巻号一覧
104 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
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Shigehiro Kamitori, Ken Hirotsu, Taiichi Higuchi, Kiyoshi Kondo, Katsu ...1988 年 104 巻 3 号 p. 317-318
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーThe crystal structure of aspartate aminotransferase of Escherichia coli was determined by X-ray structure analysis at 2.8 Å resolution. The structure was solved by the molecular replacement method and refined to an R-factor of 0.27, and it was found that the overall structure of AspAT of E. coli is similar to that of those of higher animals.抄録全体を表示PDF形式でダウンロード (213K) -
Koichi Ishiguro, Yasuo Ihara, Tsuneko Uchida, Kazutomo Imahori1988 年 104 巻 3 号 p. 319-321
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーFrom rat brain microtubule proteins, we purified a protein kinase that phosphorylated tau, one of microtubule-associated proteins. The electrophoretic mobility of the phosphorylated tau on SDS-polyacrylamide gel decreased. The enzyme was not activated by cyclic nucleotides, calmodulin, or phospholipids, and was inhibited by the calcium ions. The kinase bound to tau. The phosphorylation of tau was stimulated by tubulin under the condition of microtubule formation. From these results we propose an idea that the phosphorylation could occur concomitantly with microtubule formation in the brain. Human tau phosphorylated by the kinase carried an epitope of the paired helical filaments that accumulate in the brain in Alzheimer's disease.抄録全体を表示PDF形式でダウンロード (2352K) -
Yuji Kobayashi, Tadayasu Ohkubo, Yoshimasa Kyogoku, Satoshi Koyama, Ma ...1988 年 104 巻 3 号 p. 322-325
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーThe three-dimensional structure of a-human ANP in solution was determined through the combined use of nuclear magnetic resonance spectroscopy and distance geometry. The results are based on distance constraints determined by nuclear Overhauser effect measurements and one disulfide bond. The structure is as follows. Three separate regions, which are Ser1-Cys7, Arg11 -Ile15, and Gln18-Tyr28 each have some ordered structure. The remaining parts in the sequences of Gly9-Gly10 and Gly16-Ala17 act as hinges. And the C-terminal part is folded back toward the cyclic moiety. The conformation of α-hANP reported here is expected to give a better understanding of the relationships between its biological activities and three-dimensional structure.抄録全体を表示PDF形式でダウンロード (345K) -
Shuntaro Hara, Ichiro Kudo, Kunio Matsuta, Terumasa Miyamoto, Keizo In ...1988 年 104 巻 3 号 p. 326-328
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーThe amino acid composition and partial NH2-terminal amino acid sequence of an extracellular phospholipase A2 in human rheumatoid synovial fluid were determined. The predom-inant amino acids in the phospholipase A, were cysteine, glycine, arginine, and lysine, suggesting that it is a basic one. The NH2-terminal 34 amino acids were found to be as follows:
Asn-Leu-Val-Asn-Phe-His-Arg-Met-Ile-Lys-Leu-Thr-Thr-Gly-Lys-Glu-Ala-Ala-Leu-Ser-Tyr-Gly-Phe-Tyr-Gly-Cys-X-Cys-Gly-Val-Gly-Gly-Arg-Gly
The enzyme contains Phe-5, Met-8, Ile-9, Tyr-24, Gly-25, Cys-26, Cys-28, Gly-29, Gly-31, Gly-32, and Gly-34 residues, all of which are conserved in most of the sequenced phospholipase A2. The remarkable feature of this enzyme was the absence of Cys-11, which is conserved in the “Group I” enzyme family. This is the first report concerning partial amino acid sequences of human non-pancreatic phospholipase A2.抄録全体を表示PDF形式でダウンロード (276K) -
Yasuki Ishizak, Takashi Mikawa, Setsuro Ebash, Etsuo Yokota, Hiroshi H ...1988 年 104 巻 3 号 p. 329-332
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーBidirectional organelle movements taking place in the cytoplasm of the rhizomes of Caulerpa, a coenocytic marine green alga, have been indicated to be dependent on microtubules (Kuroda, K. & Manabe, E. (1983) Proc. Jpn. Acad. 59B, 131-134; Manabe, E. & Kuroda, K. (1984) Proc. Jpn. Acad. 60B, 118-121). However, when a crude extract of Caulerpa rhizomes was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to immunoblotting with monoclonal anti-tubulin antibody, no reacting band could be detected. This apparent absence of tubulin in the extract was found to be a result of the complete degradation of tubulin by potent intrinsic proteolytic activity. All of the commercially available protease inhibitors so far tested (p-chloromercuriphenylsulfonic acid, phenyl methylsulfonyl fluoride, 1-chloro-4pheny1-3-tosylamido-2-butanone, 7-amino-l-chloro-3-tosylamido-2-heptanone, p-tosyl-L-arginine methyl ester, soybean trypsin inhibitor, antipain, chymostatin, leupeptin, and pepstatin) failed to inhibit the activity completely. But addition of casein at the concentration of 1% (weight per volume) to the solutions used for preparation was effective in protecting tubulin from proteolytic degradation, thus making it possible to prepare tubulin from the crude extract of Caulerpa. On SDS-PAGE, the Caulerpa α-tubulin thus prepared was a little smaller in molecular weight than that of rabbit brain.抄録全体を表示PDF形式でダウンロード (2993K) -
Hayato Umekawa, Keiko Tamiya-Koizumi, Eishi Takagi, Hiroyoshi Ariga, K ...1988 年 104 巻 3 号 p. 333-336
