巻号一覧
105 巻, 6 号
選択された号の論文の38件中1～38を表示しています
- |<
- <
- 1
- >
- >|
-
Sumiko Ohshima, Hiroshi Abe, Takashi Obinata1989 年 105 巻 6 号 p. 855-857
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーAn actin-binding protein of 16 kDa was isolated from embryonic chicken skeletal muscle. The protein had the same properties as profilin, exhibited a much higher affinity for cytoskeletal (β- and γ-) actins than for sarcomeric (α-) actin in the embryonic muscle, and inhibited the polymerization of β- and γ-actins more efficiently in a physiological salt solution. These results indicate that the assembly of cytoskeletal and sarcomeric actins is regulated differently by profilin in the developing skeletal muscle, and that the former may not be involved in myofibril assembly.抄録全体を表示PDF形式でダウンロード (1139K) -
Junko Hirano-Ohnishi, Yoshio Watanabe1989 年 105 巻 6 号 p. 858-860
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーThe ciliary axoneme is the minimal structure responsible for Ca2+ -dependent modulation of ciliary movement. We demonstrated that, in Tetrahymena ciliary axonemes, β-tubulin was exclusively phosphorylated by an endogenous Ca2+/calmodulin-dependent protein kinase (s). The phosphorylation of β-tubulin also occurred in the outerdoublet microtubule fraction, suggesting that the responsible enzyme(s) was tightly associated with outer-doublet microtubules. In experiments with several types of permeabilized cilia retaining ciliary motility, Ca2+-dependent phosphorylation of β-tubulin was also found to occur exclusively. From these results, it is inferable that the phosphorylation of β-tubulin is involved in Ca2+ -dependent ciliary reversal.抄録全体を表示PDF形式でダウンロード (1726K) -
Hitoshi Manabe, Hideo Utsumi, Tsuneo Kusama, Akira Hamada1989 年 105 巻 6 号 p. 861-863
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーThe immunopotentiating property of spin-labeled lipophilic muramyl dipeptide (SL-MDP) in liposomes was studied as to the plaque-forming cell (PFC) response to glycophorin, as an antigen, in membranes. The effect of SL-MDP depended on the densities of both SL-MDP itself and the antigen. The addition of the optimal amount of SL-MDP to liposomes containing a low density (0.016 mol%) of the antigen increased the PFC response by three times, whereas the presence of SL-MDP in optimal antigen density (0.032-0.127 mol%) membranes was rather inhibitory. In these liposomes, the amounts and molecular states of SL-MDP were determined from ESR spectra and are discussed in connection with its immunopotentiating property.抄録全体を表示PDF形式でダウンロード (385K) -
Akio Yoshimoto, Shigeru Sakajo, Nobuko Minagawa, Tadazumi Komiyama1989 年 105 巻 6 号 p. 864-866
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーThe antimycin A or cyanide-dependent appearance of a 36 kDa protein in the particulate fraction was observed in L-[35S] methionine pulse-labeling experiments on cells of Hansenula anomala, in which cyanide-resistant respiration was induced. The combined addition of cycloheximide or anaerobiosis, which block the induction of cyanide-resistant respiration, repressed the synthesis of this protein. These results suggest the involvement of the particulate 36 kDa protein in cyanide-resistant respiration.抄録全体を表示PDF形式でダウンロード (1338K) -
Koichi Kato, Chigusa Matsunaga, Yoshifumi Nishimura, Markus Waelchli, ...1989 年 105 巻 6 号 p. 867-869
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーA 13C nuclear magnetic resonance study of a mouse anti-dansyl monoclonal antibody is reported. The antibody molecule was specifically labeled with [1-13C]methionine by growing hybridoma cells in serum-free medium. It was possible to observe all the carbonyl carbon resonances of the antibody. Fab and Fc fragments have been obtained from the antibody and used successfully for the assignment of each of the carbonyl resonances to either the Fab or Fc region. It has been shown that the spectrum of the intact antibody is simply those of Fab and Fc superimposed. It has also been shown that site specific assignments of carbonyl resonances can be made by means of a double labeling technique developed by Kainosho and coworkers (Biochemistry 21, 6273-6279 (1982)).抄録全体を表示PDF形式でダウンロード (366K) -
Yoshihiko Yamakita, Takayoshi Iio1989 年 105 巻 6 号 p. 870-874
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーThe fluorescence titration curve of skeletal muscle troponin containing TnI with 2-((4'- iodoacetamido)anilino)naphthalene-6-sulfonic acid-labeled Cys-48 and / or Cys-64 was composed of two transition curves. One transition occurred at the pCa region higher than 8.0, and the other between pCa 8.0 and 6.0. The transition at the lower pCa region had a midpoint of pCa 6.85, and the midpoint did not depend on Mg2+. The time course of the fluorescence change subsequent to the rapid pCa-jump of the solution was biphasic. The fast phase was due to the transition at the lower pCa region, and the rate constant of the process was characteristic of the conformational change of the protein induced by Ca2+ binding to the low affinity Ca2+ -binding sites of TnC. The slow phase was from the transition at the higher pCa region, and its rate constant was characteristic of the conformational change of the protein induced by Ca2+-binding to the high affinity Ca2+-binding sites of TnC. Therefore we can conclude that the fluorescence probe bound to Cys-48 and/or Cys-64 of TnI detects the conformational change of the Tn complex induced by Ca2+ binding to both the low and high affinity Ca2+-binding sites of TnC. The fluorescence probe bound to Cys-133 of TnI or Met residues of TnT detected the conformational change of the Tn complex induced by Ca2+ binding to the low affinity Ca2+-binding sites of TnC.抄録全体を表示PDF形式でダウンロード (508K) -
Hiroshi Hayashi, Kingo Takiguchi, Sugie Higashi-Fujime1989 年 105 巻 6 号 p. 875-877
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーMyosin, heavy meromyosin, and myosin subfragment-1 (S-1) were immobilized on the inner surfaces of glass capillary tubes, the inside walls of which had been coated with nitrocellulose. The ATPase activities of the immobilized proteins were measured using radiolabeled ATP and electrophoretic separation of the reaction products. The activity was proportional to the amount of immobilized protein. Activation by actin of the ATPase was also observed.抄録全体を表示PDF形式でダウンロード (1324K) -
