The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
99 巻, 1 号
選択された号の論文の38件中1~38を表示しています
  • Yasuo MUKOHATA, Masaharu ISOYAMA, Ayumi FUKE
    1986 年 99 巻 1 号 p. 1-8
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    Cell envelope vesicles active in ATP synthesis were prepared from Halobacterium halobium cells, which genetically lack bacteriorhodopsin, by sonication in the presence of substrates. ATP was synthesized when vesicles were illuminated to build up membrane potential through the action of halorhodopsin. The threshold value of membrane potential for ATP synthesis was about −100 mV relative to the external medium, i.e., inside-negative.
    ATP synthesis also occurred in the dark upon acidification of the external medium of a suspension of cell envelope vesicles. This base-acid transition ATP synthesis took place when the pH difference was greater than 1.6 units.
    The threshold pH difference was lowered when the base-acid transition was carried out under dim light which induced a membrane potential of about −100 mV.
    Regardless of the sort of driving force, ATP synthesis was optimum at the intravesicular pH of around 6.5 and almost nil at 8, where ATP syntheses by F0F1 type ATPases in other organisms are most active. The synthesis could be inhibited by N, N'-dicyclohexylcarbodiimide (DCCD) with a half-maximum inhibition at around 25 μM/2mg protein/ml.
    These results strongly suggest that in halobacteria a DCCD-sensitive H+-translocating ATP synthase is in operation which is driven by membrane potential and/or pH gradient, and obeys chemiosmotic energetics. The results also suggest that the ATP synthase may not be identical to F0F1 type H+-translocating ATPases found in mitochondria, chloroplasts and eubacteria.
  • Akira ITOH, Fukuichi OHSAWA, Shunji NATORI
    1986 年 99 巻 1 号 p. 9-15
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    A tumor specific cytotoxic protein produced by the murine macrophage-like cell line J774.1 in response to stimulation with Sarcophaga lectin was purified to homogeneity in three steps from the culture medium. This cytotoxin, named tumor killing factor (TKF), was a protein with a molecular weight of 15, 000, and aggregated forming an oligomer with a molecular weight of 48, 000. Its amino acid composition was similar to that of human TNF. Purified TKF had a significant effect on transplanted murine ascites tumor sarcoma 180. The biological significance of TKF in terms of ontogeny is discussed from the view point of developmental biology.
  • Torn MORITA, Yasushi NIWATA, Kazuko OHGI, Michio OGAWA, Masachika IRIE
    1986 年 99 巻 1 号 p. 17-25
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    1. In order to determine the distribution of two human urinary RNase (RNase Us and RNase UL)-like enzymes in human tissues and body fluids, enzyme immunoassay systems were established using rabbit anti-RNase sera. The sensitivity of the assay systems was of similar order to that of radioimmunoassay systems previously reported.
    2. In the enzyme immunoassay, the cross reactivities of anti-RNase UL serum towards RNase Us, bovine kidney RNase K2, bovine RNase A, and bovine seminal RNase Vs were less than 1%. The cross reactivity of anti-RNase Us-serum to-wards RNase UL was less than 0.5% and cross reactivities were minimal for RNase A, RNase K2, and RNase Vs.
    3. The RNase levels in human organs and body fluids were measured by enzyme immunoassay. In milk, semen and saliva, only RNase UL-like enzyme was found. Both RNase Us- and RNase UL-like enzymes were found in kidney, stomach, and pancreas and the RNase Us/RNase UL ratios were 0.49, 1.35, and 0.34, respectively. In lung, liver, spleen, and leukocytes, most of the RNase activity was accounted for by RNase Us-like enzyme. The activity of RNase Us-like enzyme was especially high in lung, spleen, and leukocytes.
    4. The crude extracts of several tissues and body fluids were separated by phosphocellulose column chromatography and the contents of the two urinary RNase-like enzymes were determined by enzyme immunoassay. In stomach, kidney, pancreas, and serum, both enzymes were present in multiple forms. In spleen and lung, both the major RNase (RNase Us) and minor RNase (RNase UL) existed in two forms. A single peak of RNase was found in liver and in leukocytes.
    5. The activity staining of liver, leukocytes, spleen, and lung RNases indicated that RNase Us-like enzyme in these tissues is also present in multiple forms.
  • Sumio NAKATA, Toshio EIKI, Norihiro TANAKA, Tadashi KOYAMA, Hiroto TSU ...
    1986 年 99 巻 1 号 p. 27-32
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    (i) When trinitrophenyl (TNP) myosin of either chicken breast muscle or porcine cardiac muscle was left to stand in an alkaline medium at 20°C for several hours, nitrite ions were found to be gradually produced. (ii) The nitrite production from myosin trinitrophenylated in the presence of PP1 occurred at the same rate and to the same extent as that from myosin trinitrophenylated in the absence of PP1. (iii) The nitrite production was significantly reduced when thiols of myosin were modified with 2-nitro-5-thiocyanobenzoate. iv) Four different preparations of TNP subfragment-1, S1(Aa), S1(Ab), S1(Ba), and S1(Bb), were obtained from chymotryptic digest of chicken breast myosin trinitrophenylated in the absence of PP1. When these preparations of TNP-S1 were left to stand at alkaline pH, a significant amount of nitrite was produced from S1(Ab) and S1(Bb), but very little from S1(Aa) and S1(Ba).
    In our previous report (J. Biochem. 97, 965-968, 1985), S1(Aa) and S1(Ba) were suggested to correspond to “non-burst” heads of myosin, and S1(Ab) and S1(Bb) to “burst” heads of the myosin molecule (Inoue et al. (1980) Adv. Biophys. 13, 1-194). Therefore, the present findings described above strongly suggest that the nitrite production involves some interaction of TNP groups with thiols, and that it occurs at the “burst” heads.
  • Toshio EIKI, Sumio NAKATA, Norihiro TANAKA, Shoji IKEDA, Tomohiko KANE ...
    1986 年 99 巻 1 号 p. 33-39
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    Four different preparations of skeletal subfragment-1, denoted in this report as S1(Aa), S1(Ab), Sl(Ba), and S1(Bb), and two different preparations of cardiacsubfragment-1, denoted as S1(A) and S1(B), were obtained as described in ourrecent report (J. Biochem. 97, 965, 1985).