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーA cloned plasmid, pmyc(H-K), containing sequences derived from human c-myc gene replicated in vitro in Raji nuclear extract in a semiconservative manner. Using this system, it was found that phosphatidylinositol and cardiolipin strongly inhibited the replication of pmyc(H-K) in vitro, whereas other phospholipids, i.e., phosphatidylserine, phosphatidyl-choline, phosphatidylethanolamine, phosphatidic acid, and sphingomyelin, had no appre-ciable effect. The concentrations of phosphatidylinositol and cardiolipin producing 50% inhibition of the replication were 4.6 and 5.4 μM, respectively. Phosphatidylinositol and cardiolipin inhibited the relaxation of pmyc(H-K) supercoiled DNA, but showed little or weaker effects on DNA polymerase a and topoisomerase II in Raji nuclear extract. These results suggest that phosphatidylinositol and cardiolipin antagonize the replication of pmyc(H-K) in vitro, through, at least in part, the interaction with topoisomerase I.抄録全体を表示PDF形式でダウンロード (3137K) -
Yoshio Yaoita, Masazumi Takahashi, Chihiro Azuma, Yoshiyuki Kanai, Tas ...1988 年 104 巻 3 号 p. 337-343
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーWe have examined usage of variable region gene families of the immunoglobulin heavy chain (VH gene family) in spleens of MRL/MpJ-lpr/lpr (MRL/lpr), (NZB×NZW)F1, and BXSB mice by Northern analysis using various VH probes, including the VHPAR gene which we cloned and identified as a gene encoding the heavy-chain variable region of antipoly(ADP-ribose) antibody. The amount of VHS107 family mRNA was almost constant for the same amount of splenic crude RNA in autoimmune-prone and normal mice, while concentrations of other family mRNAs were elevated in autoimmune-prone mice. For example, per splenic RNA the VHPAR family was expressed in MRL/lpr mice 10 times more than in their normal counterpart, MRL/MpJ-+/+ (MRL/ +) mice. These results indicate the bias of VH gene usage in autoimmune-prone mice. Expression of the VHS107 family was depressed from an early life stage of MRL/lpr and male BXSB mice. Furthermore, the expression of IL-4 and IL-5 were quantitatively compared, as B cell differentiation factor was thought to be produced by abnormally proliferative T cells in lymph nodes of MRL/lpr mice. We could not, however, observe overproduction of IL-4 and IL-5 mRNA in the lymph nodes.抄録全体を表示PDF形式でダウンロード (2038K) -
Chie Fujiwara, Kazunori Anzai, Yutaka Kirino, Seiji Nagao, Yoshinori N ...1988 年 104 巻 3 号 p. 344-348
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーCation channels in ciliary membrane vesicles from wild type Tetrahymena thermophila and from its mutant which does not show ciliary reversal or avoiding reaction were reconstituted into a planar lipid bilayer. Since the mutant does not produce the regenerative Ca2+ action potential, the mutation was expected to have occurred at the Ca-channel in the ciliary membrane. In the sample from the mutant, the channel most frequently observed was selective for cations over anions. The single channel conductance shows Michaelis-Menten type dependency on the cation concentration. The maximum conductance and dissociation constants (in parenthesis) for K+, Mg2+, Ca2+, and Ba2+ were 371 pS (23.3 mM), 17 pS (0.49 mM), 18 pS (0.52 mM), and 25 pS (0.82 mM), respectively, when the anion was gluconate. The properties of the corresponding channel from the wild type are similar to those from the mutant. No essential difference was detected, which indicates that the predominant channel is not the putative Ca channel responsible for the avoiding reaction of Tetrahymena. Some other channels than the predominant channel were also observed.抄録全体を表示PDF形式でダウンロード (529K) -
Nobuo Inoue, Hideo Matsui, Hiroko Tsukui, Hiroshi Hatanaka1988 年 104 巻 3 号 p. 349-354
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーImmature neurons from the cerebra of 17-day rat fetuses were cultured, and changes in Na+, K+-ATPase activity of the cells were investigated during maturation in culture. The Na+, K+-ATPase activity of the particulate fraction from the cells increased during the course of culture, up to 0.38±0.01 (μmol/min/mg protein) (=8.25±0.85 (μmol/min/mg DNA)) by day 13 in culture. The values were more than 3 and 19 times those of the 17-day fetal cerebrum on the bases of protein and DNA, respectively. The enzyme in the immature neurons was mainly a weakly digitalis-sensitive form in terms of the inhibition pattern of the Na+, K+-ATPase activity by strophanthidin. Along with the maturation in vitro of the neurons, a highly digitalis-sensitive form of the enzyme was shown to appear and increase by the biphasic inhibition patterns of Na+, K+-ATPase activity by strophanthidin and of K+ uptake activity by ouabain. The enzyme activity of the highly sensitive form overwhelmed that of the weakly sensitive form by day 13 in culture. In cultured rat cerebral astrocytes, the enzyme was judged to be only the weakly digitalis-sensitive form from the simple inhibition patterns of the Na+, K+-ATPase and K+ uptake activities by digitalis.抄録全体を表示PDF形式でダウンロード (715K) -
Yumiko Hokama, Nobuyuki Koga, Hidetoshi Yoshimura1988 年 104 巻 3 号 p. 355-361
