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Volume 101 (1987)
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- Issue 4 Pages 825-
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Volume 101, Issue 4
Displaying 1-28 of 28 articles from this issue
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Kyoko NAKAMURA, Minoru SUZUKI, Fuyuhiko INAGAKI, Tamio YAMAKAWA, Akemi ...1987 Volume 101 Issue 4 Pages 825-835
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new ganglioside showing choleragenoid-binding activity was purified from mouse spleen and characterized. From the results of sugar-composition analysis, enzymatic hydrolysis, a permethylation study, 1H-NMR spectroscopy, and negative-ion fast atom bombardment mass spectrometry, the structure of the ganglioside was determined to be as follows:
Galβ1-3GalNAcβ1-4Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1'eeramide
3-NeuGcα2
This ganglioside contains a terminal tetrasaccharide structure identical with that of II3NeuGcα-Gg4Cer (GM1 (NeuGc)). By means of a TLC-inimunobinding assay and an enzyme-linked immunosorbent assay, the ganglioside was demonstrated to have almost the same choleragenoid-binding activity as GM1. Another ganglioside, that migrated faster than the new choleragenoid-binding ganglioside, was also purified from the same source material and identified as IV4GalNAcβ, IV3NeuGcα-Gg4Cer (GalNAc-GM1b (NeuGc)). Since, in the previous study, we demonstrated the existence of IV3NeuGcα-Gg4Cer (GM1b (NeuGc)) in mouse spleen (Nakamura, K. et al. (1984) J. Biochem. 96, 949-957), the results of this study suggest that the new choleragenoid-binding ganglioside is synthesized from GM1b (NeuGc) through Ga1NAc-GM1b (NeuGc).View full abstractDownload PDF (3042K) -
Kaoru TAKEUCHI, Kyosuke NAGATA, Akira ISHIHAMA1987 Volume 101 Issue 4 Pages 837-845
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn in vitro system for the synthesis of influenza viral RNA was developed using isolated nuclei prepared from influenza virus-infected HeLa cells. In this system, two species of positive-sense RNA, i.e., mRNA and cRNA (complementary RNA to vRNA), were found to be synthesized when analyzed by RNA-RNA hybridization using a minus-strand RNA probe and high resolution gel electrophoresis. The in vitro RNA synthesis required Mg2+, GTP, CTP, UTP, a high concentration of ATP, and an ATP regenerating system. Neither actinomycin D nor α-amanitin, potent inhibitors for cellular DNA-dependent RNA polymerases, inhibited the RNA synthesis. Addition of ApG or capped RNA, well-known primers for virionassociated RNA polymerase, markedly enhanced the extent of RNA synthesis. The maximum activity was observed with nuclei isolated from cells at 5 h after infection. This system is useful for the purification and characterization of factors involved in the transcription of these two species positive-sense RNA.View full abstractDownload PDF (4467K) -
Tomoko ITOH-NASHIDA, Masao HIRAIWA, Yutaka UDA1987 Volume 101 Issue 4 Pages 847-854
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA β-D-glucosidase (linamarase) was purified 11, 700-fold from the butter bean, Phaseolus lunatus L., by means of successive procedures including extraction, ammonium sulfate fractionation, acetone treatment, and chromatographies on CMSephadex, DEAE-Sephadex, and Sephadex G-200. The final preparation gave a single protein band on both disc polyacrylamide gel electrophoresis and SDSpolyacrylamide gel electrophoresis. In spite of its electrophoretic purity, the final enzyme preparation showed four glycosidase activities; β-D-glucosidase, β-D-galactosidase, β-D-fucosidase, and β-D-xylosidase. The molecular weight of the enzyme was determined to be 124, 000±9, 000 by Sephadex G-200 gel filtration, and 59, 000±2, 400 by SDS-disc gel electrophoresis. The enzyme showed a pH optimum in the range of 5.1 to 6.0 with p-nitrophenyl β-D-glucoside, 4-methylumbelliferyl β-D-glucoside, and linamarin. Among natural substrates containing a β-glucosyl terminal, linamarin, prunasin, and salicin were hydrolyzed by the enzyme from butter beans, but amygdalin, cellobiose, gentiobiose, and laminarin were hardly hydrolyzed.View full abstractDownload PDF (1221K) -
Hiroki NAKAGAWA, Makio ASAKAWA, Noriyuki ENOMOTO1987 Volume 101 Issue 4 Pages 855-862