    (i) The four preparations were obtained from chicken breast myosin trinitro phenylated with 2, 4, 6-trinitrobenzene sulfonate in the absence of inorganic pyro phosphate (-PP1), and they were all shown to be trinitrophenylated. Addition of PP1 caused change in the absorption spectra of trinitrophenyl(TNP)-S1(Aa) and TNP-S1(Ba), but not in those of TNP-S1(Ab) and TNP-S1(Bb). (ii) The two preparations of S1 were obtained from cardiac myosin trinitrophenylated either in the absence (-) or presence (+) of PP1. S1(B) was trinitrophenylated, whereas S1(A) was not. Specifically emphasized is the observation that the yield of cardiac S1(A) was practically equal to that of cardiac SI(B). On the basis of these results, we propose the hypothesis of “two iso-myosins with non-identical heads, ” whichis essentially a combination of the hypothesis of isoenzymes and that of non-identical heads.
  • Toshiaki IMAGAWA, Takahide WATANABE, Takao NAKAMURA
    1986 年 99 巻 1 号 p. 41-53
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    The phosphorylation-induced mobility shift of the high molecular weight form of phospholamban (24, 500 daltons) in the cardiac sarcoplasmic reticulum produced on 3', 5'-cyclic AMP (cAMP)-dependent phosphorylation with 5 rum ATP was resolved into five clear steps on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and on Ca 2+-calmodulin-dependent phosphorylation into ten steps. The mobility shift of the low molecular weight form of phospholamban (<14, 400 daltons) in these reactions occurred in one step and two steps, respectively. With the two protein kinase activities, the electrophoretic pattern of the mobility shifts of the high and low molecular weight forms of phospholamban was similar to that obtained with Ca2+-calmodulin-dependent protein kinase alone. The results of pulse-chase experiments involving the centrifuge column method suggested that the site(s) of phosphorylation by cAMP- and Ca2+-calmodulin-dependent protein kinase activities are on the same phospholamban molecule. Two-dimensional tryptic peptide maps of phosphorylated phospholamban indicated that cAMP-dependent protein kinase phosphorylates at a single site, A, and Ca2+-calmodulin-dependent protein kinase phosphorylates at sites Cl and C2 in the low molecular weight form, where A is different from C1 but may be the same as C2. The high molecular weight form of phospholamban is suggested to be a pentamer of identical monomers (low molecular weight form) having one phosphorylation site for cAMP-dependent protein kinase and two for Ca2+-calmodulin-dependent protein kinase.
  • Katsuko YAMASHITA, Akira HITOI, Yoshiko MATSUDA, Tsutomu MIURA, Nobuhi ...
    1986 年 99 巻 1 号 p. 55-62
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    γ-Glutamyltranspeptidase purified from human kidneys contains 4-5 asparaginelinked sugar chains in each molecule. The sugar chains were released from the polypeptide portion of the enzyme by hydrazinolysis as oligosaccharides and separated by paper electrophoresis into one neutral and two acidic fractions. By sequential exoglycosidase digestion and methylation analysis, the neutral fraction, which comprised 69% of total oligosaccharides, was shown to be a mixture of bisected bi and triantennary complex-type sugar chains with and without a fucose on the proximal N-acetylglucosamine residue and with Galβ1→4GlcNAc and/or Galβ1→4(Fucαl→3)G1cNAc groups in their outer chain moieties. The acidic oligosaccharide fractions were mixtures of mono and disialyl derivatives of bisected triantennary complex-type oligosaccharides with Galβ1 →4G1cNAc and/or Galβ1→4 (Fucα1→3) GlcNAc group in their outer chain moieties. Some of the outer chains of the acidic oligosaccharides were considered to be sialylated X-antigenic structures.
  • Hideo IWAHASHI, Akihiko IKEDA, Youichi NEGORO, Ryo KIDO
    1986 年 99 巻 1 号 p. 63-71
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    Retinoic acid 5, 6-epoxidase activity was found in several hemoproteins such as human oxy- and methemoglobin (HbO2 and MetHb), equine skeletal muscle oxy and metmyoglobin (MbO2 and MetMb), bovine liver catalase, and horseradish peroxidase. Hematin also catalyzed retinoic acid 5, 6-epoxidation. The results suggest that the heme moiety participates in the epoxidation. However, neither horse heart cytochrome c, nor free ferrous ion nor free ferric ion exhibited the epoxidase activity.
    Some hemoproteins (HbO2, MetHb, MbO2, MetMb, catalase, peroxidase, and hematin) exhibited characteristic individual pH dependences of the activity, suggesting that the epoxidase activities of the hemoproteins are influenced by the apoenzymes to some degree. This view is also supported by the finding that preincubation of an HbO2 preparation at various temperatures (37-70°C) reduced its epoxidaseactivity with increasing temperature, whereas the activity of hematin was unaffected.
    Active oxygen scavengers such as mannitol, catalase, and superoxide dismutase exhibited no effect on the epoxidase activities of HbO2, MetHb, MbO2, and MetMb. A ligand of heme, CN-(100 mM), inhibited the epoxidase activities but N3-(100 mM) did not. The epoxidase activities were completely inhibited by NADPH, NADH, and/or 2-mercaptoethanol but not by NADP+ and/or NAD+. An intermediatein the epoxidation may be reduced by NADPH, NADH and/or 2-mercaptoethanol. Radical species can be considered as plausible candidates for the intermediate.
  • Yasuo OGAWA, Masaru TANOKURA
    1986 年 99 巻 1 号 p. 73-80
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    To improve our understanding of the physiological roles of parvalbumins, PA-1 (pI 4.78) and PA-2 (pI4.97) parvalbumins were prepared from bullfrog skeletal muscle and their calcium binding properties were examined in a medium of constant ionic strength (I=0.106, pH 6.80, at 20°C) containing various concentrations of Mg2+ by using a metallo-indicator, tetramethylmurexide. Apparent binding constants for Ca2+ in the presence of Mg2+ changed in the manner expected if Ca2+ and Mg2+ compete for two independent homogeneous binding sites. The following values were obtained: for PA-1, Kcal×107 M-1, KMg=900 M-1; for PA-2, KCa= 6×106 M-1, KMg= 830 M-1 (I=0.106, pH 6.80, at 20°C). The apparent binding constants are strongly dependent on temperature: at 10°C for PA-1, KCa=2×108 M-1, KMg=104 M-1; for PA-2, KCa=5×107 M-1, KMg=5×103 M-1 (I=0.106, pH 6.80). The dependence of the affinities for Ca2+ on ionic strength is similar to or less than that of GEDTA (EGTA). The affinities for Ca2+ and Mg2+ of parvalbumins are unchanged between pH 6.5 and 7.2.
