The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
119 巻, 3 号
選択された号の論文の31件中1~31を表示しています
  • Kyogo Itoh, Akihiro Hayashi, Masanobu Nakao, Tomoaki Hoshino, Naoko Se ...
    1996 年 119 巻 3 号 p. 385-390
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    Characterization of the MACE genes has facilitated a molecular approach to identification of the genes encoding tumor-rejection antigens expressed on human cancer cells. MAGE proteins are normal tissue antigens compartmentalized in testicular cells that play an important role in the early phase of spermatogenesis. MAGE-1, -2, -3, -4, and -6 genes are preferentially expressed in many different cancers at both the mRNA and protein levels. More than half of human cancers of various histologic type express at least one of these MAGE genes. Demethylation induces MAGE antigens in cells, suggesting that MAGE genes are important developmentally regulated genes under methylating control. Thus, genetic instability in cells causing loss of this methylating control could result in the preferential expression of MAGE genes in cancer cells. Therefore, MAGE gene products may be appropriate target molecules for development of new cancer vaccine.
  • Hikoya Hayatsu
    1996 年 119 巻 3 号 p. 391-395
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    Our studies have revealed reagents that can attack the 5, 6-double bond of pyrimidine nucleosides; potassium permanganate and bisulfite. This review is a personal account of these studies, with a discussion on the vulnerable nature of this particular double bond to external nucleophiles and oxidizing agents. The finding that N (4) aminocytidine, produced on treatment of cytidine with bisulfite and hydrazine, is a strong mutagen is also described.
  • Takeshi Kawaguchi, Toshiaki Hamanaka, Yuji Kito
    1996 年 119 巻 3 号 p. 396-399
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    Pure bovine rhodopsin pellets were prepared by removal of cholie acid from rhodopsincholie acid complex, and X-ray diffraction patterns from the internal structure of rhodopsin were obtained for the first time using wet and dry pellet samples. The peaks at around 10 and 4.3 Å spacings observed for both samples can be attributed to α-helices in rhodopsin molecule, providing direct evidence of the helical structure of rhodopsin. A shoulder peak at around 34 Å spacing was also observed for the dry pellet, which can be explained by the first-neighbor distance between rhodopsin molecules in the sample.
  • Shunji Hattori, Keiko Sakai, Kazuo Watanabe, Tadahiko Fujii
    1996 年 119 巻 3 号 p. 400-406
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    Collagen and histone H1 are stained a pink-red color with Coomassie Brilliant Blue R-250 (Coomassie R) instead of the blue color of most proteins after SDS-PAGE. Spectrophotometrically, this metachromasia was characterized by an increase in the absorbance at 535nm and a decrease in the absorbance at 600nm. The ratio of the absorbance at 535nm to that at 600nm ranged from 1.5 to 2.5 for the pink-red-stained proteins and was about 1 for the blue stained proteins. In their amino acid composition, the pink-red-stained proteins collagen and histone H1 contained less than 11% of hydrophobic amino acid residues, whereas the five blue-stained proteins examined contained more than 25% of such residues. Collagen and histone H1 also induce circular dichroism (CD) of Coomassie R in the visible region with a different CD spectrum. In the case of native collagen, a CD (+) band at 530 nm with 105 molar ellipticity was observed, while the denatured collagen showed a CD (-) band at 530nm. When the amino groups of the amino acid residues in collagen and histone H1 were converted into hydrophobic groups by fluorescamine treatment, these proteins stained bluer than pink-red and the induced CD was a lower intensity. This is the first report of the metachromasic interaction between a protein and Coomassie R that is accompanied by CD induction. This report also suggests that the induction of metachromasia and CD of Coomassie R was due to the low content of hydrophobic amino acid residues in the peptides.
  • Masanari Tsujimura, Masafumi Odaka, Shigehiro Nagashima, Masafumi Yohd ...
    1996 年 119 巻 3 号 p. 407-413
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    Nitrile hydratase (NHase) from Rhodococcus sp. N-771 exists in active and inactive forms. The inactive NHase is immediately activated by light irradiation and changes to the active form. To characterize the photoreactive center, the inactive NHase was denatured by 6M urea, and two kinds of subunits (α and β) were separated and purified by anion-exchange chromatography. In a manner similar to the native NHase, the isolated α subunit showed two absorption peaks at 280 and 370nm, which were diminished by light irradiation. However, irradiation failed to elicit the appearance of absorption peaks at around 400nm and at 710nm, which were characteristic of the activated enzyme. The β subunit seemed not to possess any photoreactive chromophore because its absorption spectrum was not altered by light irradiation. Neither of the subunits showed NHase activity before or after light irradiation, but the inactive NHase was reconstituted by incubating the two subunits together in the dark at 4°C for 1h. Light irradiation of the β subunit did not affect subsequent complex formation or NHase activity. However, the irradiated α subunit could not assemble with the β subunit, and no activity was recovered. These results demonstrate that the chromophore (s) responsible for the photoactivation of NHase are entirely located on the α subunit, and imply that light irradiation induces conformational change of the α subunit.
