The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Volume 87, Issue 6
Displaying 1-38 of 38 articles from this issue
  • Hiroshi MUNAKATA, Zensaku YOSIZAWA
    1980 Volume 87 Issue 6 Pages 1559-1565
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    The brush border fraction (Fr. P2) and the calcium chloride (10mM)-soluble fraction (Fr. S) were separated from the small intestinal mucosa of rabbit. Sulfated glycoproteins, P-SGP and S-SGP, were then purified from Fr. P2 and Fr. S by zone electrophoresis and gel filtration, respectively. P-SGP and S-SGP were both electrophoretically homogeneous, although their mobilities differed from each other. P-SGP and S-SGP contained 61.8 and 35.3% protein, 31.8 and 55.0% carbohydrate, and 1.1 and 2.7% sulfate, respectively. The major constituent sugars in P-SGP and S-SGP were galactose and glucosamine, while mannose, sialic acid, and L-fucose were minor components. In addition, galactosamine was a major sugar in S-SGP, but a minor one in P-SGP. The major amino acids of the protein moiety of P-SGP were glutamic acid, aspartic acid, glycine, and alanine, while those of S-SGP were threonine, proline, glutamic acid, aspartic acid, serine, and glycine.
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  • II. Location of the Segment Anchoring γ-Glutamyltranspeptidase to the Membrane
    Akihiko TSUJI, Yoshiko MATSUDA, Nobuhiko KATUNUMA
    1980 Volume 87 Issue 6 Pages 1567-1571
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    The amino acid compositions of the subunits of γ-glutamyltranspeptidase solubilized with Triton X-100 (T-γGTP) and papain (P-γGTP) were compared. The results showed that the light subunits of the two forms are identical in amino acid composition, but that the heavy subunit of T-γGTP contains about 52 amino acid residues more than that of P-γGTP.
    The susceptibilities of the two forms to endopeptidase and the endo-groups of the two forms were studied to determine whether the membrane binding segment was located near the amino-terminus or the carboxyl-terminus. Leucine amino peptidase released amino acids from T-γGTP, converting it to a hydrophilic form like P-γGTP without loss of activity, and the composition of the amino acids released was similar to that of the difference in the amino acid compositions of the heavy subunits of T-γGTP and P-γGTP. No amino acids were released from P-γGTP by treatment with this enzyme. The amino-terminal residues of the heavy and light subunits of T-γGTP were methionine and threonine, respectively, while those of P-γGTP were glycine and threonine, respectively. Neither T-γGTP nor P-γGTP was affected by treatment with carboxypeptidase Y. Amino acids were released from both forms by treatment with carboxypeptidase Y in the presence of sodium dodecyl sulfate, and the patterns of these released amino acids were very similar.
    These results indicate that the amino terminal portion of the heavy subunit of γ-glutamyltranspeptidase contains the portion that anchors the enzyme to the membrane.
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  • Kazuko HIRAI, Etsuko KATAOKA, Shizuko AMAGASE, Kimiko ASANO
    1980 Volume 87 Issue 6 Pages 1573-1580
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    The effect of adrenocorticotropic hormone (ACTH) on the change in adenosine 3':5'-monophosphate (cyclic AMP) binding activity and guanosine 3':5'-monophosphate (cyclic GMP) binding activity in adrenal glands of hypophysectomized rats was examined by measuring cyclic [3H] AMP-binding and cyclic [3H] GMP-binding, respectively. When ACTH1-24-retard (200 μg/100g body weight/day) was injected into rats for 3 days, the increase in cyclic AMP-binding activity (activity/100g body weight) found in the supernatants, mitochondria, and microsomes of adrenal glands was greater than the growth of adrenal glands, whereas the increase in cyclic GMP-binding activity (activity/100g body weight) was almost equal to that of adrenal size. Administration of less than 200 μg ACTH1-24-retard did not effectively stimulate both activities. During 3 days, a mixture of ACTH1-24-retard and -immediate (200 μg and 5 U/100g body weight/day) induced increases of 2.8-fold in adrenal weight, 6.0-, 1.5-, and 5.8-fold in cyclic AMP-binding activity and 1.3-, 1.3-, and 2.1-fold in cyclic GMP-binding activity in the supernatants, microsomes, and mitochondria, respectively. In regard to the effect of the ACTH1-24-mixture on the specific activity (activity/μg protein), the activity of the cyclic AMP-binding protein increased in supernatants and mitochondria but not in microsomes, whereas that of the cyclic GMP-binding protein decreased in all three subcellular fractions. The effect of administration of the ACTH1-24-mixture for 9 days was marked on the stimulation of cyclic AMP-binding activity, while the specific activity was not significantly affected. These results indicated the presence of a differential effect on the cyclic AMP- and cyclic GMP-binding activities during ACTH action, and suggested that both cyclic AMP- and cyclic GMP-binding proteins may be involved in ACTH action and play an important role in hormonal control of steroidogenesis through regulation of the adrenal cortex.
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  • Masahiro KOHASHI
    1980 Volume 87 Issue 6 Pages 1581-1586
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    Six unconjugated pteridines stored in soybean seeds were extracted with acid and isolated with a Florisil column and reversed-phase high-performance liquid chromatography (HPLC). They were identified by HPLC, TLC, and fluorescence spectra to be 6-carboxypterin, erythro-neopterin, threo-neopterin, isoxanthopterin, 6-hydroxymethylpterin, and pterin. They were eluted in that order on HPLC. A much higher density of the pteridines, excepting isoxanthopterin, was found in the axis than in the cotyledon after a 6 h-imbibition.
