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Volume 100 (1986)
- Issue 6 Pages 1403-
- Issue 5 Pages 1109-
- Issue 4 Pages 825-
- Issue 3 Pages 521-
- Issue 2 Pages 259-
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Volume 100, Issue 4
Displaying 1-39 of 39 articles from this issue
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Yukio NIIMURA, Ineo ISHIZUKA1986 Volume 100 Issue 4 Pages 825-835
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlycosphingolipids were isolated from a canine kidney cell line (MDCK) and its ouabain-resistant mutant (MDCK-OR) by solvent extraction, mild alkaline meth-anolysis, a DEAE-Sephadex column, and preparative TLC. The glycolipids were characterized by their mobilities on TLC, an analysis of carbohydrates as trimethyl-sily1 methyl glycosides and acetates of partially methylated alditols, as well as by treatment with specific glycosidases. In the neutral glycolipid fraction of both cell lines, galactosylceramide (GalCer), glucosylceramide (GlcCer), lactosylceramide (LacCer), digalactosylceramide (Ga2Cer), globotriaosylceramide (Gb3Cer), globoside (Gb4Cer), and the Forssman antigen (IV3GalNAca-Gb4Cer) were identified. The contents of Ga2Cer (4.4 nmol/mg protein), Gb3Cer (0.6), Gb4Cer (2.9), and IV3Gal-NAcu-Gb4Cer (19.5) in MDCK-OR were 1.4- to 2.1-fold higher than those in MDCK, while the concentrations of GIcCer (5.3) and LacCer (1.4) in MDCK-OR were about half of those in MDCK. Among acidic glycolipids of MDCK-OR, galac-tosyl sulfatide (GalCer-I3-sulfate) and lactosyl sulfatide (LacCer-II3-sulfate) were increased to 1.9 (2.7-fold) and 0.2nmol/mg protein (2.0-fold), respectively, as compared to MDCK. However, N-acetylneuraminosyllactosylceramide (GM3), the predominant ganglioside in both cell lines, was decreased to about one third of the level (1.5nmol/mg protein) in the parent MDCK (4.7nmol/mg protein). The fatty acid of the glycolipids in both cell lines consisted mainly of saturated acids of 16, 18, 22, and 24 carbons.View full abstractDownload PDF (1544K) -
Kaneo OHORI, Hidekazu FUKAMIZU, Kan MATSUSHITA, Kentaro HORIUCHI, Dais ...1986 Volume 100 Issue 4 Pages 837-842
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA protein having Mr of more than 900K (900, 000) was isolated by chromatography on a column of Sepharose 4B coupled with solubilized elastin, followed by sucrose density gradient centrifugation. The protein is composed of several disulfide-linked subunits. SDS-polyacrylamide gel electrophoresis and immunoblotting indi-cated that two of the subunits are identical with the heavy chains of IgM and IgG, and one is immunologically related to the heavy chain of IgA. The results sug-gested that this protein is a new protein that belongs to the category of, or is closely related to, the immunoglobulins. As described in the succeeding paper, it has cell-binding activity (Fukamizu et al. (1986) J. Blocliem. 100, 843-848). We tentatively refer to this protein as “cell-binding immunoglobulin-like protein (CIP).”View full abstractDownload PDF (1585K) -
Hidekazu FUKAMIZU, Kaneo OHORI, Yasuhisa NAITO, Kentaro HORIUCHI, Dais ...1986 Volume 100 Issue 4 Pages 843-848
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCell adhesion to plastic surfaces coated with a new high-molecular-mass immuno-globulin-like protein from normal human plasma was studied. Mouse subdermal fibroblasts, hamster kidney cells, human umbilical vein endothelial cells, and human skin fibroblasts were found to become attached to the surface, but cancer cells derived from human stomach cancer and human breast cancer did not. The ap-pearance of the attached cells differed from that of cells attached to surfaces coated with fibronectin or concanavalin A. The cell adhesion to the surfaces coated with the protein was inhibited by goat anti-human IgM. Furthermore, the binding of the protein to the cell surfaces was demonstrated by the indirect immunofluorescence method. It is concluded that this protein is a new cell-binding protein.View full abstractDownload PDF (2465K) -
Shuji OHNISHI, Shuichiro MAEDA, Kazunori SHIMADA, Tatsuyoshi ARAO1986 Volume 100 Issue 4 Pages 849-858
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSComplementary and genomic DNA clones corresponding to the human serum amvloid P component (SAP) mRNA have been isolated and analyzed. The nucleotide sequences of the cDNA and the corresponding regions of the genomic SAP DNA reported here were identical, and revealed that after coding for a signal pep-tide of 19 amino acids and the first two amino acids of the mature SAP protein, there is one small intron of 115-base pairs (bp), followed by a nucleotide sequence coding for the remaining 202 amino acid residues. The SAP gene has an ATAT-AAA sequence 29-bp upstream from the cap site, but there is no CART box-like sequence. A possible polyadenylation signal sequence, ATTAAA, was found to be located 28-bp upstream from the polyadenylation site. A comparison of the genomic SAP DNA sequence with that of human C-reactive protein (CRP) revealed a striking overall homology which was not uniform: several highly conserved regions were hounded by non-homologous regions. This comparison provides further support for the hypothesis that SAP and CRP are products of a gene duplication event.View full abstractDownload PDF (1493K) -
Hirokazu KOYAMA, Haruo SUZUKI1986 Volume 100 Issue 4 Pages 859-866
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPsuedomonas L-phenylalanine oxidase (deaminating and decarboxylating) contains two FAD molecules in one molecule of the enzyme (Koyama, H. (1983) J. Biochem. 93, 1313-1319). When the enzyme was mixed anaerobically with L-phenylalanine, β-2-thienylalanine, L-tyrosine, or L-methionine, a spectral species (purple inter-mediate) with a broad absorption band around 540 nm was observed with each substrate, and decayed slowly. From the data on the overall reaction kinetics, the rate of the L-phenylalanine oxidase reaction was expressed as follows.