  • Masahiro Iwakura, Shinya Honda
    1996 年 119 巻 3 号 p. 414-420
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    Short peptides which contained a single Cys residue were introduced into both N- and C-termini of the Cys-free mutant of DHFR (Cys85→Ala, Cys152→Ser double mutant) by a recombinant DNA method, then the terminal regions were connected through a disulfide bond by oxidation. The oxidized form and reduced form proteins have as high enzymatic activity as wild-type DHFR. There is no detectable difference between the CD spectra of the reduced and oxidized forms at low (15°C, native condition) and high temperature (80°C, unfolded condition). The thermal transition of the oxidized proteins at the concentration of 0.15mg/ml (8.5μM) is completely reversible as demonstrated by the CD spectra. No aggregated materials were detected in the oxidized protein on gel-filtration HPLC after heat treatment up to the protein concentration of 0.5mg/ml. The reduced protein, however, even in the presence of reducing agent, showed only partial reversibility, with as much as 55 and 95% of the heat-treated protein at the concentrations of 0.15 and 0.5mg/ml being eluted as the high molecular aggregated form, respectively. The apparent transition temperatures (Tm) of the oxidized forms were 5-7°C higher than those of the reduced counterparts. The oxidized protein that had been denatured with guanidine-HCl was eluted later than the denatured reduced protein on gel-filtration HPLC in the presence of 5M guanidine-HCl. The limitation of spatial movement of the termini may prevent intermolecular interaction of exposed domains during denaturation-renaturation process, giving rise to the irreversible denaturation. The flexibility of the terminal is also suggested to be an important factor for improving thermal stability of proteins.
  • Koichi Honke, Miwako Yamane, Atsushi Ishii, Takahiko Kobayashi, Akira ...
    1996 年 119 巻 3 号 p. 421-427
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    We have purified 3'-phosphoadenosine-5'-phosphosulfate: GalCer sulfotransferase [EC 2. 8. 2. 11] from a human renal cancer cell line SMKT-R3 through a combination of affinity chromatographies using galactosylsphingosine, 3', 5'-bisphosphoadenosine and heparin as ligands. The purified sulfotransferase showed a specific activity of 1.2μmol/min/mg, which is 300 times more than the highest activity among the enzyme preparations purified so far from other sources. Homogeneity of the purified sulfotransferase was supported by the facts that the enzyme preparation showed a single protein band with an apparent molecular mass of 54 kDa on reducing SDS-PAGE and that protein bands coincided with the enzyme activity on both native PAGE and nonreducing SDS-PAGE. GalCer was the best acceptor for the purified enzyme. LacCer, GalAAG, and GalDG were also good acceptors. GlcCer, Gg3Cer, Gg4Cer, Gb4Cer, and nLc4Cer did serve as acceptors although the relative activities were low. On the other hand, the enzyme could not act on Gb3Cer, which possesses α-galactoside at the nonreducing terminus. Neither galactose nor lactose served as an acceptor. These observations suggest that the sulfotransferase prefers β-glycoside, especially β-galactoside, at the nonreducing termini of sugar chains attached to a lipid moiety.
  • Thiên-Ngoc Pham, Kaeko Hayashi, Ryo Takano, Masanobu Itoh, Masah ...
    1996 年 119 巻 3 号 p. 428-434
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    The positions of the reactive site and the disulfide bridges in fungal protease inhibitor F (FPI-F) from silkworm (Bombyx mori), which has a unique amino acid sequence and inhibitory specificity, were investigated. At pH 3.0, subtilisin BPN', which is one of target proteases of this inhibitor, specifically cleaved the peptide bond of the inhibitor at Thr (29)-Val (30). The cleaved bond was regenerated by subtilisin BPN' at pH 8.0. These results indicate that the Thr (29)-Val (30) bond of the inhibitor is the reactive site. The locations of disulfide bridges were determined to be Cys (3)-Cys (35), Cys (14)-Cys (27), Cys (18)-Cys (55), and Cys (37)-Cys (49). Based on the positions of the reactive site and the disulfide bridges, FPI-F is considered to be a member of a new family of Serine protease inhibitors. We propose the designation Bombyx family for the new inhibitor family of which FPI-F is a member.
  • Norikazu Nakayama, Asako Takemae, Hirofumi Shoun
    1996 年 119 巻 3 号 p. 435-440
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    We have purified membrane-bound fatty acid (ω-1-ω-3) hydroxylase of the fungus Fusarium oxysporum MT-811 and found that the activity depends on a single polypeptide with an apparent Mr value of 118, 000. The purified hydroxylase exhibited spectral characteristics of cytochrome P450 (P450), and could catalyze the hydroxylation without the aid of any other proteinaceous components, such as NADPH-P450 reductase. These properties of the fungal hydroxylase are the same as those of bacterial P450BM3 of Bacillus megaterium, a catalytically self-sufficient fused protein of P450 and its reductase. Other properties of the two enzymes, such as molecular weight, high catalytic turnover, and the regiospecificity of the hydroxylating position, were also almost identical. Further, the fungal hydroxylase reacted with the antibody to P450BM3. It was thus shown that the fungal fatty acid hydroxylase structurally and functionally bears a close resemblance to P450BM3, although it is membrane-bound, unlike the bacterial counterpart. On the other hand, a unique phenomenon was found with the fungal hydroxylase: its NADPH-cytochrome c- or NADPH-menadione reductase activity was enhanced enormously upon binding of its substrate (fatty acid). This appears to be the first instance in which the reactivity of P450 reductase against an artificial electron acceptor was enhanced by the binding of the substrate (to be hydroxylated) to P450. These results raise interesting questions about the molecular evolution of P450. Here we term the fungal hydroxylase cytochrome P450foxy.
