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Volume 106, Issue 1
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Nobuyuki Horie, Josephine Nalbantoglu, Sumiko Kaneda, Dai Ayusawa, Tak ...1989 Volume 106 Issue 1 Pages 1-4
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSA long Li repetitive sequence (3.6 kilobase pairs) was found in the third intron of the human thymidylate synthase gene. This Li family sequence is unique in that it possesses the longest open reading frame (1.7 kilobase pairs) of all Li family members identified in sequences associated with specific genes that have been cloned thus far. Furthermore, the amino acid sequence deduced from the open reading frame of the Li sequence was found to be highly homologous (90%) to that encoded by a known human teratocarcinoma Li RNA species, and to contain several blocks of sequences homologous to ones in RNA-dependent DNA polymerases of various origins.View full abstractDownload PDF (1404K) -
Hirofumi Aiba, Fujie Nakasai, Shoji Mizushima, Takeshi Mizuno1989 Volume 106 Issue 1 Pages 5-7
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe Escherichia coli OmpR protein is an activator protein specific for the ompF and ompC genes, which respectively encode the outer membrane proteins, OmpF and OmpC. The EnvZ protein is a protein kinase specific for the OmpR protein. In this study, we compared the in vitro DNA-binding ability of the phosphorylated form of the OmpR protein with that of the non-phosphorylated form by means of non-denaturing gel retardation analysis and DNase I footprinting analysis. The results indicate that the phosphorylation of the OmpR protein results in stimulation of its in vitro DNA-binding ability as to both the ompF and ompC promoter DNAs.View full abstractDownload PDF (1958K) -
Tomoyuki Kawase, Ichijiro Ishikawa, Michiaki Orikasa, Akitoshi Suzuki1989 Volume 106 Issue 1 Pages 8-10
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe effect of NaF on prostaglandin E2 (PGE2) synthesis in a clonal osteoblast-like cell line, MOB 3-4, was examined in the presence of Al3+. The MOB 3-4 cell line, which was derived from neonatal mouse calvaria, displays many osteoblastic characteristics, including the biosynthesis of PGE2. In the absence of Al3+, 1mM NaF increased PGE2 synthesis (per well) to about 340% of the control level, whereas NaF at lower concentrations (below 0.1mM) did not show such a significant effect. In the presence of 10μM Al3+, NaF concentrations ranging from 0.01 to 1mM increased PGE2 synthesis in a dose-dependent manner, though 10μM Al3+ had no effect by itself. Similar effects were observed on alkaline phosphatase (ALP) activity per well, but a stimulatory effect of NaF on protein synthesis was observed only in the presence of 10μM Al3+. These data demonstrated that PGE2 synthesis per protein was increased by NaF alone, and this effect was markedly enhanced by the addition of AlCl3. ALP activity per protein was, however, significantly increased by NaF in the absence of AlCl3. Taken together with our previous finding that Al3+ enhances the NaF-induced Ca2+ mobilization in MOB 3-4 cells, these results suggest that F-combined with Al3+ (i. e., AlF4-) is a more potent stimulator of PGE2 synthesis in cells than F- alone, and that the AlF4--enhanced PGE2 synthesis may be caused by an increase in cytosolic free Ca2+ concentration during activation of the G protein by A1F4-.View full abstractDownload PDF (752K) -
Chikahisa Takasaki1989 Volume 106 Issue 1 Pages 11-16
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSPa ID, a long-chain neurotoxin homologue, was isolated from the venom of an Australian elapid snake, Pseudechis australis, and its amino acid sequence was determined by conventional methods. Pa ID was an acidic protein (pI=6.2) and consisted of 68 amino acid residues. It did not show binding activity to the acetylcholine receptor of an electric ray (Narke japonica) nor lethal effect on mice, though the amino acid sequence is homologous with those of long-chain neurotoxins isolated from other elapid snakes (homology, 39-51%). In the sequence of Pa ID, a structurally invariant residue (Tyr-22) and two functionally invariant residues (Val/Ala-49 and Lys/Arg-50) in snake venom neurotoxins are replaced by a cysteine, an arginine, and a methionine residue, respectively, and furthermore, four common residues in long-chain neurotoxins, Gly-17, Ala-43, Ser-59, and Phe/His-66 are replaced by a glutamic acid, a threonine, a threonine, and a valine residue, respectively. The conformational change of the protein molecule caused by these replacements and the removal of a positive charge at position 50 are probably the reasons why Pa ID has lost the lethality.View full abstractDownload PDF (1604K) -
Keith R. Jarvie, Hyman B. Niznik1989 Volume 106 Issue 1 Pages 17-22
