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Volume 102 (1987)
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- Issue 5 Pages 963-
- Issue 4 Pages 673-
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Volume 102, Issue 5
Displaying 1-39 of 39 articles from this issue
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Heinz BEITINGER, Wolfgang PROBST, Dietmar MÖBIUS, Hinrich RAHMANN1987 Volume 102 Issue 5 Pages 963-966
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of Ca2+ and temperature on mixed ganglioside-valinomycin-monolayers at the air/water interface were studied. Surface pressure-area isotherms of the pure gangliosides (GM1, GD1a) exhibited the typical monolayer characteristics. Pressure-area isotherms of the cyclodepsipeptide, valinomycin, were determined. In mixed monolayers, positive and negative deviation from the mean molecular area indicated the two components were miscible. Especially in GD1a mixtures, the addition of 0.01mM calcium exhibited, with low molar fractions of valinomycin, a demixing effect in the direction of the phase separation of the components.View full abstractDownload PDF (294K) -
Kenji SUGIYAMA, Susumu IZUMI, Shiro TOMINO, Sumi NAGASE1987 Volume 102 Issue 5 Pages 967-970
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy means of immunological screening, a cDNA clone bearing the mRNA sequence for rat transferrin was isolated from a cDNA library of rat liver mRNA. The amounts of transferrin mRNA in livers of analbuminemic rats (NAR, Nagase anal-buminemia rats) and normal rats were determined by RNA blot hybridization using a cloned transferrin cDNA probe. The level of transferrin mRNA in the NAR liver was about 1.7 times that in the normal rat liver. These findings suggest that the enhanced synthesis of transferrin in the NAR liver resulted from an increase in the transferrin mRNA level.View full abstractDownload PDF (1038K) -
Hiromi TAKANO-OHMURO, Kazuhiro KOHAMA1987 Volume 102 Issue 5 Pages 971-974
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have shown that the phosphorylation of smooth muscle regulatory myosin light chain (L20) with myosin light chain kinase (MLCK) produces faster moving bands (GMP1: heterodimer myosin with 1 unphosphorylated L20 and 1 mono-phospho-rylated L20, GMP2: homodimer myosin with 2 mono-phosphorylated L20s) on native pyrophosphate polyacrylamide gel electrophoresis (PP1 PAGE) (J. Biochem. 100, 259-268, 1986; J. Biochem. 100, 1681-1684, 1986). However, the mobility of the myosin phosphorylated, at its L20, with protein kinase C (PK-C) was the same that of the unphosphorylated myosin (GM) on PP1 PAGE. When the myosin prephos-phorylated with MLCK was further phosphorylated with PK-C, PP1 PAGE analysis showed only one band comigrating with GM, i.e., GMPI and GMP2 migrated to the same position as GM. Conversely, when the myosin prephosphorylated with PK-C was further phosphorylated with MLCK, GMP1 and GMP2 were not pro-duced. Thus the effect of L20 phosphorylated with PK-C is quite the opposite of that with MLCK, and the former predominated over the latter. We speculate that phosphorylation of L20 with PK-C “freezes” myosin in the inactive state.View full abstractDownload PDF (1858K) -
Dong-Seoung CHOI, Hisami YAMADA, Takeshi MIZUNO, Shoji MIZUSHIMA1987 Volume 102 Issue 5 Pages 975-983
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe molecular assembly of the major outer membrane lipoprotein on the peptidoglycan layer was studied using two hybrid genes coding for different OmpF-lipoprotein hybrid proteins. One gene codes for a “lipoprotein” in which the diacylglyceryl cysteine residue is replaced with the Ala-Glu residue of the NH2 terminus of the OmpF protein (hybrid protein I). The other gene codes for the lipid-free “lipoprotein” from which the COOH-terminal lysine residue was further deleted (hybrid protein II). Hybrid protein I existed as a trimer. A significant portion of it was found to be composed of only the free form, which was noncovalently associated with the peptidoglycan layer. The purified hybrid protein I trimer was dissociated into the subunit in the presence of guanidine-HCl and reassociated on dialysis. Both the native and reassociated trimers were bound to the lipoproteinfree peptidoglycan layer. No enhancement of the binding was observed when the reassociation reaction was carried out simultaneously. Hybrid protein II, on the other hand, did not exhibit association with peptidoglycan in both the cellular fractionation and in vitro binding experiments, although it existed as a trimer. It is concluded that 1) the protein domain of the lipoprotein exists as a trimer which is noncovalently as well as covalently associated with the peptidoglycan layer and 2) although the deletion of the COOH terminal lysine residue did not interfere with the trimerization, it interfered with the noncovalent interaction with the peptido-glycan layer.View full abstractDownload PDF (3584K) -
Yuzo HIROI, Yasuo NATORI1987 Volume 102 Issue 5 Pages 985-992
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat liver cytosol contains a neutral protease which degrades acetylated hemoglobin and some urea-denatured proteins maximally at pH 7.5. The enzyme was purified to homogeneity by conventional chromatographic techniques. It appears to be a metalloprotease since it is inhibited by EDTA and o-phenanthroline, the metaldepleted enzyme can be reactivated by Co2+, Zn2+, Mn2+, or Mg2+, and it is not inhibited by reagents specific for carboxyl, seryl, or thiol proteases. The enzyme has an apparent molecular weight of 200, 000 as determined on Sephacryl S-200 column chromatography, and electrophoresis in sodium dodecyl sulfate showed 3 protein bands corresponding to the molecular weights of 110, 000, 74, 000, and 40, 000.View full abstractDownload PDF (982K) -
Ryuzo SAKAKIBARA, Yoshihiro YOKOO, Kazuma YOSHIKOSHI, Nobuaki TOMINAGA ...1987 Volume 102 Issue 5 Pages 993-1001
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo determine the subcellular sites for synthesis and processing of human chorionic gonadotropin subunits in cells, first trimester placental cells were fractionated sub-cellularly on sucrose density gradients. Analysis of the subcellular fractions by immunobinding techniques revealed that the rough endoplasmic reticulum-rich fraction contained only intermediates having high-mannose oligosaccharides, but the Golgi-rich fraction contained not only intermediates but also mature forms which were resistant toendoglycosidase H but sensitive to neuraminidase. These results show that humanchorionic gonadotropin subunits are synthesized in the rough endoplasmic reticulum as forms containing high-mannose oligosaccharides, and their maturation occurs in the Golgi apparatus by trimming with endogenous glycosidases. They are then modified by addition of complex oligosaccharides and terminal sialic acid through glycosyltransferases.View full abstractDownload PDF (2907K) -
Akemichi UENO, Naokatu ARAKAKI, Seiji NISHIKAWA, Yoshiro TAKEDA1987 Volume 102 Issue 5 Pages 1003-1012