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリー3, 4, 5, 3', 4' -Pentachlorobiphenyl (PenCB), one of the most potent 3-methylcholanthrene (MC)-type inducers of hepatic enzymes in animals, caused a remarkable induction of liver microsomal monooxygenases, particularly 7-ethoxyresorufin (7-ER) O-deethylase, benzo(a)pyrene (BP) 3-hydroxylase, and testosterone 16α-hydroxylase in chickens, but not NADPH-cytochrome c(P-450) reductase and cytochrome b5. Two forms of cytochrome P-450 (P-450) in liver microsomes of PenCB-treated chickens were purified and characterized. The absorption maxima of the CO-reduced difference spectra of both enzymes (chicken P-448 L and chicken P-448 H) were at 448 nm. From the oxidized form of their absolute spectra, chicken P-448 L was a low-spin form and chicken P-448 H was a high-spin form. They had molecular masses of 56 and 54 kDa, respectively. In a reconstituted system, 7-ER O-deethylation, BP 3-hydroxylation, and testosterone 16α-hydroxylation were catalyzed at high rates by chicken P-448 L but not by chicken P-448 H. Chicken P-448 L also catalyzed N-demethylation of aminopyrine, benzphetamine, and ethylmorphine with relatively low activity. On the other hand, chicken P-448 H functioned only in catalyzing estradiol 2-hydroxylation. These results were supported by an inhibition study of microsomal monooxygenases using an antibody against each enzyme. Immunochemical studies revealed that the enzymes differ from each other but are both inducible by PenCB-treatment. Chicken P-448 L and chicken P-448 H respectively comprise about 82 and 7% of the total P-450 content in chicken liver microsomes.抄録全体を表示PDF形式でダウンロード (2427K) -
Susumu Hamajima, Masao Sakaguchi, Katsuyoshi Mihara, Shigeru Ono, Ryo ...1988 年 104 巻 3 号 p. 362-367
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーYeast porin, the major outer mitochondrial membrane protein, is synthesized without a cleavable extension peptide and post-translationally inserted into the membrane. When inserted into the membrane, it acquires resistance to externally added trypsin. To locate the sequences responsible for membrane insertion and topogenesis in the primary structure of yeast porin, we constructed several deletion and chimeric mutants of the porin cDNA. These cDNAs were expressed in vitro and the products were assayed for capacity to be correctly inserted into isolated mitochondria. It was thus found that deletion of the segment spanning residues 37-98 did not appreciably impair the insertion competence and the inserted protein became resistant to trypsin. On the other hand, the porin mutant lacking the segment consisting of residues 17-98 did not acquire the trypsin resistance, though it could bind to mitochondria specifically. Deletion of the carboxy-terminal 62 amino acid residues also abolished the capacity to be correctly inserted into mitochondria. We conclude that information required for membrane insertion and intramembranous topogenesis of the porin molecule is stored not only in the amino-terminal region but also in the carboxy-terminal portion.抄録全体を表示PDF形式でダウンロード (3174K) -
Takashi Morita, Hiroshi Kaetsu, Jun Mizuguchi, Shun-ichiro Kawabata, S ...1988 年 104 巻 3 号 p. 368-374
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーEvidence is presented for rapid, limited proteolysis of protein Z by α-thrombin. This α-thrombin-catalyzed proteolysis of protein Z occurred at a single peptide linkage, between Arg-365 and Gly-366, located in the COON-terminal portion. The resulting NH2-terminal large fragment (PZt) and the COOH-terminal peptide (C-peptide) were isolated and chemically characterized. The C-peptide consisted of 31 amino acid residues including one galactosamine-type Thr residue and was assigned to the position from Gly-366 to the COOH-terminal residue of Val-396 in protein Z. The NH2-terminal large fragment, PZt, constituted the remainder of protein Z. The abilities to bind calcium of intact protein Z, PZt, and the derivative of protein Z devoid of the NH2-terminal γ-carboxy-glutamic acid (Gla) domain (Gla-domainless), prepared with the known chymotrypsin treatment, were examined by equilibrium dialysis. The results indicated that intact protein Z and PZt contain four calcium binding sites with dissociation constants of 0.1 mM. Moreover, the Scatchard plot analysis showed positive cooperativity, suggesting the presence of at least two initial sites for calcium binding. In contrast, the Gla-domainless protein Z had no calcium binding site, indicating that the domain of protein Z functional for calcium binding occurs within the NH2-terminal Gla domain. This differed from factor X, factor IX, protein S, and protein C, all of which contain one or two calcium binding site(s) independent on their Gla-domains.抄録全体を表示PDF形式でダウンロード (2540K) -
Kenji Takahashi1988 年 104 巻 3 号 p. 375-382
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーThe complete amino acid sequence of ribonuclease N1 (RNase N1), a guanine-specific ribonuclease from a fungus, Neurospora crassa, was determined by conventional protein sequencing, using peptide fragments obtained by tryptic digestion of cyanogen bromide-treated RNase N1 and by Staphylococcus aureus V8 protease digestion of heat-denatured RNase N1. The results showed that the protein is composed of a single polypeptide chain of 104 amino acid residues cross-linked by two disulfide bonds and has a molecular weight of 11, 174:
ACMYICGSVCYSSSSISAALNKGYSYYEDGATAGSSSYPHRYNNYEGFDFPTAKPWYE
FPILSSGRVYTGGSPGADRVIFDSHGNLDMLITHNGASGNNFVACN
(Disulfide bonds: C2-C10, C6-C103)
The amino acid sequence was homologous with those of RNase T1 (65% identity) and related microbial RNases.抄録全体を表示PDF形式でダウンロード (718K) -
Tohoru Nakamura, Hideki Sato, Toshiro Shimamura, Jiro Koyama1988 年 104 巻 3 号 p. 383-387
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーThe functional roles of two distinct types of Fcγ receptors (Fcγ1/γ2R specific for both IgG1 and IgG2, and Fcγ2R specific for IgG2 alone) on the surface of guinea pig macrophages in the phagocytosis of sensitized sheep erythrocytes (EA) were investigated by the use of two Fab's of monoclonal anti- Fcγ1/γ2R and anti-Fcγ2R antibodies. The binding and subsequent ingestion of IgG1 antibody-sensitized erythrocytes (EAγ1) by macrophages were complete-ly inhibited by anti-Fcγ1/γ2R Fab', indicating that the reactions are mediated only by Fcγ1/γ2R. On the other hand, the binding and subsequent ingestion of IgG2 antibody-sensitized erythrocytes (EAγ2) were substantially inhibited by anti-Fcγ2R Fab', but not by anti-Fcγ1/γ2R Fab'. The inhibitory activities of anti-Fcγ2R Fab' were dependent upon the amount of IgG2 antibody bound on erythrocytes; increasing the amount of bound IgG2 antibody from 0.15 to 0.91 μg/2 ×108 erythrocytes resulted in a decrease in the inhibition of binding of EAγ2 by anti-Fcγ2R Fab' from 50 to 0%, and also a decrease in the inhibition of ingestion of EAγ2 from 100 to 50%. Since the binding and the ingestion of EAγ2 by macrophages were completely inhibited in the presence of both anti-Fcγ2R and anti- Fcγ1/γ2R Fab', Fcγ2R seems to preferentially operate in these reactions, and Fcγ1/γ2R does not seem to operate unless the Fcγ2R. on the same macrophage is blocked by anti-Fcγ2R Fab'. The finding that the avidity and ingestive activity of macrophages for EAγ2 are higher than those for EAγ2, also indicates that Fcγ2R functions more effectively in the binding and ingestion of EA than Fcγ1/γ2R. These different abilities of Fcγ2R and Fcγ1/γ2R do not seem to be caused by any difference in their affinities for EAγ2, since ovalbumin-complexed IgG2 antibody was found to bind to Fcγ2R with an association constant essentially equal to that to Fcγ1/γ2R.抄録全体を表示PDF形式でダウンロード (664K) -
Shinobou Gamou, Nobuyoshi Shimizu1988 年 104 巻 3 号 p. 388-396
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーEpidermal growth factor (EGF) receptor biosynthesis was examined in an oral squamous cell carcinoma line, NA, which overproduces the receptor to an even greater extent than the widely studied A431 cells. The EGF receptor of NA cells synthesized in the presence of tunicamycin had an apparent molecular weight of 130, 000. The nascent protein in untreated cells was cotranslationally glycosylated to Mr 160, 000 and further processed to Mr 170, 000. The endo-β-N-acetylglucosaminidase H (Endo H) digestion analysis revealed the presence of high mannose type oligosaccharide on the Mr 170, 000 mature receptor. We extended the analysis by correlating the biosynthesis with the acquisition of binding activity. The unglycosylated Mr 130, 000 receptor and the Mr 160, 000 receptor seen after pulse-labeling had no EGF binding activity, whereas the Mr 160, 000 receptor seen after chase-incubation and the Mr 170, 000 receptor had binding activity. Thus, not only glycosylation but also some oligosaccharide processing is apparently necessary for the EGF binding. Treatment with processing inhibitors, such as monensin, swainsonine and 1-deoxynojirimycin, affected neither receptor transport to the plasma membrane nor binding activity. Inhibition by 1-deoxynojirimycin is thought to be incomplete since the surface receptor in treated cells had the same molecular weight as that in control cells. An Mr 160, 000 receptor without binding activity accumulated in the intracellular fraction in the presence of brefeldin A, an inhibitor of intracellular transport. Thus, the EGF binding activity is thought to be acquired after the brefeldin A-sensitive process but prior to the swainsonine-sensitive mannose removal in NA cells.抄録全体を表示PDF形式でダウンロード (5885K) -
Kouki Ishitani, Shinji Ogawa, Masuko Suzuki1988 年 104 巻 3 号 p. 397-402