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSDopamine D2 receptor binding subunits of the porcine anterior pituitary were visualized by autoradiography following photoaffinity labeling with [125I] N-azidophenethylspiperone and sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The ligand binding subunit comprising the pituitary D2 dopamine receptor migrated as two distinct bands of apparent Mr≅150, 000 and 118, 000, substantially higher than neuronal D2 receptor subunits from porcine or canine brain. The glycoprotein nature of pituitary D2 receptor binding subunits was investigated by the use of exo- and endo-glycosidase treatments and peptide mapping experiments. Photoaffinity labeled polypeptides of the anterior pituitary were susceptible to both neuraminidase and α-mannosidase digestion as indexed by their increased electrophoretic mobility on sodium dodecyl-sulfate polyacrylamide gels, and suggests the presence of both complex type and terminal mannose carbohydrate residues. Moreover, the additive effects of sequential treatment with these enzymes suggests that both types of carbohydrate chains are present on each receptor peptide. N-linked deglycosylation of pituitary D2 photolabeled receptors with glycopeptidase-F produced a further increase in the mobility of the labeled protein to apparent Mrα44, 000, similar to that of deglycosylated D2 binding subunits of porcine and canine brain. Peptide mapping experiments following limited proteolysis with Staphylococcus aureus V8 proteinase and papain demonstrated that deglycosylated D2 dopamine receptors (Mrα44, 000), in different tissues and species, were homologous. Taken together, these data suggest that despite the differences in the overall molecular weight and tissue specific glycosylation pattern of pituitary D2 dopamine receptors, the primary structure of mammalian D2 receptors appears to be conserved.View full abstractDownload PDF (2904K) -
Masafumi Tsujimoto, Hideki Adachi, Shiho Kodama, Nobuo Tsuruoka, Yukio ...1989 Volume 106 Issue 1 Pages 23-28
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSRecombinant human interleukin 5 (rhIL-5) expressed in Chinese hamster ovary cells was purified and characterized. Molecular heterogeneity was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two major components of Mr around 40, 000 were detected under non-reducing conditions. However, under reducing conditions, the Mr of rhIL-5 was determined to be 22, 000 and 20, 000. After treatment with endoglycosidase F, a band with an apparent Mr of 18, 000 was observed. Treatment of rhIL-5 with 2-mercaptoethanol followed by N-ethylmaleimide resulted in its dissociation into a monomeric form. This alkylated rhIL-5 was biologically less active than intact rhIL-5. These results suggest that rhIL-5 exists as a dimer, and that the heterogeneity of rhIL-5 is mainly due to the difference in the content of carbohydrate. Moreover, the formation of disulfide bond (s) might be important for the biological activity of rhIL-5.View full abstractDownload PDF (3131K) -
Miya M. Shanahan, Eric B. Kmiec1989 Volume 106 Issue 1 Pages 29-33
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSWe have successfully uncoupled the previously interlocked activities of chromatin assembly and in vitro transcription promoted by the Xenopus oocyte S-150 cell-free extract. Our isolated fraction catalyzes extensive chromatin assembly measured both by changes in DNA topology and Micrococcal nuclease digestions. The assembly of chromatin is slowed by the exogenous addition of ATP. In the absence of exogenously added ATP, the fraction forms a chromatin template that is transcriptionally inert. Addition of small amounts of the HeLa cell extract (S-100) converts these templates into transcriptionally active ones without disrupting the chromatin structure. Our protocol defines a method for the isolation of a fraction from the Xenopus cell free extract that catalyzes the assembly of transcriptionally inactive chromatin. We characterize this reaction and establish conditions for the transcriptional activation of these inactive minichromosomes.View full abstractDownload PDF (2678K) -
Yasuo Suzuki, Kazuya Hidari, Makoto Matsumoto, Masa-aki Ikeda, Nobuo T ...1989 Volume 106 Issue 1 Pages 34-37
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSAlteration of ganglioside composition in mouse BALB/3T3 cells transformed either by DNA transfection with viral K-, H-, or cellular H-ras oncogene, or by infection with the K-ras oncogen-carrying murine sarcoma virus (Ki-KSV) was studied using a highly sensitive thin-layer chromatography/enzyme immunostaining method. Marked common decreases in the content of GD3 ganglioside and the increase of its metabolic precursor GM3, were bound in BALB/3T3 cell lines transformed by either K- or H-ras oncogenes. Moreover, a common decrease or loss in the contents of “A” series ganglio-tetraose gangliosides such as GM1a and GD1a was also found in all transformed cell lines, indicating that the alteration of cellular glycosphingolipids by ras oncogenes apparently does not depend on the type of rasconcogenes (K-and H-ras).View full abstractDownload PDF (1675K) -
Jeffrey J. Seilhamer, Shelley Plant, Waldemar Pruzanski, James Schilli ...1989 Volume 106 Issue 1 Pages 38-42
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSPhospholipase A2 (PLA2) has been purified to homogeneity from human arthritic synovial fluid. The activity resolved into multiple peaks by preparative HPLC. The most abundant peak (A) was present in synovial fluid from patients with rheumatoid arthritis, osteoarthritis, and psoriatic arthritis. A second major peak (B) was variable and lower in relative abundance, but was distinguishable from peak A by its stimulated activity in the presence of either 0.5M Tris or 0.1% sodium deoxycholate (DOC), in addition to its longer HPLC column retention time. Both peaks required Ca2+ and showed optimal activity in DOC/phosphatidylcholine (PC) mixed micelle assays between pH 8.0 and 9.0. Both peaks showed higher activity with PC as substrate than with PI, however peak A exhibited higher activity with PE than PC. Upon preparative SDS-polyacrylamide gel electrophoresis, both peaks of PLA2 activity were resolved as proteins of approximately 14, 000 Da. The N-terminal sequence obtained from purified peak A material matched that of a recent similar isolate.View full abstractDownload PDF (1907K) -