Eiko Ueno, Hideaki Sakai, Yuzo Kato, Kenji Yamamoto1989 年 105 巻 6 号 p. 878-882
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーActivation of the erythrocyte cathepsin E located on the cytoplasmic surface of the membrane in a latent form was studied in stripped inside-out membrane vesicles prepared from human erythrocyte membranes. Incubation of the vesicles at 40°C at pH 4 resulted in increased degradation of the membrane proteins, especially band 3. This proteolysis was selectively inhibited by the inclusion of pepstatin (isovaleryl-Val-Val-statyl-Ala-statine) or H 297 [Pro-Thr-Glu-Phe(CH2-NH)Nle-Arg-Leu] in the incubation mixtures, indicating that cathepsin E, as the only aspartic proteinase in erythrocytes, is responsible for the proteolysis. Two potential active-site-directed inhibitors of aspartic proteinases, pepstatin and H 297, were used to prove the occurrence of the membrane-associated active enzyme. To minimize potential errors arising from non-specific binding, the concentrations of the inhibitors used in the binding assay (pepstatin, 5×10-8M; H 297, 1×10-5M) were determined by calibration for purified and membrane-associated cathepsin E. The inhibition of the membrane-associated cathepsin E by each inhibitor, which showed the binding of the inhibitor to the activated enzyme, was temperature- and time-dependent. The binding of each inhibitor to the enzyme on the exposed surface of the membrane at pH 4 was highly specific, saturable, and reversible. The present study thus provides the first evidence that cathepsin E tightly bound to the membrane is converted to the active enzyme in the membrane-associated form, and suggests that this enzyme may be responsible for the degradation of band 3.抄録全体を表示PDF形式でダウンロード (2673K) -
Norio Matsushima, Yoshinobu Izumi, Tomoyuki Matsuo, Hidenori Yoshino, ...1989 年 105 巻 6 号 p. 883-887
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーThe technique of small-angle X-ray scattering has been employed to examine the solution conformation of calmodulin and its complexes with Ca2+ alone, and with both Ca2+ and mastoparan. The radius of gyration decreased by 3.1±0.3Â upon binding of both 4mol Ca2+/mol of protein and 1mol mastoparan/mol of protein to form the ternary complex. A smaller increase was found for the separate binding of 4mol Ca2+/mol of protein in the absence of mastoparan (0.6±0.3Â). The analyses of pair distance distribution function showed that the maximal pair distance in calmodulin complex with both Ca2+ and mastoparan decreased by 20-30% in comparison with calmodulin or its complex with Ca2+ and a shoulder near 40Â, which characterizes the dumbbell-shaped molecule of calmodulin, disappeared. These results indicate that the two globular domains of the calmodulin complex with Ca2+ and mastoparan come close together by 8.0-9.5Â on average, if the size and the overall shape of the globular domains are the same in Ca2+-calmodulin-mastoparan complex as in calmodulin or Ca2+-calmodulin complex.抄録全体を表示PDF形式でダウンロード (556K) -
Hiroyasu Nakata, Hitoshi Fujisawa1989 年 105 巻 6 号 p. 888-893
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーMouse mastocytoma P815 cell membranes were found to possess adenosine binding sites as assessed by using the adenosine agonist [3H]5'-N-ethylcarboxamideadenosine (NECA). The Kd and Bmax for the [3H]NECA binding at 0° were 380nM and 17pmol/mg of protein, respectively. The rank order of potency for inhibition of [3H]NECA binding was NECA>5' -N-cyclopropylcarboxamideadenosine>2-chloroadenosine>2', 5'-dideoxyadenosine>isobutylmethylxanthine>theophylline>N6-[(R)-1-methyl-2-phenylethyl] adenosine=N6-[(S)-1-methyl-2-phenylethyl] adenosine. Thermodynamic analyses of the adenosine receptor agonist and antagonist binding showed that all such ligands displayed negative values of both enthalpy and entropy which suggested that the driving force for the binding was enthalpic. [3H]NECA binding sites of P815 cell membranes were solubilized with sodium cholate and retaining the same ligand-binding characteristics as those of the membrane-bound form. By gel filtration on a Sepharose CL-6B column, the adenosine binding site was estimated to have a Stokes radius of approximately 6.7nm.抄録全体を表示PDF形式でダウンロード (762K) -
Atsushi Isoai, Shunsaku Harie, Keiichi Uchida, Hidematsu Hirai1989 年 105 巻 6 号 p. 894-897
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーRat liver arginase was purified and five monoclonal antibodies were produced by fusion of spleen cells from a Balb/c mouse and the myeloma cell line P3-X36-Ag-U1. One, R2D19, of five antibodies belonged to the IgG2a subclass, the other four, R1D81, R1G11, R2E10, and R2G51, were of the IgG1 type. The R1D81 cross-reacted with human liver arginase. This antibody inhibited the arginase activity, competing with argininC. These results suggest that R1D81 binds to the catalytic site of arginase. The R2D19 also inhibited the enzyme activity but acted as a noncompetitive inhibitor. With the use of R1D81 and a polyclonal anti-human liver arginase antibody conjugated with alkaline phosphatase, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of human arginase. Specificity of monoclonal antibodies for rat liver arginase was examined by means of the sandwich ELISA. Eight pairs of monoclonal antibodies could form a sandwich with the arginase. Only the R2E10 could be used for both the first and the second antibody in the sandwich system. In other cases, monoclonal antibodies could not be interchanged between solid and liquid phase.抄録全体を表示PDF形式でダウンロード (497K) -
Shiori Tamamizu, Yoshimasa Miyake, Toshiko Ito, Hyogo Sinohara1989 年 105 巻 6 号 p. 898-904