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーWe examined whether arachidonate metabolism exerted any influence on the enhancement of intracellular transglutaminase activity in mouse peritoneal macrophages. Enhancement of the intracellular transglutaminase activity was observed on stimulation of macrophages with normal sheep red blood cells (SRBC) or immunoglobulin G (IgG)-coated SRBC, and was inhibited by inhibitors of phospholipase A2 and cyclooxygenase. Moreover, prosta-glandin E2 (PGE2), a main product of the cyclooxygenase pathway, leukotriene B4 (LTB4), a product of 5-lipoxygenase, and arachidonic acid also could directly induce high levels of intracellular transglutaminase activity without stimulation of macrophages by SRBC or IgG-coated SRBC, but leukotriene C4, prostaglandin D2, and prostacyclin were unable to induce high activity of the enzyme. Enhancement of transglutaminase activity induced by LTB4 was inhibited by cyclooxygenase inhibitor, but the enzyme activity induced by PGE2 was not inhibited. Furthermore, the quantity of PGE2 released into the culture medium of macrophages stimulated with SRBC or IgG-coated SRBC correlated well with the activity of intracellular transglutaminase in macrophages. Moreover, enhancement of trans-glutaminase activity by treatment of macrophages with SRBC or IgG-coated SRBC was partially suppressed by sodium benzoate, which is a scavenger of hydroxy radical. These findings suggest that arachidonate metabolism, in particular the cyclooxygenase pathway, plays an important role in the enhancement of intracellular transglutaminase activity.抄録全体を表示PDF形式でダウンロード (701K) -
Fumitaka Oyama, Rie Kikuchi, Tsuneko Uchida1988 年 104 巻 3 号 p. 403-408
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーSynthetic, partial pre-mRNA for ovalbumin was found to prime its own cDNA with reverse transcriptase. Initiation of the cDNA occurred 36 bases upstream of the 3'-end of the RNA, probably as a result of intramolecular base pairing at this end. Inhibition of self-priming occurs following ligation of pCp or poly-adenylation at the 3'-terminus of the synthetic RNA. The secondary structure of 3'-end region of the template RNA strongly affected the ability of self-priming.抄録全体を表示PDF形式でダウンロード (2683K) -
Yuko Nagamine, Kaoru Omichi, Tokuji Ikenaka1988 年 104 巻 3 号 p. 409-415
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーThe modes of action of two isozymes of human salivary α-amylase on phenyl α-maltopentaoside, phenyl α-maltotetraoside, and their derivatives which have an iodo or an amino or a carboxyl group at their first or penultimate glucopyranosyl residues from the non-reducing-end were examined. It is conceivable that the active site of this enzyme is composed of tandem subsites (S4, S3, S2, S1, S', S2', and S3') geometrically complementary to several glucose residues, and that the glucosidic bonds of the substrates are split between S1 and S1'. Product analysis of each digest strongly suggested the presence of a hydrophobic amino acid residue at subsite S3 in the active site of the enzyme. No difference in the modes of action on the substrates was found between the two isozymes, indicating that the three-dimensional structures of their active site areas are, at the least, similar.抄録全体を表示PDF形式でダウンロード (1709K) -
Yine-Chieh Tsai, Yu-Tzu Lin, Yunn-Bor Yang, Ywan-Feng Li, Makari Yamas ...1988 年 104 巻 3 号 p. 416-420
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーThe substrate specificity of alkaline elastase Bacillus from alkalophilic Bacillus sp. Ya-B was investigated using oxidized insulin A- and B-chains. Under time-limited cleavage, the initial cleavage site of the enzyme on the oxidized insulin A-chain and B-chain was at the leucinel3-tyrosinel4 bond and the leucinel5-tyrosinel6 bond, respectively. When the cleavage was completed, three major cleavage sites and three minor cleavage sites on the A-chain, and five major cleavage sites and four minor cleavage sites on the B-chain were found. However, most of the peptides produced after complete hydrolysis of the A- or B-chain by the enzyme were composed of four to six amino acid residues. The results suggest that this enzyme cleaves the oxidized insulin A- and B-chains in a block-cutting manner.抄録全体を表示PDF形式でダウンロード (495K) -
Norio Abe, Eri Abe, Akira Yuasa1988 年 104 巻 3 号 p. 421-426
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリー5-Hydroxytryptamine UDP-glucuronyltransferase was highly purified from untreated rat liver microsomes. The specific activity towards 5-hydroxytryptamine was increased 178-fold over the starting solubilized microsomes with a final yield of 3%. The final preparation contained two major and one minor Coomassie brilliant blue staining polypep-tide bands visible after SDS-polyacrylamide gel electrophoresis. One of the major bands was identified as 3-methylcholanthrene-inducible UDP-glucuronyltransferase, so the other (molecular weight of 55, 500) appeared to be 5-hydroxytryptamine UDP-glucuronyltrans-ferase. Concanavalin A reacted with the 55, 500-dalton polypeptide. Phospholipid was indispensable for the enzyme activity. The enzyme activity in the final preparation was activated by divalent cations. Simple Michaelis-Menten kinetics was followed with respect to 5-hydroxytryptamine, but deviations from this kinetics were observed with respect to UDP-glucuronic acid and Mg2+ . As regards Mg2+ stimulation, further experiments indicated that the added Mg2+ was non-competitive with 5-hydroxytryptamine, but at low concentrations of Mg2+ it was competitive with UDP-glucuronic acid and at high concentrations of Mg2+ it was non-competitive with UDP-glucuronic acid. The final preparation showed high substrate specificity towards 5-hydroxytryptamine among endogenous substrates tested.From these results, it was concluded that the enzyme described here is a new form of UDP-glucuronyltransferase isozyme, and its activity showed a peculiar dependence on mg2+.抄録全体を表示PDF形式でダウンロード (1479K) -