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  • Purification of Three Forms of Enolase
    Fujiko SUZUKI, Yumiko UMEDA, Kanefusa KATO
    1980 Volume 87 Issue 6 Pages 1587-1594
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    Three forms of rat brain enolase (Fletcher, L., Rider, C. C., & Taylor, C. B. (1976) Biochim. Biophys. Acta 452, 245-252), which were separable by DEAE-cellulose chromatography (referred as enolase I, enolase II, and enolase III in order of the elution), were purified with a high yield by use of column chromatographies of Sephadex G-150, Blue Sepharose CL-6 B, and hydroxylapatite. About ten mg of each isozyme was obtained from 220g of the brain with a yield of 30-50%.
    Sodium dodecyl sulfate gel electrophoresis of purified enolase I and enolase III showed single bands with relative mobilities corresponding to molecular weights of 49, 000 (enolase I) or 46, 000 (enolase III), and that of purified enolase II showed two bands corresponding to the above molecular sizes.
    Amino acid analysis of three forms of enolase revealed that the amount of each amino acid of enolase II was midway between those of enolase I and enolase III.
    Antiserum to enolase I or enolase III was raised in New Zealand white rabbits. Results of immunochemical neutralization studies and immunoelectrophoresis indicated that antienolase III was specific to the subunit of enolase III and reacted with enolase II and enolase III. However, anti-enolase I reacted not only with enolase I and enolase II, but also with enolase in various other tissues.
    These results support previous reports on crude preparations that enolase II was a hybrid molecule consisting of one enolase I subunit and one enolase III subunit.
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  • IX. Biochemical Effects of Simfibrate on Precursor Incorporation into Polypeptide Associated with Peroxisome Proliferation in Rat Liver
    Takafumi WATANABE, Tetsuya SUGA
    1980 Volume 87 Issue 6 Pages 1595-1601
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    Treatment with 1, 3-propanediol-bis (2 p-chlorophenoxy-isobutyrate) (simfibrate), a hypolipidemic drug, showed little effect on liver weight and hepatic lipid level.
    When the effects of simfibrate on rat liver peroxisomal enzymes were examined, it was found that cyanide-insensitive fatty acyl-CoA oxidizing system and carnitine acetyltransferase activity increased from 0.38 U/g liver to 4.5 U/g liver and from 227 U/g liver to 6, 450 U/g liver, respectively. The activity of D-amino acid oxidase decreased from 1.05 U/g liver to 0.39 U/g liver. Other peroxisomal enzymes including catalase and urate oxidase were not significantly changed by this drug.
    Of the protein components of the light mitochondrial fraction of simfibrate-treated rat liver, a polypeptide with a molecular weight of approximately 76, 000, which has been suggested to be one of the peroxisomal proteins, increased in the treated rat liver (as estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis) and DL-[4, 5-3 H] leucine-incorporation into this particular component was also stimulated to a level about 3 times that of the control.
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  • Hideto KUWAYAMA, Koichi YAGI
    1980 Volume 87 Issue 6 Pages 1603-1607
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    Cardiac myosin liberated 1.99 mol of acetate per mol after acid hydrolysis. The myosin subfragment-1, which cannot bind g2 light chain, liberated 0.85-1.16 mol of acetate per mol. On the other hand, negligible amounts of acetate could be detected from g1 and g2 light chains. The acetates, which were measured by a microenzymic method, were derived from the acetylated N-terminal of the heavy chain. It was concluded that g2 light chain is bound to the C-terminal region of subfragment-1, which is the link between the heads and tail of myosin.
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  • Shigeo OHTA, Masamichi TSUBOI, Tairo OSHIMA, Masasuke YOSHIDA, Yasuo K ...
    1980 Volume 87 Issue 6 Pages 1609-1617
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    The catalytic and allosteric sites of proton translocating adenosine triphosphatase (ATPase) were studied by measuring the binding of nucleotides to the ATPase, and its a and β subunits purified from thermophilic bacterium PS 3, with a circular dichroic spectrometer.
    In contrast to mesophilic ATPases, this thermophilic enzyme contained no tightly bound nucleotides, and its subunits were stable after their purification. These properties were advantageous for analyzing both catalytic and allosteric sites. The former site showed rapid and loose binding, but the latter slow (t1/2_??_1 h, for ADP) and tight binding.
    When a nucleotide was bound, the g subunits showed a negative ellipticity at 275 nm corresponding to a tyrosyl residue, while the a subunits showed an ellipticity change corresponding to the absorption curve of the bound nucleotide. This difference enabled us to distinguish the binding sites in ATPase. At a low concentration, ADP selectively bound to a subunits in the ATPase, while at a high concentration, it bound to both subunits. This finding suggests that the tight binding sites are located in the α subunits. Although ADP and ATP bound to both the purified α and β subunits, CTP did not bind to β but only to α subunits, and ITP bound to β but hardly to α. These nucleotide specificities also supported the idea that the catalytic sites are located in the β subunits and the allosteric sites are located in the α subunits.
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  • Tatsuo NAKAYAMA, Yumiko KUROGI, Hisayuki MATSUO
    1980 Volume 87 Issue 6 Pages 1619-1624
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    Actinomycin D allowed 3.6% of spore formation and 4.9% of dipicolinic acid synthesis of control cultures when added at 0.4 μM at late stage (T6.5) during sporulation process of Bacillus subtilis although the drug inhibited sporulation almost completely when added at log phase and early stages. Daunorubicin added at log phase, on the other hand, did not inhibit either growth and sporulation of the bacteria up to a concentration of 2.9 μM.