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーReactions of rabbit α-2-macroglobulin with methylamine and trypsin were studied and the results were compared with those obtained for previously described 2-macroglobulins from other species. Rabbit α-2-macroglobulin was cleaved by trypsin at a number of sites, whereas the human homologue was split essentially only in the “bait” region into two fragments of similar sizes. Reaction of native or methylamine-treated rabbit α-2-macroglobulin with trypsin resulted in a substantial decrease in the intensity of fluo-rescence induced by binding of 6-(p-toluidino)-2-naphthalenesulfonate or bis(8-anilino-1-naphthalenesulfonate). Under the same conditions, the fluorescence of the human protein increased. The time course of the reaction of rabbit α-2-macroglobulin with methylamine was studied by measuring (i) the generation of thiol groups, (ii) the decrease in trypsin-inhibiting activity with remazol brilliant blue hide powder as the substrate, and (iii) the decrease in trypsin-protein amidase activity. The thiol appearance reaction exhibited a multiphasic time course. The initial phase was found to follow second-order kinetics with an apparent rate constant of 1.2M-1•s-1 Under the same conditions, the human protein showed monophasic kinetics with a rate constant of 12M-1•s-1. Both the trypsin-inhibiting activity and the trypsin-protein amidase activity concurrently decreased at a slower rate than the thiol appearance. These results indicate that rabbit α-2-macroglobulin is more stable to nucleophilic attack by methylamine but less resistant to proteolysis by trypsin than the human homologue, and that the final conformation induced by methylamine differs considerably from that induced by trypsin.抄録全体を表示PDF形式でダウンロード (3291K) -
Ikuko Ueno, Masahiro Kohno, Keiichi Mitsuta, Yukio Mizuta, Shiro Kaneg ...1989 年 105 巻 6 号 p. 905-910
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーBy employing EPR spectrometry with the aid of a spin-trapping agent, 5, 5-dimethyl-l-pyrroline-l-oxide (DMPO), the generation of superoxide anion and hydroxyl radical was reevaluated during the respiratory burst of porcine and human neutrophils. Properly prepared resting neutrophils did not generate any spin-trapped radical, and, when the cells were stimulated with phorbol myristate acetate, only DMPO-OOH, the spin-trapped adduct of superoxide anion, was detected. No formation of DMPO-OH, the spin-trapped adduct of the hydroxyl radical, was observed. DMPO-OOH was also detected principally when the neutrophils were stimulated with opsonized zymosan, a particulate stimulus. In the latter case, however, the formation of DMPO-OOH ceased shortly after the addition of zymosan and subsequent production of DMPO-OH was observed. The production of DMPO-OH was found to be associated with cell injury. DMPO at the concentration usually used for the experiment (0.045-0.09M) injured phagocytizing neutrophils, causing lysis of the cells. On the other hand, an addition of cell homogenate or glutathione-glutathione peroxidase system to the suspension of intact cells which were producing DMPO-OOH resulted in the formation of DMPO-OH. Thus, DMPO-OH was probably derived from DMPO-OOH by the action of enzymes and/or factor(s) which were released from the lysed cells.抄録全体を表示PDF形式でダウンロード (762K) -
Hiroyuki Aburatani, Akiyo Matsumoto, Takashi Ishikawa, Fumimaro Takaku ...1989 年 105 巻 6 号 p. 911-915
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーThe molecular mechanism of human intestinal apolipoprotein (apo) B-48 synthesis has been elucidated by a combination of sequencing of cloned complementary DNAs and RNase cleavage analysis of RNA heteroduplex. All intestinal cDNA clones contained a single C to T base substitution in the codon CAA encoding Gln2153 in apoB-100 cDNA, resulting in a translational stop. One of the our intestinal apoB cDNA clones was polyadenylated 106 bases downstream from the stop codon, possibly producing a 7-kb apoB message in the intestine. RNase cleavage analysis of the RNA heteroduplex between hepatic or intestinal RNA and apoB cDNA-directed anti-sense RNA showed that this single C to U substitution may occur in most of intestinal apoB mRNA. These results suggested that human apoB-48 is mostly produced by apoB mRNA with an in-frame stop codon in the intestine.抄録全体を表示PDF形式でダウンロード (4511K) -
Yumiko Iwahashi, Taro Nakamura1989 年 105 巻 6 号 p. 916-921
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーThe bulk of NADH kinase of Saccharomyces cerevisiae was recovered in the mitochondrial fraction prepared from spheroplasts. Most of the NADH kinase was localized in the inner membrane fraction, which was separated from other mitochondrial components by the combined swelling, shrinking, and sonication procedure. Treatment of mitoplasts with antiserum against the NADH kinase caused inactivation of the enzyme. On the contrary, no influence was observed upon the same treatment of intact mitochondria. p-Chloromercuribenzoate and eosin-5-maleimide inactivated the enzyme without affecting the matrix ATPase. The NADH kinase was enzymatically iodinated in mitoplasts, but not in the intact mitochodria. These results support the conclusion that NADH kinase is localized and functions at the intermembrane space side of the mitochondrial inner membrane. It is evident that the NADH kinase is encoded by nuclear gene(s) because it is synthesized in the presence of chloramphenicol or acriflavine, and a significant amount of the enzyme was detected in mitochondrial DNA-deficient mutants.抄録全体を表示PDF形式でダウンロード (1908K) -
Yumiko Iwahashi, Taro Nakamura1989 年 105 巻 6 号 p. 922-926
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーNADH kinase was reconstituted in liposomes by employing phosphatidylcholine and phosphatidylethanolamine with n-octyl-β-D-thioglucoside as a detergent. An analogous molecular organization of the NADH kinase to that in the mitochondrial inner membrane was ascertained to exist in the liposomal membrane. Michaelis constants for NADH and ATP were determined as 27 and 133 μM, respectively. Both values were lower than that of the solubilized enzyme. The catalytic center of NADH kinase was exposed on the outer surface of the reconstituted liposomes. The NADH kinase reconstituted with ADP/ATP carrier protein catalyzed the phosphorylation of exogenously supplied NADH by the use of ATP entrapped in the liposomal matrix.抄録全体を表示PDF形式でダウンロード (662K) -
Tissue-Specific DNA Binding of Nuclear Proteins That Bind to the Adenovirus Inverted Terminal RepeatKen Matsumoto, Kyosuke Nagata, Fumio Hanaoka, Michio Ui1989 年 105 巻 6 号 p. 927-932