  • Yasuo OGAWA, Masaru TANOKURA
    1986 年 99 巻 1 号 p. 81-89
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    In addition to steady-state properties of calcium binding to parvalbumins, kinetic studies are required for adequate evaluation of the physiological roles of parvalbumins. By using a dual-wavelength spectrophotometer equipped with a stoppedflow accessory, the transient kinetics of calcium binding to parvalbumins (PA-1 and 2) from bullfrog skeletal muscle was examined at 20°C in medium containing 20 mM MOPS-KOH, pH 6.80, 0.13 mM tetramethylmurexide, 25μM CaCl2, metaldeprived PA-1 or PA-2, various concentrations of Mg2+, and KCl to adjust the ionic strength of the medium to 0.106. The results can be explained in terms of the following rate constants under the conditions mentioned above when a secondorder kinetic scheme is assumed. For PA-1, the association and apparent dissociation rate constants for Ca2+ are 1.5×107 M-1•s-1 and 1.5 s-1, respectively, or more. The rate constants for Mg2+ are 7, 500M-1•s-1 and 5-6s-1, respectively. For PA-2, the rate constants for Ca2+ are 7×106 M-1•s-1 and 1.16 s-1, respectively, and those for Mg2+ are 3, 500 M-1•s-1 and 3.5-4 s-1, respectively. Increased affinities for Ca2+ and Mg2+ at 10°C are largely due to decreased apparent dissociation rate constants for these divalent cations, because no significant change in the association rate constants was found.
  • Kentaro KASAI, Hiroaki HAYASHI, Koichi IWAI
    1986 年 99 巻 1 号 p. 91-98
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    The circular dichroism spectra and the thermal denaturation profiles of the nucleosome core particles isolated by micrococcal nuclease digestion from nuclei of calf thymus and the protozoan Tetrahymena pyriformis were compared with those of the homogeneous and hybrid core particles reconstituted from calf core DNA and either calf or Tetrahymena histone octamer. The core DNA was obtained from the calf core particle, and both the histone octamers were reconstituted from the acid-extracted four core histones of calf thymus or Tetrahymena, whose amino acid sequences show the largest differences hitherto known. The reconstituted homogeneous core particle was identical in both the physical properties with the isolated calf core particle, showing that the correct reconstitution was achieved. The circular dichroism spectra of the calf and Tetrahymena core particles and the hybrid core particle showed no essential differences, indicating that the three core particles have the same overall structure. The derivative thermal-denaturation profiles, however, clearly differed; the calf core particle showed two melting transitions at 60°C and 72°C, while the Tetrahymena and hybrid core particles showed the same three transitions at 48-50°C, 60-61°C, and 72°C. Thus, the thermal denaturation properties of nucleosome core particles do not reflect the nature of DNA, but rather that of the histone octamer bound to the DNA. We conclude that the Tetrahymena histories are more weakly bound to the DNA than the calf thymus histones in the same overall structure of nucleosomes.
  • Hideo HARUKI, Keizo TESHIMA, Yuji SAMEJIMA, Saju KAWAUCHI, Kiyoshi IKE ...
    1986 年 99 巻 1 号 p. 99-109
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    The pH dependence of the binding constant of Ca 2+ to a phospholipase A2 of Agkistrodon halys blomhoffii, in which the α-amino group had been selectively modified to an α-keto group, was studied at 25°C and ionic strength 0.1 by the tryptophyl fluorescence method. The dependence was compared with the results for the intact enzyme (Ikeda et al. (1981) J. Biochem. 90, 1125-1130). The pH-dependence curve could be well interpreted in terms of the participation of the two ionizable groups Asp 49 and His 48, with pK values of 4.70 and 6.69, respectively. These values were slightly different from the respective pK values for the intact enzyme, 5.15 and 6.45. Ca2+ binding to the intact enzyme involves the participation of an additional ionizable group with a pK value of 7.30, which was thus assigned as α-amino group.
    The pH dependence of the binding constant of monodispersed n-dodecylphosphorylcholine (n-C12PC) to the α-NH2-modified enzyme was studied at 25°C and ionic strength 0.1 by the aromatic circular dichroism (CD) method. The pH-dependence curve for the modified apoenzyme was interpreted as reflecting the participation of a single ionizable group with a pK value of 4.7, which was assigned to Asp 49 (to which a Ca2+ ion can coordinate) since the curve for the Ca2+ complex lacked this transition: the binding constant was independent of pH. The pH-dependence curves for the intact apoenzyme and its Ca2+ complex involve the participation of an additional ionizable group with pK values of 7.30 and 6.30, respectively (Ikeda & Samejima (1981) J. Biochem. 90, 799-804), which was assigned as the α-amino group. The hydrolysis of monodispersed 1, 2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by the intact and the α-NH-2modified enzymes was studied by the pH stat method at 25°C, pH 8.2, and ionic strength 0.1 in the presence of 3 mM Ca2+. The Km value for the modified enzyme was found to be very similar to that for the intact enzyme: this was compatible with the results of the direct binding study on the monodispersed n-C12PC under the same conditions. However, the kcat value was about 43% of the value for the intact enzyme, suggesting that the α-keto group introduced by the chemical modification perturbed the network of hydrogen bonds in the active site.
    The pH dependence of the binding constant of micellar n-hexadecylphosphorylcholine (n-C16PC) to the α-NH2-modified enzyme was studied at 25°C and ionic strength 0.1 by the tryptophyl fluorescence method and was compared with the result for the intact enzyme (Ikeda et al. (1984) J. Biochem. 96, 1427-1436). At neutral and alkaline pH values, the binding constants for the modified apoenzyme and its Ca2+ complex were smaller than the corresponding constants for the intact enzyme (about 56% and 34%, respectively, at infinitely alkaline pH values). Nevertheless, the pH-dependence curves could be interpreted in terms of the participation of Asp 49 (and His 48) or His 48 in way similar to that for the intact enzyme, indicating no significant participation of the ionization of the α-amino group in the binding of micellar substrates: this contrasts with the result on the binding of the monodispersed substrates. In this connection, it was found that the enzymatic activity of the α-NH2-modified enzyme toward egg-yolk suspensions was much smaller than that for the intact enzyme (only about 14.5%). The α-keto groups, introduced by the chemical modification, perturbed the network of hydrogen bonds in the active site and changed not only the kcat value but also the Km value.