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy means of a simple procedure involving two gel filtrations and an ion-exchange chromatography, α-N-acetylgalactosaminidase was purified to an electrophoretically homogeneous form from skipjack liver, in which the enzyme is the dominant glycosidase. The final α-N-acetylgalactosaminidase preparation contained practically no other glycosidase activities except α-galactosidase activity, which amounted to 0.8% of the α-N-acetylgalactosaminidase activity and may be an intrinsic activity of the enzyme. The molecular weight of the enzyme was estimated to be 80, 000 at pH 4.2 and 40, 000 at pH 7.2 by molecular sieve chromatography, and to be 35, 000 by SDS-polyacrylamide gel electrophoresis. The enzyme was most active at pH 4 and was inactive above pH 7. These results suggest that skipjack α-N-acetylgalactosaminidase exists as an active dimer at acidic pH and as inactive monomer at neutral or alkaline pH. The enzyme efficiently liberated the N-acetylgalactosamine unit from ovine submaxillary glycoprotein which had been desialylated by neuraminidase. The Km value and maximum velocity were 4.28mM and 409 μmol/min•mg for p-nitrophenyl α-N-acetylgalactosaminide, and 0.0543mM and 1.19 μmol/min•mg for ovine submaxillary asialoglycoprotein.View full abstractDownload PDF (1296K) -
Pia EKMAN, Bo EK, Lorentz ENGSTRÖM1987 Volume 101 Issue 4 Pages 863-870
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTyrosine protein kinase activities have been demonstrated in transformed and normal cell systems. So far, few data on the quantity of protein-bound phosphotyrosine in intact cells have been published. A knowledge of the stoichiometric increase in phosphotyrosine in cells after hormonal induction could be of interest when evaluating the importance of the tyrosine protein kinase activities found. By the addition of a known amount of unlabeled phosphotyrosine to the precipitated protein of 32P-phosphate-labeled cells it was possible after alkaline hydrolysis to spectrophotometrically follow the phosphotyrosine during consecutive chromatographies of the material. From the specific radioactivity of the purified phosphotyrosine the initial concentration of [32P] phosphotyrosine could be calculated. The method proved to be useful for the determination of [32P] phosphotyrosine is small amounts of cells. The minimum detectable amount of [32P] phosphotyrosine was about 1 pmol, and as an example, only 2.5×106 fibroblasts were needed. By this method it was shown that platelet-derived growth factor increased protein-bound [32P] phosphotyrosine from 600 to 3, 200 pmol/g of fibroblasts, while insulin only increased the [32P] phosphotyrosine from 110 to 120 pmol/g of hepatocytes.View full abstractDownload PDF (699K) -
Masayuki KANDA, Kazuko HORI, Toshitsugu KUROTSU, Setsuko MIURA, Yoshit ...1987 Volume 101 Issue 4 Pages 871-878
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe aromatic amino acid aminotransferase was purified to a homogenous state from a gramicidin S-producing strain of Bacillus brevis. The enzyme shows a molecular weight of about 71, 000 on gel-filtration. The subunit molecular weight is about 35, 000 as determined by sodium dodecyl sulfate gel electrophoresis, indicating that the enzyme is a dimer. The enzyme exhibits absorption maxima near 425 and 330nm at neutral pH. One mole of pyridoxal phosphate is bound per subunit. The enzyme has amino donor specificity for aromatic amino acids, L-phenylalanine, L-tyrosine, and L-tryptophan, and utilizes 2-oxoglutarate as the amino acceptor. This enzyme activity was separated from both the aspartate aminotransferase activity and the branched chain amino acid aminotransferase activity by chromatography on DEAE-Sephadex.View full abstractDownload PDF (1203K) -
Ken-ichirou MOROHASHI, Kazuhiro SOGAWA, Tsuneo OMURA, Yoshiaki FUJII-K ...1987 Volume 101 Issue 4 Pages 879-887
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour independent clones containing a part of the P-450 (SCC), cholesterol desmolase, gene were isolated from human genomic libraries using bovine P-450 (SCC) cDNA as a probe. These clones covered the entire P-450 (SCC) gene except for a part of the 1 st intron. The gene is at least 20 kb long and is split into 9 exons by 8 introns. The sequence analysis revealed that the nine separated exons code for a primary structure consisting of 521 amino acids which shows 72%. homology with that of bovine P-450 (SCC). A CATT sequence and a TATAAT sequence, which are possibly a “CAT” box, and a “TATA” box, respectively, are present 129 and 91 by upstream from the initiation codon. An unusual exon/intron functional sequence that begins with GC was found in the 6th intron of the gene. A putative extension peptide consisting of 39 amino acids was found in the sequence of human P-450 (SCC) by comparison with that of the bovine counterpart. Two conserved regions were found in the extension peptide of these two forms of P-450 (SCC), suggesting a functional role of the portions in the mitochondrial localization and processing of P-450 (SCC) precursor. The mature form of human P-450 (SCC) has only one cysteine residue, which was located in the center of the HR2 region (Gotoh et al. (1983) J. Biochem. 97, 807-817). This observation established beyond doubt that the sole cysteine residue in the HR2 region is the 5 th ligand to the heme.View full abstractDownload PDF (1465K) -