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーAdenovirus (Ad) DNA replication requires nuclear factor I (NFI), a cellular sequence-specific DNA binding protein which binds to the Ad DNA inverted terminal repeat (ITR). The NFI binding consensus sequence, TGGN6-7, GCCAA, possibly includes the CCAAT-box which is often observed in regulatory elements of transcription. Adjacent to the NFI binding site on the Ad ITR is the binding site for nuclear factor III (NFIII), another cellular factor that stimulates Ad DNA replication in a cell-free system. Using gel retardation assay, we have examined the tissue specificity of these DNA binding activities in mouse. High NFI activity was detected in nuclear extracts from mouse liver, kidney, and spleen. Competition gel retardation assay with double-stranded oligonucleotides containing herpes simplex virus thymidine kinase (HSV-TK) gene or hsp70 gene CCAAT-box showed no effect on mouse NFI binding to its binding site. In the course of competitive DNA-binding assay, we detected a novel DNA binding protein in mouse kidney nuclear extracts, designated nuclear factor K (NFK). The NFK binding site is included in the NFIII binding site on the Ad ITR. NFK seems to be different from NFIII, in mode of binding to its binding site.抄録全体を表示PDF形式でダウンロード (3107K) -
Shiro Takagi, Mikihiko Kobayashi, Kazuo Matsuda1989 年 105 巻 6 号 p. 933-938
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーWater-soluble carbodiimide (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) (EDC) and glycine ethyl ester (GEE) as a nucleophile were used to modify the essential carboxyl group of phosphorylases. The inactive b form of the muscle phosphorylase was modified faster than the active a form and potato phosphorylases. Use of N, N, N', N'-tetramethyl-ethylenediamine (TEMED)-HCl buffer system (pH 6.2) resulted in a remarkable difference from the previous results obtained with phosphate and β-glycerophosphate buffer systems. That is, the substrate glucose 1-phosphate gave the best protection of the three phosphorylase activities. Glucose and glycogen were also effective to retard the inactivation of muscle phosphorylases, though glycogen was not effective for the potato enzyme. The EDC-GEE-modified phosphorylase b retained the affinity for AMP-Sepharose, though partially modified enzyme completely lost the homotropic cooperativity. Phosphorylase b was subjected to differential labeling with [14C] GEE. A labeled peptide was obtained after CNBr cleavage and peptic digestions, and corresponded to the catalytic site sequence surrounding the GEE-substituted Asp 661 and Glu 664. Either or both of these EDC-modified carboxyl residues may have an important role in the catalytic reaction.抄録全体を表示PDF形式でダウンロード (739K) -
Susumu Imaoka, Yoshitake Terano, Yoshihiko Funae1989 年 105 巻 6 号 p. 939-945
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーSpecific antibodies were prepared against cytochromes P450 PB-1, PB-2, PB-4, and PB-5 purified from hepatic microsomes of male rats treated with phenobarbital. With these antibodies, the levels of these four cytochrome P450s in hepatic, renal, and pulmonary microsomes of male rats that were untreated, treated with phenobarbital, or treated with 3-methylcholanthrene were examined. P450 PB-1 and PB-2 were present in moderate amounts in hepatic microsomes of untreated male rats and were induced 2- to 3-fold with phenobarbital. Also, the expression of these forms was suppressed by 3-methylcholan-threne. These forms were not detected in the renal or pulmonary microsomes of untreated rats or rats treated with phenobarbital or 3-methylcholanthrene. P450 PB-4 and PB-5 were found in the hepatic microsomes of untreated male rats at a low level but were induced with phenobarbital more than 50-fold. P450 PB-4 and PB-5 were not detected in renal microsomes; only P450 PB-4 or a closely related form was present in the pulmonary microsomes of untreated male rats, and its level was not changed by phenobarbital treatment. The constitutive presence of P450 PB-4 in pulmonary microsomes was confirmed by the investigation of testosterone metabolism. Purified P450 PB-4 had high testosterone 16 α- and 16 β-hydroxylation activity in a reconstituted system. The testosterone 16 β-hydroxylation activity of hepatic microsomes was induced with phenobarbital, and more than 90% of the testosterone 16β-hydroxylation activity of hepatic microsomes from rats treated with phenobarbital was inhibited by anti-P450 PB-4 antibody. The pulmonary microsomes of untreated rats had testosterone 16α- and 16β-hydroxylation activity, which was completely inhibited by anti-P450 PB-4 antibody. These results suggest that P450 PB-4 is present constitutively in pulmonary microsomes.抄録全体を表示PDF形式でダウンロード (1785K) -
Izumi Kumagai, Kin-ichiro Miura1989 年 105 巻 6 号 p. 946-948
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーTryptophan at the 62nd position (Trp62) of hen egg-white lysozyme is an amino acid residue whose action is essential for its enzymatic activity. Its indole ring may possibly come into direct contact with sugar residues of the substrate, and thus contribute significantly to substrate binding. For further elucidation of its role in catalytic processes, this amino acid was converted to other aromatic residues, such as Tyr, Phe, and His, by site-directed mutagenesis. All the mutations were found to enhance the bacteriolytic activity but to decrease the hydrolytic activity toward an artificial substrate, glycol chitin. Such a change in substrate preference appears remarkable considering the smaller size of the aromatic residue on the mutant enzyme at the 62nd position.抄録全体を表示PDF形式でダウンロード (1501K) -
Takao Matsuzaki, Chizuko Sasaki, Chieko Okumura, Hideaki Umeyama1989 年 105 巻 6 号 p. 949-952