e/v=e/Vm+A/[S]+B/[O2]
where e represents the concentration of enzyme unit, v the rate of the overall reac-tion, Vm the maximum velocity, and A and B are constants. Furthermore, the reactions of the enzyme with β-2-thienylalanine (mostly an oxygenase substrate) and L-methionine (an oxidase substrate) were analyzed by the “stopped flow” method. The following scheme for the mechanism of L-phenylalanine oxidase reaction with both substrates is proposed, based on the data obtained.
Eox+S_??_EoxS→X very slow→fully reduced state
X+O2 kox →Eox+P
Where Eox represents the oxidized form of the enzyme unit, EoxS the enzyme unit (oxidized form)-substrate compound, X the purple intermediate with a characteristic broad absorptino band around 540nm, S the substrate and P the product.View full abstractDownload PDF (573K) -
Yoshikazu SUGIMOTO, Hazime SAITÔ, Ryoko TABETA, Masahiko KODAMA1986 Volume 100 Issue 4 Pages 867-874
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to monitor the membrane fluidity of cells without perturbation by an introduced probe, we developed a method for large-scale preparation of 2H-labeled melanoma cells for a 2H NMR study by incubating melanoma cells with [18, 18, 18-2H3]stearic acid/phosphatidylcholine liposomes for 2 h at 37°C. It turned out that this treatment did not significantly change the cell viability, lipid metabolism or membrane fluidity. The 2H from C-18 of stearic acid is dominantly located at the original position of the fatty acid in the 'II-labeled membrane vesicles, as studied by a tracer experiment with [1-14C]stearic acid. We found that three to four 2H-labeled species were present at 19°C in NMR spectra of the 2H-labeled membrane vesicles prepared from B16 melanoma cells. The extent of peak-splittings due to 2H-quad-rupole interaction decreased as the temperature rose, and a definite point of phase transition was not observed. At elevated temperature, 2H-labeled lipids undergo fast exchange between the bilayer and an isotropic phase such as oil phase of triolein or inverted micelles in lipid polymorphs. We further analyzed the change of membrane organization in mouse B16 melanoma cells treated with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which strongly inhibited melanogenesis. The magnitude of the quadrupole splitting at 19°C in membranes from TPA-treated cells was sig-nificantly less (40%) than in the untreated control. This is mainly explained by decreased molecular ordering (fluidity) due to the increased amount of unsaturated fatty acids in the membranes of TPA-treated cells.View full abstractDownload PDF (639K) -
Kunio AOKI, Takashi IDEGUCHI, Tomisaburo KAKUNO, Jinpei YAMASHITA, Tak ...1986 Volume 100 Issue 4 Pages 875-882
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPolyclonal antibodies were prepared to the subunits of the spinach photosystem II fraction (PS II): p47, p43, p27, p33, p24, and p17. (The protein nomenclature refers to Mr). p47 and p43 are the subunits of reaction center complex, and p27 is light-harvesting chlorophyll protein. p33, p24, and p17 are extractable from PS II with 1M Tris, and p24 and p17 with 1M NaCI. With untreated PS II fractions, the antibody to p24 inhibited the photosynthetic oxygen-evolving activity, but not the DCPI-photoreduction activity in the presence of DPC, indicating that p24 played an important role in the former activity. Bindings of the respective anti-bodies to the PS II treated with sodium dodecyl sulfate were regarded as 100%. To untreated PS II, the bindings were 20-30% for p47, p43, and p27, about 50% for p33, and 70-80% for p24 and p17. To NaCI-washed PS II, the binding to p33 increased by 9%, indicating that p33 was adjacent or bound with p24 or/and p17. To Tris-washed PS II, the binding to p43 increased by 7%, indicating that p43 was adjacent or bound with p33. To PS II treated with 3% of Brij 58, only the binding to p27 increased appreciably. To PS II treated with 1% of octyl glucoside, the binding to p47 was still lower than 50%, whereas those to the other subunits were 74-91 %. These values could be a measure of the extents to which the subunits were exposed to the aqueous phase, because of the nature of polyclonal antibodies. These results suggest that in intact PS II, p47, p43, and p27 were in most part buried in the inside, p47 being located at the most central and p27 at the outermost part, whereas p33, p24, and p17 were exposed to the outside by 50-75%.View full abstractDownload PDF (673K) -
Noriko MOHRI, Shuji TANAKA, Tohru MIYAJIMA, Hiroshi KIHARA, Motonori O ...1986 Volume 100 Issue 4 Pages 883-893
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen phospholipase A2 from the venom of Trimeresurus flaroviridis (the Habu snake) was oxidized with N-bromosuccinimide at pH 4.0, its activity decreased linearly with increase in the extent of oxidation of tryptophan residues. Oxidation of two of the four tryptophan residues caused an apparent loss of activity. The accessibilities of the tryptophan residues were analyzed with differently oxidized phospholipase A2 preparations and were determined to be in the following order: Trp-3_??_Trp-30 > Trp-68 > Trp-108. The magnitude of the difference spectrum with a negative peak at 292 nm which is produced upon the binding of Ca2+ in the vicinity of tryptophan residue(s) decreased in a concave manner with increase in the extent of oxidation of tryptophan residues and was greatly diminished when 2mol of tryp-tophan residues were oxidized. The activity and Ca2+-induced difference spectrum are thus related to either Trp-3 or Trp-30 or both. Des-octapeptide(1-8)-phospho-lipase A2 (L-fragment) is 14% as active as phospholipase A2 and is able to give a Ca2+-induced difference spectrum which is smaller than, but similar to, that of phospholipase A2. Its activity and the magnitude of the Can -induced difference spectrum decreased along similar paths with increase in the amount of tryptophan residues oxidized, but in a manner indicating that two tryptophan residues are apparently responsible for the activity and the Ca2+-induced difference spectrum. The order of accessibility of the tryptophan residues of L-fragment was Trp-30_??_Trp-108 >Trp-68. Trp-108, however, could be excluded from the residues located in the active site by reference to the tertiary structure of homologous Crotalus atrox phospholipase A2. Thus, Trp-30 is located in the Ca2+ binding site and is respon-sible for the activity of L-fragment. It is thus concluded that in phospholipase A2 Trp-30 is located in the Ca2+ binding site. From the concave decrease of relative