Yasufumi MINAMI, Yasufumi EMORI, Hiroshi KAWASAKI, Koichi SUZUKI1987 Volume 101 Issue 4 Pages 889-895
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe cDNA fragments corresponding to the domains with four consecutive E-F hand structures in the large and small subunits of chicken and rabbit calcium-activated neutral protease (CANP) were inserted into an expression vector (pUC8 or pUC18). The resulting plasmids were used to transform E. coli, and isopropyl-1-thio-β-D-galactoside (IPTG)-inducible expression was performed. The resulting four kinds of E-F hand structure-domains (the chicken large subunit, rabbit high- and low-calcium-requiring large subunits, and rabbit small subunit) were purified and analyzed for their calcium-binding abilities and capacities by the microscale filter assay. Most of the E-F hand structures could bind calcium and 2 or 4mol of Ca2+ ions bound to the four consecutive E-F hand structures. The calcium-binding affinity of the E-F hand structures in the large subunit roughly corresponds to the calcium concentration required for its CANP activity.View full abstractDownload PDF (1780K) -
Toshiaki OHTSUKA, Masaki OZAWA, Tadao OKAMOTO, Motoyuki UCHIDA, Naoki ...1987 Volume 101 Issue 4 Pages 897-903
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSuperoxide anion (O2-) production stimulated by concanavalin A (Con A) in guinea pig polymorphonuclear leukocytes (PMNL) was suppressed by addition of methyl α-mannoside, a Con A inhibitor, and resumed upon readdition of Con A. The reversible change in the O2- production was assumed to reflect the change in NADPH oxidase activity measured for the 30, 000×g particulate fraction. The stimulation by Con A of the phosphorylation of 46 K protein (s), as observed previously with several membrane-perturbing agents in parallel with an activation of NADPH oxidase in intact guinea pig PMNL (Okamura, N., et al. (1984) Arch. Biochem. Biophys. 228, 270-277), was also suppressed by methyl-α-mannoside and resumed upon readdition of Con A. Similar parallelism between the phosphorylation and NADPH oxidase activity was also observed in the case of stimulation by N-formyl-methionyl-leucyl-phenylalanine (FMLP) and phorbol 12-myristate 13-acetate (PMA), though both processes were reversible after the stimulation by FMLP but not reversible after that by PMA. Thus, such a parallelism observed in both intact PMNL and 30, 000×g particulate fraction indicates possible involvement of the protein phosphorylation in the regulation of the production of active oxygen metabolites in PMNL.View full abstractDownload PDF (1797K) -
Masakazu TAKAHASHI, Hiroshi INANO, Bun-ichi TAMAOKI, Sumi NAGASE1987 Volume 101 Issue 4 Pages 905-910
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen androstenedione was incubated with testicular microsomes of Sprague-Dawley rats in the presence of reduced nicotinamide-adenine dinucleotide (NADH), unknown metabolites were produced, in addition to testosterone and 7α-hydroxyandrostenedione The metabolites were identified as 3β-hydroxy-4-androsten-17-one and 3β-hydroxy-5-androsten-17-one (3:1) by biochemical and radiochemical methods. These results confirmed the occurrence of the reverse reactions from androstenedione to 3β-hydroxy-4-androsten-17-one and 3β-hydroxy-5-androsten-l7-one catalyzed by the 3β-hydroxysteroid dehydrogenase and 5-ene-4-ene isomerase in the microsomal fraction of Sprague-Dawley rat testes.View full abstractDownload PDF (1198K) -
Keiko SAKAI, Hiroshi AKANUMA, Kazutomo IMAHORI, Seiichi KAWASHIMA1987 Volume 101 Issue 4 Pages 911-918
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCalf thymus histones were found to be susceptible to a calcium-activated neutral protease [CANP: EC 3. 4. 22. 17] which required a high concentration of calcium ions for its activity (mCANP). The susceptibilities of histones were in the order of relative degradation rate: H2B, H2A, and H3. The major peptide fragments released by CANP from H2A, H2B, and H3 were isolated and the cleavage sites were determined. Examination of amino acid sequences and environmental features around the cleavage site as well as kinetic analysis of the degradation process led us to the following conclusions about the mode of substrate recognition of mCANP: 1) The cleavage sites in histones could not be interpreted in terms of the primary structure around them. Thus, it seems unlikely that the specificity of CANP solely depends on its recognition of any specific amino acid residues or sequences. 2) The susceptible bonds were never located in the midst of either a hydrophobic or hydrophilic alignment of amino acid residues but in the vicinity of the boundary between hydrophilic and hydrophobic clusters. 3) Once a peptide fragment was generated by the proteolytic degradation, no further cleavage occurred even if the peptide still contained a bond corresponding to what was susceptible to CANP in an intact histone. This observation was interpreted to mean that CANP may recognize a certain higher order structure of its substrates.View full abstractDownload PDF (524K) -
