptors for the murine pulmonary surfactant protein, SP-A, and found that SP-A bound to LacCer, Gg3Cer, and Gg4Cer, but not to Gb3Cer, Gb4Cer, IV3GalNAcα-Gb4Cer, sulfatide, or several gangliosides. On TLC-blotting with 125I-labeled SP-A, the binding of SP-A to Gg3Cer and Gg4Cer was 5 times higher than that to LacCer, and on immunohistochemical staining Gg4Cer and Gg3Cer was mainly observed in the alveolar epithelium. Thus, the capacity to retain SP-A of glycolipid receptors per gram dry weight of lung at 1 week after birth was 1.6 times higher than that at 1 day after birth, and reached a maximum 3 weeks after birth. These findings suggest that the enhanced synthesis of the ganglio-series neutral glycosphingolipids 1 week after birth results in an increase in the binding capacity for SP-A on the epithelial cell surface of alveoli.抄録全体を表示PDF形式でダウンロード (4456K) -
Takashi Hashimoto, Yasuko Shindo, Masayoshi Souri, Graham S. Baldwin1996 年 119 巻 6 号 p. 1196-1201
発行日: 1996年
公開日: 2008/11/18
ジャーナル フリーThe mitochondrial enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacylCoA thiolase trifunetional protein (trifunctional protein) plays a major role in mitochondrial fatty acid oxidation. The enzyme complex consists of four molecules of α-subunit containing both hydratase and dehydrogenase domains and four molecules of β-subunit containing the thiolase domain. The primary structure of a gastrin-binding protein (GBP) was highly homologous to that of the α-subunit of the trifunctional protein. Here, we report that gastrin inhibits the hydratase, dehydrogenase, and thiolase activities of the trifunctional protein. The gastrin/cholecystokinin receptor antagonist benzotript, which inhibited binding of gastrin to the GBP, also inhibited all three activities of the trifunctional protein. In addition, benzotript inhibits the activities of multifunctional enzymes having similar structures, such as the peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein and the Pseudomonas fragi fatty acid oxidation enzyme complex. This reagent, however, hardly inhibited various monofunctional enzymes involved in fatty acid oxidation.抄録全体を表示PDF形式でダウンロード (711K)
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