  • Kunio Matsui, Sabu Kasai
    1996 年 119 巻 3 号 p. 441-447
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    Nekoflavin, which was found in cat's choroids [Matsui, K. (1965) J. Biochem. 57, 201-206], was identified as 7α-hydroxyriboflavin by comparing the physicochemical properties of nekoflavin acetate with those of chemically synthesized 7α-hydroxyriboflavin pentaacetate. 7α-Hydroxyriboflavin was synthesized from 4-chloro-2-methylbenzonitrile through 7-cyano-7-demethylriboflavin and 7α-aminoriboflavin, which are also new flavins.
  • Kuniko Akama, Hiroki Sato, Miki Furihata-Yamauchi, Yasuhiko Komatsu, T ...
    1996 年 119 巻 3 号 p. 448-455
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    The DNA binding properties of boar transition proteins 1 and 3 (TP1 and TP3) were studied by means of physicochemical techniques. The ultraviolet difference absorption spectra upon TP1 and TP3 binding to rat liver nucleosome core DNA (double-stranded DNA) showed TP1- and TP3-induced hyperchromicity at 260nm, which is suggestive of local melting of DNA. CD measurements of TP1-DNA and TP3-DNA complexes indicated that the binding of TP1 and TP3 induced different conformational changes in DNA, probably including local melting of DNA. Thermal melting studies on the binding of TP1 and TP3 to DNA showed that although at 1mM NaCl TP1 and TP3 caused slight stabilization of the DNA against thermal melting, destabilization of the DNA was observed at 50mM NaCl. From the results of quenching of the tyrosine fluorescence of TP1 and the tryptophan fluorescence of TP3 upon their binding to double-stranded and single-stranded boar liver nucleosome core DNA at 50mM NaCl, the apparent association constants for the binding of TP1 to double- and single-stranded DNA were calculated to be 8.0×104 and 1.3×105M-1, respectively, and those for the binding of TP3 to double- and single-stranded DNA to be 7.1×104 and 1.8×105M-1, respectively. These results suggest that TP1 and TP3, having higher affinity for single-stranded DNA, induce local destabilization of DNA, probably through the stacking of Tyr32 and Trp18 with nucleic acid bases, respectively.
  • Bernhard Kniep, Jasna Peter-Katalinic, Johannes Müthing, Otto Maj ...
    1996 年 119 巻 3 号 p. 456-462
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    At the IVth and Vth Workshop on Human Leukocyte Differentiation Antigens a group of monoclonal antibodies recognizing myeloid cells was found to bind to the ganglioside X3-NeuAcVII3FucnLc10Cer (VIM-2 dodecasaccharide). These antibodies were given the provisional cluster of differentiation designation CDw65. Three antibodies of this cluster (VIM-2, VIM-8, and VIM-11) have now been studied in detail at the molecular and the cellular level. Binding of VIM-2 is abolished after treatment of cells with Vibrio cholerae neuraminidase, whereas VIM-8 and VIM-11 show enhanced binding to neuraminidase-treated cells. We investigated binding of the three mAbs to glycolipid antigens with shorter carbohydrate chains. Distinct differences were observed in the binding of CDw65 antibodies to VIII3-NeuAcV3FucnLc8Cer (VIM-2 decasaccharide). VIM-2 strongly bound to this antigen, whereas no binding was observed with the other two mAbs. Conversely, the asialoganglioside of the VIM-2 decasaccharide, V3FucnLc8Cer, was not recognized by VIM-2, but this antigen bound strongly VIM-8 and VIM-11. Thus, VIM-2 and the other CDw65 antibodies represented two different antigen specificities.
  • Yuriko Komine, Makoto Kitabatake, Hachiro Inokuchi
    1996 年 119 巻 3 号 p. 463-467
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    The intracellular distribution of 10Sa RNA in Escherichia coli was investigated in cell extracts. Northern hybridization revealed that a large fraction of 10Sa RNA cosediments with 70S ribosomes. When 70S ribosomes were dissociated into 50S and 30S subunits in the presence of low levels of Mg2+ ions, almost all of the 10Sa RNA disappeared from both subunits. The extent of the association of the 10Sa RNA with ribosomes was much enhanced during the growth phase of the cells. These results suggest the possibility that 10Sa RNA might function on the ribosomes in E. coli cells.
  • Ana Kitazono, Atsuko Kitano, Tsutomu Kabashima, Kiyoshi Ito, Tadashi Y ...