Akio Iwasaki, Makoto Suda, Masanao Watanabe, Hiroshi Nakao, Yukio Hatt ...1989 Volume 106 Issue 1 Pages 43-49
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSCalphobindin II, with Mr 73, 000, is one of the human placental anticoagulant proteins. The cDNA encoding calphobindin II was obtained by screening a human placental λ gt11 cDNA library using a specific antibody as a probe. The longest cDNA insert consisted of 2, 361 nucleotides and a 64-nucleotide-long poly (A) tract. An open reading frame encoding 673 amino acids was predicted. The deduced sequence includes an 8-fold repeat of a conserved 70-amino-acid-long segment that has a high degree of sequence identity with the repeated segments in members of the Ca2+-dependent phospholipid binding protein family. The cDNA fragment including the open reading frame was introduced into the expression vector pKK223-3 and subsequently expressed in Escherichia coli JM105 cells. The resulting recombinant protein reacted with the specific monoclonal antibodies to calphobindin II and prolonged the blood coagulation time as did placental calphobindin II.View full abstractDownload PDF (2345K) -
Kaoru Ichimura, Hiroshi Kihara, Takaki Yamamura, Kazuo Satake1989 Volume 106 Issue 1 Pages 50-54
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSChicken ovotransferrin, an iron binding protein, has two metal binding sites (amino (N) and carboxy (C) terminal sites). It binds Cu (II), Al (III), Co (II), and other metals, as well as Fe (III). In this study, the selectivity and cooperativity of the N and C sites on Al (III), Co (II), and Tb (III) binding were investigated. Metals were classified into two groups according to their site preference. Co (II) and Al (III) bound to the N site more preferably than to the C site, whereas Tb (III) bound to the C site more preferably. On Fe (III) binding, the binding constant of Fe (III) becomes larger when the other site is already occupied. Thus, positive cooperativity is seen. In the present study, the binding cooperativities of Co (II), Tb (III), and Al (III) as to the N and C sites were investigated. On Co (II) and Tb (III) binding, no cooperativity was observed, as in the case of Cu (II)[Yamamura, T. et al.(1985) in Proteins of Iron Storage and Transport (Spik, G., Montreuil, J., Crichton, R. R., & Mazurier, J., eds.) pp.53-56, Elsevier Science Publ. B. V., Amsterdam]. In contrast, negative cooperativity was observed on Al (III) binding. Based on a model proposed by Yamamura et al.[Yamamura, T. et al.(1985) in Proteins of Iron Storage and Transport (Spik, G., Montreuil, J., Crichton, R. R., & Mazurier, J., eds.) pp.53-56, Elsevier Science Publ. B. V., Amsterdam], the ratio of the binding constants, Kc/KN, and the stacking coefficient, Kst, were estimated. Kc/KN is 2.2±0.4 for the Tb (III) ion, 0.5±0.1 for the Co (II) ion, and 0.12±0.02 for the Al (III) ion. Kst (=1 in a non-cooperative case) is 0.98±0.02 for the Tb (III) ion, 1.03±0.02 for the Co (II) ion, and 0.55±0.22 for the Al (III) ion.View full abstractDownload PDF (1118K) -
Tomoyoshi Kobayashi, Takashi Takagi, Kazuhiko Konishi, Sachio Morimoto ...1989 Volume 106 Issue 1 Pages 55-59
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSTroponin C is the Ca2+-receptive protein located on the thin filament of striated and cardiac muscle. We have determined the amino acid sequence of troponin C obtained from porcine cardiac muscle by sequencing and aligning the lysyl endopeptidase and Staphylococcus aureus V-8 protease peptides. It was composed of 161 amino acid residues with a blocked N-terminus. The sequence of porcine cardiac troponin C was identical with that of bovine cardiac troponin C.View full abstractDownload PDF (730K) -
Akira Tsugita, Masaharu Kamo, Carl S. Jone, Noriko Shikama1989 Volume 106 Issue 1 Pages 60-65
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe ability to analyze amino acid derivatives at the femtomole level is one of the most interesting challenges in the field of protein microsequencing. 2-Anilino-5-thiazolinone amino acids, obtained by Edman degradation, were quantitatively derivatized with fluorescent primary amines. The most fluorescent reagent tested was 4-aminofluorescein. The amino acid derivatives sensitized with this reagent were separated using reversedphase high-performance liquid chromatography and identified at the 100 attomole level. Incorporation of this method into the operation of a conventional automated sequencer is also described.View full abstractDownload PDF (1673K) -
Protective Effect of Carbohydrate Moiety on Tryptic Hydrolysis and Heat DenaturationJainxin Gu, Tsukasa Matsuda, Ryo Nakamura, Hiroshi Ishiguro, Iwao Ohku ...1989 Volume 106 Issue 1 Pages 66-70
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSHen ovomucoid was chemically deglycosylated by treatment with trifluoromethanesulfonic acid at 0°C for 60 min. About 75 mol% of the carbohydrate moiety was removed from the glycoprotein without changing its amino acid composition, and its trypsin inhibitory activity and immunoreactivity with specific antibodies remained unchanged. The deglycosylated ovomucoid was inactivated and degraded easily by an excess amount of trypsin, whereas the native glycoprotein was not. Furthermore, the biological and immunological activities of the deglycosylated ovomucoid were lowered by heat treatment more easily than those of the native ovomucoid. These results suggest that the carbohydrate moiety of ovomucoid contributes to the stability of the ovomucoid molecule against tryptic hydrolysis and heat denaturation.View full abstractDownload PDF (2031K) -
Masatoshi Hagiwara, Hiroshi Tokumitsu, Koji Onoda, Toshio Tanaka, Masa ...1989 Volume 106 Issue 1 Pages 71-75