    The joint use of both antibiotics at late stages decreased the levels of spore formation and the synthesis of dipicolinic acid allowed by the use of actinomycin D alone to 0.9% and 2.1% respectively, and also inhibited the synthesis of spore coat protein in a cooperative manner.
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  • Takahisa KANDA, Kazumasa WAKABAYASHI, Kazutosi NISIZAWA
    1980 Volume 87 Issue 6 Pages 1625-1634
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    A new endo-cellulase component of carboxymethyl cellulase (CMCase) type (En-1) was obtained by gel filtration and column chromatography from Driselase, a commerical enzyme preparation from Irpex lacteus (Polyporus tulipiferae). The enzyme behaved as a single protein on polyacrylarnide disc electrophoresis in the presence of sodium dodecyl sulfate (SDS). Its molecular weight was estimated to be 15, 500, and it contained only 0.73% carbohydrate as glucose. The pattern of its amino acid composition is similar to those of other cellulases in respect of high contents of acidic amino acids, glycine, serine, and threonine. The cellulase was most active at pH 4.0 and was very stable in the pH range of 3.0 to 6.0, but was completely inactivated by heating at 70°C for 10min. A series of cellooligosaccharides, including cellobiose, was formed by this enzyme from sodium carboxymethyl cellulose (CMC) as well as from water-insoluble celluloses. In the hydrolysis of CMC, the increase in the fluidity of the substrate was relatively large as compared with the simultaneous increase in reducing power. From this result and the pattern of hydrolysis products, En-1 was elucidated to be an endocellulase, and it showed the highest randomness among the cellulase components obtained so far from Irpex lacteus.
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  • Takahisa KANDA, Kazumasa WAKABAYASHI, Kazutosi NISIZAWA
    1980 Volume 87 Issue 6 Pages 1635-1639
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    Cotton and Valonia-celiulose (cellulose I) were readily attacked by endo-cellulase of a highly endowise-hydrolysis type, with a sharp decrease in the degree of polymerization, while the simultaneous production of reducing sugar was low. In contrast, viscose rayon and alkalicellulose (cellulose II) showed little lowering of the degree of polymerization by endo-cellulase, though the effect was slightly greater than with an exo-cellulase, while the simultaneous production of reducing sugar was very high. The synergistic effect of exo- and endo-cellulases on the hydrolysis of cellulose was much higher with cellulose I than with cellulose II. These results can be explained in terms of differences in the polarity of cellulose chains and in the crystallinity and/or ultrastructure of the cellulose fibers.
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  • Kei-ichi UEMURA, Matsuko YUZAWA-WATANABE, Nobuko KITAZAWA, Tamotsu TAK ...
    1980 Volume 87 Issue 6 Pages 1641-1648
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    Antibodies against each of three neutral glycosphingolipids, Forssman glycolipid, globoside I, and asialo GM2, were elicited in rabbits. The specificity of these antibodies was studied by liposome agglutination and liposomal membrane immune-damage assays using liposomes prepared with each glycolipid hapten. Spectrophotometric measurement of liposome agglutination was found to be effective for detecting IgG antibodies, whereas complement-dependent immune-damage assay favored the expression of IgM antibodies. Three glycosphingolipids having the same non-reducing terminal N-acetylgalactosamine residues were discriminated with IgG antibodies, while IgM antibodies showed cross-reactivity. Specifically purified anti-Forssman and anti-asialo GM2 antibodies reacted only with Forssman glycolipid and asialo GM2, respectively, but purified anti-globoside I antibodies cross-reacted with asialo GM2.
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  • Koichi TAMOTO, Jiro KOYAMA
    1980 Volume 87 Issue 6 Pages 1649-1657
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    Guinea pig peritoneal macrophages produced superoxide anions (O2-) when reacted with ovalbumin complexes of homologous IgGI and IgG 2 antibodies. In this reaction, IgG 2 complexes were about three times as active as IgG 1 complexes. But the susceptibility of IgG 1 complexes to phagocytosis by the cells appeared to be indistinguishable from that of IgG 2 complexes. The avidity of IgG 1 complexes in the antigen excess zone for Fc receptors on the cells was lower than that of the IgG 2 counterparts. The amount of IgG 1 complex bound to the cells, however, did not significantly differ from that of IgG 2 complex when compared using each complex at the equivalence zone which showed maximal effector functions on the cells. The binding of Clq to IgG 2 complexes increased markedly the amounts of complexes bound to the cells, but it reduced O2- generation. These results suggest that the difference in abilities of IgG 1 and IgG 2 complexes to promote O2- generation may be caused by different structures of the Fe parts or their antigen complexes involved in priming macrophages for O2- generation.
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  • Tatsuo SENSHU, Fusako YAMADA
    1980 Volume 87 Issue 6 Pages 1659-1668
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    The involvement of cytoplasmic soluble fraction in the assembly of nucleosomes was investigated. MH-134 SC cells were pulse-labeled with [3H] lysine just after the addition of hydroxyurea. This is known to cause accumulation of newly synthesized H 3 and H 4 histones in the cytoplasmic soluble fraction (Senshu & Ohashi (1979) J. Biochem. 86, 1259-1267). The soluble fraction was fractionated by sucrose density gradient centrifugation. Gel electrophoresis of acid-soluble proteins extracted from the fractions obtained showed that newly synthesized H 3 and H 4 histones were present as complexes that sedimented faster than their tetrameric aggregates. After incubation of the soluble fraction with mononucleosome DNA, some of the histones sedimented at a rate comparable to that of native mononucleosomes. In a simplified in vitro system, 14C-labeled mononucleosome DNA and 3H-labeled histones were mixed into the cytoplasmic soluble fraction derived from normal cells under near physiological conditions. H 3 and H 4 histones added to the soluble fraction formed complexes resembling those found in hydroxyurea-treated cells as determined by sucrose density gradient analyses. After incubation with [14C] DNA, the labeled histones and DNA co-sedimented as a broad peak at the region of native mononucleosomes. They sedimented as a sharper peak when unlabeled H 2 A and H 2 B histones were included in the incubation mixture. Not only the mixture containing all four histones, but also one devoid of H 2 A and H 2 B histones yielded DNase I digestion products resembling those derived from native nucleosomes. Omission of the soluble fraction resulted in poor formation of nucleosome-like materials. When partially purified complexes of [14C] DNA-(H 3-H 4) histones were incubated with [3H] (H 2 A-H 2 B) histones, the labeled histones co-sedimented with the preformed complexes.