    1996 年 119 巻 3 号 p. 468-474
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    The Hafnia alvei prolyl aminopeptidase gene (hpap) was cloned and sequenced, the expressed enzyme (HPAP) was purified to homogeneity and thoroughly characterized. An open reading frame of 1, 281 by was found to code for the enzyme, resulting in a protein of 427 amino acids with a molecular weight of 48, 577. HPAP resembles the Aeromonas sobria enzyme, having 45% identity and the same distinctive properties with respect to size and substrate specificities. Both enzymes show similar chromatographic behavior, and HPAP could be purified following the procedure previously described for the Aeromonas enzyme. HPAP was found to be resistant to diisopropylphosphofiuoridate as are most of the prolyl aminopeptidases hitherto described. In spite of this similarity, no inhibition by 1mM p-chloromercuribenzoate solution could be detected. Significant inhibition was, however, observed when the enzyme was incubated with 3, 4-dichloroisocoumarin. This study confirms the presence of two types of prolyl aminopeptidases, of which the Hafnia and Aeromonas enzymes constitute one group and the Bacillus, Neisseria, and Lactobacillus enzymes the other, and describes the cloning of the first prolyl aminopeptidase gene from an Enterobacteriaceae.
  • Daiji Kobayashi, Hiroko Takita, Morimichi Mizuno, Yasunori Totsuka, Yo ...
    1996 年 119 巻 3 号 p. 475-481
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    Bone sialoprotein is unique to bone and dentin, but its precise role in these tissues is still unknown, although several hypotheses have been presented. We chose ectopic chondro- and osteogenesis induced by bone morphogenetic protein (BMP) as a model system to examine the role of this protein. Partially purified bovine BMP obtained by a three-step chromatographic procedure contained all the active BMPs (natural BMP cocktail). It was combined with insoluble bone matrix and subcutaneously implanted into rats. Expression of bone sialoprotein (BSP) in the implants was followed by using a monoclonal antibody as previously reported. Immunostaining studies showed BSP in the osteoblasts lining the new bone surface at 5 weeks. Western blotting showed 53 and 30 kDa bands, instead of the 57 kDa band normally found in rat femur. These two fragments were metabolically labeled with [3H] proline. The total amount of the fragments rapidly increased after 3 weeks, and at 5 weeks was 3 times as high as that at 2 weeks and still increasing. This time-dependent change was almost parallel to that of osteocalcin. The amount of bone estimated in terms of calcium content increased until 3 weeks and was remained at a plateau thereafter. Alkaline phosphatase activity was prominent only in the first 3 weeks. It was concluded that the 53 and 30 kDa BSP fragments might contribute to maintenance or remodeling in BMP-induced ectopic bone formation.
  • Takeshi Tanaka, Masatomo Inoue, Junshi Sakamoto, Nobuhito Sone
    1996 年 119 巻 3 号 p. 482-486
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    Gram-positive thermophilic Bacilli contain quinol-cytochrome c reductase and cytochrome c oxidase as two major respiratory complexes of the electron transfer chain, and these enzymes can be extracted with mild detergents as an associated quinol oxidase supercomplex. The reductase is composed of three subunits; cytochrome b6, cytochrome c1, and FeS protein, whereas cytochrome c oxidase consists of four subunits numbered 1 through 4. In order to clarify the interactions between the subunits, the super-complex isolated from Bacillus PS3 was cross-linked with three bifunctional cross-linkers; disuccinimidyl tartrate, 3, 3'-dithiobis(succinimidylpropionate), and ethylene glycolbis(sulfosuccinimidylsuccinate). The most prominent cross-linking was observed for the combination of subunit 1 plus 2 in cytochrome c oxidase, and for that of cytochrome b6 plus cytochrome c1 in the reductase. In addition to these intra-complex cross-linkings, inter-complex linking was observed for the combination of cytochrome b6 plus subunit 1 with ethylene glycolbis(sulfosuccinimidylsuccinate), and for the combinations of cytochrome b6 plus subunit 1 and cytochrome b6 plus subunit 2 with 3, 3'-dithiobis(succinimidylpropionate). Incubation in the presence of Triton X-100, which was confirmed to cleave the two enzyme complexes, selectively reduced the inter-complex cross-linking, suggesting that the chemical crosslinking reflect the spatial arrangement of subunits in the super-complex.
  • Akiho Yokota, Akira Wadano, Hiroshi Murayama
    1996 年 119 巻 3 号 p. 487-499
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    This paper aims at clarifying the cause of the time-dependent, partial loss of the activity during reaction (so-called fallover) of ribulose 1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) from plant sources. This was done by comparing the reaction courses calculated using the reaction models constructed here based on the present conflicting two ideas on fallover with directly measured courses obtained with RuBisCO purified from spinach leaves. Since the ordinary methods with 14CO2 and indicator enzymes were not adequate for analyzing the progress of fallover, we followed the reaction by measuring the change of the light absorbance of ribulose 1, 5-bisphosphate (RuBP) at 280nm. Direct measurements of the reaction course showed that RuBisCO lost its activity with a rate constant of 6.1 to 6.5×10-3s-1 at both 0.5 and 2mM RuBP. The rate constant of the recovery of the enzyme to show the original fallover was determined as 1.2 to 1.3×10-3s-1 with RuBisCO that had just experienced fallover. These constants were used in the models. Calculation with a model assuming the binding of xylulose 1, 5-bisphosphate (XuBP) to the catalytic sites of the enzyme as the cause of fallover and using the reported dissociation constant of XuBP in the binding and the reported rate of the formation of XuBP from RuBP gave a rather linear reaction course. The minimum requirements for the model to be valid were that the rate of XuBP formation was more than once for every 600 turnovers, the dissociation constant of XuBP for the catalytic sites was less than 0.1nM, and the binding of XuBP to the sites showed a strong negative cooperativity. Inclusion of non-catalytic RuBP-binding sites in the model was essential to elucidate the course at higher RuBP concentrations. The model constructed assuming that hysteresis was the cause of fallover could calculate the measured reaction courses for the initial 20min of reaction at both 0.5 and 2mM RuBP. The rate constants of the hysteretic conformational changes of the predicted enzyme forms to others were given. The direct measurement of the long-term reaction course revealed the two phases in the decay of the activity; fast decay for the initial several minutes and subsequent slow decrease. Although the fast decay could be predicted by the hysteresis model, the slower one required the participation of inhibition by XuBP. We reasoned from these results and the reported characteristics of the binding of other sugar phosphates to the catalytic sites that the initial fast decay of the activity in fallover was due to the hysteretic property of the enzyme and the slower phase of fallover was due to the inhibition by XuBP.