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーThe three-dimensional structure of a thrombin inhibitor-trypsin complex has been determined by an X-ray analysis at 2.5 Å resolution. The result has given experimental support to the mechanisms previously proposed by the authors for the selective inhibition of trypsin, thrombin, factor Xa, and plasmin by inhibitors with an arginine or lysine backbone. The differences in the amino acid sequences at the positions corresponding to 11e63, Leu99, and Ser190 of trypsin give each enzyme different binding affinities toward inhibitors and result in the selective inhibition. Furthermore, the X-ray analysis has revealed a novel type of interaction between the inhibitor and trypsin. The hydrogen bonds between the inhibitor main chain and trypsin G1y216 play an essential role in the complex formation.抄録全体を表示PDF形式でダウンロード (416K) -
Hiroshi Takagi, Yasushi Morinaga, Haruo Ikemura, Masayori Inouye1989 年 105 巻 6 号 p. 953-956
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーSite-directed mutagenesis was employed to analyze the role of an α-helix containing catalytic Ser-221 of subtilisin E. Pro-239 located at the carboxy-terminal end of the α-helix was first replaced with Gly to examine the role of Pro-239 in the catalysis and stability of subtilisin E. The mutation was found to decrease both the catalytic rate (kcat) and the heat stability. This result strongly suggests that Pro-239 plays an important role in the maintenance of the α-helix, affecting the functioning of the active site. Various amino acid substitutions at position 239 were attempted to obtain the active subtilisins from Gly-239 subtilisin. Lys- and Arg-substitutions were found to result in more active and stable subtilisins than the Gly-239 subtilisin. In particular, the Arg-239 mutant showed enhanced heat stability compared with the wild type. These results demonstrate the important role of the α-helix containing catalytic Ser-221 in the catalysis as well as in the heat stability of subtilisin.抄録全体を表示PDF形式でダウンロード (1668K) -
Hideki Adachi, Ichiro Kubota, Nobutaka Okamura, Hiromitsu Iwata, Masaf ...1989 年 105 巻 6 号 p. 957-961
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーHuman microsomal dipeptidase (MDP, formerly referred to as dehydropeptidase-I or renal dipeptidase) [EC 3.4.13.11] was solubilized from the membrane fraction of kidney by treatment with octyl-β-D-glucoside and purified by a procedure including ion exchange chromatography and affinity chromatography on cilastatin-immobilized Sepharose. The purified human MDP was found to be homogeneous on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The apparent molecular weight (Mr) was estimated by SDS-polyacrylamide gel electrophoresis under non-reducing conditions to be 130 kDa, comprising a homodimer of two subunits. After treatment with endoglycosidase F, human MDP showed a single band with an apparent Mr of 42 kDa on SDS-polyacrylamide gel electrophoresis. Human MDP was found to bind to Con A-Sepharose and the activity was eluted with methyl-α-D-mannopyranoside, suggesting that human MDP is a glycoprotein. We also examined the substrate specificity of human MDP and found that human MDP catalyzed the hydrolysis of S(substituent)-L-cysteinyl-glycine adducts such as L-cystinyl-bis(glycine) and S-N-ethylmaleimide-L-cysteinyl-glycine, as well as the conver-sion of leukotriene D4, to leukotriene E4. These results suggest that MDP might play an important role in the metabolism of glutathione and leukotriene.抄録全体を表示PDF形式でダウンロード (2577K) -
Yuji Furukawa, Takashi Urano, Harumi Itoh, Chizuko Takahashi, Shuichi ...1989 年 105 巻 6 号 p. 962-967
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーLecithin-cholesterol acyltransferase was purified from rat plasma and the properties of this enzyme during the purification procedures and those of the purified enzyme were investigated in comparison with the human enzyme. The rat enzyme was not adsorbed on hydroxyapatite, which was employed for the purification of the human enzyme. When purified human enzyme was incubated at 37°C in 0.1 mM phosphate buffer (pH 7.4; ionic strength, 0.00025), no alteration of enzyme activity was observed for up to 6 h. In the case of the rat enzyme, however, approximately 40% of the enzyme activity was lost under the same conditions. The human enzyme and rat enzyme were both retained on a Sepharose 4B column to which HDL3 was covalently linked, in 39 mM phosphate buffer, pH 7.4. Although the human enzyme was eluted from the column in 1 mM phosphate buffer, the rat enzyme was dissociated from the column at a lower buffer concentration (0.1 mM phosphate buffer). These findings indicate that the rat enzyme effectively associated with HDL3 in 39 mM phosphate buffer, pH 7.4, but the association was more sensitive to increase of ionic strength compared with that of the human enzyme.抄録全体を表示PDF形式でダウンロード (694K) -
Fumio Fukai, Sawako Yatomi, Tadashi Morita, Sachiko Nishizawa, Toshihi ...1989 年 105 巻 6 号 p. 968-973
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーInhibition of the enzyme activity of glutathione S-transferase (GST) by a physiological concentration of bilirubin was studied using various substrates. When rat liver cytosol was used as an unfractionated GST, its GSH-conjugation activity toward 1- chloro-2, 4-dinitrobenzene was decreased to one-half by bilirubin, while the activity toward 1, 2- dichloro-4-nitrobenzene, p-nitrobenzyl chloride, or 1, 2-epoxy-(p-nitrophenoxy)propane and also the non-selenium dependent GSH-peroxidase activity toward cumene hydroper-oxide (CHPx activity) were hardly affected under the same conditions. In contrast, bilirubin inhibited each of the purified GST isozymes and no remarkable difference in bilirubin inhibition was observed with any of the substrates tested. From the chromatographic analysis of the cytosol incubated with [3H]bilirubin, it was found that a major part of the added bilirubin binds to subunit 1 (Ya) of GST isozyme, leaving not only the conjugation activity derived from 3-4 type GST but also the CHPx activity of subunit 2 (Yc) quantitatively intact. The bilirubin inhibition of both the conjugation activity of GST 3-4 and the CHPx activity of GST 2-2 was prevented almost completely by addition of a 3-fold molar excess of GST 1-1. From these results, it was assumed that the enzyme activities of both 3-4 type GSTs and subunit 2 (Yc) were protected from the inhibitory action of bilirubin by the scavenger effect of subunit 1 (Ya).抄録全体を表示PDF形式でダウンロード (670K) -
Yo Kikuchi, Yumiko Ando, Nobuko Ichimura, Akihiro Noda1989 年 105 巻 6 号 p. 974-978