    These data suggest a sequential mode of assembly of nucleosome-like particles in our unique in vitro system. It appears to proceed as follows: (1) formation of soluble complexes between H 3, H 4 histones and cytoplasmic soluble materials, (2) transfer of H 3, H 4 histones from the complexes to mononucleosome DNA to form primary nucleosome-like structures, (3) binding of H 2 A, H 2 B histones without any requirement for the involvement of cytoplasmic materials.
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  • I. Partial Purification and Some Properties
    Toshikatsu NAKABAYASHI, Hiroh IKEZAWA
    1980 Volume 87 Issue 6 Pages 1669-1680
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    Acid ATPase [ATP phosphohydrolase, EC 3. 6. 1. 3] of chicken liver lysosomes was purified from the 100, 000×g supernatant of lysosomal extract, by precipitation with acetone, column chromatographies on DEAE-cellulose and Sephadex G-150, and isoelectric focusing, with an overall recovery of 4%. and 70-fold increase in specific activity. The purified enzyme preparation showed a major band and two additional minor components on sodium dodecyl sulphate (SDS)-polyacrylamide disc gel electrophoresis.
    The enzyme thus purified had a molecular weight of about 55, 000 according to gel filtration on Sephadex G-150, or about 60, 000 according to SDS-polyacrylamide gel electrophoresis. On isoelectric focusing, two enzyme forms having isoelectric point (pI) of 5.08 and 5.18 appeared. The purified enzyme hydrolyzed not only ATP but also bis (p-nitrophenyl) phosphate. According to the distribution patterns of DEAE-cellulose and Sephadex G-150, the same enzyme hydrolyzed both ATP and bis (p-nitrophenyl) phosphate. The optimal pH of ATP hydrolysis was 5.4, whereas that of bis(p-nitrophenyl) phosphate was 7.0.
    The enzyme hydrolyzed nucleoside triphosphates, inorganic pyrophosphate and bis(p-nitrophenyl) phosphate, but did not act on nucleoside monophosphates, phosphomonoesters and sulphate monoesters. ATP was the best substrate of all compounds tested, although the value of Km was comparable to those for other nucleoside triphosphates. The products of ATP hydrolysis were ADP and inorganic phosphate. Subsequent hydrolysis of ADP to AMP was not detected by Avicel SF-cellulose thin-layer chromatography.
    ATPase activity was activated by reducing agents such as ascorbic acid, but SH blocking agents, Hg2+, p-chloromercuriphenyl sulphonic acid and PCMB were inhibitory to enzyme activity. Na+, K+, ouabain and oligomycin, which is known to affect the activities of ATPases in plasma membrane and mitochondria, were without effect.
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  • III. Hydrophobic Activators in Dipeptide Hydrolysis
    Kazufumi KURODA, Hiroshi AKANUMA, Yoshikazu SUKENAGA, Hidemitsu SUGIHA ...
    1980 Volume 87 Issue 6 Pages 1681-1689
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    Several hydrophobic compounds acted as activators in dipeptide (Bz-Gly-L-Arg-OH, Z-Gly-L-Phe-OH) hydrolysis by bovine carboxypeptidase B. These hydrophobic compounds include Bz-Gly-OH, Z-Gly-OH, Z-L-Phe-OH, and Z-L-Phe-Gly-OH. These compounds were indicated to bind to the secondary substrate binding site which is proposed to be responsible for substrate activation kinetics in dipeptide hydrolysis. Of the compounds Z-L-Phe-OH alone acted also as an inhibitor at higher concentrations, indicating that it binds to both primary and secondary sites as the dipeptide substrates do. Comparison of the activation effects of the compounds employed indicated that hydrophobic interaction played an important role in binding to the secondary site. Substrate and modifier binding constants were also determined and the results indicated that modifier binding increased both affinity and catalytic rate constant of the primary site. On the other hand, Z-Gly-OH and Z-L-Phe-Gly-OH inhibited the hydrolyses of tri and tetrapeptide substrates. This observation suggests that the secondary site is contained in the extended active center which the enzyme possibly has.
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  • IV. Oligopeptide Substrates and Extended Active Center
    Yoshikazu SUKENAGA, Hiroshi AKANUMA, Makoto YAMASAKI
    1980 Volume 87 Issue 6 Pages 1691-1701
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    Catalytic and binding properties of bovine carboxypeptidase B were studied by kinetic and affinity chromatographic methods both using several oligopeptides as substrates or immobilized ligands. These oligopeptides contained either arginines or phenylalanines at carboxy termini as well as phenylalanyl residues in one of the other positions. The chromatographic studies showed that the phenylalanyl residues in endo-positions play a significant role in binding of the immobilized peptides to the enzyme, while the kinetic studies indicated further that the presence of an internal hydrophobic residue in a substrate was advantageous for the catalytic release of the carboxyl terminal residue from the substrate. These observations support the supposition that the enzyme has an extended active center which contains an extended hydrophobic binding site. Several hydrophobic compounds, which have been shown to act as activators in dipeptide substrate hydrolyses, showed inhibitory effect on hydrolyses of oligopeptide substrates. This observation suggests that these hydrophobic compounds bind to a portion of the hydrophobic site in the active center.