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSMonoclonal antibodies raised against chicken gizzard smooth muscle myosin light chain kinase were used for immunological and structural studies of this enzyme. Epitope mapping of trypsin-digested chicken gizzard enzyme showed that MM-1, 2, 3, 4, 5, 6, and 7 bind to 65kDa (trypsin-digested) and 60kDa (chymotrypsin-digested) fragments which contain the catalytic domain of the kinase. Kinetic analysis demonstrated that MM-7 inhibited kinase activity competitively with respect to ATP and noncompetitively with respect to myosin light chain, thereby indicating that MM-7 binds at or near the ATP binding site of the enzyme. Immunoblot analysis revealed that all these antibodies (MM-1 to 12) reacted with the enzyme (130kDa) from intestinal and vascular smooth muscles, whereas 5 (MM-1, 3, 4, 6, and 9) or 3 (MM-1, 3, and 4) of 12 antibodies did not cross-react with chicken cardiac muscle or with blood platelet myosin light chain kinase (130 kDa), respectively. None of these antibodies showed cross-reactivity against skeletal muscle myosin light chain kinase. As for mammalian species, MM-11 and 12 reacted with myosin light chain kinase of vascular smooth muscle (140kDa) and MM-11 cross-reacted with the enzyme (140kDa) from cardiac muscle of rat and rabbit. These data suggest the existence of at least 4 subspecies of myosin light chain kinase in chicken tissues and the heterogeneity of tissue-and species-specific isozyme forms.View full abstractDownload PDF (2914K) -
Hiroyuki Hashimoto, Haruo Misono, Shinji Nagata, Susumu Nagasaki1989 Volume 106 Issue 1 Pages 76-80
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe activation of lysine ε-dehy drogenase [EC 1.4.1.] by L-lysine was dependent on lysine concentration and was accompanied by association of the dimeric enzymes to a tetramer. The lysine concentration required for the half-maximal activation was 0.28mM, which was lower than the Km value for L-lysine. In addition to L-lysine, several compounds, which were neither substrates nor inhibitors, activated the enzyme. The compounds which activated the enzyme have common structural characteristics: they have both a carboxyl group and a hydrophobic side chain. These activators also induced the association of the enzyme. The activation of the enzyme occurred well over the pH range 5.0 to 7.5, and the maximal activation was obtained by preincubation for 5 min at 30°C and pH 7.4, when 5mM L-lysine or 6-aminocaproate was used as an activator. NADH binding experiments indicated that about 2mol of NADH bind to 1mol of the tetrameric enzyme: the dimeric enzyme has one catalytic site. Binding experiments with n-[1-14C] heptanoate and L-[U-14C] lysine showed that approximately 2 mol of ligands bind to 1mol of the dimeric enzyme and L-lysine could not bind to the catalytic site of the enzyme in the absence of NAD+. These results indicate the presence of one catalytic site and two activator binding sites in the dimeric enzyme.View full abstractDownload PDF (1338K) -
Tuneko Baba, Kenji Kaneda, Emi Kusunose, Masamichi Kusunose, Ikuya Yan ...1989 Volume 106 Issue 1 Pages 81-86
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe effect of growth temperature on mycolic acid composition in eight strains of Mycobacterium smegmatis was investigated by gas chromatography/mass spectrometry. A change in growth temperature from 45 to 20°C caused a shift in the subclass and molecular species composition of mycolic acids. The relative amount of α'-mycolic acids to α-mycolic acids decreased, and that of hydroxy mycolic acids increased at lower temperatures. Moreover, the proportion of shorter-chain species of α-mycolic acids increased, and those of longer-chain species of α-mycolic and hydroxy mycolic acids decreased. This observation seems to be due to the changes of the chain length of meromycolates because the α-alkyl chain unit of mycolic acids was not affected. The ratio of odd to even carbon-numbered α-mycolates decreased as the growth temperature was lowered. In contrast, the molecular species composition of α'-mycolic acid was not influenced by the growth temperature.View full abstractDownload PDF (2056K) -
Mieko Katsuura, Shigeaki Tanaka1989 Volume 106 Issue 1 Pages 87-92
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe mode of interaction between human epidermal growth factor (hEGF) and its receptor has been investigated by immunochemical studies and a synthetic peptide approach. Two types of monoclonal and five different monospecific polyclonal antibodies against hEGF have been prepared, whose epitopes are regions 1-13, 13-32, 33-53, 33-43, 22-32, and discontinuous sequences of hEGF. Antibody against 22-32 (Type I) and antibody against 33-53 (PRE 4) inhibited the binding of 125I-hEGF to membrane receptor on A 431 cells more markedly than the other antibodies. When hEGF was bound to the receptor, only antibody against 13-32 (PRE 2) could bind to hEGF-receptor complex whereas antibody against 22-32 (Type I) could not. These data suggest that region 13-20 is exposed outside during receptor-binding and both region 22-32 and region 33-53 contact the hEGF receptor. The activity of synthetic peptides corresponding to the amino acid residues 1-13, 13-32, 33-53, 13-20, 22-32, and 33-43 of hEGF was also examined. Out of the six peptides, only 13-32 stimulated DNA synthesis of BALB 3T3 cells. The activity was approximately 1/106 of that of intact hEGF. All of these data suggest that region 22-32 is responsible for binding to the receptor for signal transduction and region 33-53 binds to the receptor to stabilize the ligand-receptor interaction. This dual binding model fits in well with the threedimensional hEGF structure deduced from NMR spectra [Montelione, G.T. et al.(1987) Proc. Natl. Acad. Sci. U.S. 84, 5226-5230].View full abstractDownload PDF (1781K) -
