著者

Yoshizumi Ishino Takashi Ueno Masaru Miyagi Takashi Uemori Mitsuo Imamura Susumu Tsunasawa Ikunoshin Kato

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.116, no.5, pp.1019-1024, 1994 (Released:2008-11-18)

参考文献数

18

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We cloned the pol gene from the Thermus aquaticus YT-1 strain into a plasmid vector and constructed a high-level expression system of the gene in Escherichia coli. Six codons in the translational start region were changed to simple AT-type codons or codons which are most frequently used in E. coli by the genetic engineering techniques with retention of the amino acid sequence of the native enzyme. The modified pol genes were expressed under the lac promoter of pUC-type plasmid and 266, 418 units of activity was obtained in a sonicated and heated crude extract from 2g of E. coli cells bearing one of the recombinant plasmids, pTAQ9. Highly purified protein was subjected to structural analysis using a protein sequencer and an ion-spray mass spectrometer combined with reversed-phase HPLC (LC-MS). The primary structure of the DNA polymerase was identical with the amino acid sequence deduced from the nucleotide sequence of the pol gene as far as examined (about 95% of the sequence); though, several regions where small peptides of less than 5 residues were produced by lysyl endopeptidase digestion could not be sequenced.

著者

Nobuyuki NUKINA Yasuo IHARA

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.99, no.5, pp.1541-1544, 1986-05-01 (Released:2008-11-18)

参考文献数

11

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Paired helical filaments (PHF) are unusual neuronal fibers which accumulate progressively in the brain in Alzheimer's disease (AD). The insolubility of PHF in various kinds of solvents enabled us to obtain highly purified PHF, but prevented the application of conventional analytical methods to identify their components. Here we report that antibodies against purified PHF recognize tau protein, a brainspecific microtubule-associated protein, suggesting that a portion of PHF is tau protein.

著者

Hideki Nagase Kei-ichi Enjyoji Midori Shima Kenji Kitazato Akira Yoshioka Hidehiko Saito Hisao Kato

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.119, no.1, pp.63-69, 1996 (Released:2008-11-18)

参考文献数

37

---

Our previous study has shown that depolymerized holothurian glycosaminoglycan (DHG) has two different inhibitory activities in the blood coagulation cascade: heparin cofactor II-dependent thrombin inhibition; and antithrombin III- and heparin cofactor II-independent inhibition of the intrinsic factor Xase complex [Nagase et al. (1995) Blood 85, 1527-1534]. In the present study, the effect of DHG on the activation of factor VIII and factor V by thrombin was examined with purified human components. DHG inhibited the activation of factor VIII by thrombin at concentrations exceeding 80nM, but not the activation of factor V by thrombin at concentrations of up to 8μM. On Western blot analysis, DHG inhibited the cleavage of factor VIII light chain at concentrations exceeding 0.8μM. The interaction between DHG and factors VIII and V and thrombin was examined with a DHG-cellulofine column. DHG had strong affinity for factor V and thrombin, but slight affinity for factor VIII. The interaction of DHG with thrombin was analyzed, using fluorescein isothiocyanate-labeled DHG. One mole of DHG bound 2mol of thrombin, with a dissociation constant (Kd) of 3.04×10-6M. These results suggest that DHG interferes with the interaction between thrombin and factor VIII, probably by making a binary complex through the anionic binding exosite II of thrombin.

著者

Oka Masahiro Kimata Yukio Mori Kazutoshi KOHNO Kenji

出版者

Japanese Biochemical Society

雑誌

The journal of biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.121, no.3, pp.578-584, 1997-03-01

参考文献数

36

被引用文献数

1

著者

Kinuko Kimura Yoshio Nakano Kouji Matsuoka

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.105, no.1, pp.84-87, 1989-01-01 (Released:2008-11-18)

参考文献数

12

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The nucleotide ligation site of adenylylated glutamine synthetase, which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP, was studied by digestion with three hydrolytic enzymes. The products on micrococcal nuclease digestion were adenosine and o-phosphotyrosyl glutamine synthetase. The Km for this macromolecular substrate with the nuclease was 40 μM, at pH 8.9. The glutamine synthetase activity was not affected by deadenosylation with the nuclease, in contrast to SVPDE digestion, with which the glutamine synthetase activity was markedly increased. The Km for the native adenylylated glutamine synthetase with the SVPDE was 36 μM, i. e., similar to that for the nuclease. When the isolated o-phosphotyrosyl enzyme was incubated with alkaline phosphatase at pH 7.2, the glutamine synthetase activity rapidly increased to the same level as that of the SVPDE treated enzyme. Furthermore, kinetic properties of the o-phosphotyrosyl glutamine synthetase were compared with those of the adenylylated enzyme. The optimum pH, apparent Km for each of three substrates, glutamate, ATP, and NH3, and Vmax were in good agreement, as to either Mg2+- or Mn2+-dependent biosynthetic activity. From these results we can conclude that the regulation of glutamine synthetase activitysimply requires the piosphorylation of the tyrosyl residue in each subunit, without recourse to adenylylation.

