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Volume 107 (1990)
- Issue 6 Pages 799-
- Issue 5 Pages 661-
- Issue 4 Pages 507-
- Issue 3 Pages 331-
- Issue 2 Pages 173-
- Issue 1 Pages 1-
Volume 107, Issue 4
Displaying 1-29 of 29 articles from this issue
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Masahiro Abe, Katsuhito Takahashi, Kunio Hiwada1990 Volume 107 Issue 4 Pages 507-509
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method for the co-purification of calponin and caldesmon from bovine aorta smooth muscle was established involving heat treatment, ammonium sulfate fractionation (0-30 and 30-50%), CM52 column chromatography, and Ultrogel AcA44 gel filtration. The yields of calponin and caldesmon from 200g of smooth muscle were 17 and 32mg, respectively. This simplified, fast, and efficient method for the purification of calponin and caldesmon constitutes a useful tool for studying thin filament-linked regulation of smooth muscle contraction.View full abstractDownload PDF (1126K) -
Shoichi Ishiura, Kiichi Arahata, Toshifumi Tsukahara, Ritsuko Koga, Ha ...1990 Volume 107 Issue 4 Pages 510-513
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mdx mouse is an animal model for X-linked Duchenne muscular dystrophy. A polyclonal antibody against a synthetic peptide IV equivalent to the C-terminal portion (amino acids 3495 3544) of dystrophin crossreacted with a 400kDa protein in the brain and the spinal cord of mdx mouse, as well as in the control B10 mouse. However, the protein did not crossreact with the polyclonal antibody raised against the N-terminal portion of dystrophin peptide I (amino acids 215-264). Immunofluorescent micrography revealed that the outside of the small arteries and the pia mater of the brain strongly reacted with the anti-peptide IV antibody. These results strongly suggest the presence of a crossreactive protein other than dystrophin, possibly a dystrophin-related autosomal gene product, in the pia mater.View full abstractDownload PDF (2557K) -
Takashi Kuzuhara, Yuki Nakajima, Kenji Matsuyama, Shunji Natori1990 Volume 107 Issue 4 Pages 514-518
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSapecin is a 40-residue peptide containing 6 half-cystine residues. The disulfide structure of sapecin was determined by sequencing cystine-containing peptides obtained by digesting sapecin with thermolysin. Results showed that sapecin has a vortical structure fixed by 3 disulfide bonds between cysteine residues 3 and 30, 16 and 36, and 20 and 38, respectively, and that these disulfide bonds are essential for its antibacterial activity.View full abstractDownload PDF (497K) -
Tamio Teramoto, Tsuyoshi Watanabe, Teruhiko Matsushima, Yukio Horie, H ...1990 Volume 107 Issue 4 Pages 519-525
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe characterized the lipoproteins produced by perfused rat liver in recirculating and non-recirculating systems. The apolipoprotein (apo) B of the perfusate very low density lipoprotein (VLDL) and low density lipoprotein (LDL) were labeled with a radioactive precursor amino acid in both systems, suggesting that newly synthesized apo B was secreted in association with VLDL and LDL. When the lipoproteins obtained from the non-recirculating perfusate were injected into rats in vivo, the half life of the VLDL was 13min and most of it was converted to LDL, while that of the LDL was 5.2 h, indicating that the perfusate LDL was different from the VLDL with respect to its metabolic fate. These observations suggest that both VLDL and LDL are produced as independent primary products in the liver, although the majority of LDL is derived from VLDL in vivo. The nascent lipoproteins in the non-recirculating perfusate were richer in apo E than those in the recirculating perfusate, and a part of the apo E disappeared when the VLDL was added to the recirculating perfusate. The particle sizes of the VLDL and LDL were examined by electron microscopy, which revealed that those in the non-recirculating perfusate were more homogeneous and smaller than the plasma counterparts, while those in the recirculat-ing perfusate were more heterogeneous and their mean diameter was closer to that of the plasma lipoproteins, than in the case of non-recirculating perfusate. These observations suggest that apo E secreted with the nascent lipoproteins may be picked up by the liver just after secretion, causing the heterogeneity in size, as observed in the case of plasma lipoproteins.View full abstractDownload PDF (2277K) -
