巻号一覧
103 巻, 2 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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Noriko Murakami, Akira Kumon, Sueo Matsumura, Saburo Hara, Tokuji Iken ...1988 年 103 巻 2 号 p. 209-211
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーWhile the heavy chain of rabbit skeletal muscle myosin is not phosphorylatable by casein kinase II, it turned out to be phosphorylatable after removal of all of the light chains. The phosphorylation site for the kinase was determined to be Ser-1 and/or Ser-2 at the amino terminus.抄録全体を表示PDF形式でダウンロード (1378K) -
Toshiya Osada, Takuji Sasaki, Atsushi Ikai1988 年 103 巻 2 号 p. 212-217
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーThe plasma α-macroglobulin and egg white ovomacroglobulin were purified from the sea turtle, Chelonia mydas japonica, and their structural and functional properties were studied with the aim of clarifying the degree of evolutional divergence of two homologous proteins specific to different tissues of the same animal. The concentration of α-macroglobulin in green turtle plasma was about 4mg/ml. The protein was purified from the plasma by precipitation with polyethylene glycol 6000, followed by zinc chelate chromatography and gel chromatography on Sepharose CL-6B. The concentration of ovomacroglobulin in green turtle egg white was about 0.4mg/ml. Ovomacroglobulin was purified by gel chromatography on Sepharose CL-6B. The two proteins had similar molecular weights and amino acid compositions, and both inhibited proteinases such as trypsin, chymotrypsin, papain, and thermolysin. The amino terminal sequences of the two proteins were homologous to each other but higher homologies were found between the ovomacroglobulin of turtle and chicken, and between the serum macroglobulins of the same animals. The functional difference between turtle α-macroglobulin and ovomacroglobulin became clear when they were treated with methylamine, which is known to destroy the inhibitory activity of human α2-macroglobulin by splitting internal thiolester bonds. The inhibitory activity of the turtle plasma protein was completely destroyed by methylamine but that of ovomacroglobulin was only partially affected. The number of sulfhydryl groups as titrated with 5, 5'-dithiobis (2-nitrobenzoate) before and after treatment with proteinases or methylamine was different for the two proteins. The amount of radioactive methylamine that was incorporated was also different between the two proteins. The two proteins purified in this study had no immunological cross-reactivity.抄録全体を表示PDF形式でダウンロード (4174K) -
Atsushi Ikai, Toshiya Osada, Masaaki Nishigai1988 年 103 巻 2 号 p. 218-224
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーGreen turtle plasma α-macroglobulin and ovomacroglobulin underwent conformational changes when they were treated with proteinases or methylamine. Their conformational changes were studied by HPLC gel chromatography, circular dichroism, and electron microscopy. The Stokes radii of native green turtle α-macroglobulin and ovomacro-globulin were estimated to be 84.3±0.5 Å, and 93.0±0.5 Å, respectively, by means of an HPLC experiment. After reaction with methylamine or proteinases, the Stokes radius of α-macroglobulin changed to 83.0±0.5 Å or 85.4±0.5 Å, respectively, and that of ovomac-roglobulin to 93.0±0.5 Å or 87.1±0.5 Å. The circular dichroic spectra of native α-macroglobulin and ovomacroglobulin exhibited a negative band at around 215nm, indicating the presence of β-structure. Reaction of the two macroglobulins with methylamine resulted in a slight decrease in the ellipticity and reaction with proteinases led to a slight increase. The electron micrographic images of native α-macroglobulin and ovomacro-globulin can be described as deformed rings for the former and rugby balls for the latter. A common characteristic feature of the two molecules was that the central parts of the molecules were only thinly occupied by subunit. After reaction of macroglobulins with proteinases, the void spaces became partially filled and their overall shape more rectangular. Methylamine treatment caused a structural change only in α-macroglobulin but not in ovomacroglobulin. The difference in the susceptibility of the macroglobulins to methylamine was taken as an indication of evolutional divergence of the two homologous proteins within the last 300 million years.抄録全体を表示PDF形式でダウンロード (5202K) -
Iku Utsunomiya, Sachiko Oh-ishi, Izumi Hayashi, Junji Maruhashi, Naoki ...1988 年 103 巻 2 号 p. 225-230
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーMonoclonal antibodies to rat T-kininogen were produced and 9 hybridomas were selected. Radioimmunoassay (RIA) was developed using 125I-labeled T-kininogen and cell walls of Staphylococcus aureus (Zysorbin) for the separation of bound from free ligand, when IgG2a and IgG2b were used. In the case of IgG1 monoclonals, a second antibody (goat anti-mouse IgG) and Zysorbin were used. By this RIA, 1-16 ng T-kininogen/tube showed a linear inhibition curve, and cross reactivities to rat purified LMW- and HMW-kininogens were less than 0.5%, respectively. These monoclonal antibodies were also used for the immunohistochemical staining of the liver to detect T-kininogen in hepatocytes. By using the RIA and immunohistochemical staining, the T-kininogen levels in rat plasma and liver following carrageenin-induced inflammation were estimated. At 3-5h after the carrageenin injection, when the paw swelling was at its peak, the plasma level of T-kininogen and staining of the liver were slightly increased. T-Kininogen levels in plasma and liver peaked on the 2 nd day, when the paw swelling had already decreased. The result indicates that the increase of T-kininogen level in the liver and plasma occurs with a time lag and T-kininogen is not directly involved in the increase of vascular permeability in carrageenin paw edema.抄録全体を表示PDF形式でダウンロード (3004K) -