  • Kinuko KIMURA, Kouji MATSUOKA, Hidesaburo KOBAYASHI
    1986 年 99 巻 1 号 p. 111-117
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    Glutamine synthetase from a marine enterobacterium, Photobacterium phosphoreum, was purified to homogeneity from cells grown in glycerol-yeast extract medium. The purified enzyme had a molecular weight of approximately 670, 000 and a subunit size of 56, 000, i.e. larger than that of the enzyme from E. coli. Regulation of the glutamine synthetase activity by adenylylation/deadenylylation was demonstrated on snake venom phosphodiesterase treatment. The state of adenylylation appeared to influence both the biosynthetic and γ-glutamyltransferase activities of P. phosphoreum glutamine synthetase similar to in the case of the E. coli enzyme. The enzyme activity was controlled by adenylylation and possibly in combination with feedback inhibition by alanine, serine, and glycine, metabolites which are especially effective in inhibiting P. phosphoreum glutamine synthetase.
    When either Mn2+ or Mg2+ was added to the relaxed (divalent cation-free) enzyme, similar UV-difference spectra were obtained for the enzyme, indicating that the conformational states induced by these cations were also similar. The profile of these spectra varied from those published for E. coli, and three peaks were found at 282.5, 288.5, and 298 nm.
  • Junko SOKAWA, Yoshihiro SOKAWA
    1986 年 99 巻 1 号 p. 119-124
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    The appearance and induction of (2'-5') oligoadenylate synthetase (2-5A synthetase) in chicken embryo erythrocytes during development, and the activity and molecular size of this enzyme in immature red blood cells from anemic chickens were studied. Enzyme activity first appeared in the embryos on the 15th day of incubation, a marked increase being seen 1 or 2 days after hatching. In erythrocytes from early embryos without 2-5A synthetase activity, chicken interferon (5 IU/ml at most) induced the production of a large amount of the enzyme. In immature red blood cells from anemic chickens, only a small amount of 2-5A synthetase was detected in the nuclear fraction. The cytoplasmic fraction contained the smaller enzyme (about 45 kilodaltons), but the larger enzyme (85-120 kilodaltons) was scarcely detected in either fraction. The larger enzyme may be synthesized during the maturation of red blood cells.
  • Hiroyuki ARAI, Keizo INOUE, Kiyotaka NISHIKAWA, Yoshiko BANNO, Yoshino ...
    1986 年 99 巻 1 号 p. 125-133
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    Phospholipase activity in the lysosomes of the protozoan Tetrahymena pyriformis strain NT-1 was studied using phospholipids radioactively labeled in the fatty acid moieties. Lysosomal homogenates showed high phospholipase activity with an acidic pH optimum. Unlike the phospholipases in rat liver lysosomes, almost all activity was recovered from the membranous fraction of the lysosomes. The activity was partially solubilized by treatment of the membranes with a detergent or trypsin. Using specifically labeled phospholipids revealed that phospholipase A1 and C are predominant in Tetrahymena lysosomes, no appreciable phospholipase A2 or lysophospholipase activity was detected in the fraction. There are two catabolic pathways of the hydrolysis of phospholipid: 1) Hydrolysis is initiated by deacylation at the 1-position by phospholipase A1 and the 2-acyllysophospholipid thus formed is successively attacked by (lyso)phospholipase C; 2) hydrolysis is initiated by cleavage of phosphodiester by phospholipase C and the diacylglycerol thus formed is attacked by lipase. Both pathways give the same end products, free fatty acid and 2-monoacylglycerol. The former pathway might be predominant in Tetrahymena lysosomes under physiological conditions since the pathway is independent of detergent.
    Phospholipases A1 and C activities were partially released into the medium. At least two different phospholipases C are present in the medium as judged by chromatographic behavior and their substrate specificities.
  • Shigeo OHTA, Yasuo KAGAWA
    1986 年 99 巻 1 号 p. 135-141
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    F1-ATPase is the major enzyme for ATP synthesis, and its β subunit is the catalytic site. To date, no full-length cDNA for the eukaryotic F1 gene has been reported. Human F1 was studied because of its importance in medicine and cell biology. Here we report molecular cloning of a full-length cDNA for the human F1β subunit and purification of the human F1β subunit. The HeLa cell cDNA library constructed in an expression vector λgt11 was screened with antiserum against the yeast F1β subunit. One of the positive phage DNAs containing the human F1β gene and its flanking regions (1.8 kilobase pairs) was sequenced by the dideoxy chain termination method. The open reading frame started from a putative signal presequence, which was rich in both serine and arginine. There was a homologous segment in the signal presequence of human ornithine transcarbamoylase and that of F1β. The precursor of F1β was expressed in E. coli harboring a plasmid which had been constructed with T5 promotor and the F1β cDNA. Both the precursor and mature form of F1β were detected in HeLa cells in a pulse-chase experiment. The amino acid sequence of 480 residues (51, 568.3 daltons) following the presequence was highly homologous with that of mature beef heart F1β (97.5%) and E. coli Flβ (71.7 %), but the codon usage in the human gene was very different from those of reported genes coding for F1β of other species.
  • Ashraf IMAM
    1986 年 99 巻 1 号 p. 143-152
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    The proteins from human milk-fat-globule-membrane were radioiodinated, solubilized and analyzed by SDS-polyacrylamide gel electrophoresis. The solubilized milk-fat-globule-membrane preparations contained six major size classes of components with apparent molecular weights of 155, 70, 58, 52, 42, and 39 kilodaltons. The membrane proteins were significantly more accessible to lactoperoxidase-125I in isolated membrane compared with that of whole cream. Major proteins of apparent molecular weights of 155, 70, 58, 52, 42, and 39 kilodaltons were labeled in whole cream and were extracted from the fat-globules membrane with magnesium chloride. Residual cream (after being extracted with MgCl2) showed the loss of the above proteins components. Using an indirect immunoperoxidase staining method and the antibodies to MFGM which immunoprecipitated all the six major glycoprotein components of MFGM, demonstrated their presence on the apical plasma membrane of mammary epithelial cells lining the breast duct in tissue sections. The asymmetric arrangements of proteins in the human milk-fat-globule-membranes, after secretion, is discussed.