magnitude of the Ca2+-induced difference spectrum and the linear decrease of rela-tive activity upon oxidation of phospholipase A2, it may be assumed that both Trp-3 and Trp-30 are required to produce the Ca2+-induced difference spectrum, while only Trp-30 need be intact for activity. Anomalous binding of Ca2+ was observed for oxidized phospholipase A2.View full abstractDownload PDF (811K) -
Ryuichi KATO, Yasushi YAMAZOE, Miki SHIMADA, Norie MURAYAMA, Tetsuya K ...1986 Volume 100 Issue 4 Pages 895-902
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of growth hormone and ectopic transplantation of pituitary gland on the amounts of sex-specific cytochrome P-450, P-450-male and P-450-female, and the activities of testosterone and drug hydroxylases in male rat liver microsomes were studied. Hypophysectomy decreased the content of P-450-male, without changing the total cytochrorne P-450 level. The continuous infusion of growth hormone into hypophysectomized rats and the transplantation of pituitary gland under the renal capsule caused a further decrease in P-450-male content and an expression of P-450-female. In contrast, the intermittent injection of growth hor-mone into hypophysectomized rats increased P-450-male content to the level seen in intact male rats. The activities of testosterone 2α- and 16α-, but not 6β-, 7α-, or 15α-hydroxylase, were changed in association with the level of P-450-male by these treatments. Anti-P-450-male immunoglobulin G inhibited testosterone 2α-and 16aα-hydroxylations, but not 6β-, 7α- or 15aα-hydroxylation. These results indicate that growth hormone regulates the expression of P-450-male responsible for testosterone 2α- and 16α-hydroxylations. The metabolism of 7-propoxycournarin, benzo(a)pyrene and aminopyrine also changed with the content of P-450-male, although the correlation was less than that observed with testosterone 2α- and 16a-hydroxylation.View full abstractDownload PDF (641K) -
Ken-ichi GOTOH, Mineo TAKEI, Takafumi WATANABE, Tetsuya SUGA1986 Volume 100 Issue 4 Pages 903-909
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of unsaturated fatty acids on the activities of peroxisomal enzymes of Tetrahymena pyriformis were investigated. 1) When saturated fatty acids and the corresponding unsaturated fatty acids (C18) were added to the culture medium at 0.05%, the activities of peroxisomal enzymes [fatty acyl-CoA oxidase (FAO), car-nitine acetyltransferase (CAT), isocitrate lyase (ICL), and malate synthase (MS)] were significantly increased. The order of effectiveness was linoleic acid > oleic acid > stearic acid. 2) However, a-linolenic acid and γ-linolenic acid at the same concentration were lethal to the cells. The inhibitory effect on growth disappeared upon addition of an antioxidant, a-tocopherol. 3) Lipid peroxides derived from unsaturated fatty acids induced marked cell lysis. 4) In the presence of a low concentration (0.005%) of linolenic acid the production of lipid peroxide was lower and no inhibitory effect on the growth was observed, while the activities of peroxi-somal enzymes participating in lipid metabolism and that of catalase were signifi-cantly increased. These results indicate that the peroxisomal enzyme systems related to the β-oxidations of fatty acids and the glyoxylate cycle are regulated by unsaturated long-chain fatty acids, including linolenic acid, at low concentrations, as well as by saturated fatty acid in the medium.View full abstractDownload PDF (518K) -
Chikara OHO, Haruhiko TAKISAWA1986 Volume 100 Issue 4 Pages 911-921
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of phosphatidylinositol 4, 5-bisphosphate (PtdInsP2) and inositol 1, 4, 5- triphosphate(InsP3) on the Ca2+ release from ATP-dependent Ca 2+-transporting microsomes prepared from ox platelets were investigated. Under optimal condi-tions, both PtdInsP2 and InsP3 released Ca2+ from the microsomes in a similar dose-dependent manner. However, the maximal amount of Ca2+ released by Ins3 was almost one-fourth of that released by PtdInsP2. Neither PtdInsP2 nor InsP, appeared to act as a Ca2+-ionophore since they showed no effect on the Ca2+ content of liposomes prepared from platelet microsomal lipids. InsP3-induced but not PtdInsP2-induced Ca2+ release was decreased with increasing extravesicular Ce2+ from 0.1μM to 10μM and it was completely inhibited by 10μM Ca2+. PtdInsP2- induced but not InsP3-induced Ca2+ release was markedly inhibited by Mg2+, ruthe-nium red and neomycin. In addition, InsP3 could induce no additional Ca2+ release after the accumulated Ca2+- had been maximally released by PtdInsP2. These results indicate that PtdInsP2 releases Ca2+- from platelet microsomes more effectively than InsP, by a mechanism distinct from that of InsP3-induced release, and further that InsP3-sensitive microsomes are included within the population of PtdInsP2-sensitive microsomes.View full abstractDownload PDF (858K) -
Yasuo KAGAWA, Morio ISHIZUKA, Toshio SAISHU, Shinichi NAKAO1986 Volume 100 Issue 4 Pages 923-934