Naoyuki Murakami, Tetsuya Konishi1988 年 103 巻 2 号 p. 231-236
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーThe effects of N, N'-dicyclohexylcarbodiimide (DCCD) and various ionophores on lightinduced 22Na+-transport were studied in right-side-out membrane vesicles from Halobacterium halobium R1M1. The light-induced Na+ efflux was inhibited at the same DCCD concentration (>40 nmol/mg protein) as required for inhibition of the Na+-dependent membrane potential (Δ_??_) formation. This supports our previous indication that the DCCD-sensitive, Na+-dependent transformation of pH-gradient (ΔpH) into Δ_??_ is mediated by Na+/H+-antiporter (Murakami, N. and Konishi, T. (1985) J. Biochem. 98, 897-907). FCCP or a combination of valinomycin and triphenyltin (TPT) inhibits the lightinduced Na+ efflux in accordance with the notion of protonmotive force (Δ_??_H+)-driven antiporter. However, a marked lag in initiation of the Na+ efflux occurred in the presence of valinomycin, TPMP+, or a small amount of FCCP, suggesting that a gating step is involved in the Na+ efflux. On the other hand, the ΔpH-dissipating ionophore TPT did not cause the lag. A simultaneous determination of Δ_??_, ΔpH, and Na+ efflux rate at the initial stage of illumination revealed that the antiporter is gated by Δ_??_ rather than Δ_??_H+.抄録全体を表示PDF形式でダウンロード (671K) -
Sueo Matsumura, Toshinobu Takashima, Hitoshi Ohmori, Akira Kumon1988 年 103 巻 2 号 p. 237-246
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーPurified bovine brain myosin contained approximately 1 and 3 mol of protein-bound phosphate/mol myosin in the light chains and heavy chains, respectively. Large portions of this light chain-and heavy chain-bound phosphate (about 0.8 and 2.4 mol, respectively) were removed by incubation with a brain phosphoprotein phosphatase and potato acid phosphatase, respectively. Upon phosphorylation of the dephosphorylated brain myosin with myosin light chain kinase and casein kinase II, about 1.6 and 3.0 mol of phosphate was incorporated into the light chains and heavy chains, respectively, while much lower levels of phosphate were incorporated into the non-dephosphorylated brain myosin under the same conditions. The actin-activated Mg2+-ATPase activity of brain myosin rephosphorylated with myosin light chain kinase was about twice as high as that of dephosphorylated brain myosin (about 30 and 15 nmol phosphate/mg/min, respectively). On the other hand, whereas the rephosphorylated brain myosin superprecipitated rapidly with F-actin, the rate of superprecipitation of the dephosphorylated brain myosin was extremely low. Under appropriate conditions, a loose network of tiny superprecipitates, which formed initially throughout the solution, contracted to form eventually a large and dense particle. These results indicate that phosphorylation of the light chains of brain myosin is a prerequisite for the contraction of brain actomyosin. The role of phosphorylation of the heavy chains by casein kinase II remains to be elucidated.抄録全体を表示PDF形式でダウンロード (4692K) -
Akio Saito, Hyogo Sinohara1988 年 103 巻 2 号 p. 247-253
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーTwo major forms of rabbit plasma α-1-antiproteinase, S and F, were separated by affinity chromatography on Red Sepharose, and their modes of interaction with porcine trypsin were studied. The S form interacted with trypsin much more slowly than the F form, and the resulting complex partially retained the amidolytic and proteolytic activities towards benzoyl-L-arginine P-nitroanilide and remazol brilliant blue hide powder, respectively. This S form-trypsin complex also prevented the inactivation of bound trypsin by soybean trypsin inhibitor. In marked contrast, an equimolar complex of trypsin and the F form retained neither amidolytic nor proteolytic activity. These results suggest that the F form blocks the active site of trypsin while the S form does not bind directly to the active site, thereby preserving the catalytic potential of trypsin. No similar interaction was observed, however, between the S form and either bovine chymotrypsin or porcine pancreatic elastase. Both the S and F forms inactivated these proteinases in a stoichiometric manner with differing inhibitor/proteinase binding ratios. The S form showed about twofold greater capacity to inhibit elastase than the F form, whereas the reverse was the case for chymotrypsin.抄録全体を表示PDF形式でダウンロード (4438K) -
Akio Saito, Hyogo Sinohara1988 年 103 巻 2 号 p. 254-258