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーHighly purified and commercially available preparations of reverse transcriptases from retroviruses contain a 3' to 5' exoribonuclease activity capable of hydrolyzing synthetic homopolyribonucleotides having a 3'-OH end. The exoribonuclease activity of reverse transcriptase preparations from Rous associated virus-2 was further characterized. This exoribonuclease activity cleaves poly(C) and poly(U) exonucleolytically from the 3'-OH end to produce nucleoside 5'-phosphates. Poly(A), poly(G), circular polyribonucleotide, and double-stranded polyribonucleotide were not hydrolyzed by the activity. This is a novel type of exoribonuclease activity.抄録全体を表示PDF形式でダウンロード (1806K) -
Kaeko Kamei, Yoshitaka Yamamura, Saburo Hara, Tokuji Ikenaka1989 年 105 巻 6 号 p. 979-985
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーThe amino acid sequence of chitinase from Streptomyces erythraeus was determined by the conventional method. The amino acid sequences of tryptic peptides of the reduced and S-carboxymethylated protein were determined. The tryptic peptides were aligned by overlapping the amino acid sequences of chymotryptic peptides, lysyl endopeptidase peptides and cyanogen bromide fragments. S. erythraeus chitinase consists of 290 amino acid residues with the molecular weight of 30, 400 and has two disulfide bridges at Cys(45)-Cys(89) and Cys(265)-Cys(272). The enzyme has no significant homology with other chitinases, lysozymes, and other proteins.抄録全体を表示PDF形式でダウンロード (663K) -
Atsushi Takeda, Hiroyuki Kaji, Kazuyasu Nakaya, Yasuharu Nakamura, Tat ...1989 年 105 巻 6 号 p. 986-991
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーWe have studied the primary structure of human cystatin As from epidermis, liver, spleen, and leukocytes. These molecules were indistinguishable on PAGE in the presence and absence of SDS, by fast protein liquid chromatography (FPLC) chromatofocusing on a Mono P column, and in amino acid composition. The NH2- and COOH-terminal amino acid sequences of human cystatin As from epidermis, liver, and spleen were identical with those of human leukocyte cystatin A previously reported except for the lack of the NH2-terminal methionine residue in human epidermal cystatin A. The peptides obtained upon digestion of four human cystatin As with Achromobacter protease I (AP) showed identical peptide maps on HPLC except for different retention times of the NH2-terminal peptides. Further-more, the amino acid compositions of corresponding separated peptide quartets were identical. We also determined the complete amino acid sequence of human epidermal cystatin A by sequencing peptides obtained from AP digestion and cyanogen bromide (CNBr) cleavage.It consisted of 97 amino acid residues, and was identical with those of human cystatin As from liver, spleen, and leukocytes except for the lack of the NH2- terminal methionine residue.抄録全体を表示PDF形式でダウンロード (1959K) -
Nobuaki Tominaga, Ryuzo Sakakibara, Yoshihiro Yokoo, Masatsune Ishigur ...1989 年 105 巻 6 号 p. 992-997
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーThe molecular sizes of human chorionic gonadotropin (hCG) subunits in the native state in normal first trimester placental extracts were determined by gel filtration on Sephacryl S-300, followed by SDS-polyacrylamide gel electrophoresis, protein blotting, and im-munobinding analysis using anti-α and -β antibodies. Mature forms of hCG subunits in the extracts were only found in the same fraction as that which contained standard urinary hCG, indicating an αβ dimer. On the other hand, immature forms were detected with a wide range of molecular weights, which were higher than that of standard hCG, suggesting oligomerization of associated or non-associated immature subunits. In order to determine the associated state of these subunits, various forms of associated subunits (hCGαβ) in placental extracts were immunoprecipitated with anti-hCG antiserum, which only recognized hCGαβ, and Protein A-Sepharose. They were then analyzed by SDS-polyacrylamide gel electrophoresis under reducing and non-reducing conditions, followed by immunobind-ing assaying. It has been suggested that there are three kinds of hCGαβs (one mature and two immature). To confirm the above results and to clarify the existence of free subunits, placental extracts were subjected to two-dimensional SDS-polyacrylamide gel electropho-resis. With this technique, high molecular weight forms of immature hCG subunits were found to be present in placental cells as an oligomer of not only the αβ dimer but of each subunit as well.抄録全体を表示PDF形式でダウンロード (4617K) -
Yoshiki Takesue, Kunio Yokota, Shigetoshi Miyajima, Ryo Taguchi, Hiroh ...1989 年 105 巻 6 号 p. 998-1001
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーThe larval midgut epithelial cell of the silkworm, Bombyx mori, has two forms of alkaline phosphatase and trehalase, soluble and membrane-bound. Alkaline phosphatase and trehalase of the latter form are found in the brush border membrane and the basolateral membrane, respectively. In this work we studied the membrane anchors of these membrane-bound enzymes. Alkaline phosphatase was solubilized by phosphatidyl-inositol-specific phospholipase C, but not by papain. Conversely, trehalase was released from the membrane by papain, but not by phosphatidylinositol-specific phospholipase C. Both enzymes were solubilized in an amphiphilic form with 0.5% Triton X-100 plus 0.5% sodium deoxycholate (pH 7.0). The detergent-solubilized alkaline phosphatase and tre-halase were converted to hydrophilic form on incubation with phosphatidylinositol-specific phospholipase C and papain, respectively. The effects of papain on solubilization and conversion of trehalase were completely inhibited by leupeptin. These results suggest that, in the silkworm larvae, alkaline phosphatase is anchored in the brush-border membrane via a glycosyl-phosphatidylinositol, while trehalase is associated with the basolateral membrane through a hydrophobic segment of the polypeptide.抄録全体を表示PDF形式でダウンロード (489K) -
Haruo Misono, Hiroyuki Hashimoto, Haruhiko Uehigashi, Shinji Nagata, S ...1989 年 105 巻 6 号 p. 1002-1008