  • Hiroshi NAKASHIMA, Ken NISHIKAWA, Tatsuo OOI
    1986 年 99 巻 1 号 p. 153-162
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    The folding types of 135 proteins, the three-dimensional structures of which are known, were analyzed in terms of the amino acid composition. The amino acid composition of a protein was expressed as a point in a multidimensional space spanned with 20 axes, on which the corresponding contents of 20 amino acids in the protein were represented. The distribution pattern of proteins in this composition space was examined in relation to five folding types, α, β, α/β, α+β, and irregular type. The results show that amino acid compositions of the α, β, and α/β types are located in different regions in the composition space, thus allowing distinct separation of proteins depending on the folding types. The points representing proteins of the α+β and irregular types, however, are widely scattered in the space, and the existing regions overlap with those of the other folding types. A simple method of utilizing the “distance” in the space was found to be convenient for classification of proteins into the five folding types. The assignment of the folding type with this method gave an accuracy of 70% in the coincidence with the experimental data.
  • Shinji TAMURA, Toshihiro SUGIYAMA, Yuzo MINAMI, Seiichiro TARUI, Mitsu ...
    1986 年 99 巻 1 号 p. 163-171
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    Debromination of l, 2-dibromoethane (DBE) by a rabbit liver microsomal preparation and a reconstituted cytochrome P-450 enzyme system was investigated. The reaction was performed in our newly constructed reaction vessel, in which a bromide electrode was installed. During the reaction, the liberated bromide ion was continuously measured by the bromide electrode, and the amount was recorded. In the microsomal preparation, the DBE-debromination rate per nmol cytochrome P-450 was enhanced by phenobarbital-pretreatment of rabbits compared with the untreated microsomes, whereas it was diminished by 3-met hylcholanthrene-pretreatment. The debromination reaction was reconstituted in a purified enzyme system containing phenobarbital-inducible rabbit liver microsomal cytochrome P-450 (P450PB), NADPH-cytochrome P-450 reductase, and NADPH. The optimum conditions required the presence of dilauroylphosphatidylcholine and cytochrome b5. Cytochrome b5 was found not to be an obligatory component for the DBE-debromi nation in the reconstituted system, but it stimulated the activity about 3.4-fold. Preincubation of the reconstituted mixture with guinea pig anti-cytochrome P-450PB antiserum markedly inhibited the debromination reaction.
  • Takashi SASAKI, Takanobu KIKUCHI, Ichiro FUKUI, Takashi MURACHI
    1986 年 99 巻 1 号 p. 173-179
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    Three new tripeptidyl chloromethyl ketones, Leu-Leu-XCH2Cl, with X representing Phe, Tyr, or Lys, were synthesized and their potencies to inactivate calpains I and II were compared. They were designed to fulfil the specificity requirement of cal-pains established recently. When compared in terms of the dose for 50% inactivation, Leu-Leu-PheCH2Cl was the strongest inactivator, being 500-600 times more effective than tosyl-PheCH2Cl and 5-14 times more than N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl] agmatine (E-64). The potency toward calpain, either I or II, decreased in the order Phe>Tyr>Lys derivatives>E-64, whereas that toward papain was E-64>Lys>Phe>Tyr derivatives. From the determined kinetic parameters, the Phe derivative was 18.3 and 16.6 times more effective than E-64 on calpains I and II, respectively. Likewise, the rate of the alkylation reaction by these chloromethyl ketones with calpain I was 2-4 times greater than that with calpain II. Leu-Leu-PheCH2Cl and its N-dansylated product should be useful for highly selective affinity labeling of calpains I and II.
  • Tetsuro ISHII, Loyal G. TILLOTSON, Kurt J. ISSELBACHER
    1986 年 99 巻 1 号 p. 181-189
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    Photoaffinity labeling with [3H] cytochalasin B detects two D-glucose-sensitive proteins in the chicken embryo fibroblast (CEF) plasma membrane, which accumulate under conditions of glucose starvation and are probably involved in the glucose transport system (Pessin, J. E., et al. (1982) Proc. Natl. Acad. Sci. U. S. 79, 2286-2290). The two labeled components, designated as peak I (Mr 45, 000) and II (Mr 52, 000) com-ponents, were separated by preparative gel electrophoresis in the presence of sodium dodecyl sulfate. The fractions were digested with S. aureus V8 or papain, and the radioactive products were analyzed by one-dimensional gel electrophoresis. The peptide maps showed that they have different peptide structures. Peptide maps of authentic actin, a possible contaminant of the peak I fractions, were quite different from those of the peak I component. Rous sarcoma virus-transformed CEF have two components similar as to apparent molecular size and peptide maps to those present in glucose-starved cells. The peak I and II components show minimal affinity to agarose-bound Ricinus communis agglutinin which binds the human erythrocyte glucose transporter quite well. The peak II component was more susceptible to proteolysis than the peak I one or the human erythrocyte glucose transporter. However, the peptide maps of the peak II component were similar to those of the human erythrocyte glucose transporter.
  • Keiko KITAGISHI, Keitaro HIROMI
    1986 年 99 巻 1 号 p. 191-197
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    The interaction between thermolysin and its specific inhibitor, PLT (N-phosphoryl-L-leucyl-L-tryptophan), has been investigated by steady-state inhibitory kinetics analysis, fluorometric titration, and the stopped-flow method.
    The inhibitor constant of PLT, K1, and the dissociation constant of therm-olysin(E)-PLT(I) complex, Kd, are found to be smaller by a factor of 4 to 300, depending on pH, resulting in stronger binding, than those of talopeptin and phos-phoramidon, but all of them show similar pH dependence. The dependence of the apparent first-order rate constant, kapp, on the inhibitor concentration is consistent with a minimum two-step mechanism, including a fast bimolecular step followed by a slow unimolecular step, E+I_??_EItr_??_EI. The values of K-1 (the dissociation constant of the intermediate EItr) and k-2 (the backward rate constant in the unimolecular step) are not so significantly different between PLT and talopeptin, while the k+2 (forward rate constant in the unimolecular step) value for PLT is about 14 times larger than that of talopeptin (pH 5.5). These facts suggest that the forward rate of the isomerization step, EItr→EI, is much larger in the absence of the sugar moiety of talopeptin, and hence it induces the stronger binding of PLT to thermolysin than that of talopeptin.