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSF1-ATPase is the major enzyme for ATP synthesis in mitochondria, chloroplasts, and bacterial plasma membranes. Fl-ATPase obtained from thermophilic bac-terium PS3 (TF1) is the only ATPase which can be reconstituted from its primary structure. Its β subunit constitutes the catalytic site, and is capable of forming hybrid F1's with E. coli a and γ subunits. Since the stability of TF1 resides in its primary structure, we cloned a gene coding for TF1, and the primary structure of the β subunit was deduced from the nucleotide sequence of the gene to compare the sequence with those of β's of three major categories of F1's; prokaryotic mem-branes, chloroplasts, and mitochondria. The following results were obtained. 1) Homology: The primary structure of the TF1β subunit (473 residues, Mr=51, 995.6) showed 89.3% homology with 270 residues which are identical in the β subunits from human mitochondria, spinach chloroplasts, and E. coli. It contained regions homologous to several nucleotide-binding proteins. 2) Secondary structure: The deduced a-helical (30.1 and β-sheet (22.3%) contents were consistent with those determined from the circular dichroism spectra. Residues forming reverse turns (Gly and Pro) were highly conserved among the F1β subunits. 3) Substituted residues and stability of TF1: We compared the amino acid sequence of the TF1β subunit with those of the other F1β subunits mentioned above. The observed substitutions in the thermophilic subunit increased its propensities to form secondary structures, and its external polarity to form tertiary structure. 4) Codon usage: The codon usage of the TF1β gene was found to be unique. The changes in codons that achieved these amino acid substitutions were much larger than those caused by minimal mutations, and the third letters of the optimal codons were either guanine or cyto-sine, except in codons for GIn, Lys, and Glu.View full abstractDownload PDF (870K) -
Naoto OKU, Naoto YAMAGUCHI, Nobuko YAMAGUCHI, Sayumi SHIBAMOTO, Fumiak ...1986 Volume 100 Issue 4 Pages 935-944
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of synthetic polycations, polyallylamine, and polyethylenimine, on lipo-somes containing phosphatidylserine was investigated along with that of polylysine and divalent cations. The addition of polycations caused aggregation of sonicated vesicles composed of phosphatidylserine and phosphatidylcholine (molar ratio 1 : 4) as determined by measuring the turbidity changes. Liposomal turbidity increased 10 times compared with that of control liposomes at charge ratios of polymer/vesicle from 0.23 (polylysine) to 2.5 (linear polyethylenimine), while the turbidity was unchanged by the addition of Ca2+ or Mg2+ at charge ratios up to 500. These polycations also induced intermixing of liposomal membranes as indicated by resonance energy transfer between fluorescent lipids incorporated in lipid bilayers, without inducing drastic permeability changes as determined from the calcein release. Fifty percent intermixing of liposomes (0.05mM as lipid concentration) was induced by these polycations at charge ratios of around 1.0. However, the highest resonance energy transfer was produced by the addition of polyallylamine, which caused multi-cycles of membrane intermixing between vesicles. Polycation-induced membrane intermixing and permeability changes of phosphatidylserine liposomes were also investigated. At charge ratios of around 1.0, these polymers caused resonance energy transfer of fluorescent lipids incorporated in separate vesicles; however, pollallylamine and branched polyethylenimine also caused permeability increases of liposomal membranes. Membrane intermixing and permeability changes of phos-phatidylserine vesicles induced by polyallylamine were dependent on the polymer/vesicle charge ratio, and were different from those induced by Ca2+ since the latter caused half-maximal membrane intermixing or permeability change of phosphatidyl-serine vesicles at about 1mM at the liposomal concentrations investigated.View full abstractDownload PDF (847K) -
Masanao KATAGIRI, Hiroko MURAKAMI, Yoshiyasu YABUSAKI, Toshihiro SUGIY ...1986 Volume 100 Issue 4 Pages 945-954
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nucleotide sequence of the mRNA for NADPH-cytochrome P-450 reductase from rabbit liver was determined from a full-length cDNA clone (pFP105). The clone contains 2.269 nucleotides complementary to rabbit liver reductase mRNA. The single open reading frame of 2, 037 nucleotides codes for a 679-amino acid polypeptide with a calculated molecular weight of 76, 583 daltons. The cloned cDNA contains the complete 3'-noncoding region of 193 nucleotides, including 68 nucleo-tides of poly (A), and 39 nucleotides of the 5'-noncoding region. The nucleotide sequence in the coding region of cDNA of rabbit reductase (pFP105) showed 85 homology to that of rat reductase (Porter, T. D. & Kasper, C. B. (1985) Proc. Natl. Acad. Sci. U. S. 82, 973-977, and Murakami, H. et al. (1986) DNA 5, 1-10). Rabbit reductase has one more amino acid residue than the rat enzyme, and the amino acid compositions of the two enzymes are similar. The amino acid sequence of the rabbit enzyme showed 91 % identity with that of the rat enzyme. The segment related to binding of FMN and FAD was well conserved among rabbit, rat, and pig reductases. The sequence related to AMP moiety-binding was also conserved among these species, and was found in the amino acid sequence of NADH-cytochrome b5 reductase, another flavoenzyme in the microsomal electron transport system.View full abstractDownload PDF (1312K) -
Seiji INOUE, Hiroaki INOUE, Toshiki HIROYOSHI, Hiroshi MATSUBARA, Tate ...1986 Volume 100 Issue 4 Pages 955-965