著者

Tamio MIZUKAMI Seiga ITOH

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.101, no.5, pp.1307-1310, 1987 (Released:2008-06-30)

参考文献数

14

---

A useful vector, pAGE103, has been developed for the expression of cDNA in animal cells using the simian virus 40 (SV40) expression signals. cDNA could be expressed easily by inserting it into the multiple cloning sites (HindIII, SalI/AccI, XbaI, BamHI, SmaI/XmaI, KpnI/Asp718, SacI and EcoRI) of the vector, which are located between the SV40 early promoter and the SV40 early RNA processing signals for splicing and polyadenylation. In addition to the above transcription unit, pAGE103 contains the replication origin of ColE1, and a dual KmR/G418R selective gene. Several unique restriction sites are located on the boundaries between the above-mentioned three components of the vector, allowing the easy substitution or insertion of other genetic elements. The human interferon-β gene was inserted into pAGE103 and shown to be expressed transiently in COS-1 cells and stably in several animal cell lines.

著者

Sakamoto Taiichi Kim Mi Hee Kurihara Yasuyuki SASAKI Nobuyuki NOGUCHI Tomoaki KATAHIRA Masato UESUGI Seiichi

出版者

社団法人 日本生化学会

雑誌

The journal of biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.121, no.2, pp.288-294, 1997-02-01

参考文献数

48

被引用文献数

1

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The properties of a mutant hammerhead ribozyme system, which consists of two RNA oligomer strands and in which stem II is deleted (replaced with a UUUU loop), are described. The effects of temperature, pH, and metal ions on the cleavage reaction were similar to those for the parent ribozyme with stem II. The mutant ribozyme showed a much lower cleavage rate (k_cat=0.04min^-1) in the presence of 10mM MgCl₂, where the parent ribozyme showed full cleavage activity. However, increasing the concentration of MgCl₂ from 10 to 100mM restored the cleavage activity of the mutant ribozyme to the original level (k_cat=0.2min^-1). CD titration experiments with MgCl₂ using a noncleavable substrate were carried out. Deletion of stem II resulted in an about 20-fold reduction of the apparent Mg²⁺ binding affinity when the Mg²⁺ concentrations of half-saturation are compared. The results were analyzed by curve-fitting analysis and compared with those for the parent ribozyme. The analysis showed that the Mg²⁺ concentration dependence data in CD and cleavage experiments for the mutant enzyme can be explained by a two Mg²⁺ ion binding mechanism. These results suggest that stem II is important for maintaining the conformation of the catalytic core suitable for Mg²⁺ binding.

著者

KARAZAWA SUGIZO

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.10, no.3, pp.389-394, 1929

著者

Kong Xiangfei Gan Huachen Hao Yuqing CHENG Chunming JIANG Jianhai HONG Yi YANG Junwu ZHU Hao CHI Yayun YUN Xiaojing GU Jianxin

出版者

Japanese Biochemical Society

雑誌

The journal of biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.146, no.3, pp.417-427, 2009-09-01