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーHuman plasma α-1-antiproteinase interacted with porcine trypsin in two different manners. One was a well known interaction, which resulted in inhibition of the proteolytic activity of the trypsin. The other has not been described to date, and resulted in retention of the amidolytic activity of the trypsin towards benzoyl-L-arginine P-nitroanilide in the presence of soybean trypsin inhibitor. The latter, so-called trypsin-protein amidase, activity is essentially the same as that observed with vertebrate α-macroglobulin and rodent murinoglobulin under similar conditions. All attempts to separate the two different activities as well as to abolish either activity by means of chemical or physical modifications were unsuccessful. The proteolysis-inhibiting interaction, which was virtually completed within 5min, was predominant over the amidolysis-retaining interaction, when the inhibitor/trypsin molar ratio was less than 1. On the other hand, the amidolysis-retaining interaction, which proceeded much more slowly, became evident when the molar ratio was greater than 1.抄録全体を表示PDF形式でダウンロード (1626K) -
Kitae Ito, Hirotaka Yamamoto, Michinao Mizugaki1988 年 103 巻 2 号 p. 259-262
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーAMP deaminase from sheep brain was purified to homogeneity on SDS-PAGE and its general properties were investigated. The native enzyme has a molecular weight of approximately 350, 000 as estimated by gel filtration and it is composed of four identical subunits with a molecular weight of 85, 000 each. The purified enzyme had a specific activity of 500 units/mg protein and shows a sigmoid-shaped AMP saturation curve in the presence of 100mM KCl. This deaminase is strongly activated by ATP and inhibited by GTP. It slightly catalyzes the hydrolysis of adenosine monosulfate (AMS), dAMP, and adenosine phosphoramidate (APA). These catalytic properties resemble those of AMP deaminase froin human liver.抄録全体を表示PDF形式でダウンロード (1516K) -
Makio Hayakawa, Ichiro Kudo, Motowo Tomita, Keizo Inoue1988 年 103 巻 2 号 p. 263-266
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーPhospholipase A2 was solubilized from rat platelet membrane by 1M KCl and purified to near homogeneity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC. The characteristics of the purified membrane-bound enzyme were compared with those of phospholipase A2 released from thrombin-stimulated rat platelets (Horigome, K., Hayakawa, M., Inoue, K., & Nojima, S. (1987) J. Biochem. 101, 625-631). The molecular weights, elution profiles on reversed-phase HPLC, and NH2-terminal sequences were identical for the two enzymes. Other characteristics of the two enzymes, such as specific activity, substrate specificity, pH optimum, Ca2+ requirement, heat lability, and sensitivity to P-bromophenacyl bromide were also indistinguishable. These findings suggest that both enzymes share a common structure.抄録全体を表示PDF形式でダウンロード (1398K) -
Kazuko Ohgi, Akihiro Sanda, Yoshio Takizawa, Masachika Irie1988 年 103 巻 2 号 p. 267-273
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーTwo acid RNases were purified from bovine spleen by means of ammonium sulfate fractionation, chromatographies on•phospho-cellulose, heparin-Sepharose CL-6B, poly G-Sepharose, and 2', 5'-ADP-Sepharose, and gel filtration on Toyopearl HW 55 F. Both purified preparations were homogeneous as judged by disc electrophoresis at pH 4.3. They were designated as RNase BSP1 and RNase BSP2 in the order of elution from a phospho-cellulose column. RNase BSP2, was immunologically indistinguishable from RNase K2 from bovine kidney. RNase BSP1 was a typical pyrimidine base-specific, uridylic acidpreferential RNase and had very sharp pH optimum at 6.5. RNase BSP1 thus obtained was a glycoprotein giving two major bands on SDS-slab electrophoresis. Although the apparent molecular weight of RNase BSP1 was distributed in the range of 27, 000-20, 000, it decreased to about 17, 000-18, 000 after endoglycosidase F digestion. The N-terminal amino acid sequence up to the 20 th amino acid had no homology to those of RNase K2 and RNase A.抄録全体を表示PDF形式でダウンロード (3014K) -
Katsuhiro Yamamoto1988 年 103 巻 2 号 p. 274-280
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーH-protein is a component of the thick filaments of skeletal myofibrils. Its effects on the assembly of myosin into filaments and on the formation of light meromyosin (LMM) paracrystals at low ionic strength have been investigated. H-protein reduced the turbidities of myosin filament and LMM paracrystal suspensions. Electron microscopic observation showed that the appearances of the filaments prepared in the presence and absence of H-protein were different. The filament length was not substantially changed by H-protein, but the diameter of the myosin filament was markedly reduced. H-protein bound to LMM and co-sedimented with it at low ionic strength upon centrifugation. Two types of paracrystals, spindle-shaped and sheet-like, were observed in LMM suspensions. H-protein altered the structure of the LMM paracrystals, especially the spindle-shaped ones. The thickness of the spindle-shaped paracrystals was reduced when H-protein was present during LMM paracrystal formation. On the other hand, periodic features along the long axis of the sheet-like paracrystals were retained even at high ratios of H-protein to LMM. However, there were fewer sheet-like paracrystals in the LMM suspensions containing H-protein than in the control. These results suggest that H-protein interferes with self-association of myosin molecule into filaments due to its binding to the tail portion of the myosin. However, H-protein does not have a length-determining effect on the formation of myosin filaments.抄録全体を表示PDF形式でダウンロード (5525K) -