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  • A Spin Label Study
    Takeo SUDA, Nobuji MAEDA, Takeshi SHIGA
    1980 Volume 87 Issue 6 Pages 1703-1713
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    The effect of cholesterol on the membrane fluidity of human erythrocytes has been studied by electron spin resonance (ESR) spectroscopy, sensing the motion of androstane and fatty acid spin labeles in the cell membrane and in vesicles made from extracted phospholipids.
    1. Androstane spin label (ASL) was incorporated from ASL-containing phospholipid vesicles into the erythrocyte membrane, essentially by a partition mechanism in proportion to their phospholipid contents.
    2. On increasing the cholesterol or ASL content in the cell membrane, the spin label was gradually immobilized.
    3. ASL motion in the cell membrane seemed to be primarily determined by the cholesterol/phospholipid molar ratio, regardless of the membrane protein-lipid interaction, as judged from the temperature effects on the ESR spectra of both membranes.
    4. However, glutaraldehyde pretreatment induced considerable changes of the cholesterollipid interaction in the cell membrane, i.e., strong immobilization and cluster formation of ASL were observed.
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  • Toshitsugu YUBISUI, Masazumi TAKESHITA, Yoshimasa YONEYAMA
    1980 Volume 87 Issue 6 Pages 1715-1720
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    The reduction of methemoglobin by NADPH-flavin reductase of human erythrocytes through flavin was studied under various conditions using a reconstituted methemoglobin reductase system. The reduction of methemoglobin by the reconstituted enzyme system could be easily detected with flavin at the physiological concentration (e.g., 0.1-1.0 μM), and the rates obtained with 0.1 and 1.0 μM FMN were 0.19 and 2.2 nmol heme reduced per min per ml, respectively, in the absence of oxygen. FMN was more effective than FAD in reduction by the reconstituted enzyme system, and oxygen decreased the rate of the reduction. The reduction of methemoglobin by the reconstituted enzyme system with flavin at a physiological concentration proceeded as a zero order reaction.
    These results apparently suggest that the NADPH-flavin reductase system is able to reduce methemoglogin in erythrocytes at a moderate speed with about 1 μM flavin, and the reduction was estimated to vary from less than 1% to about 20% of that by the NADH-cytochrome b5 reductase system with 1 μM cytochrome b5, depending on the uptake of flavin by human erythrocytes.
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  • Junshi SAKAMOTO, Yuji TONOMURA
    1980 Volume 87 Issue 6 Pages 1721-1727
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    The inhibition of Ca2+-dependent ATPase from SR [EC 3. 6. 1. 3] by ADP was of mixed type under both low Ca2+ and high Mg2+ concentrations and high Ca2+ and low Mg2+ concentrations. On the other hand, the inhibition of Na+, K+-dependent ATPase [EC 3. 6. 1. 3] by ADP was of competitive type in the presence of low and high K+ concentrations. These results suggest that ADP is released before P1 from the phosphoenzyme with bound ADP (EADPP) in the case of Ca2+-ATPase, but that P1 is released before ADP in the case of Na+, K+-ATPase.
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  • Ryoji TOKUOKA, Nobuo OKABE, Ken-ichi TOMITA
    1980 Volume 87 Issue 6 Pages 1729-1734
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    The Cotton effects induced by the interaction of thyroxine (T4) with human serum albumin (HSA) have been examined at various molar ratios of T4 to HSA. The results suggested the following three distinct modes of interaction, depending on the molar ratio (n).
    1. The first mode of interaction appeared at n=0-1. A large difference CD amplitude attributable to T4 was observed in the region between 280 nm and 325 nm. This suggests that the T4 molecule binds tightly to a highest affinity site of HSA. The difference CD spectra of HSA in the presence and absence of T4 bound at the highest affinity site showed that the peak at 292 nm might be induced by an intrinsic dissymmetry owing to an asymmetric carbon atom in the T4 molecule, while a CD band at 325 nm might be due to perturbation of the β-ring chromophore of T4 by certain intrinsic loci of HSA at the binding site.
    2. As the second mode of interaction at n=1_??_5-8, T4 molecules may bind loosely to HSA with various conformations, because the difference CD amplitude at 325 nm was decreased.
    3. The third mode was detected at n⟩5-8. The difference CD patterns is quite distinct from the others and is very similar to the CD band of T4: L-lysine mixture.
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  • Takashi OSUMI, Takashi HASHIMOTO, Nobuo UI
    1980 Volume 87 Issue 6 Pages 1735-1746
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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  • Wataru SHINKAI, Toshiharu HASE, Tatsuhiko YAGI, Hiroshi MATSUBARA
    1980 Volume 87 Issue 6 Pages 1747-1756
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
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    The complete amino acid sequence of a tetrahemoprotein, cytochrome c3 isolated from Desulfovibrio vulgaris, Miyazaki, was determined to be Ala-Pro-Lys-Ala-Pro-Ala-Asp-Gly-Leu-Lys -Met -Asp-Lys-Thr-Lys-Gln-Pro-Val-Val-Phe-Asn-His-Ser-Thr-His-Lys-Ala-Val-Lys-Cys-Gly-Asp-Cys-His-His-Pro-Val-Asn-Gly-Lys-GI u-Asn-Tyr-Gln-Lys-Cys-Ala-Thr-Ala-Gly-Cys-His-Asp-Asn-Met-Asp-Lys-Lys-Asp-Lys-Ser-Ala-Lys-Gly-Tyr-Tyr-His-Ala-Met-His-Asp-Lys-Gly-Thr-Lys-Phe-Lys-Ser-Cys-Val-Gly-Cys-His-Leu-Gl u-Thr-Ala-Gly-Ala-Asp-Ala-Ala-Lys-Lys-Lys-Glu-Leu-Thr-Gly-Cys-Lys-Gly-Ser-Lys-Cys-His-Ser. The highest homology was found between the sequence of cytochrome c3 of D. vulgaris, Miyazaki, and that of D. vulgaris, Hildenborough, on comparison among various cytochrome c3's. These two consist of 107 amino acid residues and they differ by 14 residues.