Akira Nakamura, Takao Arai, Yoshiyuki Kondo, Toshihiro Fujii1989 Volume 106 Issue 1 Pages 93-97
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe effects of poly (L-aspartic acid)(PLAA) on microtubule assembly and microtubuleassociated protein (MAP) 2-actin interaction were examined in vitro. PLAA inhibited assembly of rat brain microtubules and induced rapid disassembly of already formed microtubules. Inhibition was stronger by PLAA with a high molecular weight than that by low molecular weight. The ratios of 47 kDa PLAA to microtubule proteins causing 50% inhibition of the assembly and disassembly were 0.015 and 0.04 (w/w), respectively. Both MAP 1 and MAP 2 were bound to a PLAA-Sepharose 4B affinity column, while tubulin was not retained by the column. PLAA caused selective dissociation of MAP 1 and MAP 2 from microtubules polymerized by taxol. It is therefore concluded that PLAA interacts specifically with MAPs. PLAA also inhibited the MAP 2 induced cross-linking of actin filaments, showing an almost complete inhibition at a PLAA to MAP 2 ratio of 1: 5, 000 (w/w). Binding experiments of PLAA with digested MAP 2 by chymotrypsin using affinity chromatography and sedimentation experiments showed that PLAA was preferentially bound to a 35 kDa fragment which includes the microtubule-and actin-binding domain of the MAP 2 molecule. These results suggest that PLAA suppressed the functions of MAP 2 through a domain which is located in the 35 kDa fragment.View full abstractDownload PDF (2434K) -
Kenji Horiuchi, Shoji Tajima, Masakazu Menju, Akira Yamamoto1989 Volume 106 Issue 1 Pages 98-103
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe mouse apolipoprotein E gene was isolated from a genomic library by screening with a cDNA probe. DNA including apolipoprotein E gene plus segments 2.5 kilobases upstream and 0.3 kilobase downstream of the coding region was transfected into NIH3T3 cells. The cells expressed the same-size apolipoprotein E mRNA and protein as those produced by mouse endogenously. The nucleotide sequence of the gene plus 5' and 3' flanking regions (one kilobase each) was determined. The sequence of the mouse apolipoprotein E gene was highly homologous to that of the rat gene, not only in the coding regions but also in the non-coding and intron regions. The mouse and the human apolipoprotein E genes were homologous in the 5' proximal flanking region up to about 200 nucleotides as well as in the four exons. This proximal region was highly conserved for the genes of mouse, rat and human; the relative positions of the “TATA box” and the two copies of “GC box” were identical.View full abstractDownload PDF (2055K) -
Kazuyo Ohashi, Kensuke Ishikawa, Koscak Maruyama1989 Volume 106 Issue 1 Pages 104-109
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSI-protein was mixed with myosin before or after myosin filaments were reconstituted. In both cases, I-protein seemed to accelerate the myosin assembly. The binding of I-protein to myosin filaments was tested by sedimentation experiments and SDS-polyacrylamide gel electrophoresis. In a low ionic strength solution at pH 6.5, the binding ratio of I-protein to myosin was 1: 40 by molar ratio when the I-protein molecules highly specifically bound to myosin filaments. I-protein could maximally bind to myosin filaments at the molar ratio of 1: 2.7. In this case, excess I-protein molecules remained in the supernatant after sedimentation, although the unbound I-protein could still bind to myosin filaments. Electron microscopic observations revealed that I-protein bundled myosin filaments in the low ionic strength solution (pH 6.5). Cage-like structures which were very similar to the Mgparacrystals of non-muscle myosins were formed at pH 7.2. In gel filtration, the apparent molecular mass of I-protein was 100 kDa, while it was 50 kDa in SDS gel electrophoresis. Therefore, I-protein is regarded to be a homodimer of a 50 kDa subunit and can divalently bind to myosin molecules.View full abstractDownload PDF (4344K) -
Kazuyo Ohashi, Koscak Maruyama1989 Volume 106 Issue 1 Pages 110-114
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSUsing an improved procedure, Z-protein was prepared from myofibrils of chicken breast muscle. The yield of pure Z-protein increased to 10mg per kg of muscle. The chain weight of Z-protein was 55, 000 in the presence of SDS. However, Z-protein was eluted before aldolase (Mr 158, 000) in Sephacryl S-400 column chromatography, and, therefore, it appeared to exist as a tetramer in a physiological salt solution. Z-protein had at least four isopeptides whose isoelectric points ranged from pH 6.0 to 6.4. Anti-Z-protein antiserum reacted equally with these isopeptides. The extinction coefficient of Z-protein at wavelength 278nm was 4.2 (1%; light path, 1cm). Z-protein which was purified according to this improved method did not bind to F-actin and α-actinin in a physiological salt solution.View full abstractDownload PDF (2685K) -
Kazuyo Ohashi, Koscak Maruyama1989 Volume 106 Issue 1 Pages 115-118
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSAntiserum against Z-protein, which is one of the components of myofibrillar Z-lines, strongly stained the epithelial cells of chicken small intestine at the apical tips of the microvilli and the junctional complexes. Immunoblot tests with anti-Z-protein antiserum showed that there is a peptide of the same antigenicity as myofibrillar Z-protein in the components of the epithelial cells of chicken small intestine. Hence it seems that the apical tip of the microvillus contains a constituent corresponding to the Z-protein of myofibrillar Z-line.View full abstractDownload PDF (2173K) -