Shuzo Otani, Isao Matsui-Yuasa, Hitoshi Goto, Seiji Morisawa1990 Volume 107 Issue 4 Pages 526-529
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Ca2+ ionophore A23187 induced small increases in ornithine decarboxylase activity and ornithine decarboxylase mRNA in guinea pig lymphocytes. 1, 2-Dioctanoylglycerol potentiated the A23187-induced ornithine decarboxylase activity and the accumulation of mRNA for this enzyme. Dibutyryl cAMP also potentiated the enzyme activity, but had little effect on the accumulation of mRNA. 1, 2-Dioctanoylglycerol and 12- O-tetradecanoylphor-bol-13-acetate potentiated ornithine decarboxylase activity that had been increased by treatment with both A23187 and dibutyryl cAMP with a consistent increase in the ornithine decarboxylase mRNA. However, dibutyryl cAMP augmented ornithine decarboxylase activity that had been increased by the combination of A23187 and 1, 2-dioctanoylglycerol without affecting the ornithine decarboxylase mRNA level. These results suggest that the protein kinase C and cyclic AMP pathways are involved in the enhancement of ornithine decarboxylase activity in guinea pig lymphocytes, but that the mechanisms of the enhance-ment differ for each pathway, the former increasing the ornithine decarboxylase mRNA level, but not the latter.View full abstractDownload PDF (3199K) -
Jae-Eon Jung, Hyoungman Kim1990 Volume 107 Issue 4 Pages 530-534
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe water-soluble form of apoproteolipid (APL) from bovine brain myelin was found to bind with phosphatidylcholine (PC)/phosphatidylethanolamine (PE) (6 : 4) vesicles below pH5.The protein bound to vesicles was photoactively labeled with 3-(trifluoromethyl)-3- (m- [125I]iodophenyl)diazirine (125ITID) and was digested with trypsin.A[125I]TID-labeled fragment with an apparent molecular weight of approximately 2, 500 was extracted. An APL fragment with an identical Mr value was also obtained from the tryptic digest of APL/vesicle complex without prior labeling with [125I]TID. Determination of amino acid composition and the identification of the N-terminal amino acid residue of this unlabeled fragment showed that this protected segment covers the amino acid residues from Met-205 to Lys-228. In another experiment, the[125I] TID-labeled APL obtained from the above experiment without the proteolysis step was extracted and reconstituted into PC vesicles. Subsequent tryptic digestion of the exposed segment and comparison of the elution profile of the extracted polypeptides on a Sephadex LH-60 column with the published profile of these polypeptides indicated that the membrane-inserted segment of the water-soluble form of APL when bound to vesicles is the C-terminal region of this apoprotein within the amino acid residues between Met-205 and Lys-268.View full abstractDownload PDF (1891K) -
Tomoko Kozu, Kaoru Nikaido, Tatsuo Yagura1990 Volume 107 Issue 4 Pages 535-538
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe molecular masses of two of the four DNA polymerase α-primase complex subunit peptides from various mammalian cells have been compared through the use of specific monoclonal antibodies. One monoclonal antibody (E4) binds to 77-kDa peptide from HeLa cells and cognate peptides from other mammalian cells (monkey, mouse, bovine, Indian muntjac, and hamster). Another monoclonal antibody (A5) binds the 180-kDa type peptide and its degradation product (160-kDa peptide) of the mammalian DNA polymerase α-primase complexes. Neither of these antibodies reacts with DNA polymerase α-primase complex from chicken cells. Comparative immunoblot analysis indicates that the molecu-lar masses of the two main peptides of DNA polymerase α-primase complex isolated from the various mammalian sources are in excellent agreement with each other, except for the 77-kDa type peptide from bovine and Indian muntjac cells which was found to be significantly smaller (68 kDa) in these cases. The small molecular mass of bovine 77-kDa type peptide is not attributable to the action of a protease which may be present in the extract of bovine cells.View full abstractDownload PDF (3113K) -