Takashi Shirai, Satoshi Shiojiri, Hirataka Ito, Shuji Yamamoto, Hirosh ...1988 年 103 巻 2 号 p. 281-285
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーThe gene coding for human thrombomodulin, a thrombin receptor on endothelial cells and a cofactor for the activation of anticoagulant protein C zymogen, was isolated from a human genomic library by employing human thrombomodulin cDNA as a probe. The nucleotide sequences of the gene and the adjacent 5' and 3' flanking regions were then determined. The nucleotide sequence of this gene with approximately 3.7 kilobase pairs was identical to that of the cDNA, indicating that the gene for human thrombomodulin is free of introns. Hybridization data showed that there is only a single thrombomodulin gene in the human genome.抄録全体を表示PDF形式でダウンロード (1924K) -
Setsuko Katoh, Terumi Sueoka1988 年 103 巻 2 号 p. 286-289
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーThe 6-lactoyl tetrahydropterin (C1'-keto PH4) isomerase activity of sepiapterin reductase, which was found in our recent work (Katoh and Sueoka (1987) J. Biochem. 101, 275-278) as a novel activity of the enzyme, i.e., the conversion of C1'-keto PH4 to 6-1'-hydroxy-2'-oxopropyl tetrahydropterin (C2'-keto PH4) without coenzymes, could be enhanced by a small amount of NADPH or NADP+. The concentration of NADP+ required for the maximal stimulation was approximately the same as the concentration of the enzyme subunit. When NADP+ was added with the enzyme and C1'-keto PH4 at pH 8.6, the reaction sequence of C1'-keto PH4→C2'-keto PH4→tetrahydrobiopterin (BH4) was observed in the presence of dithioerythritol. These observations suggest that the coenzyme stimulating the isomerase function of sepiapterin reductase may be involved in the two sequential reductions, from pyruvoyl tetrahydropterin to BH4, by causing internal rearrangement of the keto group of the first intermediate, C1'-keto PH4, to form the second one, C2'-keto PH4.抄録全体を表示PDF形式でダウンロード (391K) -
Kensuke Hayashi, Kiyokazu Agata, Makoto Mochii, Sadao Yasugi, Goro Egu ...1988 年 103 巻 2 号 p. 290-296
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーEmbryonic chicken pepsinogen is an aspartyl proteinase that is specifically secreted during the embryonic period in the chicken proventriculus (glandular stomach). To learn the phylogeny of this pepsinogen, we isolated a cDNA clone by screening a λgt11 library of embryonic proventricular cDNAs with an antiserum to the embryonic chicken pepsinogen. We obtained a 200-base pair cDNA clone which encoded 18 amino acids that had high sequence homology with the carboxyl termini of other pepsinogens. Northern blot analysis revealed that this cDNA clone hybridized to a mRNA of 1, 600 bases in the embryonic proventriculus but not to the mRNA in the adult proventriculus. The almost complete nucleotide sequence of embryonic chicken pepsinogen-cDNA was determined by sequencing longer cDNAs obtained by screening the same library with the 200-base pair cDNA and primer extension with a synthetic primer. The cDNA consisted of 1, 281 nucleotides and encoded 383 amino acids for prepepsinogen. The predicted amino acid sequence was compared with the sequences of other aspartyl proteinases: pepsinogen A of human, monkey, pig, and chicken, progastricsin of monkey and rat, and bovine prochymosin. The phylogenetic tree constructed for them indicates the possibility that embryonic chicken pepsinogen diverged from prochymosin, after prochymosin and pepsinogen A had diverged from each other.抄録全体を表示PDF形式でダウンロード (3012K) -
Takashi Kumazaki, Asamichi Fujitani, Kumiko Terasawa, Kiyohito Shimura ...1988 年 103 巻 2 号 p. 297-301
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーAnhydrochymotrypsin immobilized on Sepharose specifically adsorbed various peptides containing L-tryptophan, L-tyrosine, or L-phenylalanine residues at their carboxy-termini. These peptides correspond to the specific products of chymotryptic cleavage of polypeptides. A mixture of the chymotryptic peptides once adsorbed on the column could be effectively separated by eluting them with a pH gradient. This adsorbent, on the other hand, showed no substantial affinity toward the substrate-type peptides that contain the aromatic amino acid (s) within the peptide chain, or toward the C-terminal leucine peptides. By taking advantage of this unique property of anhydrochymotrypsin-Sepharose in combination with reversed-phase high-performance liquid chromatography, we have succeeded in separating the C-terminal peptides from chymotryptic digests of reduced and S-carboxymethylated bovine insulin and from tryptic digests from reduced and S-carboxymethylated Streptomyces subtilisin inhibitor.抄録全体を表示PDF形式でダウンロード (629K) -
Akira Hata, Teruhisa Tsuzuki, Kazunori Shimada, Masaki Takiguchi, Masa ...1988 年 103 巻 2 号 p. 302-308