Daisaku Muno, Takamitsu Sekine1988 年 104 巻 3 号 p. 427-432
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーWe reported in the preceding paper [Muno, D., et al. (1987) J. Biochem. 101, 661-669] that the dinitrophenyl group exclusively introduced to SH1, on the 20-kDa fragment of myosin subfragment 1 was cross-linked to the 50-kDa fragment by irradiation, and that limited trypsinolysis of the cross-linked S1 generated an 83-kDa peptide, a cross-linking product between the 20- and 50-kDa fragments. This paper will deal with the location of the cross-linked residue on the 50-kDa fragment. When the 83-kDa fragment labeled at SH2, with a fluorogenic SH reagent was subjected to bromocyanolysis, a main fluorescent band, which implied a cross-linked peptide, appeared in the position with an apparent molecular mass of 18.5-kDa on SDS-PAGE. On the other hand, another cross-linked peptide was obtained from a complete tryptic digest of a 83-kDa fragment rich fraction. Amino acid sequence analysis of the two cross-linked peptides revealed that the DNP moiety attached at SH1, was cross-linked with a residue in the segment of the heavy chain spanning the 485-493 region from the N-terminus of the heavy chain.抄録全体を表示PDF形式でダウンロード (3240K) -
Motohisa Oobatake, Tatsuo Ooi1988 年 104 巻 3 号 p. 433-439
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーThermodynamic properties associated with hydrated water of proteins of known three-dimensional structure were computed and average values of hydration free energy, enthalpy, and heat capacity of unfolding for every amino acid residue were obtained. Each amino acid residue had characteristic values; in particular, the quantities for a side chain reflected the character of the amino acid, while those for the main chain were more or less the same except for glycine, alanine, and proline. The major contribution to the quantities was from the end group(s) of a side chain. The following interesting features were found. 1) The hydration quantity of unfolding derived from the native and extended conformations for a protein was approximately equal to the sum of the corresponding average quantities of component amino acid residues in the protein. 2) The profile of a quantity such as hydration free energy of unfolding along the sequence computed from the accessible surface areas of the native and extended conformations showed a strong correlation with the profile obtained by allocating the average value for the amino acid residue at every position on the sequence. The correlation coefficients between two profiles for unfolding quantities of hydration, i. e., free energy, enthalpy, heat capacity, and free energy of side chain are 0.72, 0.62, 0.80, and 0.75, respectively. Thus, every amino acid residue in the native conformation of a globular protein seems to be located in such a position that a thermodynamic quantity for each residue is approximately equal to its average value.抄録全体を表示PDF形式でダウンロード (756K) -
Tatsuo Ooi, Motohisa Oobatake1988 年 104 巻 3 号 p. 440-444
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーThe structural aspects of protein functions, e. g., molecular recognition such as enzymesubstrate and antibody-antigen interactions, are elucidated in terms of dehydration and atomic interactions. When a protein interacts with some target molecule, water molecules at the interacting regions of both molecules are removed, with loss of the hydration free energy, but gaining atomic interactions between atoms of the contact sites in both molecules. The free energies of association originating from the dehydration and interactions between the atoms can be computed from changes in the accessible surface areas of the atoms involved. The free energy due to interactions between atomic groups at the contact sites is estimated as the sum of those estimated from the changes in the accessible surface area of 7 atomic groups, assuming that the interactions are proportional to the change of the area. The chain enthalpies and entropies evaluated from experimental thermodynamic properties and hydration quantities at the standard temperature for 10 proteins were available to determine the proportional constants for the atomic groups. This method was applied to the evaluation of association constants for the dimerization of proteins and the formation of proteolytic enzyme-inhibitor complexes, and the computed constants were in agreement with the experimental ones. However, the method is not accurate enough to account quantitatively for the change in the thermal stability of mutants of T4 lysozyme. Nevertheless, this method provides a way to elucidate the interactions between molecules in solution.抄録全体を表示PDF形式でダウンロード (575K) -
Masazumi Miyazaki, Masahiro Uritani, Hideto Kagiyama1988 年 104 巻 3 号 p. 445-450