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequences of cytochromes c purified from the fruit fly Drosophila melanogaster and the flesh fly Boettcherisca peregrina were determined. In contrast with the case of the housefly, isocytochromes c were not detected in these flies at any developmental stage. The sequence of fruit fly cytochrome c differed from that reported previously but was identical with that predicted from the nucleotide sequence of the fruit fly cytochrome c gene (DC4) (Limbach, K. J. & Wu, R. (1985) Nucl. Acids Res. 13, 631-644). Isocytochrome c of the fruit fly, reported to be encoded by the DC3 gene, was not detected as a functional cytochrome c molecule.View full abstractDownload PDF (1442K) -
Katsuyoshi KONDO, Kazuko OBA1986 Volume 100 Issue 4 Pages 967-974
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS3-Hydroxy-3-methylglutaryl coenzyme A reductase (NADPH) was solubilized by trypsin digestion from sliced potato tuber microsomes, and purified to apparent homogeneity in the absence of detergent with a recovery of 1.8%. The enzyme had a specific activity of 7, 910nmol of mevalonate formed per min per mg of protein. On molecular-sieving high-performance liquid chromatography, the activity was coincident with the single protein peak corresponding to a molecular weight of approximately 110kDa. On SDS-polyacrylamide gel electrophoresis, the purified enzyme showed only one protein staining band corresponding to a molecular weight of approximately 55kDa. The apparent Km value for S-HMG-CoA was 6.4μM and that for NADPH was 25μM.View full abstractDownload PDF (928K) -
Shoji ODANI, Takehiko KOIDE, Teruo ONO1986 Volume 100 Issue 4 Pages 975-983
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA number of trypsin inhibitors were isolated from wheat germs by affinity chromatography on immobilized trypsin, gel-filtration, and ion-exchange and reverse-phase chromatography. These inhibitors were classified into two groups, inhibitors I (Mr=14, 500) and II (Mr=7, 000), based on their molecular sizes. Inhibitors I and II inhibited bovine trypsin stoichiometorically at an enzyme to inhibitor ratio of 2 and 1, respectively. Sequence analysis of these inhibitors indicated a high degree of homology and that inhibitors I had a duplicated structure of inhibitors II. They are highly homologous to double-headed proteinase inhibitors (Bowman-Birk inhibitors) of Leguminosae plants. Inhibitors II are the first example of single-headed inhibitor corresponding to one inhibitory domain of the Bowman-Birk type double-headed inhibitors, which suggests that inhibitors It are relic of an ancestral single-headed inhibitor before the gene-duplication that led to the formation of present-day Bowman-Birk type inhibitors.View full abstractDownload PDF (695K) -
Tsukasa MATSUDA, Izumi NAKASHIMA, Ryo NAKAMURA, Kaoru SHIMOKATA1986 Volume 100 Issue 4 Pages 985-988
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe reactivity of IgG and IgE antibodies from egg allergy patients to ovornucoid and its domain fragments was examined quantitatively, and compared with that of rabbit and mouse IgG anti-ovomucoid antibodies. The human antibodies reacted with the carbohydrate-containing domain fragments more strongly than the animal antibodies. In contrast, the human antibodies did not react with the carbohydrate-free third domain, whereas the animal antibodies definitely reacted. These results suggested that the sites and structures recognized by allergic individuals as ovomucoid antigenic determinants are not necessarily the same as those recognized by laboratory animals.View full abstractDownload PDF (328K) -
Kazuhiro HAYASHIDA, Yoshihiro TSUCHIYA, Satoshi KUROKAWA, Masahira HAT ...1986 Volume 100 Issue 4 Pages 989-993
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat α2-macroglobulin (α2M) is a typical acute phase protein, the concentration of which in serum increases more than 100-fold after inflammation. It is also known that the protein increases during pregnant (and neonatal) stages. Using a specific cDNA probe, expression of the α2M gene during pregnancy was studied at the mRNA level. During inflammation, the liver is almost the only organ producing α2M, but during pregnancy the placenta and uterus were found to be major organs producing a large amount (70-80 of that of inflamed liver) of α2M mRNA at days 12-15. The yolk sac, maternal liver and fetal (or neonatal) liver also produced a small but significant amount (5-20% of that of inflamed liver) of the mRNA. Southern blotting analysis showed that only one copy of the α2M gene was present in a haploid rat genome. These results indicated that a single α2M gene has the ability to respond to two completely-different physiological states.View full abstractDownload PDF (2217K) -
Hiroshi INUI, Kazutaka MIYATAKE, Yoshihisa NAKANO, Shozaburo KITAOKA1986 Volume 100 Issue 4 Pages 995-1000
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSShort chain-length specific trans-2-enoyl-CoA reductase (reductase I), which contributed to mitochondrial fatty acid synthesis, was purified about 200-fold from crude extract of mitochondria in Euglena gracilis. It had a molecular weight of 39, 000, and consisted of two dissimilar subunits with molecular weights of 15, 000 and 25, 000. The enzyme utilized crotonyl-CoA as the most active substrate and showed negative cooperativity in the reaction with the substrate. NADH was the sole electron donor. Some divalent cations were inhibitory to the enzyme when incubated with the enzyme prior to the start of the reaction. The reductase apparently contained loosely bound FAD.View full abstractDownload PDF (880K) -
Masatake OOSAWA, Koscak MARUYAMA1986 Volume 100 Issue 4 Pages 1001-1008