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn insulin-stimulating peptide derived from bovine serum albumin by digestion with trypsin was shown to inhibit insulin degradation. Addition of this peptide (1.2 μM) to the medium of isolated rat adipocytes markedly inhibited the degra-dation of insulin in the medium, but had a little effect on degradation of cell-asso-ciated insulin. Moreover, this peptide did not prevent dissociation of cell-associated insulin, suggesting that it is a bacitracin-type, not a chloroquine-type inhibitor of insulin degradation. The peptide also potentiated the stimulations by insulin mimickers of glucose oxidation byratadipocytes, strongly indicating that it has some other effects besides inhibition of insulin degradation. Therefore, the effect of the peptide on activation of pyruvate dehydrogenase (PDH), one of the postbinding actions of insulin, was studied. Addition of the peptide (4 μM) to adipocytes was found to activate PDH in the absence or presence of insulin. This stimulatory effect of the peptide on PDH was dose-dependent and was observed in both whole cells and subcellular fractions of rat adipocytes. The peptide also stimulated PDH in a subcellular system of either plasma membranes and mitochondria or mito-chondria only. Sodium fluoride, an inhibitor of phosphatase, blocked the action of the peptide almost completely, suggesting that the stimulatory effect of the peptide on PDH activity is at least partly due to its activation of PDH phosphatase. The mechanisms of action of the peptide are discussed. The peptide should be useful in studies on modulation of the action of insulin.View full abstractDownload PDF (832K) -
Ken-ichi TSUTSUMI, Reiko TSUTSUMI, Kiichi ISHIKAWA1987 Volume 102 Issue 5 Pages 1013-1021
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChromatin structures of the aldolase B gene locus in repressed and derepressed states were examined by DNase I digestion. Within the gene locus, several structural features were observed with respect to the sensitivity to DNase I; hypersensitive sites, relatively resistant regions, and preferential cleavage sites within the resistant regions. The hypersensitive sites and the resistant regions are tissue- or cell-specifically distributed, but are not simply related to the active or inactive state chromatin. Among these structural features, however, a DNase I-hypersensitive site located about 0.3 kilobase pairs (kb) upstream from the transcription-initiation site is characteristic only in transcriptionally active tissues or cells (liver, kidney and Morris hepatoma 5123D). In addition, analysis with nuclei of fetal liver cells indicated that this hypersensitive site is constructed prior to the transcriptional activation of the aldolase B gene during development. These results may indicate that the structural alteration in chromatin at the 0.3kb upstream site is related to the regulation of the aldolase B gene expression.View full abstractDownload PDF (3386K) -
Shoichi ISHIURA, Takeshi YAMAMOTO, Mari YAMAMOTO, Michio NOJIMA, Takaa ...1987 Volume 102 Issue 5 Pages 1023-1031
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo major aminopeptidases, an aminopeptidase B and an aminopeptidase M-like enzyme, were purified from human skeletal muscle by DEAE-cellulose, HPLC gel filtration, and hydroxyapatite column chromatographies. The purified aminopep-tidase B exhibits a molecular weight of 76, 000 under both native and denaturing conditions. The activity of the aminopeptidase B is regulated by Cl ions and other anions in vitro. On the other hand, the aminopeptidase M-like enzyme is a mono-meric protein having a molecular weight of 96, 000. It is capable of significantly cleaving Phe-, Leu-, Arg-, and Ala-aminoacyl bonds in the presence of 2-mercapto-ethanol. The pH optima for both enzymes are around 7.0, and bestatin is an effective inhibitor of both enzymes.View full abstractDownload PDF (1177K) -
Masahide TONE, Reiko KIKUNO, Akiko KUME-IWAKI, Tamotsu HASHIMOTO-GOTOH1987 Volume 102 Issue 5 Pages 1033-1041
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have isolated three cDNA clones for human a2-plasmin inhibitor (a2-PI). Two clones are from human hepatoma cell line, Hep G2, and cover the entire protein coding region plus the 3'-flanking region up to the poly(A) sequence, and the other clone is from human liver and contains the carboxyl-terminal half. The total length of the cDNAs is 2.29 kb, corresponding to more than 95% of the full-length mRNA. a2-PI seems to consist of 452 amino acid residues plus 39 amino acid residues for the signal peptide. The amino acid sequence shows 23 to 28 % homology to those of five other protease inhibitors, plasminogen activator inhibitor (PAI), pro-tein C inhibitor (PCI), arantitrypsin (a1-AT), antithrombin III (AT III), and aranti-chymotrypsin (a1-AC). a2-PI seems to be the most distantly related among these in-hibitors. Comparison of the phylogenetic trees of proteases and their inhibitors indi-cates that four proteases, namely elastase (or trypsin), chymotrypsin, plasminogen activator, and thrombin, may have evolved concurrently with the corresponding inhibitors. However, a2-PI and PCI seem to have evolved asynchronously from their substrates. The data suggest that a2-PI may originally have inhibited some protease other than plasmin, and protein C may have had an inhibitor different from the pre-sent one early in its evolutionary history.View full abstractDownload PDF (785K) -
Tsunehiro MUKAI, Hitomi YATSUKI, Yuji ARAI, Keiichiro JOH, Sachiko MAT ...1987 Volume 102 Issue 5 Pages 1043-1051
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe chromosomal gene encoding human aldolase B was isolated. The gene is composed of nine exons interrupted by eight introns and spans 15 kb, and a single copy of it occurs per haploid human genome. The initiation of transcription occurs at three different sites. Two minor sites, m1 and m2, start at 49 and 21 nucleotides, respectively, upstream from the major site, M. The gene also carries poly(A) addition signals at two different sites, thereby another two distinct mRNA species are produced. We examined the sequences required for mRNA 3'-end formation in this gene carrying multiple poly(A) addition sites. By constructing deletion mutants as to the region distal to the poly(A) addition site and then assaying through transfection into COS-1 cells, we demonstrated that 8 nucleotides distal to the site of poly(A) addition is sufficient for proximal polyadenylation, but is not sufficient for distal polyadenylation.View full abstractDownload PDF (3793K) -
Yoshihisa NAKANO, Yoshihiro URADE, Reiko URADE, Shozaburo KITAOKA1987 Volume 102 Issue 5 Pages 1053-1063
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe pellicle was isolated from the cell homogenate obtained on sonication of Euglena gracilis z grown aerobically under illumination and purified by a combination of differential and sucrose density gradient centrifugations. The purity and homogeneity of the pellicle fragments were determined by an electron microscopic method and biochemical analysis of the components. The protein, lipid, and sugar con-tents of the purified pellicle were 68.7, 17.9, and 13.5 %, respectively. The equilibrium density of pellicle fragments was 1.21g/cm3. SDS-polyacrylamide gel electrophoresis revealed that the pellicle contained 50mol % of nonpolar amino acids. The constituents of the lipid and sugar were very different from those of the cell membrane of other organisms.View full abstractDownload PDF (1358K) -