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  • Yoki MORI, Emiko UEDA, Tsugio TAKEUCHI, Sachiko TANIUCHI, Jiro KOYAMA
    1980 Volume 87 Issue 6 Pages 1757-1763
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    When rabbit C_??_ purified by affinity chromatography on IgG-Sepharose 6 B was chromatographed on DEAE-cellulose in the presence of ethylenediaminetetraacetate, C_??_s was isolated as two forms, C_??_s (I) and C_??_s (II), having different molecular weights. On the other hand, incubation of the C_??_ with soybean trypsin inhibitor before the chromatography resulted in the isolation of C_??_s (I) alone, indicating that, during the purification, C_??_s (II) was derived from C_??_s (I) by proteolytic cleavage of C_??_s (I) by a contaminating protease, probably plasmin [EC 3. 4. 21. 7]. In fact, C_??_s (I) was completely converted to C_??_s (II) or a C_??_s (II)-like fragment by highly purified plasmin. Analysis of the polypeptide chain structures revealed that C_??_s (I), which consisted of H and L chains with molecular weights of 70, 000 and 36, 000, respectively, was converted to C_??_s (II) by cleavage of the H chain, since C_??_s (II) consisted of two chains each with a molecular weight of 37, 000. This conversion proceeded without any alteration in C_??_ esterase activity, but was accompanied by loss of the ability to form C_??_r-C_??_s complex.
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  • Hironobu SUNADA, Yutaka NAGAI
    1980 Volume 87 Issue 6 Pages 1765-1771
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    A rapid micro-assay method for gelatinolytic activity has been developed using 3H-labeled heat-denatured polymeric collagen (gelatin) as a substrate to investigate enzymes involved in the post-collagenase catabolism of collagen. The method is based on the incubation of gelatin with enzyme followed by determination of the enzyme digestion products soluble in 67% dioxane.
    It is sensitive enough to detect microgram levels of gelatin fragments, and can be employed over wide ranges of pH and ionic strength. By applying the method to an embryonic chick skin culture system, three gelatinolytic enzyme fractions which showed high, limited and no caseinolytic activities were demonstrated to be separable by gel chromatography.
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  • Akitaka SHIBUYA, Shigeo HORIE
    1980 Volume 87 Issue 6 Pages 1773-1784
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    Making use of the difference in oxygen affinity between sulfite oxidase [EC 1. 8. 3. 1] and the mitochondrial sulfite oxidase system, the composition of the sulfite oxidase system of rat and mouse liver mitochondria was studied polarographically.
    Cyanide showed a profound inhibition of sulfite oxidation by mitochondria at lower oxygen concentrations, but did not show any inhibition at higher oxygen concentrations. Sonicated mitochondria and also a crude supernatant fraction from the sonicated mitochondria showed relatively high sulfite oxidation activity at higher oxygen concentrations but the activity diminished as the oxygen concentration decreased. The addition of cytochrome c caused a marked increase in the reaction velocity at lower oxygen concentrations. By gel filtration of a concentrate of the crude supernatant, a finely dispersed particulate fraction containing cytochrome oxidase [EC 1. 9. 3. 1] and a soluble fraction containing sulfite oxidase were separated. Both of these fractions and also cytochrome c were necessary for the reconstitution of the system with high affinity for oxygen. A high oxygen affinity system was also reconstituted in the presence of catalase [EC 1. 11. 1. 6] with cytochrome oxidase, cytochrome c, and a more highly purified preparation of sulfite oxidase. Catalase exerted a protective effect on this system against inactivation during catalysis.
    Carefully prepared phosphorylation-coupled mitochondria often, but not always, showed respiratory control when sulfite was used as a substrate. The respiratory control rate observed was about the same as that of ascorbate-tetramethyl-p-phenylene diamine (TMPD), a known electron donor system for cytochrome c. Experimental results suggested that the reduction of cytochrome c was the rate-limiting or nearly rate-limiting step in oxidative phosphorylation of the sulfite oxidase system.
    These results provide further support and more direct evidence for the recent view that, in intact mitochondria, electrons are transferred from sulfite oxidase to oxygen via cytochrome c and cytochrome oxidase.
    Sulfite inhibited the state 3 oxidation of glutamate by mitochondira, but not those of succinate and malate.