Masafumi TSUJIMOTO, Shoji TANAKA, Yayoi SAKURAGAWA, Nobuo TSURUOKA, To ...1987 Volume 101 Issue 4 Pages 919-925
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBiological activities of human tumor necrosis factor (TNF) and its derivatives were compared. In cytotoxicity assay with L 929 cells, one derivative, designated as TNF (Asn), showed significantly lower activity than any other TNF examined. In binding assay, this derivative was also shown to have lower affinity for TNF receptors on L 929 cells, suggesting that the cytotoxic activity of TNFs on L 929 cells correlates with their affinity for receptors. We also found that the cytotoxic activity of TNF on A673 cells and its inhibitory effect on lipoprotein lipase were parallel with the cytotoxic activity on L 929 cells, but the growth-enhancing activity on FS-4 cells and the cytotoxic activity on endothelial cells were not. It was also shown that TNF (Asn) had lower affinity than any other TNF for receptors on these target cells tested. These results suggested that there might be at least two types of cellular responses to TNF; one might correlate with the receptor-binding affinity of TNFs and the other not.View full abstractDownload PDF (524K) -
Lucia GANDINO, Giorgio GRIBAUDO, Pietro GHEZZI, Santo LANDOLFO1987 Volume 101 Issue 4 Pages 927-932
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIncubation of quiescent tumor cells with fetal calf serum induced ornithine decarboxylase (ODCase) activity concomitantly with mitogenic stimulation. Pretreatment of cells with highly purified natural or recombinant murine interferon-gamma (MuIFN-γ) for 5 h caused a dose-dependent increase of ODCase activity induced by fetal calf serum (FCS). Pretreatment of target cells with IFN-γ for 5 h in absence of FCS stimulation did not induce ODCase activity. When pretreatment of cells with natural or recombinant MuIFN-γ was prolonged for 18 h both ODCase activity and DNA synthesis induced by FCS were suppressed. By contrast when a mixture of MuIFN-α and -β was used, ODCase activity was significantly suppressed after 5 h pretreatment compared to untreated controls. These results suggest that IFN-γ exerts a differential effect on mitogen-stimulated events depending on the dose and the time of addition.View full abstractDownload PDF (442K) -
Transformation of Macrophages into Foam Cells In Vitro Induced by Cholesteryl Oleate Liquid CrystalsMasayasu ENOMOTO, Keiji NAKAGAMI, Shoji OHKUMA, Tatsuya TAKANO1987 Volume 101 Issue 4 Pages 933-938
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTransformation of macrophages into foam cells after the uptake of cholesteryl oleate anisotropic liquid crystals was studied. A new technique to enhance the uptake of the liquid crystals by macrophages using an inverted petri dish was developed. Uptake of lipid droplets was found to increase in parallel with the amount of liquid crystals in the medium. A lysosomal enzyme was shown (by using lysosomotropic chloroquine) to be involved in the hydrolysis of the liquid crystals. About 47 and 72% of the [3H] cholesterol in liquid crystal-laden cells had disappeared after chase for 24 and 48 h, respectively. Thus the 50% clearance time of the liquid crystals by the macrophages was about 24 h, which was longer than that of denatured lipoprotein. A possible model of transformation of macrophages to foam cells is discussed.View full abstractDownload PDF (1142K) -
Noboru MOTOYAMA, Kouichi TAKIMOTO, Masato OKADA, Hachiro NAKAGAWA1987 Volume 101 Issue 4 Pages 939-947