Michio YAZAWA, Koichi YAGI, Kenji SOBUE1987 Volume 102 Issue 5 Pages 1065-1073
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA calmodulin binding portion was separated from chicken gizzard caldesmon by chymotryptic digestion and it was purified through two column chromatography steps on calmodulin-Sepharose and Ultrogel AcA 44. The isolated fragMent has an estimated molecular weight of 35, 000 (35K) and it was possibly derived from the C-terminal portion of caldesmon. The affinity of the 35K fragment for calmodulin was determined by using the characteristic calmodulin-dependent mobility shift in polyacrylamide gel electrophoresis. The 35K fragment retained the actin binding site of caldesmon. The interaction of the 35K fragment with actin was released in the presence of Ca2+ and calmodulin.View full abstractDownload PDF (1964K) -
Toshio YASUMORI, Sumie KAWANO, Kiyoshi NAGATA, Miki SHIMADA, Yasushi Y ...1987 Volume 102 Issue 5 Pages 1075-1082
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSP-450 human-2 is a human cytochrome P-450 that is immunochemically related to a constitutive male-specific cytochrome P-450 (P-450-male) and the phenobarbital-inducible P-450b/e in rat liver. By screening a human liver cDNA library in bac-teriophage λgt11, we isolated a clone with an insert length of 1, 847 bases (pHY13). The clone was sequenced and shown to code for a protein of 487 amino acids. The N-terminal 11-amino-acid sequence was in agreement with the protein sequence of P-450 human-2. The nucleotide sequence of pHY13 showed less than 50% similarity with those of human cytochrome P-450s, pHP-450(1), HLp, P-45ONF, P1-450, P-450 4, and P3450, but the nucleotide sequence of pHY13 is 80% similar to the reported sequence of rat cytochrome P-450, P-450(M-1). In addition, the coding sequence of pHY13 showed close similarity to that of MP-8, which was recently reported as the sequence corresponding to human cytochrome P-450mp, although no apparent similarity was observed in their 3' non-coding sequences except for the first 75 bases and the expected length of the complete sequences. These results, together with the immunochemical data, indicate that P-450 human-2 is closely related, but not identical, to P-450mp, and may belong to the category of developmentally regulated constitutive cytochrome P-450s.View full abstractDownload PDF (1141K) -
Hiroyuki KOJIMA, Kazuhiko TAKAHASHI, Fumio SAKANE, Jiro KOYAMA1987 Volume 102 Issue 5 Pages 1083-1088
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNADPH-cytochrome c reductase [NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4] was highly purified from the membrane fraction of porcine polymorpho-nuclear leukocytes by column chromatographies on DEAE cellulose DE-52, 2', 5'-ADP-agarose, Sephacryl S-300, and Bio-gel HTP. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, the purified preparation gave a main band with a molecular weight of 80, 000. The enzyme contained 0.79 mol of FAD and 0.88 mol of FMN per mol, and was capable of exhibiting a benzphetamine N-demeth-ylation activity in the presence of cytochrome P-450 purified from rabbit liver microsomes and dilauroylphosphatidylcholine, as is the case with liver NADPH-cytochrome P-450 reductase. The cytochrome c reductase activity of the polymor-phonuclear leukocytes (PMN) enzyme was precipitated with rabbit anti-guinea pig liver NADPH-cytochrome P-450 reductase IgG followed by addition of guinea pig anti-rabbit IgG antibody. The biochemical and immunological properties of the PMN enzyme so far examined were similar to those of the liver enzyme, although its function in leukocytes has not yet been determined.View full abstractDownload PDF (468K) -
Kazuhiro KIYONO, Keiji MIURA, Youichi KUSHIMA, Takeshi HIKIJI, Miyuki ...1987 Volume 102 Issue 5 Pages 1089-1100
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn open reading frame of 828 base pairs was found in the CH01 gene region of Saccharomyces cerevisiae by nucleotide sequencing analysis. Its enhanced expression with the aid of the PHO5 regulatory sequence resulted in an overproduction of a protein with a molecular weight of approximately 30, 000, which in turn was con-verted by proteolysis to active phosphatidylserine synthase, whose molecular weight was approximately 23, 000. The larger protein was concluded to be the primary product of the CH01 gene, since its amino-terminal sequence was identical to that deduced from the nucleotide sequence of the above open reading frame, except for the terminal methionine residue. A partial homology in primary structures was noticed between this yeast enzyme and phosphatidylglycerophosphate synthase of Escherichia coli which also uses CDP-diacylglycerol as a substrate. The overpro-duced phosphatidylserine synthase in both microsomal and extensively purified fractions displayed two different Km values for L-serine, i.e., 0.14 mM at low L-serine concentrations and 9.5 mM at high L-serine concentrations. This may indicate a negatively cooperative regulation of this enzyme activity or the presence of two active components with different affinities for L-serine.View full abstractDownload PDF (2206K) -
Hiromu MUROFUSHI, Susumu KOTANI, Hiroyuki AIZAWA, Shohei MAEKAWA, Hiko ...1987 Volume 102 Issue 5 Pages 1101-1112
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA heat-stable microtubule-associated protein (MAP) with a molecular weight of 190, 000, termed 190-kDa MAP, has been purified from bovine adrenal cortex (Murofushi, H. et al. (1986) J. Cell Biol. 103, 1911-1919). Immunoblotting experiments using an antibody against this MAP revealed that several kinds of culture cells derived from human tissues contain proteins with an apparent molecular weight of 180, 000 reacting with the antibody. Indirect immunofluorescence microscopic observation of HeLa cells showed that the immunoreactive protein co-exists with microtubules, indicating that the protein is one of the HeLa MAPs. A heat-stable MAP with a molecular weight of 180, 000, termed here HeLa 180-kDa MAP, was purified by the taxol-dependent procedure (Vallee, R. B. (1982) J. Cell Biol. 92, 435-442) and successive co-polymerization with brain tubulin. This protein was the most abundant MAP in HeLa cells, suggesting that the MAP is identical to the major HeLa MAP previously reported by Bulinski and Borisy (Bulinski, J. C. & Borisy, G. G. (1980) J. Biol. Chem. 255, 11570-11576) and Weatherbee et al. ((1980) Biochemistry 19, 4116-4123). It was shown that, like bovine adrenal 190-kDa MAP, yet distinct from brain MAP2 and tau, purified HeLa 180-kDa MAP does not interact with actin filaments. This common characteristic of the two MAPs along with the same heat-stability strongly suggests that they are members of the same group of MAPs. The fact that HeLa 180-kDa MAP reacts with an antibody against bovine adrenal 190-kDa MAP means that they share common epitopes, in other words, common local amino acid sequences. However, the limited proteolytic patterns of the two MAPs with S. aureus V8 protease and chymotrypsin were distinct from each other, suggesting the presence of large differences in the overall primary structures between bovine adrenal 190-kDa MAP and HeLa 180-kDa MAP.View full abstractDownload PDF (4402K) -