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーLysine ε-dehydrogenase, which has been purified to homogeneity from the extract of Agrobacterium tumefaciens ICR 1600, had a molecular weight of approximately 78, 000 and consisted of two subunits identical in molecular weight (about 39, 000). The enzyme showed a high substrate specificity. In addition to L-lysine, S-(β-aminoethyl)-L-cysteine was deaminated by the enzyme, but to a far lesser extent. NAD+ and some NAD+ analogs (deamino-NAD+ and 3-acetylpyridine-NAD+) served as a cofactor. The pH optimum was at about 9.7 for the deamination of L-lysine. Although the NAD+ saturation curve was hyperbolic, a sigmoid saturation curve for L-lysine was obtained with the diluted enzyme solution, in which the dimeric enzyme was predominant. The reversible association of the enzyme to the tetramer was induced either by increasing the enzyme concentration or by addition of L-lysine. The preincubation of the enzyme with 5mM L-lysine resulted in a 2-fold increase in the activity and gave a hyperbolic saturation curve for L-lysine. Upon modification of SH groups of the enzyme with DTNB, neither the interconversion between the dimer and the tetramer nor the activation by L-lysine occurred. These results indicated that the dimeric enzyme was activated by L-lysine and the activation resulted from the association of two dimeric enzymes to form a tetramer.抄録全体を表示PDF形式でダウンロード (1972K) -
Tokuji Maruyama, Kaoru Kometani, Kazuhiro Yamada1989 年 105 巻 6 号 p. 1009-1013
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーThe effect of ethylene glycol on the contractile properties of skeletal muscles was studied using glycerinated rabbit psoas muscle fibers. Measurements were made at an ionic strength of 0.2 M, pH 7.0, and at 10°C. Ethylene glycol reversibly reduced isometric tension, active stiffness, the tension-to-stiffness ratio, and the shortening velocity at zero load (V0) in a dose-dependent fashion. Ethylene glycol also reduced the Ca sensitivity for contrac-tion. The extent of the reduction in V0 by ethylene glycol was much larger than that in the actomyosin ATPase activity reported by Travers and Hillaire (Eur. J. Biochem. 98, 293-299 [1979]). Although ethylene glycol reduced tension and V0, the MgATP concentration dependence of these two quantities was almost unaffected. These results suggest that in the presence of ethylene glycol, force produced by crossbridges in the principal force-producing state is reduced and/or the relative population of the attached crossbridges in the low-force state increases. The results also suggest that the reduction in V0 by ethylene glycol is caused not only by a reduction in the actomyosin ATPase activity but also by a reduction in the shortening distance per mole of ATP split.抄録全体を表示PDF形式でダウンロード (634K) -
Michiyasu Itoh, Yoshiyuki Shibata, Yasumi Ohshima1989 年 105 巻 6 号 p. 1014-1023
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーIn vitro splicing of 11 different RNA transcripts carrying two introns was performed to determine whether the removal of an intron was dependent on its position or removal of the other intron. The original RNA transcript carried two identical units, each consisting of the first exon-first intron-a major part of the second exon of the human β-globin gene. Five transcripts carrying a mutation in one of the two introns of the original RNA and one carrying mutations in both introns were prepared. One of the other RNA transcripts had a shorter middle exon than did the original transcript. The remaining three RNAs had at least one shortened form of the human β-globin second intron. The results can be summarized as follows. 1) There was no obligatory order in the excision of the two identical or different introns, although the 5' proximal intron may be removed more rapidly in some cases. The efficiency of removal of the 5' proximal intron seems to be higher than or similar to that of the distal intron. 2) Excision of a mutated intron was inhibited, but no mutation significantly affected the excision of the other intact intron. 3) Except in an RNA transcript with two mutated introns, significant splicing between exon 1 and exon 3 (exon jumping) did not take place. 4) These results support the proposal that the two introns are basically independent of each other as to their removal.抄録全体を表示PDF形式でダウンロード (7172K) -
Fusao Watanabe, Kiyoshi Fukui, Kyoko Momoi, Yoshihiro Miyake1989 年 105 巻 6 号 p. 1024-1029
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーIn order to evaluate the possible contributions of Lys-204, Tyr-224, Tyr-228, and His-307 in porcine kidney D-amino acid oxidase [EC 1.4.3.3] (DAO) to its catalytic function, we constructed four point mutant cDNAs encoding enzymes possessing Glu-204, Phe-224, Phe-228, and Leu-307 by oligonucleotide-directed in vitro mutagenesis. The four mutant cDNAs and the wild type cDNA could be expressed in vitro with similar efficiencies and about 200 ng of each enzyme protein was produced from 5μg of the respective capped RNA. The electrophoretic mobilities of the in vitro synthesized mutant enzymes on SDS-polyacrylamide gel were almost identical with that of the wild type DAO, and the molecular weight was calculated to be 38, 000. The Glu-204 and Phe-224 mutant DAOs showed comparable enzyme activities to that of the wild type enzyme, and were inhibited strongly by sodium benzoate, a potent competitive inhibitor of DAO. The kinetic parameters of the two mutant DAOs were also comparable to those of the wild type DAO. On the other hand, the Phe-228 and Leu-307 mutant DAOs showed no detectable activity. The results indicate that Tyr-228 and His-307 play important roles as to the constitution of the active site or participate in the reaction directly, while Lys-204 and Tyr-224 are not essential in the enzyme reaction.抄録全体を表示PDF形式でダウンロード (1708K) -
S. Marvin Friedman, Tairo Oshima1989 年 105 巻 6 号 p. 1030-1033