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA unique phosphatase that selectively hydrolyzed phosphotyrosine and 2'-AMP at alkaline pH and p-nitrophenylphosphate at neutral pH was isolated from a cytosolic fraction of rat brain. The purified enzyme appeared homogeneous on SDS polyacrylamide gel electrophoresis and its molecular weight was estimated to be 42, 000. The molecular weight of the native enzyme was 45, 000 as determined by molecular sieve chromatography. These findings indicate that the native enzyme is a monomer protein. At pH 8.6, the enzyme hydrolyzed L-phosphotyrosine, D-phosphotyrosine, 2'-AMP, p-nitrophenylphosphate, 3'-AMP, 2'-GMP, and 3'-GMP; the ratio of its activities with these substrates was 100:96:115:68:39:25:16. Its Km values for L-phosphotyrosine, 2'-AMP, and p-nitrophenylphosphate were 0.8×10-4M, 1.4×10-4M, and 1.7×10-4M, respectively. At pH 7.4, the enzyme hydrolyzed p-nitrophenylphosphate, L-phosphotyrosine, and D-phosphotyrosine; the ratio of its activities with these compounds was 100:17:17, and its Km values for L-phosphotyrosine and p-nitrophenylphosphate were 1.8×10-4M and 2.0×10-4M, respectively. The enzyme activity was dependent on Mn2+ or Mg2+, and was strongly inhibited by 5'-nucleotides, pyrophosphate, and Zn2+. The 5'-nucleotides caused competitive inhibition of the binding of substrates. The enzyme was not sensitive to inhibitors of some well-characterized phosphatases such as NaF, molybdate, L (+) tartrate, tetramisole, vanadate, and lithium salt. The physiological role of the enzyme is discussed with respect to its activities toward phosphotyrosine, 2'-AMP, and p-nitrophenylphosphate.View full abstractDownload PDF (1188K) -
Takamitsu HORI, Noritaka NAKAMURA, Harumi OKUYAMA1987 Volume 101 Issue 4 Pages 949-956
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFatty acid synthetase (FAS) preparations from Saccharomyces cerevisiae cells grown at either 35 or 10°C produced the same products at different temperatures and showed quite similar temperature-dependencies in Arrhenius plots, with break points at 25°C. This break point does not appear to reflect a phase transition of phospholipids present in the purified FAS preparations but rather is associated with protein conformational changes. S. cerevisiae cells grown at 35°C and then shifted to 10°C produced fatty acids with a shorter average chain length than those fatty acids synthesized at 10°C by cells already adapted to 10°C (hyper response). Acetyl-CoA carboxylase activity was relatively higher in the cells grown at 35°C than in the cells grown at 10°C; moreover, fatty acids with longer average chain lengths were synthesized in vitro at higher malonyl-CoA concentrations, which was consistent with the difference in the average chain lengths of newly synthesized fatty acids in cells grown at 35 and 10°C. However, the activity levels of acetyl-CoA carboxylase and fatty acid synthetase alone did not account for the hyper response phenomena.View full abstractDownload PDF (594K) -
Kazuo OKAMURA, Toshiyuki MIYATA, Sadaaki IWANAGA, Ken-ichiro TAKAMIYA, ...1987 Volume 101 Issue 4 Pages 957-966
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of cytochrome c551 isolated from an aerobic photosynthetic bacterium, Erythrobacter sp. strain OCh 114, was determined. The cytochrome molecule was composed of a total of 119 amino acid residues and its molecular weight including heme was calculated to be 13, 235. The sequence was EGDIEAGEKAFNKCKSCHQIVSDAGEEIVKGGRTGPNLYG VLGRQAGTADFRYGDDLVAAGEAGLVWDADNFV EYVTDPRAFLRAYLDDS KAKSKMAYKLRSGGEDIAAYLASVSGSSS. Its molecular weight indicates that this cytochrome is of the L-type. Sequence alignment with other bacterial cytochromes c shows that this cytochrome is similar to cytochromes c of Rhodobacter capsulatus, Rhodobacter sphaeroides, and Paracoccus denitrificans, which were grouped into the a-3 subcluster from the 16 S rRNA sequence analysis.View full abstractDownload PDF (628K) -
Atsushi MORIKAMI, Kenzo NAKAMURA1987 Volume 101 Issue 4 Pages 967-976
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have characterized four pea mitochondrial DNA segments carrying the F1ATPase α-subunit coding sequences. These four types share a common 1.7-kb repeat sequence flanked by four combinations of two different left- and right-hand sequences. These results suggest that the α-subunit genes locate at the homologous recombination sites in the pea mitochondrial genome and that homologous recombination between two of these loci generates the other two types of structures. The uninterrupted α-subunit coding sequence of 1, 521 bp is present in two of these loci. A rearrangement of 965 by 3' to the ATG initiation codon generates two copies of pseudogenes where the C-terminal two-thirds of the α-subunit coding sequence is replaced with an unidentified coding frame. The other border of sequence divergence is located 733 by upstream of the ATG initiation codon. Although multiple forms of α-subunit gene transcripts are present in mitochondria, the pseudogenes do not seem to express any α-subunit-related polypeptide.View full abstractDownload PDF (1938K) -
Yukikazu SAEKI, Atsunori KASHIWAGI, Noriyoshi UEHARA1987 Volume 101 Issue 4 Pages 977-985