Shozo SHOJI, Junichi OHNISHI, Takayuki FUNAKOSHI, Kohji FUKUNAGA, Eish ...1987 Volume 102 Issue 5 Pages 1113-1120
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe phosphorylation sites of myelin basic protein from bovine brain were deter-mined after phosphorylation with Ca2+-calmodulin-dependent protein kinase. Four phosphorylated peptides were selectively and rapidly separated by reversed-phase high-performance liquid chromatography. Partial sequencing of the phosphorylated peptides by automated Edman degradation revealed that Ca2+-calmodulin-dependent protein kinase phosphorylated serine-16, serine-70, and threonine-95 specifically, as well as serine-115, which is located on the experimental allergic encephalitogenic determinant of the protein. Of the four amino acid sequences determined, two sequences surrounding phosphorylated amino acids, -Lys-Tyr-Leu-Ala-Ser(P)16-Ala-and -Arg-Phe-Ser(P)115-Trp-Gly-, have both sides of each phosphoserine residue occupied by hydrophobic amino acids, and a basic amino acid, arginine or lysine, is located at the position 2 or 4 residues amino-terminal to the phosphoserine residue. In contrast, the two other sequences surrounding phosphorylated amino acids, -Tyr-Gly-Ser(P)70-Leu-Pro-Glu-Lys- and -Ile-Val-Thr(P)95-Pro-Arg-, have a basic amino acid at the position 2 or 4 residues carboxyl-terminal to the phosphoamino acid residue.View full abstractDownload PDF (581K) -
Yoichi NAKAMURA, Satoshi OGIHARA, Sachiya OHTAKI1987 Volume 102 Issue 5 Pages 1121-1132
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn H2O2-generating fraction was prepared from porcine thyroid homogenate by differential and Percoll-density gradient centrifugations. The fraction consisted of mainly fragmented plasma membranes as judged by marker enzyme analysis and electron microscopy. The fraction produced H202 by reaction with NADPH only in the presence of Ca2+. The Ca2+ concentration for half-maximal activation (KCa) was about 0. 1 μiM and the Hill coefficient was 2. Sr2+ also activated the reaction whereas Mn2+, Zn2+, and Cd2+ inhibited it. The reaction was enhanced about twice by addition of ATP but not ADP, and inhibited by addition of hex-okinase together with glucose to remove ATP. The Km value for NADPH was 35μM and was less than 1/12 that for NADH. The NADPH oxidation rate was measured and the KCa and the Km were similar to those for the H202 production. The stoichiometry between the oxidation and the H202 formation was essentially 1. Superoxide dismutase (SOD) and KCN did not affect H2O2, production. The fraction catalyzed NADPH-cytochrome c reduction but the activity was SOD-insensitive. These results suggest that H202 was not generated through superoxide anion formation. NADPH-dichloroindophenol (DCIP) reductase activity was also observed and DCIP inhibited the production of H202. The cytochrome c and DCIP reductase activities were not influenced by Ca2+ or ATP. A unique electron transport system regulated by Ca2+ and ATP exists in the thyroid plasma membrane that produces H202. The concentrations of Ca2+ and ATP in thyroid cells may regulate hormone synthesis through activation of the production of H202, a sub-strate for peroxidase.View full abstractDownload PDF (1605K) -
Masaru TANOKURA, Kumi GOTO, Yukiko TOYOMORI, Kazuhiro YAMADA1987 Volume 102 Issue 5 Pages 1133-1139
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe major isotype of parvalbumin has been isolated from the skeletal muscle of the toad, Bufo bufo japonicus. Unlike the skeletal muscle of every frog so far examined (Rana esculenta, Rana temporaria, and Rana catesbeiana), which contains two major isotypes of parvalbumins, toad skeletal muscle has been shown to contain only one isotype, but the content of parvalbumin in toad skeletal muscle was similar to the sum of those of the two isotypes in skeletal muscles of frogs. This feature of toad skeletal muscle is advantageous to clarify the physiological role of parvalbumin. The relative molecular mass of toad parvalbumin was estimated to be 12, 200 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was determined to be 4. 81 by polyacrylamide gel isoelectric focusing. The amino acid composition indicated that toad parvalbumin corresponds to bullfrog (R. catesbeiana) pI 4. 97 parvalbumin, showing that toad parvalbumin is genetically an a-parvalbumin. It was also revealed by the amino acid composition that toad parvalbumin is distinctly different from any of the parvalbumins from frogs. The ultraviolet spectrum of toad parvalbumin is consistent with its amino acid composition. The ultraviolet differ-ence spectrum of the Ca2+-loaded form vs. the metal-free form indicates that some Phe residues in the toad parvalbumin molecule are affected by a conformational change associated with Ca2+ binding. On electrophoresis in polyacrylamide gel in 14 mM Tris and 90 mM glycine, the metal-free and Mg2+-loaded forms of toad parvalbumin migrated twice as fast as the Ca2+-loaded form. The fact that toad parvalbumin shows decreased mobility in the Ca2+-loaded form is similar to the cases of bullfrog parvalbumins or calmodulin but is different from the case of tropo-nin C.View full abstractDownload PDF (1175K) -
Tetsuo MAITA, Kunihiko KONNO, Shinsaku MARUTA, Hajime NORISUE, Genji M ...1987 Volume 102 Issue 5 Pages 1141-1149
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of the essential light chain (abbreviated as SHLC) of adductor muscle myosin from Ezo giant scallop (Patinopecten yessoensis) was deter-mined by conventional methods. The light chain was composed of 156 amino acid residues with proline and lysine as its amino and carboxyl termini, respectively. Comparing this sequence with that of the SHLC from bay scallop (Aquipecten irradians), only 5 amino acid substitutions were recognized. The sequence homology between scallop and squid SHLCs was 53. 7%. On the other hand, a partially fragmented SHLC “modified SHLC'” reported by Konno and Watanabe (J. Bio-chem. 98, 141-148 (1985)) was prepared bychymotryptic digestion of the scallop myosin in the presence of EDTA, and was assigned as the carboxyl-terminal 106-residue peptide of the SHLC. This may suggest that the regulatory light chain covers the amino-terminal region of the SHLC in the myosin molecule.View full abstractDownload PDF (568K) -
Tetsuo MAITA, Hirofumi TANAKA, Kunihiko KONNO, Genii MATSUDA1987 Volume 102 Issue 5 Pages 1151-1157
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of the regulatory light chain of mantle muscle myosin from squid (Todarodes pacifieus) was determined by conventional methods. It was: xA-E-E-A-P-R-R-V-K-L-S-Q-R-Q-M-Q-E-L-K-E-A-F-T-M-I-D-Q-D-R-D-G-F-I-G-M-E-D-L-K-D-M-F-S-S-L-G-R-V-P-P-D-D-E-L-N-A-M-L-K-E-C-P-G-Q-L-N-F-T-A-F-L-T-L-F-G-E-K-V-S-G-T-D-P-E-D-A-L-R-N-A-F-S-M-F-D-E-D-G-Q-G-F-I-P-E-D-Y-L-K-D-L-L-E-N-M-G-D-N-F-S-K-E-E-I-K-N-V-W-K-D-A-P-L-K-N-K-Q-F-N-Y-N-K-M-V-D-I-K-G-K-A-E-D-E-D.