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーComplementary and genomic DNA clones corresponding to the human ornithine transcar-bamylase (OTC) [EC 2. 1. 3. 3] mRNA have been isolated and analyzed. The OTC gene is about 73 kilobase pairs (kb) long and contains 10 exons interrupted by 9 introns of highly variable sizes. The smallest intron is 80 base pairs and the largest, 21.7 kb. The 5'-and 3'-flanking regions, entire exons and all the exon/intron boundaries were sequenced. The nucleotide and deduced amino acid sequences of isolated OTC cDNAs as well as the corresponding regions of the genomic DNA were compared with those of human OTC eDNA (Horwich, A. L., Fenton, W. A., Williams, K. R., Kalousek, F., Kraus, J. P., Doolittle, R. F., Koningsberg, W., & Rosenberg, L. E. (1984) Science 224, 1068-1074). We found 20 nucleotide substitutions among these sequences, of which 6 related to amino acid changes. The nature of these nucleotide substitutions is discussed.抄録全体を表示PDF形式でダウンロード (1866K) -
Warawadee Nirdnoy, Prayad Komaratat, Prapon Wilairat1988 年 103 巻 2 号 p. 309-312
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーSarcoplasmic reticulum Ca2+-ATPase from rabbit skeletal muscle has an Arrhenius curve of enzyme activity with a discontinuity at about 20°C. Preparations treated with FeSO4 and ascorbic acid and from a vitamin E-deficient dystrophic rabbit have 22% of the normal activity and a linear Arrhenius curve (Promkhatkaew, D., Komaratat, P., & Wilairat, P. (1985) Biochem. Int. 10, 937-943). All three preparations were cross-linked to the same extent by dimethyl suberimidate and copper-phenanthroline reagent at temperatures above and below the temperature of the Arrhenius discontinuity. Both iron-ascorbate-treated Ca2+-ATPase and that from a vitamin E-deficient animal had 50% of the normal sulfhydryl content, but the disulfide and free amino contents were unaltered. These observations suggest that loss of sulfhydryl groups through lipid peroxidation, both in vivo and in vitro, resulted in reduction of Ca2+-ATPase activity and loss of the break in the Arrhenius plot. Changes in Ca2+-ATPase polypeptide aggregational state could not account for the discontinuity in the Arrhenius curve as revealed by the similar extent of crosslinking of the three enzyme preparations at temperatures above and below the temperature of the Arrhenius discontinuity.抄録全体を表示PDF形式でダウンロード (396K) -
Kenji Takahashi, Junji Hashimoto1988 年 103 巻 2 号 p. 313-320
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーThe complete amino acid sequence of ribonuclease U1 (RNase U1), a guanine-specific ribonuclease from a fungus, Ustilago sphaerogena, was determined by conventional protein sequencing, using peptide fragments obtained by several enzymatic cleavages of the performic acid-oxidized protein. The oxidized protein was first cleaved by trypsin and the resulting peptides were purified and their amino acid sequences were determined. These tryptic peptides were aligned with the aid of overlapping peptides isolated from a chymotryptic digest of the oxidized protein. The amino acid sequence thus deduced was further confirmed by isolation and analysis of peptides obtained by digestion of the oxidized protein with lysyl endopeptidase. The location of the disulfide bonds was deduced by isolation and analysis of cystine-containing peptides from a chymotryptic digest of heat-denatured RNase U1. These results showed that the protein is composed of a single polypeptide chain of 105 amino acid residues cross-linked by two disulfide bonds, having a molecular weight of 11, 235, and that the NH2-terminus is blocked by a pyroglutamate residue. It has an overall homology with other guanine-specific or related ribonucleases, and shows 48% identity with RNase T1 and 38% identity with RNase U2.抄録全体を表示PDF形式でダウンロード (722K) -
Takemitsu Mizunaga, Masayuki Izawa, Kaoru Ikeda, Yoshiharu Maruyama1988 年 103 巻 2 号 p. 321-326
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーThe role of mannan chains in the formation and secretion of active acid phosphatase of yeast (Saccharomyces cerevisiae), a repressible cell surface mannoprotein, was studied in yeast protoplast systems by using tunicamycin at various temperatures. AT 30°C, tunicamycin-treated protoplasts did not produce active acid phosphatase; however, at 25 or 20°C they formed and secreted active enzyme. This form of acid phosphatase gave 59-, 57-, and 55-kDa bands on SDS-PAGE which neither bound to concanavalin A Sepharose, nor changed in molecular weight upon treatment with endoglycosidase H, indicating that the peptides are nonglycosylated. The nonglycosylated form, like its glycosylated counterpart, is a dimer on the basis of gel permeation chromatography. The Km for para-nitrophenyl-phosphate and K1 for inorganic phosphate of both glycosylated and nonglycosylated acid phosphatases were almost the same. These results suggested that 1) the conformation of the nonglycosylated acid phosphatase secreted at low temperatures is probably identical with that of the glycosylated one, and 2) the conformation of acid phosphatase is very important for its secretion. The rate of intracellular transport of nonglycosylated acid phosphatase is about one-fourth that of the glycosylated enzyme, indicating that glycosylation facilitates the transport of acid phosphatase proteins.抄録全体を表示PDF形式でダウンロード (3022K) -
Takahiro Tsujita, Tomihiro Miyada, Hiromichi Okuda1988 年 103 巻 2 号 p. 327-331