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe acute effects of insulin on glucose utilization in isolated rat quiescent cardiac myocytes were studied. Insulin (80 nM) increased the rate of glucose clearance by 2-3 times in the presence of glucose ranging from 0.3 μM to 5.5mM. Glucose transport, which was measured in terms of both D-glucose uptake in the presence of 0.3 μM D-glucose and initial rate of uptake of 3-O-methylglucose, was stimulated 3-fold in the presence of insulin. At higher glucose concentrations (>100 μM), a decrease in glucose clearance rate due to a shift of the rate-limiting step from glucose transport to a post-transport step in the pathway of glucose metabolism was observed. At the physiological concentration of glucose (5.5mM), about 73% of glucose was metabolized into lactate, about 10% was oxidized into CO2 and the rest (17%) remained inside the cells. The pentose phosphate pathway did not contribute to the glucose metabolism in these cells. Insulin (80 nM) significantly increased the uptake of glucose (112%), and the conversions of glucose into lactate (16%), glycogen (64%), and triglyceride (18%), but not into CO2 (3%). Insulin transiently increased the percentage of I-form of glycogen synthase by 16% above basal, but did not affect the percentage of a-form of glycogen phosphorylase. The content of glucose 6-phosphate in the cells was increased by 46% above the basal value in the presence of insulin. These results indicate that insulin has different acute stimulatory effects on various steps in the metabolic pathway of glucose in isolated quiescent cardiac myocytes. Insulin has a marked influence on the steps of glucose transport and glycogen synthesis, of which the latter is stimulated not only by the large increase in glucose 6-phosphate content but also by the small increase in the percentage of I-form of glycogen synthase.View full abstractDownload PDF (1466K) -
Jun HIRABAYASHI, Hiroshi KAWASAKI, Koichi SUZUKI, Ken-ichi KASAI1987 Volume 101 Issue 4 Pages 987-995
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe properties of a previously purified β-galactoside-binding lectin of human placenta were studied in detail. Isoelectric focusing gave multiple bands around pH 4.9, although the lectin preparation was homogenous in SDS-polyacrylamide gel electrophoresis. High-performance gel chromatography suggested that the lectin exists mainly as the monomer and that a small fraction forms a dimer. From all the criteria examined, human placenta lectin resembles one of the chick lectins obtained from embryonic skin or adult intestine (subunit molecular weight: 14, 000). The lectin was inactivated by thiol-modifying reagents, p-chloromercuribenzoic acid and N-ethylmaleimide. Reduced and carboxymethylated lectin contained five carboxymethylated cysteines per subunit, and five free thiol groups were titrated by using 5, 5'-dithio-bis (2-nitrobenzoic acid) Preliminary sequence analysis showed the presence of a region highly homologous to the corresponding region of the chick lectin (13 identical residues out of 18 from number 70 to 87 of the chick lectin), suggesting a close evolutionary relation between these lectins and the importance of this conserved region in the function of the lectins.View full abstractDownload PDF (1468K) -
Masateru NISHIHARA, Yosuke KOGA1987 Volume 101 Issue 4 Pages 997-1005
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe usual Bligh and Dyer method could extract only a small part of the lipids of Methanobacterium thermoautotrophicuin. When the water in the solvent was replaced by 5% trichloroacetic acid, the lipid recovery reached the maximum level, which was 6 times higher than that by the former method. The use of HCI (2M) or disruption of cells was also effective but prolonged extraction with the HCl-containing solvent caused degradation of some phosphoglycolipids. Twenty-three spots of polar lipids were detected on a thin-layer chromatogram of the total lipid. These were 10 phospholipids (18%), 6 aminophospholipids (17%), 3 aminophosphoglycolipids (15%), 2 phosphoglycolipids (31%), and 2 glycolipids (19%). The predominant polar lipids were a highly polar phosphoglycolipid (PGL1, 30%) and a glycolipid (GL1a, 16%). The other major lipids included an aminophospholipid (PNL1a, 9%), and an aminophosphoglycolipid (PNGL1, 7%). The complete structure determination of PNL1a, GL1a, and PNGL1 is described in the accompanying paper. Acetolysis of the total lipids followed by acid methanolysis was required for the complete cleavage of polar head groups, releasing core residues of diphytanyl glycerol diether (C20 diether) and dibiphytanyl diglycerol tetraether (C40 tetraether). A densitometric assay of a thin-layer chromatogram showed that the ratio of C20 diether and C40 tetraether was 1:14. GLC analysis of alkyl chlorides prepared from the total lipid by BCI3 treatment showed that phytanyl (C20), biphytanyl (C40), and unidentified alkyl chains accounted for 10, 83, and 7 mol% of the total alkyl chains, respectively. Strong acid hydrolysis of the macromolecular residue obtained after lipid extraction gave a significant amount of C40 tetraether, which had probably been bound covalently to other substances in the cells.View full abstractDownload PDF (739K) -