  • Keiichi YAMAMOTO, Takamitsu SEKINE
    1986 年 99 巻 1 号 p. 199-206
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    To elucidate the difference between subfragment-1 and heavy meromyosin in their interaction with F-actin, we used limited tryptic digestion and cross-linking with 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide. The binding of actin to subfrag-ment-1 lowers the susceptibility of the 50K-20K junction of its heavy chain to tryptic digestion. At a molar ratio of one actin to one subfragment-1, all the sites were gradually cleaved by trypsin whereas the sites were completely protected in the presence of a 2-fold molar excess of actin over subfragment-1. In the case of heavy meromyosin, nearly half of the sites were protected completely by the presence of an equimolar amount of actin to its heads suggesting that the two heads of heavy meromyosin bound actin in a different manner. The rate of the cross-linking reaction between subfragment-1 heavy chain and actin with 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide also depended on the molar ratio of actin to subfragment-1. The rate was maximum at a molar ratio of about 5 actin to 1 subfragment-1. When heavy meromyosin was cross-linked to actin, the maximum rate was observed at a molar ratio of about 3 actin to 1 heavy meromyosin head, the level being about 60% that for subfragment-1 and actin. It was suggested that the presence of the subfragment-2 portion of heavy meromyosin caused these differences by restricting the motion of the two heads.
  • Toshiko OMATA, Hitoshi HORIE, Shusuke KUGE, Nobumasa IMURA, Akio NOMOT ...
    1986 年 99 巻 1 号 p. 207-217
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    Complementary DNA to the genome of the Sabin 1 strain of poliovirus was prepared by reverse transcription with oligo(dT)10 as a primer and separated into six classes of DNA by their size. Each class of the DNA, after digestion with restriction endonuclease HaeIII, was analyzed by two-dimensional polyacrylamide gel electrophoresis. Comparison of the patterns of the restriction fragments led us to compose a possible arrangement of the restriction fragments on the viral genome. Sequence analysis of these fragments indicated that the arrangement was consistent with the known total nucleotide sequence of the genome. In the determined sequences, two bases were observed to differ from those of a cloned complementary DNA of the Sabin 1 genome. This suggested that the sequence of the cloned DNA reflected that of a mutated virus genome that was a minor component in the virus inoculation stock. The genomes of defective interfering particles generated from the Sabin 1 strain were also analyzed by this technique. The results suggested that the RNAs lacked an internal region of the Sabin 1 RNA encoding viral capsid proteins. The location of the deletion was further confirmed by determination of the nucleotide sequence of a cloned complementary DNA copy of the defective interfering particle RNA. Thus, the method described here is useful for mapping and sequencing of RNAs and for knowing whether cloned cDNAs represent the major population of RNA molecules or not.
  • Kazuo NAKAMURA, Shizuo HANDA
    1986 年 99 巻 1 号 p. 219-226
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    A simple and rapid method for the preparation of N-methylamides (-CONHCH3) of sialic acids in gangliosides and biochemical properties of the modified gangliosides are described. The sialic acid carboxyl groups of gangliosides were esterified with CH3I-dimethylsulfoxide, followed by heating with monomethylamine. The modified gangliosides were chemically identified by TLC, IR spectroscopy, GLC-mass spectrometry and NMR spectroscopy.
    The N-methylamide derivative of GM1 produced a high titer IgG antibody. The antibody weakly cross-reacted with the methylester of GM1 and its reductive derivative but did not react with the intact GM1.
    A monoclonal antibody (M2590) specific for GM3 did not react with carboxyl-modified GM3 (methylester, N-methylamide, and reduced GM3), but it reacted with modified GM3 which contains the C7-analog of the sialic acid.
    Clostridium perfringens and Arthrobacter ureafaciens sialidases did not hydrolyze the N-methylamide derivatives, methylesters or reductive derivatives of the ganglio-sides and, furthermore, these derivatives did not inhibit the actions of these sialidases.
  • Toshiro SHIMAMURA, Tohoru NAKAMURA, Jiro KOYAMA
    1986 年 99 巻 1 号 p. 227-235
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    The variety and properties of Fc receptors (FcR's) for homologous IgG on guinea pig peritoneal macrophages were investigated with the use of a mouse monoclonal antibody, VIA2 IgG1, prepared by fusion of splenic cells of a mouse immunized with guinea pig macrophages with a mouse myeloma cell line. VIA2 IgG1 completely inhibited the formation of macrophage rosettes with IgG1 antibody-sensitized erythrocytes, but not that with IgG2 antibody-sensitized erythrocytes. The Fab' of VIA2 IgG1 also completely inhibited the bindings of both monomeric and ovalbu-min-bound IgG1 antibodies to macrophages. On the other hand, the Fab' did not affect the binding of monomeric IgG2 antibody to macrophages, although it partially inhibited that of ovalbumin-bound IgG2 antibody. These results show that at least two distinct types of FcR are present on guinea pig macrophages; one (FcR1, 2) binds monomeric IgGI antibody and also antigen-bound IgG1 and IgG2 antibodies, and the other (FcR2) binds monomeric and antigen-bound IgG2 antibodies alone, and also that VIA2 IgG1 binds specifically to FcR1, 2.
    When FcR1, 2 was isolated by affinity chromatography on F(ab')2 of VIA2 IgG1 coupled to Sepharose, it gave a main band with a molecular weight of 55, 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was indistinguishable from the main band isolated with the IgG1 immune complex. The number of FcR1, 2 per macrophage cell was estimated to be 2×105 by measuring the binding of 125I-Fab' of VIA2 IgG1.