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーATP (GTP) hydrolysis was clearly demonstrated by using at most 16 pmol of yeast peptide elongation factor 3 (EF-3) in the absence of ribosomes. However, the highly active yeast ribosomes (up to 48 pmol) displayed virtually no ATPase (or GTPase) activity in the absence of EF-3. Several lines of evidence indicated that both the catalytic and binding sites of the ATPase reside in the elongation factor itself, not on the ribosomes. The patterns of protection by various nucleoside triphosphates against tryptic digestion of EF-3, reflecting the wide substrate specificity of the ATPase, confirmed that the active center of the endogenous ATPase is located on the factor itself and not on contaminants. The intrinsic activity was stimulated up to two orders of magnitude by the presence of the yeast ribosomes fully active in polyphenylalanine synthesis. The activation was achieved by enhancing the catalytic activity (kcat) to a much greater extent than the binding affinity (Km). On the other hand, the ribosome-activated ATPase activity was revealed to inherit its wide substrate specificity from the intrinsic property of EF -3, which shows an affinity to various XTPs, including pyrimidine- and purine-nucleoside triphosphates, irrespective of 2' - hydroxylation of the sugar moiety. From experiments on protection against tryptic digestion, we determined that intricate conformational changes of the factor molecule occur upon interaction with the substrate XTP and ribosomes.抄録全体を表示PDF形式でダウンロード (2699K) -
Norio Yoshida, Shigeru Tsuruyama, Kyoko Nagata, Kouichirou Hirayama, K ...1988 年 104 巻 3 号 p. 451-456
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーA protease was purified 163-fold from Pronase, a commercial product from culture filtrate of Streptomyces griseus, by a series of column chromatographies on CM-Toyopearl (Fractogel), Sephadex G-50, hydroxyapatite, and Z-Gly-D-Phe-AH-Sepharose 4B using Boc-Ala-Ala-Pro-Glu-pNA as a substrate. The final preparation was homogeneous by polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and gel isoelectric focusing. Studies on the substrate specificity with peptide p-nitroanilides revealed that this protease preferentially hydrolyzed peptide bonds on the carbonyl-terminal side of either glutamic acid or aspartic acid. It was most active at pH 8.8 for the hydrolysis of Boc-Ala-Ala-Pro-Glu-pNA. The molecular weight of the protease was estimated to be 20, 000 by gel filtration on Sepharose 6B using 6M guanidine hydrochloride as an eluent, and 22, 000 by SDS-PAGE in the presence of 2-mercaptoethanol. The isoelectric point of the enzyme was 8.4. The enzyme was inactivated by diisopropyl phosphofluoridate (DFP) but not by p-cbloromercuribenzoate (PCMB) or EDTA.抄録全体を表示PDF形式でダウンロード (1626K) -
Hiroyasu Nakata, Hitoshi Fujisawa1988 年 104 巻 3 号 p. 457-460
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーRat pheochromocytoma PC 12 cell membranes were shown to possess A2-like adenosine binding sites as assessed by using 5'-N-ethylcarboxamide[3H]adenosine ([3H]NECA). Specific [3H]NECA binding to PC 12 cell membrane at 0°C was saturable and showed a monophasic saturation profile. In contrast, [3H]NECA binding to PC 12 cell membrane at 30°C exhibited a biphasic profile suggesting the presence of two specific binding site. The rank order of potency for inhibition of [3H]NECA binding at 0°C was NECA>2-chloro-adenosine > 2', 5'-dideoxyadenosine > isobutylmethylxanthine_??_phenylisopropyladenosine. These adenosine binding sites were solubilized with sodium cholate and the solubilized portion retained the same ligand binding characteristics as those of the membranebound form. Gel filtration experiments indicated an apparent Stokes radius of 6.7nm for these adenosine binding sites/detergent complexes.抄録全体を表示PDF形式でダウンロード (451K) -
Satoshi Nagura, Takashi Kawasaki, Takahisa Taguchi, Michiki Kasai1988 年 104 巻 3 号 p. 461-465
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーThe effects of SH reagents on Ca2+ release from sarcoplasmic reticulum (SR) vesicles were examined by the tracer method using 45Ca2+. Among the various SH reagents tested, 4, 4'-dithiodipyridine (PDS) was found to induce Ca2+ release most specifically from the heavy fraction of SR vesicles. Further, the following results were obtained. (i) PDS bound covalently to proteins in the SR membrane and induced Ca2+ release. (ii) The Ca2+ release was further enhanced by ATP and caffeine, but inhibited by procaine, ruthenium red and various divalent cations. (iii) PDS enhanced the Ca2+ release in the whole range of Ca2+ concentrations tested. (iv) Choline permeability was also enhanced by PDS. Further, the electrical conductance of the Ca2+ -induced Ca2+ release channels was studied by incorporating them into lipid bilayers and it was found that PDS increased the probability of opening of the channels. These results suggest that PDS binds to certain SH groups of the Ca2+ -induced Ca2+ release channels in the SR membrane and thus induces Ca2+ release.抄録全体を表示PDF形式でダウンロード (562K) -
Tatsuo Horiuchi, Toshiko Kurokawa1988 年 104 巻 3 号 p. 466-471