  • Manabu Sugimoto, Yukio Suzuki
    1996 年 119 巻 3 号 p. 500-505
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    A cDNA encoding Mucor javanicus α-glucosidase was cloned and sequenced by the reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends methods. The cDNA comprised 2, 751bp, and included an open reading frame which encodes a polypeptide of 864 amino acid residues with a molecular mass of 98, 759 Da. The deduced amino acid sequence showed homology to fungal and mammalian α-glucosidases and related enzymes, and the sequence around the putative active site was well conserved among these enzymes. The cloned gene was expressed in Escherichia coli cells to produce the α-glucosidase, which hydrolyzes not only maltose, but also soluble starch.
  • Akiko Sakai, Mie Kato, Masashi Fukasawa, Masatsune Ishiguro, Eisuke Fu ...
    1996 年 119 巻 3 号 p. 506-511
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    Two independent cDNA clones encoding fructose 6-phosphate, 2-kinase/fructose 2, 6-bisphosphatase were isolated from a human placental cDNA library. The deduced amino acid sequences showed that one of the clones, 2K-1, was almost identical to the rat testis isozyme and the other, 2K-3, was different from any known isozymes expressed in mammalian tissues. The results of Southern blot analysis suggested that clones 2K-1 and 2K-3 were encoded as single copy genes and located in different parts of the genome. Since open reading frames of the cDNA clones were not complete, we obtained the 5'-end of the clone 2K-3 cDNA using the 5'-rapid amplification of cDNA end method. The entire cDNA (HP; 1, 756bp) had a coding capacity of 519 amino acids (Mr=59, 410), and putative phosphorylation sites for protein kinases A and C on the C terminus. Northern blot analysis using a fragment of the HP as a probe showed that a major band of 5.4kb, significantly different in size from known isozyme mRNAs such as liver (2.1kb), muscle (1.9kb), heart (4.0kb), and testis (2.0kb), was present in poly (A)+RNA preparations of human first trimester and term placentae. These results strongly suggested that this 5.4kb mRNA codes a novel isozyme of fructose 6-phosphate, 2-kinase/fructose 2, 6-bisphosphatase.
  • Retsu Miura, Yasuzo Nishina, Shigeru Fujii, Kiyoshi Shiga
    1996 年 119 巻 3 号 p. 512-519
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    The charge-transfer interaction in the complex of pig kidney medium-chain acyl-CoA dehydrogenase (MCAD) with acetoacetyl-CoA was investigated by 13C-NMR spectroscopy and molecular orbital treatment. The acyl carbons of acetoacetyl-CoA were separately 13C-labeled and 13C-NMR spectra of the complexes of MCAD with the 13C-labeled acetoace-tyl-CoA were measured. Each 13C-carbon atom was observed as a distinct peak and easily distinguished from the protein background. The chemical shift values for free acetoacetyl-CoA were 198.5, 59.9, 208.8, and 32.8ppm for C (1), C (2), C (3), and C (4), respectively, which shifted to 181.3, 103.4, 192.3, and 29.9ppm, respectively, when acetoacetyl-CoA was complexed with MCAD. While C (4) underwent a small upfield shift, the other carbons experienced significant shifts; both the C (1) and C (3) carbonyl carbons shifted upfield by about 17ppm, and the C (2) carbon was observed as a very broad peak at a position shifted downfield by more than 40ppm. These results were compared with 13C-NMR spectra of the keto-, enol-, and enolate forms of ethyl acetoacetate labeled with 13C at the acyl carbons, and interpreted with reference to the charge-transfer model based on the optimum overlap between the lowest unoccupied molecular orbital (LUMO) of flavin and the highest occupied molecular orbital (HOMO) of the enolate state of the acetoacetyl moiety of acetoacetyl-CoA. The C (2) carbon of acetoacetyl-CoA takes on the sp2 configuration in the bound form, indicating that one of the protons at C (2) of acetoacetyl-CoA is abstracted when bound to MCAD. C (1)=O is substantially polarized in the bound form of acetoacetyl-CoA, implying the presence of a machinery that polarizes this carbonyl group at the binding site, which thereby lowers the pKa value of the α-proton at C (2). This machinery is of fundamental importance in the initial step of MCAD catalysis.