Seiji Tsuboi, Mariko Kobayashi, Mayumi Nanba, Sanae Imaoka, Shinji Ohm ...1990 Volume 107 Issue 4 Pages 539-545
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe contents of S-(1, 2-dicarboxyethyl)glutathione (DCE-GS) in several tissues of rat were determined by HPLC. The peptide was present at concentrations (nmol/g tissue) of 119 in lens, 71.6 in liver, and 27.4 in heart. It was, however, not detected in spleen, kidney, cerebrum, or cerebellum. In rat liver, DCE-GS was located primarily in the cytosolic fraction. The substrates for the enzymic synthesis of DCE-GS were GSH and L-malate. In rats, the DCE-GS- synthesizing activity was found to be highest in the liver and in the cytosol of rat liver subcellular fractions. The DCE-GS-synthesizing enzyme was partially purified from rat liver cytosolic fraction by ammonium sulfate fractionation, Phenyl Superose chromatography, hydroxyapatite chromatography, and gel filtration. The molecular mass of the enzyme was estimated to be 53 kDa by gel filtration and SDS-PAGE, showing it to be a monomeric protein. The Km values for GSH and L-malate were 2.3 and 4.0 mM at 37°C, respectively. The enzyme did not utilize 1-chloro-2, 4-dinitrobenzene, 1, 2-dichloro-4-nitrobenzene, p-nitrophenyl bromide, trans-4-phenyl-3-buten-2-one, or p-nitrobenzyl chloride, which were substrates for previously characterized glutathione S-transferases. The isolated enzyme preparation showed no fumarase activity, which supported the conclusion that the formation of DCE-GS was not the result of a nonenzymic reaction following the synthesis of fumarate from L-malate by the isolated enzyme. The N-terminal amino acid of this polypeptide was presumably blocked since no sequence was obtained by automatic sequencing after electro-blotting onto a siliconized-glass fiber (SGF) sheet.View full abstractDownload PDF (1738K) -
Kaoru Omichi, Kouichi Shiosaki, Kenichi Matsubara, Tokuji Ikenaka1990 Volume 107 Issue 4 Pages 546-549
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman non-salivary, non-pancreatic α- amylase (yHXA) is the gene product of a newly found human α- amylase gene expressed in yeast. Its mode of action on a fluorogenic derivative of p-nitrophenyl α- maltopentaoside, FG5P (FG-G-G-G-G-P), was examined at various pH values to elucidate the difference between yHXA and pancreatic or salivary α- amylase. The product analysis of the digests by HPLC showed that the enzyme hydrolyzed FG5P to FG3 (FG-G-G) and p-nitrophenyl α- maltoside (G-G-P) and to FG4 (FG-G-G-G) and p-nitrophenyl α- glucoside (G-P), and the ratio of the two reactions changed with pH. The three enzymes differed from each other in the mode of action at pH 5.5. The molar ratio of FG4 to FG3 in the digest with yHXA was the largest. This suggested that the expression of the new gene in human can be detected by the use of FG5P as the substrate in the α- amylase assay.View full abstractDownload PDF (406K) -
Satoshi Kimura, Sandra Higham, Buena C. Robison, Naomi Kraus-Friedmann1990 Volume 107 Issue 4 Pages 550-553