  • Kunio TSURUGI, Kikuo OGATA
    1986 年 99 巻 1 号 p. 237-241
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    A neutral protease, named protease B in the previous report (Tsurugi, K. & Ogata, K. (1982) J. Biochem. 92, 1369-1381), was partially purified from rat liver chromatin by gel filtration through Sepharose 6B followed by DE-Sephadex column chromatography. The proteolytic activity on total histones of the partially-purified protease B was increased about two fold by addition of DNA and again increased by further addition of 2M urea. Analysis of the hydrolysed products showed that out of five species of histones, only H1 was degraded in the presence of an amount of DNA equivalent to the amount of histones, whereas core histones were also degraded in the absence or presence of one-tenth amount of DNA. Urea accelerated the selective degradation of H1 histone because H1 histone was preferentially degraded in the presence of even a low amount of DNA. In contrast, core histones became resistant to the protease B in the presence of DNA and/or urea. Heat-denatured DNA stimulated the degradation of H1 histone even in the absence of urea to almost the same extent that native DNA did in the presence of urea. Thus, protease B efficiently degrades H1 histone when its association with DNA is destabilized by either addition of urea or pretreatment of DNA with heat.
  • Tomoko ITOH, Yutaka UDA, Hiroki NAKAGAWA
    1986 年 99 巻 1 号 p. 243-250
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    An α-galactosidase [EC 3.2.1.22] was isolated from the fruit of the watermelon, Citrullus battich. The enzyme was purified by procedures including extraction, ammonium sulfate precipitation, and chromatographies on DEAE-Sephadex, CM-Sephadex and Sephadex G-100. The final preparation was found to be fairly homogeneous on disc and SDS-polyacrylamide gel electrophoresis, and sufficiently free from other glycosidase activities. The molecular weight of the enzyme was estimated to be 45, 000 by Sephadex G-100 column chromatography and SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 4.5 for natural substrates and at 5.9 for artificial substrates. The enzyme liberates the α-galactose units from oligosaccharides of the raflinose series and ceramide trihexoside, and the hemagglutination-inhibiting activities of human ovarian cyst B-glyco-protein and blood group B-type ghosts were abolished by the enzyme.
  • Yumiko OKADA, Tadao HASHIMOTO, Yukuo YOSHIDA, Kunio TAGAWA
    1986 年 99 巻 1 号 p. 251-256
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    Two proteinous factors, 15K and 9K proteins, which acted together to stabilize the inactivated yeast F1F0-ATPase-inhibitor complex [Hashimoto, T., et al. (1984) J. Biochem. 95, 131-136] were hardly distinguishable from the δ and ε subunits, respectively, of yeast F1-ATPase by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. However, they were clearly distinguishable from these subunits by analyses of the sequences at their amino terminals and by immunoblotting combined with SDS polyacrylamide gel electrophoresis. The two stabilizing factors and an ATPase inhibitor existed in mitochondria in equimolar ratios to F1-ATPase. These three protein factors were not present in purified F1-ATPase or in F1F0-ATPase preparations, but remained in the mitochondrial membranes after extraction of F1F0-ATPase with Triton X-100. These observations strongly suggest that the two stabilizing factors and the ATPase inhibitor form a regulatory substructure of mitochondrial ATP synthase, in addition to the F1 and F0 subunits.
  • Yasushi TANAKA, Ryohei MIYAKE, Ushio KIKKAWA, Yasutomi NISHIZUKA
    1986 年 99 巻 1 号 p. 257-261
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    Protein kinase C is generally accepted to be a receptor protein of tumor-promoting phorbol esters. The binding of [3H] phorbol-12, 13-dibutyrate to protein kinase C can be assayed by a rapid filtration procedure using a glass-fiber filter that has been treated with a cationic polymer, polyethylenimine. The phorbol ester specifically binds to the protein kinase only in the presence of phosphatidylserine and calcium. Non-specific binding is less than 10%, at most, of the total binding. The binding is linear with respect to the concentration of protein kinase C, is dependent on the concentrations of phorbol ester and phosphatidylserine in a saturative manner, and is inhibited by diacylglycerol (an endogenous activator of the protein kinase).
  • Mitsuo TORII, Setsuko OGAWA, Kazuhito WATABE, Tomihiko KOSHIKAWA, Mari ...
    1986 年 99 巻 1 号 p. 263-267
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    Cross-reactions of four synthetic branched glucans (3-O-α-D-glucopyranosyl-(1→6)-α-D-glucopyranans: V39, V17, V37, and V32, each containing one unit glucose branches amounting to 11-12%, 33-43%, 50-54%, and 71-100%, respectively) with rabbit anti-N4 dextran were examined. All four samples precipitated antibodies raised in rabbits by injecting N4 dextran-concanavalin A conjugate. The ability of glucans to precipitate antibody depended on the quantity of branches, samples with more branches precipitating less antibody nitrogen under the same conditions. This may indicate an inhibitory effect of the branches on precipitation. Oligosaccharide inhibition assay showed that the precipitation reactions were specific for (1→6)-α-D-glucopyranosyl linkages, and the maximum size of the α-(1→6)-specific antibody combining site corresponded to isomaltopentaose. Determination of antibody nitrogen and glucan in the precipitates indicated that the ratios of one combining site of antibody to numbers of glucose residues were 1:9 (V39), 1:11 (V17), and 1:16 (V37) in the extreme antibody excess region. A synthetic sample of manno-glucan ((1→6)-α-D-glucopyranan containing about 27% of randomly linked 3-O-α-D-mannopyranosyl side chains) also reacted with the same antibody.
  • Kenichi ONO, Tomofumi JITSUKAWA, Hideki ISHIHARA, Akio FUKUDA
    1986 年 99 巻 1 号 p. 269-279
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    Three monoclonal antibodies against human liver ferritin were selected to study antigenic determinants (epitopes) of human isoferritins. These monoclonal antibodies were found to form immunoprecipitin lines with ferritin in double diffusion tests (Ouchterlony), indicating multiple epitopes on a single ferritin molecule. The antibodies revealed high species specificity as well. Monoclonal antibodies MA301 and MA311 appeared to recognize different epitopes, since they did not inhibit each other in competitive enzyme-linked immunosorbent assay (ELISA). However, MA309 recognized both epitopes for MA301 and MA311 with similar competitive inhibition. These epitopes were not detectable when ferritin was treated with 8 M urea (pH 2.5) and were detectable upon reconstruction by dialysis against 2 M urea (pH 7.2), suggesting that these monoclonals recognize epitopes in the tertiary structure of the ferritin molecule. As a matter of fact, these monoclonals react preferentially with intact ferritin molecule and only negligibly with subunits. Isoelectric focusing patterns of human ferritins demonstrated that liver, spleen, placenta, and hepatoma cells (Li-7) transplanted in nude mice contained basic isoferritins, whereas HeLa cells (carcinoma), Wa cells (EB virus-transformed B cells), and Raji cells (Burkitt's lymphoma) contained acidic isoferritins. Human heart ferritin displayed a somewhat intermediate pattern between liver and HeLa ferritins. In spite of the heterogenous population of human isoferritins, the dissociation constants (Kd) of the three monoclonal antibodies to liver, HeLa, and heart isoferritins were quite similar.