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe rate of actin polymerization gradually decreased without changing the final level of polymerization, when incubated in the presence of 0.2mM ATP at pH 8.0 and 25°C. This change was much faster in Mg2+-actin than Ca2+-actin, and Mg2+-actin became denatured and unpolymerizable on prolonged incubation. The drop in the polymerization rate was due both to weakened nucleation and a slowed elongation rate in the incubated actin. The change in the polymerization rate was partially reversible by storing the sample at 0°C. When the rate of polymerization dropped markedly on prolonged incubation, a gel filtration profile showed that Ca2+-actin existed as monomer not as oligomer. On the other hand, Mg2+-actin formed dimers, and other oligomers, as revealed by crosslinking analysis. There were changes in fluorescence intensities due to tyrosine and/or tryptophan residues of the actin molecule, and in difference absorption spectra, suggesting that conformational changes intermediate between native and denatured states occurred during incubation.View full abstractDownload PDF (1470K) -
Isao ADACHI, Chihiro SUMI, Kazuya FURUHASHI, Hirokazu ADACHI, Masaharu ...1986 Volume 100 Issue 4 Pages 1009-1014
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo manipulate the intracellular milieu of rabbit platelets, permeabilization was performed using digitonin. Permeabilized platelets showed dose-dependent release of histamine, which was stored in granules of rabbit platelets, in response to extra-cellular calcium ion. As PMA stimulated the release reaction in digitonin-perme-abilized platelets, the protein kinase C system, which regulates metabolic processes and cell reactions in intact platelets, was revealed to be working. Cupric phenanthroline also released histamine from permeabilized rabbit platelets dose-dependently, and dithiothreitol inhibited the release strongly. Since cupric phenanthroline is a mild oxidant which catalyzes the formation of disulfide bridges, as in the case of Ca2+-ATPase of sarcoplasmic reticulum, the results suggested that protein crosslinking is implicated in the regulation of the release reaction in permeabilized rabbit platelets.View full abstractDownload PDF (461K) -
Peng WANG, Satoshi TOYOSHIMA, Toshiaki OSAWA1986 Volume 100 Issue 4 Pages 1015-1022
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMembrane-bound and cytosolic phosphatidylinositol (P1)-specific phospholipases C in murine thymocytes have been partially purified and characterized. The membrane-bound enzyme was extracted from microsomes with sodium cholate and purified by sequential column chromatographies on Sephadex G-100, heparin-Sepharose CL-6B, and Sephadex G-100. The cytosolic enzyme was purified from the cytosol by sequential column chromatographics on Sephadex G-100 and FPLC-Mono S. Specific activities of the membrane-bound enzyme and the cytosolie enzyme increased more than 1, 800- and 1, 400-fold, respectively, compared with those of rnicrosomes and the cytosol. The molecular weights of the both enzymes were estimated to be about 70, 000 by gel filtration. These purified enzymes also hydrolyzed phospha-tidylinositol 4, 5-bisphosphate (PIP2). At neutral pH and low Ca2+ concentrations, the membrane-bound enzyme hydrolyzed PIP2, in preference to PI and showed higher activity than the cytosolic enzyme. These activities were also affected differently by various lipids. For PIP2 hydrolysis, all lipids investigated except lysophosphaiidyl-choline enhanced the activity of the membrane-bound enzyme, while phosphatidyl-choline (PC) and phosphatidylserine (PS) did not significantly affect the activity of the cytosolic enzyme. PC, PE, and PS inhibited the activities of the membrane-bound and cytosolic enzymes for PI hydrolysis. The physiological implications of these results are discussed.View full abstractDownload PDF (617K) -
Hajime NAGASHIMAI1986 Volume 100 Issue 4 Pages 1023-1029
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLong bipolar filaments synthesized from rabbit skeletal myosin have been visualized directly under a dark-field light microscope. It was also possible to observe movements of single myosin filaments in the presence of F-actin, which was invisible. When the two kinds of filaments were mixed (final concentrations: 17μg/ml myosin and 0.6mg/ml actin) in a medium containing 0.13M KC1, 3mM MgCl2, and 1mM ATP, myosin filaments did not aggregate for a long period of time but were distributed separately in the field of microscope. Under these conditions, myosin filaments intermittently moved along their length in a bidirectional manner with a maximum instantaneous velocity of 5μm/s. Individual filaments alternated, at random, between motile and nonmotile periods of time, with the motile fraction of filaments at any moment being roughly 10%. The in vitro movement of myosin filaments may be caused by their active sliding relative to F-actin in the background.View full abstractDownload PDF (2866K) -
Shinobu WATARAI, Tatsuji YASUDA, Misao ONUMA, Shizuo HANDA1986 Volume 100 Issue 4 Pages 1031-1042
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe analyzed glycosphingolipids from normal lymph node cells of seven cattle and lymph node cells of eight cattle with enzootic bovine leukosis. The neutral glyco-sphingolipids and gangliosides were analyzed by thin-layer chromatography. Both normal and tumorous lymph node cells had GlcCer, LacCer, and GbOse3Cer as major neutral glycosphingolipids. In the ganglioside fraction, GM3 was the predominant component in both normal and tumorous lymph node cells, and another component, ganglioside Gx fraction, was also prominent in tumorous lymph node cells. The structure of this ganglioside Gx fraction was elucidated by thin-layer chromatography, sugar analysis, neuraminidase digestion, and permethylation studies. This ganglioside Gx fraction was found to be a mixture of four ganglioside species. The structures of individual gangliosides Gx (1 to 4) were characterized as follows. 1: GD3, NeuAca2-8NeuAca2-3Gall-4Glc-Cer. 2: GD3, NeuAca2-8NeuGca2-3Gall-4G1o-Cer. 3: GD3, NeuGca2-8NeuAct2-3Gall-4Gle-Cer. 4: GD3, NeuGca2-8NeuGca2-3 Gall-4GIc-Cer. These GD3 species may be formed as a result of the induced synthesis in association with malignant transformation.View full abstractDownload PDF (3291K) -