Masateru NISHIHARA, Hiroyuki MORII, Yosuke KOGA1987 Volume 101 Issue 4 Pages 1007-1015
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe structures of three of the major polar lipids (PNL1a, GL1a, and PNGL1) of Methanobacterium thermoaulotrophicum were elucidated. These lipids are derivatives of dibiphytanyl diglycerol tetraether (C40 tetraether; the proposed name is caldarchaeol). PNL1a is a C40 tetraether analog of phosphatidylethanolamine (proposed name: caldarchaetidylethanolamine). GL1a was identified as β-D-glucopyranosyl- (1-6) -β-D-glucopyranosyl C40 tetraether (diglucosyl caldarchaeol). PNGL1 has the polar head groups of both PNL1a and GL1a; one of the free hydroxyls of this tetraether is esterified with phosphoethanolamine while the other is linked to a glueosylglucose residue with the same structure as that of GL1a (proposed name: diglucosyl caldarchaetidylethanolamine). That is, PNL1a (aminophospholipid), GL1a (glycolipid), and PNGL1 (aminophosphoglycolipid) form structurally a quartet of lipids with the bare caldarchaeol. We propose a new systematic nomenclature of archaebacterial polar lipids in the “DISCUSSION, ” because the alternative names are too lengthy and laboratory designations of these lipids are not at all systematic. This nomenclature starts with giving the names archaeol and caldarchaeol to dialkyl diether of glycerol or other polyol and tetraether of glycerol or other polyol and alkyl alcohols, respectively, because these lipids are specific to archaebacteria. Phospholipids with a phosphodiester bond were named as derivatives of archaetidic acid or caldarchaetidic acid (phosphomonoesters of archaeol and caldarchaeol) by analogy with phosphatidic acid.View full abstractDownload PDF (1581K) -
Yoshiko OKAMOTO, Eiko MUTA, Shoshi OTA1987 Volume 101 Issue 4 Pages 1017-1024
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMugiline β isolated from mature sperm nuclei of the Formosan grey mullet, belonging to Perciformes, was fractionated into seven components (M 1-M 7), by chromatography on CM-Sephadex C-25. The amino acid sequences of the two major components (M 6 and M 7) were then determined. M6 contained 33 amino acid residues per molecule: Arg, 21; Thr, 1; Ser, 1; Glu, 1; Pro, 3; Ala, 2; Val, 2; Met, 0.3 and Ile, 1.7. The amino acid sequence of M 6 is: Pro-Arg-Arg-Arg-Arg-Glu-Thr-Ser-Arg-Pro-Ile-Arg-Arg-Arg-Arg-Arg-Ala-Arg-Arg-Ala-Pro- Ile (Met)-Arg-Arg-Arg-Arg-Arg-Val-Val-Arg-Arg-Arg-Arg. Isoleucine at position 22 is partially replaced by methionine. M 7 had an amino acid sequence similar to that of M 6 except that glutamic acid at position 6 of M 6 was replaced by glutamine. A high degree of homology in the sequences was found between mugiline β from mullet and thynnine from tuna fish, which also belongs to Perciformes.View full abstractDownload PDF (1647K) -
Toshiyuki KANEMASA, Kenzou BANBA, Michiki KASAI1987 Volume 101 Issue 4 Pages 1025-1032
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVoltage-gated anion channels in vesicles prepared from the electric organ of Narke japonica were studied using two methods. Ionic permeability was measured by the light scattering method, which could be used to measure the ion permeation of whole vesicles but only at a time scale of slower than about 0.1 s. The single channel conductances and permeability ratios for various ions were measured after fusing the vesicles to phospholipid bilayers. Both sets of results coincided, indicating that the anion channels observed with the planar bilayer method are the major route for anion passage in these vesicles. The channels showed anion selectivity and did not allow the permeation of cations such as K+ and choline+. The single channel conductance was 18 pS in 0.1M Cl-. SCN- inhibited the conductance in a voltagedependent reversible manner on both sides of a channel. SCN- may bind to the Cl- binding site in a channel and thus block it. 4, 4'-Diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS) blocked a channel on the cis (extracellular) side irreversibly. The number of anion channels per vesicle was estimated to be about 50. It was also shown that all anion channels in the vesicles were open at the very instance of fusion with planar membranes.View full abstractDownload PDF (633K) -