参考文献数

50

著者

SAKUMA FUTOSHI

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.12, no.2, pp.247-279, 1930

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1. The distribution of glyoxalase in animal tissues was ex-amined. Generally speaking, the glyoxalase content was highest in the liver, the other tissues containing 30-60% of that of the liver. There was no noticeable fluctuation in the glyoxalase distribution in the tissues, so long as the animal was kept under normal conditions. The main difference between warm- and cold-blooded animals was that the liver of the latter was 50% inferior to that of the former in its glyoxalase content.<br> 2. The glvoxalase contents of the animal tissues decreased definitely when the tissues were subjected to continuous deprivation of sugar. The liver lost the enzyme most slowly in comparison with other tissues.<br> 3. The relation between the glyoxalase content and the process of germination of the soy-bean was studied. Glyoxalase of the bud increased rapidly up to the 7th or 8th clay of germinationn, and was succeeded by a subsequent declination. This increase in gly oxalase may be attributed to the increase of co-enzyme rather than to that of the enzyme itself.<br> 4. The dry glyoxalase sample was prepared by the successive treatment of animal tissues with alcohol and ether. The powder retained 70% activity of the original fresh tissue and could be kept for at. least four weeks without' loss of its power. The extraction of the enzyme from the powder was achieved most effectively by shaking it with a neutral liquid for two hours at room temperature or for an hour at 37°.<br> 5. When glyoxalase was dialysed through collodion membrane against distilled water, its activity disappeared after 5 hours. The lost activity was restored definitely by the addition of boiled tissue juice.<br> 6. Glucose and polysaccharides, which contain glucose in the molecules, accelerated the activity of dialysed and non-dialysed glyoxayase. The hexoses other than glucose were quite indifferent to the enzyme. Inorganic phosphate inhibited the glyoxalase action. Guanine and its derivatives reacted directly with methylglyoxal. When mnet.hlyglyoxal was brought into contact with amino acids, rather a rapid disappearance of methylglyoxal was observed, coupled with the production of NH₃ and CO₂. The amount of liberated NH₃ and CO₂ was 70-80% of the value computed from the quantity of amino acids involved in the reaction.<br> 7. Fosters pancreatic powder which, according to her statement, inhibits the lactic acid formation in chopped muscle and differs from "antiglyoxalase" in some respects, seems to be identical with "antiglyoxalase" in every point.

著者

Hiromi Sakai Yohei Masada Shinji Takeoka Eishun Tsuchida

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.131, no.4, pp.611-617, 2002-04-01 (Released:2008-06-30)

参考文献数

49

---

Hemoglobin-vesicles (HbV) have been developed for use as artificial O2 carriers in which a purified Hb solution is encapsulated within a phospholipid bilayer membrane. In this study, bovine Hb (BHb) was tested as a source of HbV instead of human Hb (HHb). We compared the preparation process and characteristics of BHbV with those of HHbV. The purification of BHb was effectively performed simply with an ultrafiltration system including a process for removing virus and scrapie agent. The removal ratio of the phospholipid components of bovine red blood cells was over 99.99%, and the protein purity was over 99.9%. The deoxygenated and carbonylated BHb showed denaturation transition temperatures at 83 and 87°C, respectively, which are higher than those of HHb (80 and 78°C, respectively), and resistant to pasteurization (60°C, 10h). The purified BHb was concentrated to over 40g/dl, and encapsulated in a phospholipid bilayer membrane to form BHbV with a diameter of about 280nm. The O2 affinity (P50) of the BHbV was regulated by coencapsulation of an appropriate amount of Cl- (as NaC1), which binds to BHb as an allosteric effector, in the range 16-28 Torr, comparable to human blood (P50=28 Torr). This is quite simple in comparison with HHb which requires phosphate derivatives such as pyridoxal 5'-phosphate as a replacement for 2, 3-diphoshoglyceric acid. The viscosity and colloid osmotic pressure of the BHbV when suspended in 5% human serum albumin are 3.5cP and 20 Torr, respectively, comparable to those of human blood. In conclusion, BHb can be used as a source for the production of HbV, not only because of its abundance in the cattle industry, but also because of the physicochemical advantages of the purification process, thermal stability, and regulation of 02 affinity in comparison with HHb.

著者

Yutaka ORII Masayuki MORITA

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.81, no.1, pp.163-168, 1977-01-25 (Released:2008-11-18)

参考文献数

17

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A method was established to estimate the pH change of several buffer solutions on freezing by using a combination of pH indicators. Among more than 30 buffer solutions examined, almost half exhibited a pH change in the temperature range between freezing point and 220°K; the results were tabulated. Glycerol was found to suppress the pH changes because of its “salt buffer” effect.