  • Yasushi Kamisaka, Toro Nakahara
    1996 年 119 巻 3 号 p. 520-523
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    Diacylglycerol acyltransferase (DGAT), which catalyzes the final step in triacylglycerol (TG) biosynthesis, is crucial for lipid accumulation and formation of lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora. Since solubilization of DGAT in the lipid body fraction from this fungus with 0.5% Triton X-100 gave very low recovery of the activity, some activation factors for solubilized DGAT activity were investigated. Addition of phospholipids, especially anionic phospholipids such as phosphatidic acid and phosphatidylserine, to the assay mixture greatly increased DGAT activity. The activation by these phospholipids was most prominent when 0.2% Triton X-100 was added to the assay mixture. The effect of phosphatidic acid was reproduced using DGAT fraction obtained by 0.5M KCl elution on Mono S column chromatography. The results provide new insight on activation of DGAT during TG accumulation as well as optimal DGAT assay conditions for solubilized fractions.
  • Fumihiko Matsuno, Shoaib Chowdhury, Tomomi Gotoh, Katsuro Iwase, Hirom ...
    1996 年 119 巻 3 号 p. 524-532
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    The synthetic glucocorticoid, dexamethasone, and glucagon cooperatively elevated the level of mRNA for the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) in primary-cultured rat hepatocytes. In response to dexamethasone and/or glucagon, C/EBPβ mRNA started to increase as early as at 30min, reached a maximum within 2h, and then gradually decreased. The administration of cycloheximide, a protein synthesis inhibitor, led rather to an increase in C/EBPβ mRNA, which suggested that a labile negative protein factor (s) is involved in regulation of the C/EBPβ mRNA level. Cyclohex-imide further augmented the increases in C/EBPβ mRNA by dexamethasone and/or glucagon. Therefore, C/EBPβ mRNA accumulation in response to these hormones is apparently independent of ongoing protein synthesis. The elevation of the C/EBPβ mRNA level by these hormones was accounted for by increases in the rate of transcription of the C/EBPβ gene, as deduced on nuclear run-on analysis. Gel mobility shift analysis revealed that the DNA-binding activity of C/EBPβ was increased cooperatively by dexamethasone and glucagon. These results suggest that the C/EBPβ gene is primarily induced by glucocorticoids and/or glucagon and that the accumulated C/EBPβ protein is then involved in secondary activation of target genes in response to these hormones in the liver.
  • Rikako Uenaka, Masamichi Kuwajima, Akira Ono, Yuji Matsuzawa, Jun-ichi ...
    1996 年 119 巻 3 号 p. 533-540
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    The juvenile visceral steatosis (JVS) mouse is a novel mutant animal for studying systemic carnitine deficiency. The importance of the model has been pointed out in carnitine-deficient cardiac hypertrophy, since cardiomyopathy has been often improved after oral carnitine therapy in human systemic carnitine deficiency. To understand the effects of carnitine deficiency on gene expression in the heart, we tried to find the genes regulated by carnitine by means of a modified differential display procedure. Carnitine palmitoyltransferase I (CPT I) was one of the isolated genes. The level of CPT I gene expression in the ventricles of the JVS mice was at least three- to sixfold that of normal mice as judged by reverse transcription-polymerase chain reaction (RT-PCR). When the JVS mice were treated with carnitine, CPT I gene expression was repressed to the level of normal mice. Therefore, the increased expression of the CPT I gene was associated with carnitine deficiency.
  • Shin-ichi Ohnuma, Masatada Watanabe, Tokuzo Nishino
    1996 年 119 巻 3 号 p. 541-547
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    Geranylgeranyl diphosphate is an important precursor of archaebacterial ether-linked lipids, and it has been thought that all of this compound is “de novo” synthesized by geranylgeranyl diphosphate synthase. We studied the phosphorylation of geranylgeraniol, which seems to be related to the salvage pathway of biosynthesis of archaebacterial ether-linked lipids, in the Archaebacterium Sulfolobus acidocaldarius. Activities of geranylgeraniol kinase and geranylgeranyl phosphate kinase were detected in a cell lysate of S. acidocaldarius. The two enzymes were easily separated by ultracentrifugation. The membrane fraction and the cytosolic fraction contained geranylgeraniol kinase activity and geranylgeranyl phosphate kinase activity, respectively. Geranylgeraniol kinase, which requires divalent cation such as Mg2+, Co2+, and Mn2+ and NTP (ATP, GTP, CTP, UTP), catalyzes monophosphorylation of (all-E) -geranylgeraniol to produce geranylgeranyl phosphate. (all-E)-Farnesol, (all-E)-hexaprenol, and (all-E)-octaprenol were also active as substrates, though they were less effective than (all-E)-geranylgeraniol. However, neither geraniol nor (2E, 6E, 10Z, 14Z, 18Z, 22Z, 26Z, 30Z, 34Z, 38Z)-undecaprenol was active. This enzyme is extremely thermostable and its pH optimal is between 6.5 and 8.5. The Michaelis constants for (all-E)-geranylgeraniol and ATP are 27nM and 650μM, respectively.