  • Shuji TANAKA, Noriko MOHRI, Hiroshi KIHARA, Motonori OHNO
    1986 年 99 巻 1 号 p. 281-289
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    The amino acid sequence of phospholipase A2 from the venom of Trimeresurus flavoviridis (the Habu snake) was determined. The enzyme subunit has a molecular weight of 13, 764 and consists of a single polypeptide chain of 122 amino acids and seven disulfide bonds. The fragmentation was conducted by digesting the reduced and S-carboxymethylated derivative of the protein with Achromobacter protease I, chymotrypsin, and trypsin, respectively. Achromobacter protease I peptides were used for alignment and to establish overlaps over chymotryptic and tryptic peptides. The automated Edman degradation of the S-carboxymethylated protein, which was extended to the N-terminal 30 amino acid residues, supplemented the deletions found with the enzymatic peptides alone. T. flavoviridis phospholipase A2 was found to be highly (65-67 %) homologous in sequence to the enzymes from T. okinavensis, Crotalus adamanteus, and Crotalus atrox (viperid family) and less (35-44%) homologous to those from elapid snakes and mammalian pancreas. The T. flavoviridis enzyme appears to be similar in secondary structure composition to the C. atrox enzyme.
  • Kaoru OMICHI, Tokuji IKENAKA
    1986 年 99 巻 1 号 p. 291-294
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    The complete hydrolysis of a fluorogenic derivative of p-nitrophenyl α-maltopentaoside, FG5P, by human salivary α-amylase, resulted in a 5-fold increase in fluorescence. This is due to disruption of the intramolecular quenching of the fluorescence of the 2-pyridylamino residue by the p-nitrophenyl residue by separation of the two residues. This change of fluorescence accompanying the cleavage of the glucosidic bond was exploited to develop a fluorometric rate assay of α-amylase in human serum.
  • Osamu NISHIKAWA, Shinji YOKOYAMA, Takashi KURASAWA, Akira YAMAMOTO
    1986 年 99 巻 1 号 p. 295-301
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    [14C] Cholesteryl ester was directly incorporated into human plasma low-density lipoproteins (LDL) for the purpose of preparing a tracer substrate for investigation of the cholesteryl ester transfer reaction between plasma lipoproteins. The radiolabeled cholesteryl oleate was sonicated with egg phosphatidylcholine to form cholesteryl ester-containing liposomes. The liposomes were incubated with plasma fraction of density>1.006 at 37°C in the presence of dithionitrobenzoic acid. When the distribution of the radiolabeled cholesteryl ester was equilibrated among liposomes and lipoprotein fractions, the mixture was applied to an affinity chromatography column of dextran sulfate-cellulose (LA01) (Arteriosclerosis 4, 276-282). LDL was eluted by increasing the NaCl concentration and was finally isolated as a floating fraction by ultracentrifugation at a solvent density of 1.063 (adjusted with NaCl). The chemical composition, electrophoretic mobility and density of the labeled LDL were consistent with those of the native LDL. Radioactivity in this preparation was present exclusively in cholesteryl ester. Apolipoprotein B100 was preserved intact throughout the procedure. When the rate of cholesteryl ester transfer was measured between LDL and high-density lipoproteins by using this labeled LDL, the kinetics was consistent with the equilibrium transfer model, but the apparent rate measured was slightly higher than that measured with the labeled LDL prepared by the method using the intrinsic cholesterol esterification reaction of plasma.
  • Masataka ISHINAGA
    1986 年 99 巻 1 号 p. 303-306
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    The effect of phosphatidylethanolamine (PE) molecular species on the reconstitution of partially purified glycerophosphate acyltransferase of Escherichia coli was investigated. The acyltransferase activity was abolished by 1, 2-di-unsaturated (U-U) PE, but not by 1-saturated-2-unsaturated (S-U) PE or 1-saturated-2-cyclopropanoyl PE. Since both the U-U and S-U PE used in the present work are in a fluid state at temperatures above about 30°C, the differential effect cannot be accounted for in terms of the membrane fluidity. Therefore, the inactivation of the reconstituted enzyme was attributed to the large amount of the 1, 2-di-cis-vaccenoyl species of PE.
  • Saori TAKAHASHI, Retsu MIURA, Chikao YUTANI, Yoshihiro MIYAKE
    1986 年 99 巻 1 号 p. 307-310
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    By means of a new rapid and small scale purification method, human kidney renin has been purified from a single kidney in a homogeneous state, as judged on SDS-PAGE. The kidney which showed unusually high renin activity was from a patient with cardiomyopathy. 8, 000-fold purification was attained by means of only pep-statin-aminohexyl-Sepharose chromatography and FPLC on a Mono Q column, and the yield was 34%. The specific activity was 5.63 mg angiotensin I per mg protein per h at 37°C and pH 6.5 with porcine angiotensinogen as the substrate. The molecular weight was estimated to be 37, 000 by SDS-PAGE and 38, 000 by HPLC on a TSK G-3000 SW column. The preparation showed three bands on isoelectric focusing. The molecular weight and the profile on isoelectric focusing of the purified renin agreed with those found for the extracts of both the patient's kidney and a kidney with the usual low renin activity.
  • Tamao NOGUCHI, Joong-Kyun JEON, Osamu ARAKAWA, Haruo SUGITA, Yoshiaki ...
    1986 年 99 巻 1 号 p. 311-314
    発行日: 1986年
    公開日: 2008/11/18
    ジャーナル フリー
    Vibrio sp. isolated from a xanthid crab, Atergatis floridus, was cultured, and tetrodotoxin (TTX) and anhydro TTX were indicated to be present in several fractions of the cell extract and the culture medium by reverse phase HPLC. The presence of the C9-base in alkaline hydrolyzates of these fractions was confirmed by GC-MS and UV spectrometry. These results showed the production of TTX and anhydro-TTX in the Vibrio sp., thus indicating one of the origins of TTX in nature.
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