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーA new enzyme, N-acyl-D-mannosamine dehydrogenase, was purified to apparent homogeneity from a cell-free extract of Flavobacterium sp. 141-8 and some of its properties were investigated. The enzyme showed optimum activity at pH 8.0-9.5. N-Acetyl- and N-gly-colyl-D-mannosamine were oxidized but other commonly existing sugars, such as N-acetylglucosamine, N-acetylgalactosamine, amino sugars, neutral hexoses, and pentoses, were not oxidized. NAD+ was specifically utilized as an effective hydrogen acceptor. The apparent Km values for N-acetyl- and N-glycolyl-D-mannosamine, and NAD+ were 1.0, 13.3, and 0.41mM, respectively. Thestoichiometry data showed that 1 mol each of N-acetyl-D-mannosamine and NAD+ were converted to 1mol each of N-acetyl-D-mannosaminic acid and NADH, respectively. Although the formation of lactone was detected in the enzyme reacton mixture, the reverse reaction of the enzyme, the reduction of N-acetyl-D-mannosamino-lactone, was not observed. The enzyme activity was strongly inhibited by Hg2+ and SDS, but metal-chelating reagents and sulfhydryl-group-blocking reagents had almost no effect. The molecular weight of the enzyme was estimated to be 120, 000 on gel filtration and 29, 000 on SDS-polyacrylamide gel electrophoresis. Its isoelectric point was at pH 4.8. On trial application of the enzyme, it was indicated that N-acetylneuraminic acid can be determined quantitatively with the combined enzyme system involving the new enzyme and N-acetylneuraminic acid aldolase.抄録全体を表示PDF形式でダウンロード (2932K) -
Kenji Wada, Tadashi Tanabe1988 年 104 巻 3 号 p. 472-476
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーCathepsin B was purified, 400-fold, to homogeneity from chicken liver. The enzyme comprised a mixture of two kinetically indistinguishable forms (approximately 1:1), which were separated on concanavalin A (Con A)-Sepharose; one consisting of Mr 25, 500
and 5, 000 polypeptide chains bound to Con A-Sepharose but the other, composed of Mr 24, 500 and 5, 000 polypeptide chains, did not. N-terminal amino acid sequence analyses of a mixture of the Mr 25, 500 and 24, 500 polypeptide chains, and of the Mr 5, 000 polypeptide chain revealed single amino acid sequences, respectively. These amino acid sequences were homologous to those of the heavy and light chains of mammalian enzymes, respectively. The chicken liver and mammalian cathepsin B were similar in structure and properties.抄録全体を表示PDF形式でダウンロード (861K) -
Yoshikazu Tanaka, Toshihiko Ashikari, Yuji Shibano, Teruo Amachi, Haji ...1988 年 104 巻 3 号 p. 477-480
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーVarious in vitro mutated human cytochrome c genes which encode displaced amino acid
residues at the 14th, 17th, 28th, 37th, 38th, 56th, and/or 84th residues were constructed, and their degrees of complementation of yeast CYC1 deficiency were examined. Invariant Cys-17 and Arg-38 could not be replaced by alanine and tryptophan, respectively, without function impairment. Cytochrome c containing Ala-14 instead of conserved Cys-14, Gly-38 or Lys-38 instead of Arg-38, and Ser- 84 instead of invariant Gly-84 were partly functional. These results indicate that these invariant or conserved residues are important. Cytochromes c containing Cys-56 instead of native Gly-56 was partly functional. Cytochrome c containing Arg-37 and Gly-38 instead of Gly-37 and Arg-38 was slightly functional. Replacement of variable Thr-28 and Gly-37 by Ile-28 and Arg-37, respectively, produced no effects. Our results are as a whole consistent with the view that conserved residues are important and variable residues are less important for cytochrome c to function.抄録全体を表示PDF形式でダウンロード (448K) -
Susumu Imaoka, Yoshitake Terano, Yoshihiko Funae1988 年 104 巻 3 号 p. 481-487
発行日: 1988年
公開日: 2008/11/18
ジャーナル フリーThe cytochrome P-450 that was purified from hepatic microsomes of male rats treated with phenobarbital and designated P450 PB-1 (Funae and Imaoka (1985) Biochim. Biophys. Acta 842, 119-132) had high testosterone 6β-hydroxylation activity (turnover rate, 13.5 nmol of product/ min/nmol of P-450) in a reconstituted system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5, and a 1:1 mixture of lecithin and phosphatidylserine in the presence of sodium cholate. In ordinary conditions in the reconstituted system with cytochrome P-450, reductase, and dilauroylphosphatidyl-choline, P450 PB-1 had little 6β-hydroxylase activity. The catalytic activities toward testosterone of two major constitutive forms, P450 UT-2 and P450 UT-5, were not affected by cytochrome 65, phospholipid, or sodium cholate. P450 PB-1 in rat liver microsomes was assayed by immunoblotting with specific antibody to P450 PB-1. P450 PB-1 accounted for 24.4±5.6% (mean±SD) of the total spectrally-measured cytochrome P-450 in hepatic microsomes of untreated adult male rats, and was not found in untreated adult female rats. P450 PB-1 was induced twofold with phenobarbital in male rats. P450 PB-1 was purified from untreated male rats and identified as P450 PB-1 from phenobarbital-treated rats by its NH2-terminal sequence, peptide mapping, and immunochemistry. These results showed that P450 PB-1 is a constitutive male-specific form in rat liver. There was a good correlation (r=0.925) 0.925) between the P450 PB-1 level and testosterone 6β-hydroxylase activity in rat liver microsomes. Furthermore, testosterone 6β-hydroxylation activity of hepatic microsomes was completely inhibited by anti-P450 PB-1 antibody. The conclusion is that P450 PB-1 is the major or sole constitutive catalyst of testosterone 6β-hydroxylase in rat liver.抄録全体を表示PDF形式でダウンロード (2038K)
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