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーCarboxylesterase was purified from rat kidney in an electrophoretically homogeneous form by acetone precipitation, followed by successive chromatographies on DEAE-cellulose and hydroxyapatite and then isoelectric focusing. The purified enzyme catalyzed the hydrolyses of monoacylglycerols and short-chain triacylglycerols, such as tributyrin, but not the hydrolysis of long-chain triacylglycerol. Its optimum pH with methyl butyrate as a substrate was 8.0. The relation of its activity to the methyl butyrate concentration differed from those for pancreatic lipase and liver esterase, and also from those for lipolytic enzymes from various other tissues. The relations of methyl butyrate-hydrolyzing activity with methyl butyrate concentration were compared among various carboxylester hydrolyzing enzymes. Based on the results, these enzymes were classified into four classes.抄録全体を表示PDF形式でダウンロード (1413K) -
Noriyuki Morita, Sumihiro Hase, Kazuhiro Ikenaka, Katsuhiko Mikoshiba, ...1988 年 103 巻 2 号 p. 332-335
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーPyridylamino asialo-agalacto-biantennary sugar chain (PA-acceptor), prepared from human α1-acid glycoprotein, was incubated with bovine milk galactosyltransferase. Transfer of galactose residues to PA-acceptor was detected by HPLC analysis, and thus PA-acceptor was shown to be useful for galactosyltransferase assay. Moreover, three species of products, i.e. PA-acceptor monogalactosylated on the Man α1-3 branch of the trimannosyl core, PA-acceptor monogalactosylated on the Man α1-6 branch, and digalactosylated PA-acceptor, were separated and identified by reversed-phase HPLC, so we could simultaneously determine the branch specificity (the ratio of galactosylation on Man α1-3 branch to that on Man α1-6 branch) of the galactosyltransferase. We fractionated the bovine milk galactosyltransferase on a DEAE-5PW column and confirmed that there was a heterogeneity in this enzyme preparation. Each fraction was assayed for acceptor specificity (the ratio of the activity towards N-acetylglucosamine to that towards PA-acceptor) and branch specificity using the PA-acceptor. However, we could not detect differences in the specificities among the fractions. In addition, we found that α-lactalbumin stimulated the galactosyltransferase activity towards PA-acceptor.抄録全体を表示PDF形式でダウンロード (487K) -
Makoto Miyata, Toshiaki Arata, Akio Inoue1988 年 103 巻 2 号 p. 336-341
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーThe reaction intermediates formed by the two heads of smooth muscle myosin were studied. The amount of myosin-phosphate-ADP complex, MADPP, formed was measured from the P1-burst size over a wide range of ATP concentrations. At low concentrations of ATP, the P1-burst size was 0.5 mol/mol myosin head, and the apparent Kd, value was about 0.15 μM. However, at high ATP concentrations, the P1 burst size increased from 0.5 to 0.75 mol/mol myosin head with an observed Kd value of 15 μM. The binding of nucleotides to gizzard myosin during the ATPase reaction was directly measured by a centrifugation method. Myosin bound 0.5 mol of nucleotides (ATP and ADP) with high affinity (Kd_??_1 μM) and 0.35 mol of nucleotides with low affinity (Kd=24 μM) for ATP. These results indicate that gizzard myosin has two kinds of nucleotide binding sites, one of which forms MADPP with high affinity for ATP while the other forms MADPP and MATP with low affinity for ATP. We studied the correlation between the formation of MADPP and the dissociation of actomyosin. The amount of P1-burst size was not affected by the existence of F-actin, and when 0.5 mol of ATP per mol of myosin head was added to actomyosin (1mg/ml F-actin, 5 μM myosin at 0°C) most (93%) of the added ATP was hydrolyzed in the P1-burst phase. All gizzard actomyosin dissociated when 1 mol of ATP per mol myosin head was added to actomyosin. However, the extent of actomyosin dissociation at 0.5ml of ATP per mol myosin head depended on the experimental conditions. In 0.35mg/ml F-actin and at 0°C, where myosin binds weakly to F-actin, more than, half of the myosin molecule dissociated from F-actin. Under conditions where myosin binds tightly to F-actin (1mg/ml F-actin and at 20°C), only less than 1/4 of myosin dissociated from F-actin. These results indicate that smooth muscle myosin forms MADPP with a high affinity for ATP in one of the two heads of the myosin molecule and that the formation of MADPP in the high affinity site results in a remarkable weakening of the binding of the other head to F-actin.抄録全体を表示PDF形式でダウンロード (770K) -
Koichi Mishima, Yoshinori Tanigawa, Nobumasa Hara, Mikako Tsuchiya, Ta ...1988 年 103 巻 2 号 p. 342-347
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーWe investigated the ADP-ribosylation of bradykinin by hen liver nuclear ADP-ribosyltransferase. Two Arg residues of the peptide were modified by this enzyme. Arg1 was preferentially modified as compared to Arg9; the Vmax/Km for Arg1 was 3 times higher than that for Arg9. These results were given support by data observed in experiments with des-Arg1 and des-Arg9 bradykinin; the Vmax/Km for des-Arg9 bradykinin was 3 times that for des-Arg1 bradykinin. ADP-ribosylation suppressed the biological activity of bradykinin, as related to both binding and contractile activities. The extent of ADP-ribosylation-induced suppression of both activities was higher in the case of the modification of Arg1 than that of Arg9. In view of the observation of ADP-ribosyltransferase activity in skeletal, cardiac, and smooth muscles (Soman, G. et al. (1984) Biochem. Biophys. Res. Commun. 120, 973-980; Shimoyama, M. et al. (1987) in The 8 th International Symposium on ADP-Ribosylation, Texas, abstract p. 13), bradykinin functioning in the contraction of smooth muscle may be modified in this way in vivo.抄録全体を表示PDF形式でダウンロード (653K) -