The a-amino group of this light chain was blocked, and a typical calcium-binding structure was recognized at the sequence of residue 26 to residue 37, like those in other myosin regulatory light chains.View full abstractDownload PDF (427K) -
Takashi SHIMIZU1987 Volume 102 Issue 5 Pages 1159-1165
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe substrate specificity of the 22S dynein ATPase from Tetrahymena cilia was investigated. The 22S dynein exhibited a high specificity for ATP in terms of both apparent Km and Vmax: naturally occurring nucleoside triphosphates other than ATP were hydrolyzed slowly with an apparent Km of 0.25-1 mM, a sharp contrast to that of ATP hydrolysis (1-4 μM). Pyrophosphate was a poor inhibitor for the dynein ATPase, indicating weak affinity. Since dynein binds ATP tightly and hydrolyzes it at a high rate, a method to determine a trace amount of ATP in the presence of other nucleoside triphosphates has been developed by taking advantage of this enzymatic characteristic of dynein. The effect of P1, P5-di(adenosine-5'-)- pentaphosphate (Ap5A) on the 22S dynein ATPase was also investigated. Ap5A acted as a weak competitive inhibitor of the ciliary 22S dynein ATPase and the non-linearity of the double-reciprocal plot of the ATPase was confirmed in the presence of Ap5A.View full abstractDownload PDF (599K) -
Bruno VENERANDO, Amelia FIORILLI, Luigi CAIMI, Guido TETTAMANTI1987 Volume 102 Issue 5 Pages 1167-1176
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytosolic sialidase A, obtained from pig brain and purified, interacts with ganglio-side GT1b giving two catalytically inactive enzyme-ganglioside complexes. Treat-ment of these complexes with Triton X-100 under given conditions (1% detergent ; 1 h at 37°C; 0.1M acetic acid-sodium acetate buffer, pH 4.8) leads to the liberation of part of the enzyme (about 47%) in a free and fully active form. Reversible inactivation of cytosolic sialidase requires the presence of homogeneous micelles of GT1b or of mixed micelles (for instance Triton X-100 and GT1b) with a high GT1b content. Triton X-100/ganglioside mixed micelles with a molar ratio above 50, as well as small unilamellar vesicles of egg yolk lecithin and GT1b (7-15 mol%), did not inactivate the enzyme at all; on the contrary these forms of ganglioside dispersion behaved as excellent substrates for the enzyme. It is to be concluded that under in vitro conditions the ability of ganglioside to interact with cytosolic sialidase, giving rise to catalytically inactive complexes or to Michaelis-Menten enzyme-substrate complexes, depends on the supramolecular organization of the ganglioside molecules. Arrangements of tightly packed molecules with strong side-side interactions facilitate the formation of complexes with the enzyme; arrange-ment with separated and loosely interacting molecules facilitates binding at the catalytically active site of the enzyme.View full abstractDownload PDF (845K) -
Shigenori KAIDA, Toshiyuki MIYATA, Yukio YOSHIZAWA, Shun-ichiro KAWABA ...1987 Volume 102 Issue 5 Pages 1177-1186
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe entire staphylocoagulase gene of Staphylococcus aureus strain BB was cloned on a MboI restriction endonuclease fragment inserted into pAT153 plasmid vector. The staphylocoagulase was expressed in Escherichia coli, as judged by the formation of a fibrin halo on an agar plate containing rabbit plasma and bovine fibrinogen. We have determined the complete nucleotide sequence of the staphylocoagulase gene by the dideoxynucleotide chain termination method. The deduced amino acid sequence consisted of 715 residues including a signal peptide of 26 residues.Therefore, the predicted molecular weight of the mature protein was 77, 337. This sequence was corroborated by reference to the amino acid compositions of 30 lysyl endopeptidase peptides and the sequences of 12 of these peptides isolated from the purified staphylocoagulase. The 5'-flanking region was found to contain a putative Shine-Dalgarno sequence and a putative “-10” element for transcription. The COOH-terminal stretch of 216 amino acids of staphylocoagulase was composed of 8 tandem repeats each consisting of 27 amino acid residues. The amino acid se-quence of staphylocoagulase derived from strain BB showed 57% identity with that of the chymotryptic 43-kDa fragment of staphylocoagulase isolated previously from strain 213 (Kawabata, S., Miyata, To., Morita, T., Miyata, Ta., Iwanaga, S., & Igarashi, 1-1. (1986) J. Biol. Chem. 261, 527-531).View full abstractDownload PDF (708K) -
Paratropomyosin, a New Myofibrillar Protein, Weakens Rigor Linkages Formed between Actin and MyosinlKoui TAKAHASHI, Minoru YAMANOUE, Tomoyuki MURAKAMI, Takanori NISHIMURA ...1987 Volume 102 Issue 5 Pages 1187-1192
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have used an enzymatic technique to determine the weakening effect of paratro-pomyosin, a new myofibrillar protein, on rigor linkages formed between actin and myosin, and to clarify the distinct function of paratropomyosin, as to that of tropo-myosin. Paratropomyosin inhibited the Mg-ATPase activity and enhancedtheK-ATPase activity of reconstituted actomyosin stoichiometrically, and its maximal binding to actin was estimated to occur at a molar ratio of 1: 12.5. Paratropomyosin also inhibited the myofibrillar Mg-ATPase activity by 49% and enhanced the myo-fibrillar K-ATPase activity to 126%, while tropomyosin had no effect on these ATPases. These results indicate that paratropomyosin is able to bind to thin filaments of myofibrils, because the binding site for paratropomyosin on F-actin is different from that for tropomyosin, and that, due to its greater affinity for the myosin binding site on actin, paratropomyosin competes for the binding site and helps weaken rigor linkages.View full abstractDownload PDF (432K) -
Yumiko MIZUSHIMA, Hiroshi KOSAKA, Shozo SAKUMA, Keiko KANDA, Kazuyuki ...1987 Volume 102 Issue 5 Pages 1193-1201