著者

Ono Yasuko Kinouchi Tadatoshi Sorimachi Hiroyuki ISHIURA Shoichi SUZUKI Koichi

出版者

社団法人 日本生化学会

雑誌

The journal of biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.121, no.3, pp.585-590, 1997-03-01

参考文献数

39

---

Alzheimer's disease amyloid precursor protein (APP) generates a &beta;-amyloid protein (A&beta;) that is a main component of the senile plaques found in the brains of Alzheimer's disease patients. APP is thought to undergo proteolysis via two different pathways, the amyloido-genic pathway which produces A&beta;, and the non-amyloidogenic pathway which releases a large N-terminal fragment into the medium. The proteases that mediate these processes remain unidentified. The physiological function of APP is not clear yet. Therefore, the cytoplasmic region of APP has attracted much interest, because this region is highly conserved among species, and members of the amyloid precursor-like protein (APLP) family. Several potentially functional sequences exist in the region, including signal sequences for protein sorting and a Go-protein binding sequence. We constructed two mutants, 695<i>&Delta;</i>NPTY and 695<i>&Delta;</i>GYEN. They lack potential endosome/lysosome targeting signals, NPTY and GY, in the cytoplasmic domain of APP695, respectively. The mutant APPs had longer half-lives and were secreted more easily into the medium than the wild type, suggesting that these sequences are important for the secretion and metabolism of APP.

著者

Takahiro Yamashita Akihisa Terakita Yoshinori Shichida

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.130, no.1, pp.149-155, 2001 (Released:2008-11-18)

参考文献数

45

---

G protein-coupled receptors identified so far are classified into at least three major families based on their amino acid sequences. For the family of receptors homologous to rhodopsin (family 1), the G protein activation mechanism has been investigated in detail, but much less for the receptors of other families. To functionally compare the G protein activation mechanism between rhodopsin and metabotropic glutamate receptor (mGluR), which belong to distinct families, we prepared a set of bovine rhodopsin mutants whose second or third cytoplasmic loop was replaced with either the second or third loop of Gi/Go-or Gq-coupled mGluR (mGluR6 or mGluR1). Among these mutants, the mutants in which the second or third loop was replaced with the corresponding loop of mGluR exhibited no G protein activation ability. In contrast, the mutant whose third loop was replaced with the second loop of Gi/Go-coupled mGluR6 efficiently activated Gi but not Gt: this activation profile is almost identical with those of the mutant rhodopsins whose third loop was replaced with those of the Gi/Go-coupled receptors in family 1 [Yamashita et al. (2000) J. Biol. Chem. 275, 34272-34279]. The mutant whose third loop was replaced with the second loop of Gq-coupled mGluR1 partially retained the Gi coupling ability of rhodopsin, which is in contrast to the fact that all the rhodopsin mutants having the third loops of Gq-coupled receptors in family 1 exhibit no detectable Gi activation. These results strongly suggest that the molecular architectures of rhodopsin and mGluR are different, although the G protein activation mechanism involving the cytoplasmic loops is common.

著者

Takeo Imai Katsuhiko Taguchi Yoko Ogawara Daijiro Ohmori Fumiyuki Yamakura Hidenori Ikezawa Akio Urushiyama

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.130, no.5, pp.649-655, 2001 (Released:2008-11-18)

参考文献数

32

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An extremely thermostable [4Fe-4S] ferredoxin was isolated under anaerobic conditions from a hyperthermophilic archaeon Thermococcus profundus, and the ferredoxin gene was cloned and sequenced. The nucleotide sequence of the ferredoxin gene shows the ferredoxin to comprise 62 amino acid residues with a sequence similar to those of many bacterial and archaeal 4Fe (3Fe) ferredoxins. The unusual Fe-S cluster type, which was identified in the resonance Raman and EPR spectra, has three cysteines and one aspartate as the cluster ligands, as in the Pyrococcus furiosus 4Fe_ferredoxin. Under aerobic conditions, a ferredoxin was purified that contains a [3Fe-4S] cluster as the major Fe-S cluster and a small amount of the [4Fe-4S] cluster. Its N-terminal amino acid se-quence is the same as that of the anaerobically-purified ferredoxin up to the 26th residue. These results indicate that the 4Fe ferredoxin was degraded to 3Fe ferredoxin during aerobic purification. The aerobically-purified ferredoxin was reversibly converted back to the [4Fe-4S] ferredoxin by the addition of ferrous ions under reducing conditions. The anaerobically-purified [4Fe-4S] ferredoxin is quite stable; little degradtion was observed over 20 h at 100°C, while the half-life of the aerobically-purified ferredoxin is 10h at 100°C. Both the anaerobically-and aerobically-purified ferredoxins were found to function as electron acceptors for the pyruvate-ferredoxin oxidoreductase purified from the same archaeon.