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  • Separation and Characterization of Monomer Subunits and Studies on Sulfhydryl Groups
    Takashi TAKAGI, Takayuki NEMOTO
    1980 Volume 87 Issue 6 Pages 1785-1793
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    Hemocyanin monomers were isolated from Tachypleus tridentatus hemolymph by gel chromatography on Sephadex G-100. The isolated monomers were separated into four fractions by DEAE-Sephadex chromatography; they comprised 6 different subunits, designated as a-ξ chains. All of them showed the same molecular weight, 70, 000, on SDS-gel electrophoresis. The ξ chain was eluted ahead of the other subunits during gel chromatography. It tended to dimerize during prolonged dialysis or purification procedures. The a and ξ chains were isolated in pure form. The γ and δ chains, and also the β and ε chains, were obtained as mixtures but were not separated from each other. All the subunits showed different antigenicities. The amino-terminal portions of the α through ε chains have the same sequence, Thr-Ile•Leu-Lys-Glu-Lys-Gln. The ξ chain has a different amino-terminal sequence, Val-Leu-Asp-X-Ile/Leu-Glu-Lys. The ξ chain contained only 1 mol of Cu/mol of protein, whereas the other chains contained 2 mol of Cu/mol of protein. No free sulfhydryl group was detected in the absence of guanidine. However, 3 mol of SH/mol of protein was detected immediately after the addition of 6M guanidine-HCI. These SH groups were very unstable and disappeared on standing.
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  • Osamu OHARA, Sho TAKAHASHI, Tatsuo OOI
    1980 Volume 87 Issue 6 Pages 1795-1803
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    The cross-linking reaction of α-tropomyosin with dimethyl adipimidate yielded a dimer of the α-subunit of tropomyosin as a major product, which was isolated by gel filtration on Sephadex G-150 in the presence of urea. Amino acid analyses revealed that the cross-linked α-tropomyosin contained about two adipimidate cross-links per molecule. Selective cleavage of the cross-linked molecule at the cysteinyl residue, Cys 190 (a single cysteinyl residue in the α-subunit), gave a new band at a position corresponding to a molecular weight of 48, 000 on SDS-gel electrophoresis, suggesting that the cross-links were incorporated in the N-terminal fragment. When the cross-linked molecule was cleaved with CNBr, two large fragments from residue 11 to 127, and from 142 to 281, were obtained, as in the case of the intact molecule. Therefore, it is inferred that the location of the intersubunit cross-links is in the region from residue 2 to 8 and/or from 128 to 141. These results indicate that the arrangement of α-subunits of tropomyosin in solution must be in parallel and in register. Although the exact positions of the reactive sites could not be determined in the present study, stereochemical examination of the coiled-coil model suggests that the most probable sites of cross-linking are Lys 5 of one subunit and Lys 7 of the other.
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  • Hiroshi NAKATANI, Keitaro HIROMI
    1980 Volume 87 Issue 6 Pages 1805-1810
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    Signal amplitudes of reaction curves obtainable by the stopped-flow method depend on the reaction mechanism as well as the optical properties of reactants, products and transient intermediates. Equations were derived for the concentration dependence of signal amplitudes expressed as absorbance changes for three typical reaction mechanisms which appear frequently in enzyme-ligand interactions. These relationships can be conveniently used to monitor experimentally observed signal amplitudes and characterize the optical properties of the transient intermediates. A useful method for accurately determining the dead-time of a stopped-flow apparatus is also described.
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  • Hisao FUJISAKI, Hiroshi ASAI
    1980 Volume 87 Issue 6 Pages 1811-1820
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    We have established a method to estimate the values of various kinetic parameters of actoheavy meromyosin (acto-HMM) ATPase, using a fluorescent ATP analog, β-naphthyl triphosphate (β-NapP3); from the fluorescence intensity change accompanying β-NapP3 hydrolysis, the various kinetic parameters of β-NapP3 hydrolysis, including its product inhibition, were obtained. β-NapP3 hydrolysis is inhibited competitively by ATP, resulting in different time courses of fluorescence intensity change in the presence and absence of ATP. From this difference, the values of kinetic parameters of ATP hydrolysis, including its product inhibition, can be estimated. By extending this method to the acto-HMM system, seventeen parameters in a reaction scheme for the concurrent hydrolysis of ATP and β-NapP3, including association constants between F-actin and substrate-free or substrate-bound HMM, were obtained. The kinetic parameters estimated for ATP hydrolysis were in good agreement with those in the literature.
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  • Michihiro TAKAMA, Yoshiaki NOSOH
    1980 Volume 87 Issue 6 Pages 1821-1827
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    6-Phosphoglucose isomerase [EC 5. 3. 1. 9] was purified from Bacillus caldotenax. The isomerase shared many common properties with the isomerase from B. stearotherinophilus, i.e., pH and temperature optima, thermostability, competitive inhibition by 6-phosphogluconate and P1, and amino acid composition. The enzyme activity of the former, however, was lower than that of the latter. The molecular weight of the B. caldotenax isomerase was estimated to be 202, 000-204, 000 by gel filtration and electrophoresis of the enzyme cross-linked with dimethyl adipimidate (DMA). The enzyme was shown to consist of four subunits of equal molecular weight (50, 600), and the four subunits were concluded to be identical based on the results of dansylation and cyanogen bromide cleavage of the enzyme. The interaction between the subunits were shown to be isologous by cross-linking with DMA.