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe in vitro stimulation of murine splenic T lymphocytes with concanavalin A (Con A) produced interleukin 2 (IL2). The addition of cyclosporin A (CsA) to the culture resulted in complete inhibition of IL2 production. The Con A stimulation of T lymphocytes induced the breakdown of phosphatidylinositol into inositol trisphosphate and diacylglycerol, each of which could function as the second mes-sengers in the subsequent signal transduction pathway. CsA did not inhibit the production of inositol (poly)phosphates. Further, CsA did not affect Ca2+-calmodulin functions; a) the redistribution of various cytoskeletal proteins as well as Con A-receptor aggregation, and b) the cytosolic Ca2+-calmodulin-dependent en-zyme activities. Moreover, the activity of protein kinase C, which has been accepted to be the target of diacylglycerol, was not influenced in the presence of CsA. While the above steps of signal transduction are bypassed by synergy between calcium ionophore and phorbol ester, T lymphocyte activation which was induced by such stimuli was completely inhibited by CsA. These results indicate that CsA does not influence early steps of T lymphocyte activation as bypassed by calcium ionophore and phorbol ester, but rather inhibits later step(s) subsequent to the activation of protein kinase C and Ca2+-calmodulin.View full abstractDownload PDF (782K) -
Yoshichika KITAGAWA, Eiji OKUHARA1987 Volume 102 Issue 5 Pages 1203-1212
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe formation of the triple helix of poly(A)•poly(U)•poly(U)was studied by using antibodies specific to poly(A)-poly(U)•poly(U). The 10-11 base chain length for oligo(A) and the 20-30 base chain length for oligo(U) may be the minimum sizes required to maintain a stable triple helix. Double-stranded poly(A)•poly(U) which was the core of triple-stranded poly(A)•poly(U)•poly(U) could bind poly(U) and produce an analogue of poly(A)•poly(U)•poly(U) reactive with the antibodies even if the poly(A) or poly(U) was brominated or acetylated to the extent of 35-55%. However, brominated or acetylated poly(U) did not produce a stable triple helix with double-stranded poly(A)•poly(U).View full abstractDownload PDF (747K) -
Osamu MINOWA, Yoichi OHBA, Yusuke MIZUNO, Hiroyuki SHIOKAWA1987 Volume 102 Issue 5 Pages 1213-1220
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of chicken muscle acylphosphatase isozyme Chl was determined. The protein consists of 102 amino acid residues, does not contain histidine, and the NH2-terminus is acetylated: Ac-Ser-Ala-Leu-Thr-Lys-Ala-Ser-Gly-Se-Leu-Lys-Ser-Val-Asp-Tyr-Glu-Val-Phe-Gly-Arg-Val-GIn-Gly-Val-Cys-Phe-Arg-Met-Tyr-Thr-GIu-Glu-Glu-Ala-Arg-Lys-Leu-Gly-Val-Val-Gly-Trp-Val-Lys-Asn-Thr-Ser-GIn-Gly-Thr-Val-Thr-Gly-Gln-Val-Gln-Gly-Pro-Glu-Asp-Lys-Val-Asn-Ala-Met-Lys-Ser-Trp-Leu-Ser-Lys-Val-GIy-Ser-Pro-Ser-Ser-Arg-Ile-Asp-Arg-Thr-Lys-Phe-Ser-Asn-Glu-Lys-Glu-Ile-Ser-Lys-Leu-Asp-Phe-Ser-Gly-Phe-Ser-Thr-Arg-Tyr-OH. This sequence differs in 44% of the total positions from the other isozyme (Ch2) of chicken muscle acylphosphatase (Ohba et al., the accompanying paper). The sequence of Chl has three substitutions from that of turkey muscle acylphospha-tase; these are Ser from Ala at position 9, Ser from Arg at 47, and Lys from Asn at 83. The sequence has about 80% homology with those of mammalian muscle acylphosphatases.View full abstractDownload PDF (562K) -
Yoichi OHBA, Osamu MINOWA, Yusuke MIZUNO, Hiroyuki SHIOKAWA1987 Volume 102 Issue 5 Pages 1221-1229
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of one, Ch2, of the two isozymes of chicken muscle acyl-phosphatase was determined. It consists of 98 amino acid residues with N-acetyl-alanine at the amino(N)-terminus and contains no cysteine: Ac-Ala-Gly-Ser-Glu-Gly-Leu-Met-Ser-Val-Asp-Tyr-Glu-Val-Ser-Gly-Arg-Val-Gln-Gly-Val-Phe-Phe-Arg-Lys-Tyr-Thr-G1n-Ser-Glu-Ala-Lys-Arg-Leu-Gly-Leu-Val-Gly-Trp-Val-Arg-Asn-Thr-Ser-His-Gly-Thr-Val-Gln-Gly-Gln-Ala-Gln-Gly-Pro-Ala-Ala-Arg-Val-Arg-Glu-Leu-Gin-Glu-Trp-Leu-Arg-Lys-Ile-Gly-Ser-Pro-Gln-Ser-Arg-Ile-Ser-Arg-Ala-Glu-Phe-Thr-Asn-Glu-Lys-Glu-Ile-Ala-Ala-Leu-Glu-His-Thr-Asp-Phe-Gln-Ile-Arg-Lys-COOH. The sequence differs in 44% of the total positions from the other isozyme, Chl. Comparison of the sequence and the predicted conformational profile of Ch2 with those of Chl suggests that they share a common evolutionary origin and appear to have retained similar conformations throughout their evolutionary development.View full abstractDownload PDF (679K) -
Sumio ISHIJIMA, Masaaki TAGUCHI, Keisuke HIRAI, Katsura IZUI, Yuziro N ...1987 Volume 102 Issue 5 Pages 1231-1240
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwelve hybridoma clones which secrete monoclonal antibodies (mAb) against purified phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli K-12 were obtained. These 12 mAb were prepared from the ascites fluids of mice. Six among the 12 mAb formed precipitin lines with the enzyme on immunodiffusion. Four mAb inhibited the activity of the enzyme and 2 mAb enhanced it. Four mAb altered the sensitivity of the enzyme to allosteric effectors. Competitive enzyme-binding experiments among the 12 different mAb were also performed. The results showed that the 12 mAb can be classified into at least 8 groups.View full abstractDownload PDF (1334K) -
Ayae HONDA, Kenji UÉDA, Kyosuke NAGATA, Akira ISHIHAMA1987 Volume 102 Issue 5 Pages 1241-1249