著者

HITOSHI TAKAHASHI

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.63, no.6, pp.789-791, 1968-06-25 (Released:2008-11-18)

参考文献数

8

著者

Tomofumi Fujino Tomohiro Tada Toshiki Hosaka Masatoshi Beppu Kiyomi Kikugawa

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.127, no.2, pp.307-313, 2000 (Released:2008-11-18)

参考文献数

42

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Oxidized protein hydrolase (OPH), an 80 kDa serine protease whose activity is inhibited by diisopropyl fluorophosphate (DFP), has been isolated from human erythrocytes [Fujino, T. et al. (1998) J. Biochem. 124, 1077-1085]. The presence of OPH in various biological samples was examined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting using an anti-OPH antibody raised against OPH purified from human erythrocytes, and by [3H] DFP-labeling and successive SDS-PAGE/fluorography. Solubilized samples of human cell lines including K-562 cells, THP-1 cells and Jurkat cells, and rat tissues including brain, heart, liver, kidney, and testis, inhibited the anti-OPH antibody binding to OPH in ELISA. Immunoblotting of lysates of K-562 cells, THP-1 cells and Jurkat cells showed four immunoreactive protein bands including an 80 kDa protein. Immunoprecipitation of the [3H] DFP-labeled K-562 cell lysate and successive SDS-PAGE/fluorography showed the presence of only the 80 kDa DFP-reactive protein with OPH antigenic activity. The level of the 80 kDa immunoreactive protein in K-562 cells rose as the cells differentiated toward erythrocytes. Immunoblotting of human and rat plasma showed two immunoreactive protein bands, including the 80 kDa protein, and SDS-PAGE/fluorography of [3H] DFP-labeled rat and human plasma showed the presence of only the 80 kDa DFP-reactive protein. The results indicate that OPH is present in a wide variety of biological samples.

著者

Hayakawa Hiroshi Kumura Keiko Sekiguchi Mutsuo

出版者

社団法人 日本生化学会

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.84, no.5, pp.1155-1164, 1978

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Uracil-DNA glycosylase, which acts specifically on uracil-containing DNA, was purified 250-fold from an extract of <i>Escherichia coli</i> 1100. The enzyme releases free uracil from DNA, producing alkali-labile apyrimidinic sites in the DNA. The enzyme is active on both native and heat-denatured DNA of phage PBS1, which contains uracil in place of thymine. &phi;X174 DNA which had been treated with bisulfite and then at alkaline pH was susceptible to the action of uracil-DNA glycosylase. Since DNA treated with bisulfite alone was less susceptible to the enzyme, it is likely that the enzyme recognizes deaminated cytosine, namely uracil, but not bisulfite adducts of uracil and cytosine in the treated DNA. DNA treated with nitrite or hydroxylamine was not attacked by the enzyme. Enzyme activity acting on bisulfite-treated DNA was absent from an extract of <i>E. coli</i> mutant BD10 (<i>ung</i>). The mutant exhibited higher sensitivity to bisulfite than did the wild-type strain and was unable to reactivate phage T1 pre-exposed to bisulfite and weak alkali.

著者

Akira ISHIHAMA Masayoshi ENAMI Yoshikazu NISHIJIMA Toshikazu FUKUI Eiko OHTSUKA Morio IKEHARA

出版者

The Japanese Biochemical Society

雑誌

The Journal of Biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.87, no.3, pp.825-830, 1980-03-01 (Released:2008-11-18)

参考文献数

23

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The effects of 2'-substitutions of ATP on the substrate and inhibitor properties for RNA synthesis were studied in the poly(dAT)-dependent reaction of Escherichia coli RNA polymerase. In the presence of UTP, 2'-deoxy-2'-azidoadenosine 5'-triphosphate (AzTP) was incorporated into an acid-insoluble fraction at one-tenth of the rate of ATP incorporation; it thus acts as a competitive inhibitor for poly(AU) synthesis. On the other hand, another ATP analog, 2'-deoxy-2'-fluoroadenosine 5'-triphosphate (AfTP), was co-polymerized with UTP into acid-insoluble materials at a rate less than 1% of that of ATP incorporation; in addition, it exerted a strong but mixed-type inhibition on poly(AU) synthesis. Different modes of action of the two ATP analogs are discussed in connection with the specificity of substrate recognition by RNA polymerase.

著者

Inoue Masahiro Okamoto Kenta Uemura Haruki

出版者

Japanese Biochemical Society

雑誌

The journal of biochemistry (ISSN:0021924X)

巻号頁・発行日

vol.158, no.1, pp.49-60, 2015-07