Ken NISHIKAWA, Tatsuo OOI1986 Volume 100 Issue 4 Pages 1043-1047
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe location measure of a residue in a globular protein is defined as the number of C° atoms surrounding the residue located within a sphere of the radius of 14Å. This quantity is a measure of the exposure of a residue to solvent, and is related closely to the distance from the center of mass of a protein. In this work, the experimental value for each residue of a protein is obtained from the X-ray crystal-lographic data, and the quantity is also calculated from the amino acid sequence data by applying an empirical parameter set to it. The correlation between the experimental and computed quantities is as high as 0.50 on the average over 92 proteins of known three-dimensional structure. Therefore, the location measure of every residue in a globular protein is predictable with good accuracy from the sequence.View full abstractDownload PDF (370K) -
Masayoshi TAKEUCHI, Kotaro NINOMIYA, Kumi KAWABATA, Naoki ASANO, Yukih ...1986 Volume 100 Issue 4 Pages 1049-1055
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA membrane-bound glucoside 3-dehydrogenase [EC 1.1.99.13], which oxidizes validoxylamine A to the 3-keto derivative, was solubilized from the membrane fraction of Flavobacterium saccharophilum by Triton X-100 and purified about 280-fold with an overall yield of 30% from the membrane fraction by column chromatography on DEAE- and CM-Sepharose CL-6B and gel filtration on Sephacryl S-300. The purified enzyme exhibited a single protein band on disc gel electrophoresis, and FAD was shown to be the prosthetic group. The enzyme had a molecular weight of 270, 000 as determined by gel filtration on Sephacryl S-300 and consisted of 4 identical subunits each with a molecular weight of 66, 000. The enzyme reacted with various artificial electron acceptors such as 2, 6-dichlorophenolindophenol (DCIP), phenazine methosulfate, and ferricyanide. The optimum pH for DCIP reductase activity was 6.0. The enzyme was inhibited by Hg2+ and p-chloromer-curibenzoate. D-Glucose and methyl-α- and β-D-glucoside showed the highest susceptibility to the enzyme, and were converted to the corresponding 3-keto sugars.View full abstractDownload PDF (1097K) -
Masachika IRIE, Kazuko OHGI, Makiko YOSHINAGA, Tamami YANAGIDA, Yoshio ...1986 Volume 100 Issue 4 Pages 1057-1063
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1) In order to investigate the roles of Lys1, and Lys7, of RNase A in the enzymatic activity, four S-peptide derivatives were prepared and their abilities to activate S-protein were measured. They are 1-norleucine-S-peptide, 7-norleucine S-peptide, 1, 7-di-norleucine-S-peptide, and tri-N-acetyl S-peptide. 2) From the analyses of the relative activity and kinetic parameters of RNase S' derivatives with UpU, UpU > p, and UpUpU > p, it was concluded that Lys7, of RNase A is a binding site for 3'-phosphate of UpU>p and the modification or substitution of Lys1, affects the binding of trinucleotide substrate.View full abstractDownload PDF (594K) -
Taira OHNISHI, Akira WADA, Yasuki NONAKA, Toshihiro SUGIYAMA, Toshio Y ...1986 Volume 100 Issue 4 Pages 1065-1076
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBovine adrenocortical calmodulin was purified and its general properties were examined. The latter were similar to those of bovine brain calmodulin. When added to a cytochrome P-45011β-reconstituted system in the presence of dilauroyl-phosphatidylcholine, calmodulin decreased the rate of aldosterone production from corticosterone from 0.8 to 0.1nmol/(min.nmol P-450), while it increased the rate of 18-hydroxycorticosterone production from 1.8 to 4.6mol/(min.nmol P-450). This effect of calmodulin on steroid production was maximum at a concentration of 1μM, when 1μM cytochrome P-45011β was used. The effect was dependent on the presence of Ca2+, and maximal response was observed at less than 1μM Ca2+. There was essentially no difference in the effect when bovine brain calmodulin was used. Calmodulin induced a change in the activity of cytochrome P-45011β in the presence of a wide concentration range of corticosterone as a substrate. As for 18-hydroxycorticosterone production, calmodulin increased both the maximal activity and the apparent Km, for corticosterone, but it decreased the apparent Km for adrenodoxin. Adrenodoxin at a concentration of less than 20μM did not fully abolish the effect of calmodulin. A small type I difference spectrum appeared when calmodulin was added to cytochrome P-45011β. The difference spectrum increased significantly in the presence of both Ca2+ and adrenodoxin. These results suggest that calmodulin interacts with cytochrome P-45011β in the presence of adrenodoxin and then modulates the activity of aldosterone synthesis catalyzed by cytochrome P-45011β.View full abstractDownload PDF (998K) -
Tsutomu TSUJI, Toshiaki OSAWA1986 Volume 100 Issue 4 Pages 1077-1085