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding sites for influenza viral RNA polymerase on genome RNA segments were investigated. Ribonucleoprotein (RNP) cores containing the RNA polymerase were isolated from detergent-treated virions by glycerol gradient centrifugation. On ApG-primed in vitro transcription by the isolated RNP cores, different levels of RNA transcripts were synthesized for the eight RNP cores, suggesting an uneven distribution of the RNA polymerase. 3'-Terminal labeling of the RNP cores with the use of [32P] pCp and T4-RNA ligase indicated a reciprocal correlation between the levels of the RNA-3' label and RNA synthesis. Centrifugation of detergent-treated virions in a double gradient of cesium trifluoroacetate (or cesium chloride) and glycerol yielded RNA polymerase-RNA complexes devoid of NP, the major RNA-bound protein, but the pattern of RNA-3' labeling remained virtually unaffected. All these observations together indicated that the RNA polymerase is associated near the 3' termini of some viral RNA segments, thereby preventing the in vitro labeling of the RNA-3' ends. The results of foot-printing experiments using RNase V1 and RNase T2 were in agreement with this model.View full abstractDownload PDF (3482K) -
Yoko OKAMOTO, Yoshiko MINAMI, Hiroshi MATSUBARA, Yasutomo SUGIMURA1987 Volume 102 Issue 5 Pages 1251-1260
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA photosynthetic c-type cytochrome, cytochrome c6, was extracted from a green alga, Bryopsis maxima, by cutting and immersing the frozen thalli in phosphate buffer, pH 7.0, and purified by acrinol treatment, ammonium sulfate fractionation, DEAE-Sephacel chromatography and Bio-Gel P-10 gel filtration. The ferrocytochrome c6 has absorption maxima at 553.5 (α), 523 (β), 417 (γ), 318 (δ), and 275 nm, and the ferricytochrome at 695, 528, and 411 (γ). The molecular weight was estimated to be about 10, 000 from Sephadex G-75 gel filtration and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The midpoint redox potential for the cytochrome was determined by equilibrium titration with a ferroand ferricyanide system to be 0.385 volt at pH 7.0. Isoelectric points for ferroand ferricytochromes were determined by density gradient isoelectric focusing electrophoresis to be at pH 3.91 and 4.02, respectively. The complete amino acid sequence of the cytochrome was determined by Edman degradation and by carboxypeptidase digestions of the Cm-cytochrome, 6 staphylococcal protease peptides and 5 lysyl endopeptidase peptides. The cytochrome contained 88 amino acid residues, giving a molecular weight of 9, 904 including 1 mol of heme c. The sequence is as follows: GGDLEIGADVFTGNCAACHAGGANSVEPLKTLNKED-V T KY LD G G LSIEAITSQVRNGKGA MPAWSDRLDDEEIDGVVAYVFKNINE-GW. A phylogenetic tree of 13 algal cytochromes c6 was constructed by comparing the amino acid differences.View full abstractDownload PDF (1204K) -
Akio IWASAKI, Makoto SUDA, Hiroshi NAKAO, Takao NAGOYA, Yushi SAINO, K ...1987 Volume 102 Issue 5 Pages 1261-1273
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn inhibitor of blood coagulation, a new protein with an apparent molecular weight of 34, 000 and an isoelectric point of 4.9, was purified from human placental tissue by EDTA extraction. Five cDNA clones were isolated from the human placental λgt11 cDNA library using the mouse monoclonal antibody raised against the coagulation inhibitor as the probe. The longest insert consists of 1, 566 nucleotides, and contains 960 nucleotides entirely encoding the 320 amino acids of the inhibitor, and a poly A tail. The deduced amino acid sequence was corroborated by chemical analyses of the protein. The entire amino acid sequence shows homology to those of lipocortin I, lipocortin II, andendonexin-related proteins. The cDNA for the inhibitor was expressed in Escherichia coli under the regulation of the trc promotor of the plasmid pKK233-2. The resulting recombinant protein manifested inhibitory activities against both blood coagulation and phospholipase A2 activity, as did the coagulation inhibitor isolated from human placenta.View full abstractDownload PDF (1605K) -
Peng WANG, Satoshi TOYOSHTMA, Toshiaki OSAWA1987 Volume 102 Issue 5 Pages 1275-1287
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA soluble inositolphospholipid-specific phospholipase C (PI-phospholipase C) has been purified 5, 800-fold from the cytosolic fraction of calf thymocytes. The puri-fication was achieved by sequential column chromatographies on DEAE-Sepharose CL-6B, heparin-Sepharose CL-6B, Sephacryl S-300, Mono S, and Superose 12, followed by column chromatography on Sephadex G-100 in the presence of 1% sodium cholate. The enzyme thus purified was found to be homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The mo-lecular weight of the enzyme was estimated to be 68 kDa by SDS-PAGE. The enzyme is specific for inositol phospholipids. Phosphatidylinositol and phospha-tidylinositol 4, 5-bisphosphate (PIP2) were hydrolyzed, but phosphatidylcholine and phosphatidylethanolamine were not affected by the enzyme. GTPγS-binding activity was detected in the enzyme fractions after all the purification steps, but not in the final enzyme preparation. The PI-phospholipase C and GTPγS-binding activities in the partially purified enzyme preparation could be separated by the column chro-matography on Sephadex G-100 only in the presence of 1 % sodium cholate. Thus, the soluble PI-phospholipase C has affinity to a GTP-binding protein. SDS-PAGE of the GTP-binding fractions eluted from the Sephadex G-100 column gave three visible bands of 54, 41, and 27 kDa. The 54 kDa polypeptide was specifically ADP-ribosylated by pertussis toxin. Furthermore, it was found that GTP and GTPγS (10μM and 1 mM) could enhance the PIP2 hydrolysis activity of the partially purified enzyme in the presence of 3 mM EGTA, but the purified enzyme after separation from the GTP-binding activity was not affected by GTP and GTPγS. The soluble PI-phospholipase C of calf thymocytes may be not only physically but also functionally associated with a GTP-binding protein.View full abstractDownload PDF (2265K) -
Hiroshi SAKAMOTO, Mutsuhito OHNO, Kunio YASUDA, Kiyohisa MIZUMOTO, Yos ...1987 Volume 102 Issue 5 Pages 1289-1301