  • Kazuko Ohgi, Mitsuaki Takeuchi, Masanori Iwama, Masachika Irie
    1996 年 119 巻 3 号 p. 548-552
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    In order to determine the role of Asp5l of RNase Rh from Rhizopus niveus, enzymes with mutations at the 51st position, D51N, D51E, D51Q, D51S, D51T, D51A, and D51K, were prepared, and their enzymatic properties were investigated as to specific activity and base specificity. All the mutant enzymes showed relatively high activity toward poly I and poly C, and markedly reduced activity toward poly A and poly U. In particular, the enzymatic activities toward poly I of D51T and D51S were higher than that of RNase RNAP Rh. Among the mutant enzymes, D51N, D51S, and D51T showed more than ca. 30% of the activity of RNase Rh, when RNA, poly I and poly C were used as substrates, respectively. The substitution of Ala, Glu, or Lys at Asp51 is unfavorable for enzymatic activity. Among XpGs (X=A, G, U, or C), D51N, D51S, and D51T showed higher activity toward GpG then CpG. Therefore, AspSl in RNase Rh plays a critical role in the adenylic acid preference of RNase T2 family enzymes. Our results obtained with a protein engineering technique provide basic insights into the control of the base specificity of RNase Rh.
  • Han Geuk Seo, Noriko Fujiwara, Hideaki Kaneto, Michio Asahi, Junichi F ...
    1996 年 119 巻 3 号 p. 553-558
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    Nitric oxide (NO) is synthesized from L-arginine by three isoforms of NO synthase (NOS). It is essential to suppress the function of the inducible isoform (macNOS) for amelioration of some inflammatory diseases in which the cytotoxic effect of NO is involved. S-Ethylisothiourea (S-EIU) was reported to be a potent and specific inhibitor of macNOS. We also confirmed that it rather specifically inhibited the activity of the purified macNOS and the formation of nitrite by RAW264.7 cells compared to NG-monomethyl-L-arginine (L-NMA) and NG-nitro-L-arginine (L-NNA), the other isoforms being less effective. S-EIU suppressed the release of nitrite and lactate dehydrogenase from rat vascular smooth muscle cells treated with interleukin-1β and forskolin more potently than L-NMA or L-NNA. S-EIU also slightly suppressed internucleosomal DNA cleavage in pancreatic β-cells induced by NO produced by macNOS. Intravenous administration of either S-EIU at 0.1mg/kg/min or L-NMA at 1mg/kg/min increased the blood pressure but decreased the heart rate in normal rabbits, while aminoguanidine at 1mg/kg/min affected neither cardiovascular function. These inhibitors at these doses caused recovery of the blood pressure in lipopolysaccharide-treated rabbits that exhibited lowered blood pressure similar to that in the case of septic shock. Although S-EIU seemed not to be an adequate inhibitor for therapeutic use in vivo due to its side effects on cardiovascular functions, it is one of the most potent inhibitors of macNOS among reported inhibitors in vitro.
  • Axel Raisig, Glenn Bartley, Pablo Scolnik, Gerhard Sandmann
    1996 年 119 巻 3 号 p. 559-564
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    The phytoene desaturase gene from Rhodobacter capsulatus was expressed in Escherichia coli and the resulting protein was purified. The purification steps involved were ammonium sulfate precipitation and ion exchange chromatography, leading to a homogenous protein of 57 kDa with high specific enzymatic activity. The purified enzyme was characterized with respect to substrate specificity and product formation. In addition to phytoene, the intermediates, phytofluene and ζ-carotene, were both converted to neurosporene, the end product of the reaction. Furthermore, 1, 2-epoxy phytoene was a suitable substrate whereas the C30 diapophytoene was not. The Km, values for phytoene and ζ-carotene were determined to be 33.3 and 16.6μM, respectively. The desaturation reaction is dependent on the cofactor FAD. Oxidized nicotine nucleotides or ATP had no positive effect. The Km value for FAD was 4.9μM. Inhibition of the desaturation reaction was observed with diphenylamine.
  • Yasushi Uchida, Naomi Kondo, Tadao Orii, Takashi Hashimoto
    1996 年 119 巻 3 号 p. 565-571
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    It is generally accepted that there are two different acyl-CoA synthetases in rat liver peroxisomes. One is long-chain acyl-CoA synthetase, and the other very-long-chain acyl-CoA synthetase. Nowadays, the nature of long-chain acyl-CoA synthetase is wellknown, but that of very-long-chain acyl-CoA synthetase remains unclear. Very-long-chain acyl-CoA synthetase has been extracted from the washed membrane fraction of frozen rat liver peroxisomes with a buffer containing a detergent, and has been purified by chromatography on Ultrogel AcA 34, calcium phosphate gel/cellulose, blue dextran-Sepharose 4B and DEAE-Toyopearl. The molecular masses of the native enzyme and the subunit were estimated to be 235 and 70 kDa, respectively. This enzyme showed marked differences in behavior from long-chain acyl-CoA synthetase during purification. The carbon chain length specificity of very-long-chain acyl-CoA synthetase differed from that of long-chain acyl-CoA synthetase. Very-long-chain acyl-CoA synthetase was active toward long- and very-long-chain fatty acids, but more active toward very-long-chain fatty acids compared with long-chain acyl-CoA synthetase. Antibodies against long-chain acyl-CoA synthetase did not cross-react to very-long-chain acyl-CoA synthetase. Based on these data, the final enzyme preparation is judged to be highly purified very-long-chain acyl-CoA synthetase from rat liver peroxisomes.