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman platelet membrane glycoprotein IV (GPIV), one of the major glycoprotein components, was purified by successive affinity chromatographies on columns of Lens culinaris agglutinin-Sepharose and wheat germ agglutinin-Sepharose followed by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). On SDS-polyacrylamide gel electrophoresis in either the presence or absence of dithiothreitol, GPIV gave a single band with an apparent molecular weight of 97, 000, suggesting that GPIV is composed of a single polypeptide chain without interchain disulfide bonds. Compositional analysis showed that GPIV contains large amounts of acidic and hydroxy amino acids, but only very small amounts of cystine and methionine, and 28.1% (w/w) carbohydrate consisting of galactose, glucosamine, and sialic acid as the principal sugars with smaller amounts of fucose, mannose, and galactosamine. This suggested that GPIV contains both N-linked and O-linked sugar chains. The O-linked sugar chains isolated from GPIV, together with those from GPIb and glycocalicin, were comparatively analyzed on a Bio-Gel P-4 column after neuraminidase treatment. The results indicated that all three glycoproteins have two common species of carbohydrate chains, a disaccharide, Gal-GaINAc, and a tetrasaccharide, Gal-GlcNAc-(Gal-)GaINAc. The ratio of the tetrasaccharide to the disaccharide in GPIV was found to be some-what different from that in GPIb or glycocalicin.View full abstractDownload PDF (1808K) -
Facile Release of NADPH Oxidase from Polymorphonuclear Leukocyte Membrane by Mild Pressure TreatmentTeruaki KATAYAMA, Naoki OKAMURA, Sadahiko ISHIBASHI1986 Volume 100 Issue 4 Pages 1087-1089
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNADPH oxidase, a complex enzyme system in the cell membrane responsible for the bactericidal function of polymorphonuclear leukocytes through the production of superoxide anion, was facilely released by mild treatment with a press. At the pressure where almost all NADPH oxidase activity was released, releases of the activities of lactate dehydrogenase, 5'-nucleotidase, lysozyme, and N-acetyl-β-glucosaminidase, and of the amount of total protein were negligible. This method can be useful for the elucidation of NADPH oxidase.View full abstractDownload PDF (231K) -
Tomoh MASAKI, Hisao IIZUKA, Hiromi TAKANO-OHMURO, Junko OKAMOTO, Takas ...1986 Volume 100 Issue 4 Pages 1091-1094
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA distinct difference in the 3-methylhistidine (3-MeHis) content and the pH-depend-ency curve for calcium-activated adenosine triphosphatase (Ca-ATPase) activity was observed between chicken and mammalian cardiac ventricular myosins. The 3-MeHis content and pH dependency of the Ca-ATPase activity of myosins from adult and embryonic chicken cardiac ventricular muscles and chicken fast white and slow red muscles were almost the same.View full abstractDownload PDF (287K) -
Yasushi YAMAZOE, Miki SHIMADA, Norie MURAYAMA, Sumie KAWANO, Ryuichi K ...1986 Volume 100 Issue 4 Pages 1095-1097
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe relationship between growth hormone and testosterone (T) 6β-hydroxylase was investigated in normal and hypophysectomized (hypox) male rats. The administration of human growth hormone (hGH) by either intermittent injections or continuous infusion to normal and hypox rats decreased the activity of T 6βΒ-hydroxylase in hepatic microsomes. Hypophysectomy did not reduce, but rather increased the 6β-hydroxylase activity, while the 16α-hydroxylase activity decreased. These results, together with the data from a Western blot, indicate that growth hormone acts as a repressive factor for the expression of T 6β-hydroxylase in a manner different from the regulation of T 16α-hydroxylase.View full abstractDownload PDF (227K) -
Hyeun Wook CHANG, Ichiro KUDO, Shuntaro HARA, Ken KARASAWA, Keizo INOU ...1986 Volume 100 Issue 4 Pages 1099-1101
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn extracellular phospholipase A2 was detected in the peritoneal cavity of rats injected intraperitoneally with casein. This activity required Ca2+ ion and had a pH optimum of 9. The increase of phospholipase A2 activity in the peritoneal fluid as a function of time roughly paralleled the increase in the number of poly-morphonuclear (PMN) leukocytes found in the peritoneal cavity. Phospholipase A2 activity was also observed in rat PMN homogenates, and its characteristics were similar to those of the enzyme found in cell-tree peritoneal fluid fraction. PMN leukocytes may release non-lysozomal phospholipase A2 into the extracellular space as ‘inflammation reactions’ proceed.View full abstractDownload PDF (255K) -
Hiroyoshi ARIGA, Masanobu NARUTO, Hiroko KAWANO, Hirofumi NAKANO1986 Volume 100 Issue 4 Pages 1103-1106
Published: October 01, 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOf the several DNA fragments present in the human lung cancer gene, 1.1 and 2.0 kilobase (kb) fragments corresponding to the intron of this gene were hybridized to a half part of the 27 nucleotides perfect palindrome present in the initiation part of replication in simian virus 40 (SV40) DNA. These two fragments cloned in pBR322 had good template activity, and the initiation of DNA replication started from the region of these fragments in an in vitro system, in which the initiation of DNA replication occurs on cloned DNA containing SV40 origin of DNA replication as described previously. Furthermore, these two clones could replicate autonomously in nuclei of SV40 transformed Cos cells, producing SV40 T antigen constitutively when the clones were transfected into Cos cells. These results show that functional SV40 origin-like sequences are present in human genomes, and they can replicate autonomously within the cells which are producing SV40 T antigen.View full abstractDownload PDF (1776K) -
1986 Volume 100 Issue 4 Pages 1107c
Published: 1986
Released on J-STAGE: November 18, 2008
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1986 Volume 100 Issue 4 Pages 1107a
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (31K) -
1986 Volume 100 Issue 4 Pages 1107b
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (31K) -
1986 Volume 100 Issue 4 Pages 1107d
Published: 1986
Released on J-STAGE: November 18, 2008
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