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn in vitro splicing system was constructed using portions of chicken δ-crystallin pre-mRNA synthesized in vitro and a HeLa nuclear extract. Analysis of the reaction products revealed that about 25 of the pre-mRNA was precisely spliced at 30°C in 2h under the standard conditions. The other major products of the reaction detected were a 5'-exon fragment and three RNA species showing unusual electrophoretic mobilities on polyacrylamide gels. Structural analyses showed that these three RNAs contain a branch (lariat) structure as seen in the in vitro splicing reactions of human β-globin, adenovirus, and yeast pre-mRNAs. In addition, methylation at the N-7 position of the blocking guanosine of the 5'-terminal cap structure of pre-mRNA has been suggested to play an important role in the splicing reaction.View full abstractDownload PDF (5751K) -
Naoto OKU, Ryuichi ARAKI, Hiroko ARAKI, Sayumi SHIBAMOTO, Fumiaki ITO, ...1987 Volume 102 Issue 5 Pages 1303-1310
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDepartment of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University The effect of human tumor necrosis factor (TNF) on the permeability properties of liposomal membranes was investigated. TNF caused an increase in permeability of liposomes containing phosphatidylserine at pH 5-6, as demonstrated by the calcein efflux. However, it did not induce any permeability change in such liposomes at neutral pH. The TNF-induced calcein efflux was also observed when an other acidic lipid was used as a component of the liposomes, i.e., phosphatidic acid or dicetyl phosphate. On the other hand, liposomes composed of neutral phospholipids such as phosphatidylcholinc, phosphatidylethanolamine, and sphingomyelin showed little increases in permeability when incubated with TNF above pH 5.0. The TNF-induced permeability change was inhibited by the addition of polyaspartic acid, while it was not affected by the presence of 0.5 mM calcium ions. These data suggest that the negative charges on the liposomal surface trigger the interaction between TNF and liposomes. However, when the pH of the reaction mixture was decreased to 4.5, TNF-inducedcalcein efflux was observed even from neutral liposomes. When TNF wasincubated with 8-anilinonaphthalene-1 -sulfonic acid, the fluorescence intensityof this fluorophore increased with a decrease in the pH of the solution from 7 to 5, and a drastic increase in fluorescence was observed at pH 4.5. These data suggest that the hydrophobic region of TNF is also important for liposomal damage. Furthermore, the potencies of TNF and its derivative as to the induction of theView full abstractDownload PDF (568K) -
Retsu MIURA, Kozo YAMAICHI, Kunio TAGAWA, Yoshihiro MIYAKE1987 Volume 102 Issue 5 Pages 1311-1320
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLimited proteolysis of brewer's yeast old yellow enzyme (OYE) was carried out with bovine pancreatic α-chymotrypsin. The reaction proceeded with a decrease of the NADPH oxidase activity, generating specifically two peptides (designated as 34K and 14K fragments) with apparent molecular weights of 34, 000 and 14, 000, respec-tively. The same proteolytic treatment of apo OYE resulted in rapid and complete digestion of the protein. The 34K and 14K fragments are so intimately associated with each other that the isolation of each peptide from the other in the native form was unsuccessful. However, the complex of the two fragments was separated from the intact OYE and termed “nicked OYE.” Nicked OYE still retained FMN and showed a visible-absorption spectrum slightly modified from that of intact OYE. Nicked OYE showed decreased affinity toward p-bromophenol as compared to intact OYE. Nicked OYE exhibited lower Km. and Vmax values than intact OYE in theNADPH oxidase reaction. The 34K and 14K fragments could be separated from each other by reversed-phase FIF'LC under denaturing conditions and the amino acid sequences of the two fragments and intact OYE in the amino terminal regions were determined. The N-terminal sequence of the 34K fragment coincided with that of intact OYE, indicating that the 34K fragment lies in the N-terminal side of OYE. The N-terminal sequence of the 14K fragment was found to show homology with the site of flavodoxin where it forms an electron-transfer complex with cyto-chrome c. The characteristic feature of this region is the presence of acidic residues and is shared by the FMN domain of NADPH-cytochrome P-450 reductase. We interpret these findings as indicating that OYE has a physiological role as an electron transfer component.View full abstractDownload PDF (2216K) -
Hiromi TAKANO-OHMURO, Yuichi OGASAWARA, Takashi OBINATA1987 Volume 102 Issue 5 Pages 1321-1327
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel embryo-specific myosin light chain of 23 kDa molecular weight (L23) was found previously in embryonic chicken skeletal, cardiac, and smooth muscles (Takano-Ohmuro et al. (1985) J. Cell Biol. 100, 2025-2030). When we examined myosin in embryonic and adult brain by two-dimensional electrophoresis, 23 kDa myosin light chain present in brain (Burridge & Bray (1975) J. Mol. Biol. 99, 1-14) comigrated with L23. Two monoclonal antibodies, EL-64 and MT-185d, were applied to clarify the identity of the brain 23 kDa myosin light chain and the chicken embryonic muscle L23. The two antibodies recognize different antigenic deter-minants in the L23 molecule; the former antibody is specific for L23, whereas the latter recognizes the sequence common to fast skeletal muscle myosin light chains 1 and 3, and also L23. The immunoblots combined with two-dimensional gel electrophoresis showed that both EL-64 and MT-185d can bind to the brain 23 kDa myosin light chain as well as the chicken embryonic muscle L23. These results indicate that chicken brain and chicken embryonic muscles contain a common myosin light chain of 23 kDa molecular weight.View full abstractDownload PDF (2274K)
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