Analysis of the Small-Angle Scattering DataSatoru Fujiwara, Yoji Inoko, Tatzuo Ueki1989 Volume 106 Issue 1 Pages 119-125
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSSmall-angle X-ray scattering experiments were carried out on rat thymus chromatin in “native” and “H1 -depleted” states at various NaCl concentrations using synchrotron radiation. From the analysis of cross-sectional Guinier plots, the radius of gyration of the cross section (Rc) and the mass per unit length (Mc) of native chromatin were evaluated. In the absence of NaCl, the cross section of chromatin filament has a radius of gyration of 3.44nm, suggesting the structure corresponding to the “10 nm” filament. With increasing NaCl concentration, the Rc, value increases steeply to 6.74 nm at 5 mM NaCl and then gradually to 8.82 nm at 50 mM NaCl, whereas the Mc value, which is determined relative to that of tobacco mosaic virus (TMV), increases steadily from 1.58 nucleosomes per 10 nm in the absence of NaCl to 7.66 nucleosomes per 10 nm at 50 mM NaCl. However, since calibration with TMV tends to overestimate the Mc value, the actual Mc values may be less than those values. Above about 40 mM NaCl, aggregation of chromatin is suggested. Similar analysis of H1-depleted chromatin confirmed that H1-depleted chromatin takes a more disordered structure than native chromatin at low ionic strength and does not undergo a definite structure change upon further addition of NaCl.View full abstractDownload PDF (2329K) -
Akira Hara, Kouji Mouri, Makoto Nakagawa, Mitsuhiro Nakamura, Toshihir ...1989 Volume 106 Issue 1 Pages 126-132
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSIndanol dehydrogenase was purified to apparent homogeneity from monkey liver cytosol. The enzyme was a monomer with a molecular weight of 36, 000 and pI of 8.7. The amino acid composition was determined. The enzyme oxidized alicyclic alcohols including transdihydrodiols of benzene and naphthalene in the presence of both NADP+ and NAD+, and reduced several xenobiotic carbonyl compounds in the presence of NADPH, the 4-pro-R hydrogen atom of which was transferred to the substrate. The results of fluorometric binding and kinetic studies are consistent with an ordered sequential mechanism with NADP+ binding first. The enzyme was inhibited competitively versus NADP+ and uncompetitively versus 1-indanol not only by chelating agents such as 1, 10-phenanthroline and 2, 2'-bipyridine but also by a nonchelating isomer, 4, 4'-bipyridine, which suggests hydrophobic interaction of the aromatic compounds with the enzyme, which did not contain zinc. The enzyme was also inhibited by Cibacron blue dye, synthetic estrogens, and Δ4-3-ketosteroids. The inhibition by Cibacron blue was competitive versus NADP+ and noncompetitive versus 1-indanol, whereas those by hexestrol, medroxyprogesterone acetate, and progesterone were uncompetitive versus NADP+ and competitive versus 1-indanol, corraborating the ordered addition of the coenzyme prior to 1-indanol.View full abstractDownload PDF (2256K) -
Christiane Levrat, Pierre Louisot, Renée Morelis1989 Volume 106 Issue 1 Pages 133-138
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe trypsin sensitivity of the mitochondrial N-acetylglucosaminyl and mannosyltransferase activities involved in the N-glycoprotein biosynthesis through dolichol intermediates as well as the N-acetylglucosaminyl-transferase activity involved in direct N-glycosylation were examined in mitochondria and isolated outer mitochondrial membrane preparations. The trypsin action on mitochondrial membrane was checked by measuring the activities of marker enzymes (rotenone-insensitive NADH cytochrome c reductase, adenylate kinase, and monoamine oxidase). Glycosyl-transferase activities of both N-glycosylation pathways were insensitive to trypsin action and consequently were located in the outer mitochondrial membrane. Based on the activator effect of the trypsin on these enzyme activities, the results suggested two distinct orientations of their active sites. As regards the N-glycoprotein biosynthesis pathway through dolichol intermediates, the dolicholphosphoryl-mannose and dolichol-pyrophosphoryl-di-N-acetylchitobiose synthases would be oriented outside while the oligomannosyl-synthase and the oligomannosyltransferase would be rather oriented inside in the outer membrane. The N-acetylglucosaminyl-transferase involved in the direct transfer of N-acetylglucosamine from its nucleotide donor to a proteinic acceptor would be oriented outside in the outer membrane.View full abstractDownload PDF (1360K) -
Jesús Vázquez, Gonzalo Ortega, Fernando Valdivieso, Fede ...1989 Volume 106 Issue 1 Pages 139-142
Published: July 01, 1989
Released on J-STAGE: June 07, 2011
JOURNAL FREE ACCESSGangliosides seem to play an important role in the interaction of the neurotoxic pigment bilirubin with the synaptosomal plasma membrane (Vázquez et al.[1988] J. Biol. Chem. 263, 1255-1265). In this report, a further characterization of the bilirubin-ganglioside interaction is presented. The interaction is fast, and it is observed at any pH in the range 7.0-9.0. The characteristics of the interaction are different from those observed with other membrane lipids, including sphingomyelin. A model of binding to a single population of sites is able to adequately fit the experimental data. This model predicts a decrease in the tendency of bilirubin to interact with gangliosides and an increase in the binding capacity as the pH is decreased from 8.0 to 7.0. Our data would suggest a role for gangliosides in explaining the preferential accumulation of bilirubin in some areas of the brain and the toxic effect of this pigment in neuronal membrane-related functions.View full abstractDownload PDF (975K) -