Hiroshi HOMMA, Ichiro KUDO, Keizo INOUE, Shoshichi NOJIMA1987 Volume 101 Issue 4 Pages 1033-1039
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have previously demonstrated the activity(ies) of phospholipid transacylase in Escherichia coli extract (Homma & Nojima (1982) J. Biochem. 91, 1093-1101), which catalyzed a new type of reaction of acyl transfer from diacylphospholipids to lysophospholipids. In this communication we report the specificities and characteristics of this enzyme activity. The activity catalyzed a reversible transfer of an acyl group between diacylphospholipids and lysophospholipids. The acyl group in the 1-position of the glycerol backbone was selectively transferred, and palmitic acid was the only fatty acid species transferred. Presumably, neutral lipids do not serve as substrates. The transacylase was firmly associated with the envelope fraction of E. coli. Neither potassium chloride nor urea was effective in solubilization of the activity and only about half of the activity was solubilized with Triton X-100. This observation was consistent with the equal distribution of the activity between the outer membrane and the inner membrane of E. coli. Functional aspects of this phospholipid transacylase are also discussed.View full abstractDownload PDF (557K) -
Kyo YAMASU, Tatsuo SENSHU1987 Volume 101 Issue 4 Pages 1041-1049
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have examined critically whether or not new and old histones mix in the octameric units of nucleosomes during chromatin replication. MH-134SC cells were densitylabeled by culturing with amino acid mixtures enriched with dense isotopes 2H, 13C, and 15N. Mononucleosomes obtained from labeled cells were fractionated by rate zonal sedimentation through a sucrose gradient in heavy water (Senshu et al. (1985) Eur J. Biochem. 150, 575-580). The fractionation can be performed under conditions that do not destabilize nucleosomes. Density-labeling yielded heterogeneous mononucleosome species which showed higher sedimentation rates than normal mononucleosomes. However, they were indistinguishable with respect to their protein compositions, electrophoretic mobilities, electrophoretic patterns of singlestranded DNA fragments liberated by DNase I digestion, electrophoretic mobilities and sedimentation velocities of the DNA moieties, and metabolic stabilities of the histone moieties. These data suggest that the heterogeneity of density-labeled mononucleosomes resulted from the formation of histone octamers density-sub-stituted to different degrees. This would be an inevitable consequence of mixing of new and old histones in the octameric unit of nucleosomes.View full abstractDownload PDF (1887K) -
Yoshinobu KIMURA, Sumihiro HASE, Yuji KOBAYASHI, Yoshimasa KYOGOKU, Gu ...1987 Volume 101 Issue 4 Pages 1051-1054
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe structures of sugar chains from two lectins in seeds of the castor bean (Ricinus communis) were identified. The sugar chains were liberated from the lectins by hydrazinolysis. After N-acetylation, the reducing-end residues of the sugar chains were coupled with 2-aminopyridine, The pyridylamino derivatives thus obtained were purified by gel filtration and HPLC. The structures of the purified derivatives were identified by component sugar analysis, stepwise exoglycosidase digestion, partial acetolysis, and 500 MHz 1H-NMR spectroscopy. A new processing pathway for sugar chains in plant glycoproteins was proposed on the basis of the structures of the sugar chains.View full abstractDownload PDF (229K) -
Shigeru NAKASHIMA, Toyohiko TOHMATSU, Hiroaki HATTORI, Akiyoshi SUGANU ...1987 Volume 101 Issue 4 Pages 1055-1058
Published: April 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGTP or GTPγS alone caused low but significant liberation of arachidonic acid in saponin-permeabilized human platelets but not in intact platelets. GTP or GTPγS also enhanced thrombin-induced [3H] arachidonic acid release in permeabilized platelets. Inhibitors of the phospholipase C (neomycin)/diacylglycerol lipase (RHC 80267) pathway for arachidonate liberation did not reduce the [3H] arachidonic acid release. The loss of [3H]arachidonate radioactivity from phosphatidylcholine was almost equivalent to the increase in released [3H] arachidonic acid, suggesting the hydrolysis of phosphatidylcholine by phospholipase A2. The effect of GTPγS was greater at lower Ca2+ concentrations. These data indicate that the release of arachidonic acid by phospholipase A2 in saponin-treated platelets may be linked to a GTP-biding protein.View full abstractDownload PDF (258K)
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