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  • Anti-Galactocerebroside Antibodies
    Tsutomu UCHIDA, Yoshitaka NAGAI
    1980 Volume 87 Issue 6 Pages 1829-1841
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    Galactocerebroside was derivatized for use as the ligand for affinity chromatography in a high yield. The derivatization was performed by ozonolysis of the double bond of the sphingosine moiety in Baeyer-Villiger solvent, pinacolone, and by the following oxidation. The carboxy group of the derivative was coupled to amino-alkyl glass beads with N', N'-carbonyldiimidazole as the condensation reagent and the affinity adsorbent coupled with 2-hydroxy-3-N-acylamido-4-O-β-galactosyl butyric acid was obtained. The antibodies tightly bound to the adsorbent could be eluted with a weak chaotropic reagent, 1.0M NaI, and successively with a stronger chaotrope, 3.0M NaSCN. The antibody eluted with 3.0M NaSCN was characterized by complement fixation assay using the liposome system. The antibody showed strong affinity for galactocerebroside and a weak cross-reaction with galactosyl (β1→4) glucosyl ceramide (CDH), but no reactions were observed with the other structurally related glycosphingolipids such as glucocerebroside, galactocerebroside-3´-sulfate (CSE), galactosyl (α1→4) galactosyl (β1→4) glucosyl ceramide (CTH) and galactosyl (β1→3) N-acetylgalactosaminyl (β1→4) N-acetylneuraminyl (α2→3) lgalactosyl (β1→4) glucosyl ceramide (GM1 ganglioside) or with lecithin-cholesterol, suggesting that the antibody recognizes not only the galactose moiety but also the β-anomeric configuration and possibly the polar ceramide portion of galactocerebroside. Lecithin and cholesterol as auxiliary lipids were essential for the complement fixation reaction, where the concentration of cholesterol in the liposome was particularly important. The optimal ratio for the maximal reactions among compositional lipids of the antigen liposome was limited to a narrow range. The reactivity of the purified anti-galactocerebroside antibody showed that this antibody could be classified as the type X antibody that Rapport, Cavanna and Graf had reported.
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  • Significance of Lipid Matrix
    Tsutomu UCHIDA, Yoshitaka NAGAI
    1980 Volume 87 Issue 6 Pages 1843-1849
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    The significance of the lipid matrix in the reaction of liposomal antigen, antibody and complement (Ag-Ab-C) was analyzed using purified anti-galactocerebroside antibody and synthetic lipids and the following results were obtained.
    1. For the optimal Ag-Ab-C reaction it was necessary that galactocerebroside (gal•CMH) and lecithin molecules were well dispersed by virtue of cholesterol (Chol) and that the molar ratio of cholesterol to the sum of galactocerebroside and lecithin was more than one.
    2. The Ag-Ab-C reactivity changed depending upon the chain length of lecithin, and the maximal reaction was observed in the case of dilauroylphosphatidylcholine. When the fatty acyl chain of lecithin was either shorter or longer than that of dilauroylphosphatidylcholine, the reactivity was reduced.
    3. The Ag-Ab-C reactivity was increased by elongation of the fatty acyl chain of galactocerebroside and an abrupt change was found to be around the carbon number 8 to 10 of the fatty acyl chain.
    4. The Ag-Ab-C reactivity was elevated by the increase in unsaturation of fatty acyl moiety.
    5. There is a tendency that an increase in the charge of the lipid matrix leads to the reduction of the Ag-Ab-C reactivity.
    6. The results suggest that the physicochemical properties of lipids and especially the lipid-lipid interaction in the hydrophobic region of the lipid matrix play an important role in the Ag-Ab-C reaction.
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  • Masatoshi MAKI, Masaaki HIROSE, Hideo CHIBA
    1980 Volume 87 Issue 6 Pages 1851-1854
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    Glucocorticoid receptor activities in cytosol from lactating rat mammary glands were greatly stabilized by molybdate against inactivation during incubation at 25°C. The sedimentation coefficient was 9-10 S in the presence of 10mM Na2MoO4, but 6-7 S in the absence of the ion. These data strongly suggest that alterations in receptor conformation induced by direct interaction with molybdate are involved in the stabilization process.
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  • Hidemi ISHII, Shuichi HORIE, Tetsuya SUGA
    1980 Volume 87 Issue 6 Pages 1855-1858
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    In the livers of fasted rats, the activity of peroxisomal palmitoyl-CoA oxidation (NADH production) was increased more rapidly and markedly than that of mitochondrial carnitine palmitoyltransferase, which is the rate limiting enzyme of mitochondrial β-oxidation. The peroxisomal oxidizing activity was about twice that of the control throughout the period of fasting (1-7 days). Carnitine acetyltransferase activity was increased to a similar extent in both peroxisomes and mitochondria. A possible physiological role of liver peroxisomes may thus be as an effective supply of NADH2, acetyl residues and short and medium-length fatty acyl-CoA in the cells on the enhancement of peroxisomal β-oxidation of the animals under starvation; these substances thus produced may be transported into the mitochondria as energy sources.
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  • Susumu ANDO, Kazuo KON, Yasukazu TANAKA, Sumi NAGASE, Yoshitaka NAGAI
    1980 Volume 87 Issue 6 Pages 1859-1862
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    The mutant analbuminemia rat (NAR) was recently reported by Nagase (1) to spontaneously develop hypercholesterolemia. A detailed lipid analysis was carried out to characterize the hyperlipidemia. The concentrations of major phospholipids as well as cholesterol were increased in plasma, whereas the triacylglycerol level remained unchanged. Free fatty acid and lysophosphatidylcholine levels, on the contrary, declined. The liver lipids were not accumulated, but appeared to be depleted.
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  • Ichiro HARA, Mitsuyo OKAZAKI, Yoshimi OHNO
    1980 Volume 87 Issue 6 Pages 1863-1865
    Published: June 01, 1980
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
    A simple and rapid method for the determination of cholesterol in human serum lipoproteins (VLDL, LDL, HDL2, and HDL3) was described.
    The contents of cholesterol of lipoprotein fractions from a very small amount of serum (20 μl) can be determined by the enzymatic assay after separation by high performance liquid chromatography in less than 50min.
    The method is very useful for studying lipoprotein metabolism and related diseases.
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  • 1980 Volume 87 Issue 6 Pages 1867
    Published: 1980
    Released on J-STAGE: November 18, 2008
    JOURNAL FREE ACCESS
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