Roland Schauer, Gerd Reuter, Sabine Stoll, Ashok K. Shukla1989 Volume 106 Issue 1 Pages 143-150
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSFrom bovine brain an esterase was purified 2, 600-fold in an overall yield of 5.6%. For the isolation ion-exchange chromatographies, gel filtration, and preparative isoelectric focusing were used. The molecular mass is 56 kDa after gel chromatography on Sephacryl S-200 and 51 kDa after HPLC, the pH-optimum at 7.4, and the isoelectric point in the range of pH 5.8-6.1, as estimated from preparative isoelectric focusing. The substrate specificity of this enzyme was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. Besides aromatic acetyl esters such as e. g.α-naphthyl acetate, the highest preference was for N-acetyl-9-O-acetylneuraminic acid, followed by N-acety1-4-O-acetylneuraminic acid. Other primary acetyl esters such as 6-O-acetylated D-glucose and 2-acetamido-2-deoxy-D-mannose were not hydrolyzed. The 9-O-acetyl derivative of the naturally occurring unsaturated sialic acid 2-deoxy-2, 3-didehydro-N-acetylneuraminic acid, however, is a substrate for this esterase. Where as N-acetyl-9-O-acetylneuraminic acid as a component of sialyllactose is nearly as well hydrolyzed as the corresponding free sialic acid, O-acetylated sialoglycoconjugates with high molecular weights (mucins, serum glycoproteins, gangliosides) are not hydrolyzed by this esterase. N-Acetylated sialic acids are better substrates than the analogous N-glycoloylderivatives. Esterification of the carboxyl function of sialic acids prevents the action of the esterase on the O-acetyl groups. The enzyme has no carboxyl esterase or amidase activity, and does not act on acetylcholine. It hydrolyzes almost exclusively acetyl esters. Inhibition studies suggest that it has a catalytically active serine residue. Based on its substrate specificity and its preference for sialic acids among the natural substrates, the bovine brain esterase can be classified as sialate O-acetylesterase [EC 3. 1. 1. 53].View full abstractDownload PDF (2321K) -
Hiroyuki Takeya, Mitsue Arakawa, Toshiyuki Miyata, Sadaaki Iwanaga, Ta ...1989 Volume 106 Issue 1 Pages 151-157
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe complete amino acid sequence of and the locations of disulfide bridges in H2-proteinase, a major non-hemorrhagic proteinase isolated from the venom of the habu Trimeresurus flavoviridis, have been determined and compared with those of HR2a, one of the hemorrhagic metalloproteinases in this venom. The strategy involved consisted of structural analysis of peptides in digests with cyanogen bromide, lysyl endopeptidase, trypsin, Staphylococcus aureus V8 protease and thermolysin. Peptides were purified by gel filtration followed by reversed-phase HPLC. H2-proteinase is a non-glycosylated single chain polypeptide consisting of 201 amino acids with an amino-terminal pyroglutamic acid, a calculated molecular weight of 22, 991 and a net charge of + 14 at neutral pH. There was no evidence of heterogeneity of the sequence. H2-proteinase has a typical zinc-chelating sequence and its overall sequence identity with HR2a is 73.6%. The 3 disulfide bridges in H2-proteinase link Cys-117 to Cys-196, Cys-158 to Cys-180, and Cys-160 to Cys-163, in the same manner as in the case of HR2a. In striking contrast to HR2a, it contains en extra free cysteine residue at position 94 which becomes reactive to a sulfhydryl reagent in the presence of a denaturant.View full abstractDownload PDF (2128K) -
Masaaki Yamaguchi, Seiichiro Kinoshita, Norio Suzuki1989 Volume 106 Issue 1 Pages 158-162
Published: July 01, 1989
Released on J-STAGE: June 07, 2011
JOURNAL FREE ACCESSGastrullation of sea urchin embryos is arrested in sulfate-free sea water. This developmental arrest has been considered to be due to lack of sulfation of glycosaminoglycans in the extracellular matrix of the embryos. In the present study, we characterized a dermatan sulfate type component formed in gastrula-stage embryos of the sea urchin Clypeaster japonicus and examined the effects of sulfate deprivation on the formation. Glycosaminoglycans were prepared from gastrula-stage embryos incubated with [3H] acetate in normal and sulfate-free sea water. Enzymatic analyses indicated that embryos formed a glycosaminoglycan of the dermatan sulfate type which contained an N-acetylgalactosamine-6-sulfate-containing disaccharide as a major unit, plus a minor unidentified component. Under sulfate-free conditions, embryos formed an under-sulfated chondroitin/dermatan sulfate copolymer which mainly consisted of non-sulfate, glucuronic acid-containing (chondroitin) disaccharide units. These results suggest that sulfate deprivation diminishes not only the degree of sulfation but also the formation of L-iduronic acid-containing (dermatan) disaccharide units in dermatan sulfate in sea urchin embryos.View full abstractDownload PDF (3366K) -
Akikazu Hirashima, Saeko Sawaki, Takafumi Mizuno, Nicole Houba-Herin, ...1989 Volume 106 Issue 1 Pages 163-166