Ole Some1988 年 103 巻 2 号 p. 348-353
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーMaking freshly isolated rat hepatocytes permeable by 0.4g/liter digitonin doubled the number of binding sites for α2-macroglobulin-trypsin complex without changing the affinity. Thus, digitonin unmasked a receptor pool, probably of intracellular origin. The total cellular binding capacity was measured in the presence of digitonin, the surfaceexposed in its absence. Upon preincubation of the cells at 37°C, the total cellular binding capacity for α2-macroglobulin•trypsin decreased over a 2-h period to 0.26 of the initial value. By contrast, the surface-exposed binding capacity initially increased in response to a preincubation at 37°C, reached after 20min a peak value 1.74 times that at 0 time, followed by a decrease. Neither the increase in nor the loss of surface-exposed binding capacity was influenced by inhibitors of lysosomal functions, protein synthesis and glycosylation. Colchicine abolished the increase in surface-exposed binding capacity but not the disappearance. By contrast, phenylarsine oxide (inhibitor of endocytosis), N-ethylmaleimide, and phenylmethanesulphonyl fluoride inhibited the receptor loss, suggesting that the loss occurred by proteolysis. The insulin receptor concentration, studied in parallel, remained practically constant in the investigated period in the presence and absence of digitonin. Thus, the hepatic receptor for α2-macroglobulin•protease complexes is regulated independently of other specialized plasma membrane proteins.抄録全体を表示PDF形式でダウンロード (795K) -
Shigetoshi Sugio, Takashi Amisaki, Hirofumi Ohishi, Ken-ichi Tomita1988 年 103 巻 2 号 p. 354-366
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーThe three-dimensional X-ray structure of the RNase T1 [EC 3. 1. 27. 3] -2'GMP complex crystallized at low pH value (4.0) was determined, and refined to 1.9 Å resolution to give a final R value of 0.203. The refined model includes 781 protein atoms, 24 inhibitor atoms, and 43 solvent molecules. The imidazole rings of His 27 and His 40 interact with the carboxyl side chains of Glu 82 and Glu 58, respectively, whereas that of His 92 is in contact with the main chain carbonyl oxygen of Ala 75. In the complex, the ribose ring of the 2'GMP molecule adopts a C2'-endo puckering, and the exocyclic conformation is gauche(-)-gauche (+). The glycosyl torsion angle is in the syn range with an intramolecular hydrogen bond between N3 and 05', and the 2'-phosphate orientation is trans-gauche (-). The guanine base of the inhibitor is tightly bound to the base recognition site with five hydrogen bonds (N1- -Glu46Oε2, N2- - -Asn98O, 06- - -Asn44N, and N7- - -Asn43Nδ2/Asn43N) and is sandwiched between the phenolic ring portions of Tyr42 and Tyr45 by stacking interactions. The 2'-phosphate group interacts with Arg77Nη2, Glu58Oε2, and Tyr38Oε but not with any of the histidine residues. Arg77Nε2 also interacts with Tyr38Oε. There is no interaction between the ribose moiety of the inhibitor and the enzyme.抄録全体を表示PDF形式でダウンロード (1282K) -
Kazuyo Ohashi, Kensuke Ishikawa, Koscak Maruyama1988 年 103 巻 2 号 p. 367-369
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーIon-exchange column-purified I-protein was labeled by fluorescein isothiocyanate (FITC) at an equimolar ratio. When FITC-labeled I-protein was reacted with glycerinated myofibrils of chicken breast muscle in a phosphate-buffered saline, fluorescence was observed at the A-band and/or the Z-line of the sarcomere. However, FITC-labeled I-protein did not stain freshly prepared myofibrils. When FITC-I-protein was reacted with a nitrocellulose paper sheet on which muscle proteins were blotted after SDS-polyacrylamide gel electrophoresis, some peptide bands, including connectin and nebulin, were fluorescent. These facts can explain why anti-I-protein antibodies stain the A-I junctional region of fresh myofibrils and A-bands and/or Z-lines of glycerinated myofibrils. It is very likely that I-protein is transferred from the A-I junctions of myofibrils and translocates to A-bands and Z-lines, where some components that can bind to I-protein are localized, as myofibrils are degraded during the glycerination.抄録全体を表示PDF形式でダウンロード (2475K) -
Takanori Nakamura, Fuminori Tokunaga, Takashi Morita, Sadaaki Iwanaga1988 年 103 巻 2 号 p. 370-374