  • Satoshi Tsubuki, Yumiko Saito, Masanori Tomioka, Hisashi Ito, Seiichi ...
    1996 年 119 巻 3 号 p. 572-576
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    To explore membrane-permeable synthetic inhibitors that discriminate between endogenous calpain and proteasome in cells, we examined the inhibition profiles against calpain and proteasome in vitro and in vivo of peptidyl aldehydes possessing di-leucine and tri-leucine. The tripeptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal (ZLLLal) strongly inhibited calpain and proteasome activities in vitro. The concentration required for 50% inhibition (IC50) of the casein-degrading activity of calpain was 1.25μM, and the IC50s for the succinyl-leucyl-leucyl-valyl-tyrosine-4-methylcoumaryl-7-amide (SucLLVY-MCA)- and benzyloxycarbonyl-leucyl-leucyl-leucine-4-methylcoumaryl-7-amide (ZLLL-MCA)-degrading activities of proteasome were 850 and 100nM, respectively. On the other hand, the synthetic dipeptide aldehyde benzyloxycarbonyl-leucyl-leucinal (ZLLal) strongly inhibited the casein degrading activity of calpain (IC50) 1.20μM), but the inhibition of proteasome was weak (IC50s for SucLLVY-MCA- and ZLLL-MCA-degrading activities were 120 and 110μM, respectively). Thus, while calpain was inhibited by similar concentrations of ZLLal and ZLLLal, the inhibitory potencies of ZLLLal against the ZLLL-MCA-and SucLLVY-MCA-degrading activities in proteasome were 1, 100 and 140 times stronger than those of ZLLal, respectively. To evaluate the effectiveness of these inhibitors on intracellular proteasome, the induction of neurite outgrowth in PC12 cells caused by proteasome inhibition was examined. ZLLLal and ZLLal initiated neurite outgrowth with optimal concentrations of 20nM and 10μM, respectively, again showing a big difference in the effective concentrations for the proteasome inhibition as in vitro. As for the effect on intracellular calpain, the concentrations of ZLLLal and ZLLal required for the inhibition of the autolytic activation of calpain in rabbit erythrocytes were 100 and 100μM or more, respectively. The almost equal inhibitory potencies of ZLLLal and ZLLal were in agreement with the inhibition of calpain in vitro. These differential effects of inhibitors against calpain and proteasome are potentially useful for identifying the functions of calpain and proteasome in cell physiology and pathology.
  • Ken Hashimoto, Takashi Tobe, Jun-ichi Sumiya, Yoshihiro Sano, Nam-Ho C ...
    1996 年 119 巻 3 号 p. 577-584
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    The pig counterpart of human IHRP has been isolated from pig serum, and cDNA clones encoding this counterpart were isolated and characterized. The amino acid sequence of pig IHRP predicted from the nucleotide sequence of its cDNA shows reasonable homology to that of human IHRP. The nucleotide sequence of pig IHRP cDNA is identical to that of the mRNA partially determined to be the heavy chain of pig ITI [Buchman et al. (1990) Surgery 108, 560-566] ; it is one of the major mRNAs induced in pig liver on cardiogenic shock. Thus we concluded that the reported mRNA should code for IHRP and not for the heavy chain of ITI. The pig IHRP also seems to be identical to pig-MAP, which was recently reported to be a major acute phase serum protein in pig [Gonzalez-Roman et al. (1995) FEBS Lett. 371, 227-230]. The results suggest that IHRP might be involved in acute phase reactions.
  • Chantragan Srisomsap, Jisnuson Svasti, Rudee Surarit, Voraratt Champat ...
    1996 年 119 巻 3 号 p. 585-590
    発行日: 1996年
    公開日: 2008/11/18
    ジャーナル フリー
    A glycosidase enzyme with both β-glucosidase and β-fucosidase activities has been purified from the seeds of Dalbergia cochinchinensis Pierre (Thai Rosewood) by ammonium sulfate fractionation, preparative isoelectric focusing, and Sephadex G-150 chromatography. The enzyme has molecular weights of 330, 000 in the native state and 66, 000 in the denatured state. Hydrolysis of p-NP-β-D-glucoside and p-NP-β-D-fucoside showed pH optimum at pH 5.0 and was inhibited by δ-gluconolactone, HgCl2, and p-chloromercuribenzoate. The Km and kcat values of the purified enzyme were 5.4mM and 307s-1 for p-NP-β-D-glucoside and 0.54mM and 151s-1 for p-NP-β-D-fucoside, so that the latter had by far the higher kcat/Km ratio. p-NP-β-D-galactoside, p-NP-β-D-xyloside, and p-NP-α-L-arabinoside were hydrolyzed more slowly. Hydrolysis of sophorose, laminaribiose, and gentiobiose were also rather slow, and hydrolysis of cellobiose was even slower. No hydrolysis of the cyanogenic glucosides linamarin or prunasin, but some hydrolysis of amygdalin and salicin was found. Further studies are required to identify the natural substrates of the enzyme. However, high yields, ease of purification, and storage stability of the enzyme make it a useful candidate for various applications, such as study of oligosaccharide synthesis by reversal of hydrolysis.
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