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーSeveral strains of the sulfur-dependent archaebacterium, Sulfolobus, were analyzed for their polyamine content. Caldine (norspermidine), spermidine, and thermine were found to be major components in all of the cells tested. The most abundant polyamine in all cultures examined was spermidine. The Langworthy strain had the highest spermine content, whereas S. acidocaldarius strain no. 7 was devoid of this polyamine. Cultures of strain no. 7 grown at 70°C were rich in spermidine and caldine (triamines) and the thermine : spermidine ratio was much lower than that of cultures grown at 78°C. Equal amounts of thermine and spermidine were present in strain DSM 1616. Preincubation of Langworthy strain extracts at 10°C did not overcome the requirement for polyamines in protein synthesis. Putrescine exerted a concentration-dependent inhibition of the spermineinduced stimulation of protein synthesis at 70°C. Increasing concentrations (6 and 9 mM) of spermine and thermine progressively inhibited poly(U)-dependent phenylalanine incorporation at 45°C to about the same extent, whereas the same concentrations of these polyamines had little effect on the reaction at 70°C. Although 3mM spermine had only a slight stimulatory effect on the attachment of phenylalanine to tRNA at 65°C, this polyamine had a pronounced effect on the formation of 70S ribosomes in a standard buffer containing 10mM Mg2+. Increasing the Mg2+ concentration to 30mM in the absence of spermine was even more effective in causing the reassociation of subunits to form 70S particles.抄録全体を表示PDF形式でダウンロード (496K) -
Shigeto Yamamoto, Sumihiro Hase, Hiroshi Yamauchi, Tadao Tanimoto, Tok ...1989 年 105 巻 6 号 p. 1034-1039
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーSugar chains of interferon-γ(IFN-γ) from human peripheral-blood lymphocytes (PBL) were liberated by hydrazinolysis. After N-acetylation, the reducing end residues of the sugar chains were tagged with 2-aminopyridine and the pyridylamino (PA-) derivatives were purified by gel filtration and reversed-phase HPLC. Five major PA-sugar chains were obtained. The structure of each PA-sugar chain was estimated by comparing its elution times on anion exchange, reversed-phase, and size-fractionation HPLC with those of PA-sugar chains of IFN-γ from the human myelomonocyte cell line HBL-38 (Yamamoto, S. et al. (1989) J. Biochem. 105, 547-555) as standards, and also by comparison of their elution times after partial desialylation. The results showed that IFN-γ(PBL) contained monoand disialo-biantennary structures with 0 or 1mol of fucose residue, as found for IFN-γ(HBL-38), but the N-acetylneuraminy1α2-6 linkage was dominant in IFN-γ(PBL), unlike IFN-γ(HBL-38), which contains both N-acetylneuraminy1α2-3 and α2-6 linkages.抄録全体を表示PDF形式でダウンロード (586K) -
Shoji Oda, Masahiro Sato, Satoshi Toyoshima, Toshiaki Osawa1989 年 105 巻 6 号 p. 1040-1043
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーA 45-60 kDal Gal/Ga1NAc-specific macrophage lectin was found to participate in the interaction between tumor cells and tumoricidal macrophages activated by an antitumor streptococcal preparation, OK-432, and in the tumoricidal activity of the activated macrophages. The binding between OK-432-elicited activated macrophages and murine mastocytoma P-815 cells was inhibited on preincubation of the macrophages with a neoglycoprotein (Gal-BSA) or a complex-type glycopeptide (unit B) which was a specific inhibitor of the macrophage lectin. This binding of the macrophages to P-815 cells was also inhibited on the addition of anti-macrophage lectin antiserum. Contrary to the case of OK-432-elicited macrophages, the binding of thioglycolate-elicited (responsive) macro-phages to P-815 cells was inhibited only a little by Gal-BSA and unit B, and not inhibited by the antiserum. Furthermore, the tumoricidal activity of the activated macrophages was inhibited by the addition of the anti-macrophage lectin antiserum. These results suggest that the binding of activated macrophages to tumor cells through the Gal/Ga1NAc-specific macrophage lectin is an important part of the tumor cell killing mechanism.抄録全体を表示PDF形式でダウンロード (468K) -
Keizo Teshima, Kiyoshi Ikeda, Takehiro Miyake, Mitsuo Imamura, Seiji I ...1989 年 105 巻 6 号 p. 1044-1051
発行日: 1989年
公開日: 2008/11/18
ジャーナル フリーBindings of the phospholipase A2 from Trimeresurus tlavoviridis to the monodispersed and micellar n-alkylphosphorylcholines (n-CnPC) were studied at 25°C and ionic strength 0.2 by the aromatic CD and tryptophyl fluorescence methods, respectively. The bindings to micelles of the substrate analog were analyzed by assuming that the micellar surface has multiple binding sites for the enzyme and that these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of the substrate (monomer) molecules, N=9-13. The binding constant to the micelle was about 40 times greater than it was to the monodispersed substrate. The binding constant to the micellar substrate analog increased on the binding of Ca2+ to the enzyme and decreased on modification of the N-terminal α-NH2 group, whereas the binding to the monodispersed substrate analog was independent of pH, of the Ca2+ binding, and of the chemical modification of the α-NH2, group. The kinetics of the hydrolyses of monodispersed and micellar dihexanoylphosphatidylcholines (diC6PC) were studied at 25°C and ionic strength 0.2 by the pH-stat method in the presence of saturating amounts of Ca2+. The catalytic center activity, kcat, as well as the binding constant, 1/Km, for the micellar substrate, were found to be much greater than those for the monodispersed substrate. The binding constant, 1/Km of the monodispersed substrate was independent of pH; this was in good agreement with that of the substrate analog described above. The pH-dependence curve of kcat for the monodispersed substrate exhibited two transitions, one below pH 6.5 and the other above pH 9.5. The analysis indicated that the deprotonation of an ionizable group (pK 5.7) and the protonation of another group (pK 10.0) were essential to the catalysis. The former ionizable group was assigned to the catalytic group, His 48 on the basis of its pK value, which had been determined from the pH dependence of the binding constant of Ca2+ (Miyake et al. [1989] J. Biochem. 105, 565-572). The latter group was tentatively assigned to the invariant Tyr 52 located in close proximity to the imidazole ring of His 48.抄録全体を表示PDF形式でダウンロード (886K)
- |<
- <
- 1
- >
- >|