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーThe interaction between lipopolysaccharide (LPS) and an LPS-sensitive serine protease zymogen, factor C, purified from horseshoe crab (Tachypleus tridentatus) hemocytes, was investigated to elucidate the LPS-mediated activation of factor C. The rate of activation of the zymogen factor C was highly dependent on the concentration of LPS and on temperature, and the curve of amount of LPS versus activation showed saturation at 37°C. Moreover, a high-molecular-mass complex formed between factor C and LPS was found in a gelfiltration experiment on a Sepharose 4 B column. This complex formation was also confirmed by double diffusion analysis on agarose plates. Triton X-100, which destroys LPS micelles, strongly inhibited the LPS-mediated activation of factor C but not activated factor _??_. These results indicate that the binding of factor C with LPS is required for its activation and that only LPS-associated factor C generates the active factor _??_. On the other hand, the LPS-mediated activation of factor C was strongly inhibited by the S-alkylated heavy chain derived from factor C. In contrast, the S-alkylated factor C-light chain did not show any inhibitory effect on the activation of factor C, suggesting that the heavy chain located in the NH2-terminal portion of factor C contains an LPS-binding region.抄録全体を表示PDF形式でダウンロード (1496K) -
Dae-Seok Byun, Takeshi Kasama, Takao Shimizu, Hiroshi Yorifuji, Yousuk ...1988 年 103 巻 2 号 p. 375-379
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーIn order to elucidate the mechanism of xanthoma formation in cerebrotendinous xanthomatosis, mice were fed for 32 weeks with a diet rich in 5α-cholestan-3β-ol (cholestanol) (1%, w/w). The concentrations of sterols in the serum, liver, and cerebellum were determined using high performance liquid chromatography. In the cholestanol-fed mice, the cholestanol concentrations in the serum and liver reached maxima in the first 2 to 4 weeks; the levels were about 30- to 100-fold higher than in the control diet mice. The cholestanol concentrations declined thereafter, finally to 50-60% of the maxima. Cholesterol concentrations were slightly lower in the cholestanol-fed mice throughout the experiments than in the control diet mice. On the other hand, the levels of cholestanol in the cerebellum increased almost linearly in parallel to the feeding time, and no decline was observed. These results suggest that the capacity of the liver to remove or degrade cholestanol was increased by long-term intake of this compound, whereas the cerebellum had no such feed-back regulation. Histological examinations using an electron microscope revealed the enlargement of lysosomal granules in the liver of the cholestanol-fed mice.抄録全体を表示PDF形式でダウンロード (1778K) -
Mikiharu Yoshida, Koichi Yagi1988 年 103 巻 2 号 p. 380-385
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーTwo kinds of myosin phosphatases were purified from fresh chicken gizzard smooth muscle. Alkaline phosphatase (CGP-a), which requires Mg2+, was most active at pH 8.6 with 2 to 4mM Mg2+, and was essentially the same as the phosphatase we reported previously (J. Biochem. 99, 1027-1036 (1986)). On the other hand, neutral phosphatase (CGP-b), was most active at pH 7.5 with 0 to 2mM Mg2+, and was similar to SMP-IV reported by Pato and Kerc (J. Biol. Chem. 260, 12359-12366 (1985)). Although both phosphatases showed similar Vm, (4.8 to 13 μmol/mg/min) using phosphorylated myosin head as the substrate under optimal conditions, CGP-b had a smaller Km (3.7 to 6.7 μM) than CGP-a by about 4-fold. CGP-b showed a lower Vm (1.9 to 8.4 μmol/mg/min) for the isolated myosin light chain than myosin itself, while CGP-a showed rather higher Vm (17 to 32 μmol/mg/min). Although the activity of CGP-a decreased monotonically with increase of ionic strength, that of CGP-b increased slightly with increase in NaCl until 0.1M and then decreased. Those results suggest that CGP-b may be the effective myosin phosphatase in vivo. The analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that both phosphatases were composed of a single polypeptide having a molecular weight of 37, 000. The tetrameric structure was assumed for both phosphatases, because the molecular weight in the native state was estimated as 140, 000 or 145, 000 for CGP-a or CGP-b, respectively. A dimeric form of each enzyme was also found and purified, and showed the same enzymatic properties as the corresponding tetramer form. We assume tentatively that CGP-b and CGP-a may be active and inactive forms of the myosin phosphatase, respectively, which are interconvertible by some mechanism, e. g., phosphorylation or methylation.抄録全体を表示PDF形式でダウンロード (1759K) -
Kunihiko Konno1988 年 103 巻 2 号 p. 386-392
発行日: 1988年
公開日: 2008/06/30
ジャーナル フリーThe chymotryptic digestion of G-actin in the presence of calcium produces not only a C-terminal 33 kDa “core, ” spanning from residue 68 to the C-terminus, but also an N-terminal Cys-lO-containing fragment (10 kDa fragment), spanning from the N-terminus to the 44 th residue. The minimum calcium concentration required for producing just these two structures is 10-7.5M. In a Ca medium, the 10 kDa fragment remains attached to the 33 kDa core, and the 10 kDa fragment detaches when the divalent cation is removed from the complex, as was proved by Sephacryl S-200 gel filtration. We conclude that 10 and 33 kDa form a complex that is calcium-sensitive. The Cys-10 in the 10 kDa moiety of the complex reacts with 5-iodoacetamide fluorescein in the presence of calcium ion, whereas Cys-257 is practically inert. The removal of calcium allows Cys-257 also to react with the reagent. Therefore, the complex seems to retain the calcium binding site. The nucleotide binding ability of the complex was also demonstrated.抄録全体を表示PDF形式でダウンロード (5553K)
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