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSWe previously reported the utilization of antisense RNA in the development of a novel immune system against RNA coliphage SP proliferation (Hirashima et al.[1986] Proc. Natl. Acad. Sci. U. S. 83, 7726-7730). We attempted to determine the most effective (i. e., those eliciting antiviral activity) sequences for targeting micRNAs within the 5'-terminal noncoding region of 54 nucleotides (nt). It was found that a 30-nt micRNA against the sequence from base 32 to 61 exhibited nearly complete inhibition of phage production. Upon further dissection of this sequence, it was concluded that the most effective micRNA against phage SP production should contain the sequences complementary to the Shine-Dalgarno (SD) sequence of the first gene and its 13-nt upstream sequence. The addition of downstream sequences had little effect. These results suggest that the micRNA functions by preventing the binding of ribosomes to the SD sequence of the first gene. The addition of further upstream sequences had a significant negative effect on the micRNA function, indicating that the removal of such impeditive sequences from a micRNA is an important strategy for the development of a potent micRNA immune system.View full abstractDownload PDF (1026K) -
Ken-ichi Miyamoto, Tatsuzo Oka, Takeru Fujii, Masahiro Yamaji, Hisanor ...1989 Volume 106 Issue 1 Pages 167-171
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSOrnithine decarboxylase (ODC) was induced in rat small intestine by treatment with hypotonic solution in vitro and purified by two procedures, a conventional procedure and an immunoaffinity procedure. SDS-polyacrylamide gel electrophoresis showed that the molecular weight of the preparation purified by the immunoaffinity procedure (Mr=53, 000) was slightly larger than that of the preparation obtained by the conventional procedure (Mr=52, 000). Values for the Km for L-ornithine (0.1mM), the isoelectric point (5.4), and the final specific activity (5.1-5.5×105 nmol CO2/mg protein/30min) of the two preparations were similar to those reported for the rat liver ODC. Addition of a protease inhibitor (limabean trypsin inhibitor) to the crude extract prevented the appearance of the smaller enzyme (Mr=52, 000) obtained by the conventional purification procedure. Our result indicates that the large enzyme is native ODC and the smaller one is a partial proteolysis product of native ODC.View full abstractDownload PDF (2076K) -
Purification and CharacterizationHiroshi Abe, Takashi Obinata1989 Volume 106 Issue 1 Pages 172-180
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSIn embryonic skeletal muscle, a large amount of non-polymerized actin exists in the cytoplasm (Shimizu and Obinata [1986] J. Biochem. 99, 751-759). A 19-kDa protein (called 19K protein) which binds to G-actin was purified by sequential chromatography on DNase I-agarose, hydroxylapatite, SP-Sephadex, and Sephadex G-75, from the sarcoplasmic fraction of embryonic chicken skeletal muscle. This protein decreased the extent of actin polymerization at a steady state and increased the monomeric actin in a concentrationdependent fashion; it also caused quick depolymerization of F-actin, as determined by spectrophotometry at 237 nm, viscometry, DNase I inhibition assay, and electron microscopy. The molar ratio of 19K protein and actin interacting with each other was estimated to be 1: 1. From these results, 19K protein was regarded as being actin depolymerizing protein. The amount of 19K protein in muscle decreased during development. The inhibitory action of 19K protein was removed by myosin or heavy meromyosin, and actin filaments were formed on the surface of myosin filaments when myosin filaments were added to a mixture of actin and 19K protein in a physiological salt solution. We propose that actin assembly is dually controlled in the developing muscle by the inhibitor (s) and an accelerator (myosin); this mechanism may enable the ordered assembly of actin and myosin in the early phase of myofibrillogenesis.View full abstractDownload PDF (5862K) -
Submitochondrial Localization of Cytochrome c1 Whose Presequence is Replaced by the Amino-Terminal Region of a 70 kDa Outer Membrane ProteinMasato Nakai, Masayuki Harabayashi, Toshiharu Hase, Hiroshi Matsubara1989 Volume 106 Issue 1 Pages 181-187
Published: July 01, 1989
Released on J-STAGE: January 25, 2011
JOURNAL FREE ACCESSThe amino-terminal region of a 70 kDa mitochondrial outer membrane protein of yeast and the presequence of cytochrome c1, an inner membrane protein exposed to the intermembrane space, are thought to be responsible for localizing the proteins in their final destinations after synthesis in the cytosol. Gene fusion experiments were used to identify signals that are responsible for protein sorting between the outer and inner mitochondrial membranes. The submitochondrial localization of cytochrome c1 whose presequence was replaced by the amino-terminal region of the 70 kDa mitochondrial outer membrane protein has been investigated. We have also used an in vivo complementation assay to determine whether or not a 70k-cyt c1 fusion protein is functional. Both the first half and all of the presequence of cytochrome c1 can be replaced by the amino-terminal 12 or 29 residues of the 70 kDa protein for transport to the inner membrane and functional assembly into succinate-cytochrome c reductase. However, replacements by the amino-terminal 61 residues of the 70 kDa protein result in exclusive localization of the fusion proteins to the outer membrane, and the fusions cannot be assembled into the enzyme complex. These data indicate that a mitochondrial targeting signal alone is sufficient to direct cytochrome c1 of mature size to the inner membrane.View full abstractDownload PDF (3059K)
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