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mechanism by which GTP induces Ca2+ release from Ca2+-preloaded rat hepatic microsomes was studied. In the same concentration range as that for Ca2+ release, GTP inhibited the initial rate of ATP-driven Ca2+ uptake. It also inhibited the formation by ATP of the phosphorylated intermediate of Ca2+-ATPase, which had previously been identified by us as a 97-116 kDa protein (Fleschner, C.R., et al. (1985) Biochem. J. 226, 839). Vanadate, an inhibitor of Ca2+ -ATPase, also caused Ca2+ release in a similar fashion, but its effect was not additive to that of GTP. Although the non-metabolizable GTP analogues, GMPPNP and GTPγS, did not cause Ca2+ release by themselves, GTPγS completely and GMPPNP partially blocked the effect of GTP. Pretreatment of vesicles with either cholera or pertussis toxin did not alter the responsiveness to GTP. These results indicate that GTP inhibits microsomal Ca2+-ATPase, independently of the Gs and G1, proteins. Because a decrease in Ca2+ uptake results in a net increase in Ca+ release, this effect of GTP seems to account, at least partially, for the GTP-induced Ca+ release from microsomes.View full abstractDownload PDF (1497K) -
Stefan Highsmith1990 Volume 107 Issue 4 Pages 554-558
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe concentration dependences of the activation of myosin subfragment-1 (S1) Mg-ATPase by the detergents CHAPS and Cl2E8 were determined at 23°C in 25 mM Tris (pH 7.0), 250 μM EDTA, 5 mM MgCl2, and 100μM ATP. At detergent concentrations expected to bind hydrophobic S1 surface areas equally, C12E8 caused an 8.5-fold greater increase in activity than CHAPS, which suggets that detergent binding to the surface of S1 is not the mechanism of activation. At detergent concentrations above their critical micelle concentrations, C12E8 was also much more effective than CHAPS, suggesting that micelles are not involved. A series of n-alcohols (which do not form micelles) with from 3 to 10 carbons all increased S1 Mg-ATPase activity as much or more than C12E8. The largest increase (5.7-fold) was caused by n-hexanol. The more hydrophobic alcohols activated S1 at lower concentrations. A linear plot of the alcohol concentration that caused 50% of maximum activity versus the number of carbons in the alcohol, indicated the apparent free energy of binding per CH2-group was-0.600±03 kcal/mol. There were two indications that alcohol binding caused an S1 conformational change. The intrinsic fluorescence increase of S1 during steady-state activity was reduced from 17.5 to 12.8%, and the apparent hydrodynamic rotational mobility of fluorescently labeled S1 was decreased 25% by the present of n-hexanol. The data suggest that S1 activation by C12E8 and by n-alcohols is due to hydrophobic binding to S1 at non-surface sites, which causes an S1 structural change.View full abstractDownload PDF (614K) -
Eiji Suzuki, Munehiro Maeda, Satoru Kuki, Martin C. Steward, Hiroshi T ...1990 Volume 107 Issue 4 Pages 559-562
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLongitudinal relaxation times (T1) of phosphorus compounds in the perfused rat heart and erythrocytes were measured using the 31P Driven-Equilibrium Single-Pulse Observation of T1 relaxation (DESPOT) method at 33°C. Both creatine phosphate in the heart and the three phosphate groups of adenosine triphosphate (ATP) in erythrocytes showed single-exponential relaxation. The three phosphate groups of ATP in the heart, however, had two T1 components. The T1 values of the short and the long T1 components of the β-phosphate of ATP were ca. 0.4 and 14 s, respectively. The fraction with the long T1 represented ca. 30% of the total ATP content. These results suggested that there were two major pools of intracellular ATP in the rat heart which could be determined by 31P NMR spectroscopy.View full abstractDownload PDF (430K) -
Shigeo Wakabayashi, Toshiaki Imagawa, Munekazu Shigekawa1990 Volume 107 Issue 4 Pages 563-571
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen a purified preparation of sarcoplasmic reticulum Ca2+-ATPase was labeled with 0.3 mM 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) in 1 mM AMPPNP and 1 mM CaCl2 at 25°C and pH 7.0 for 60 min and then was treated with 10 mM dithiothreitol for 7 min, about 1 mol of NBD was incorporated per mol of the enzyme, and this inhibited the enzyme activity by 90 to 95%. The modified residue was identified as Cys-344 that is located near the phosphorylation site of the ATPase, Asp-351. The NBD- inhibition of enzyme activity could be reversed by treatment with membrane-acting agents such as C12E8, suggesting that Cys-344 is not directly involved in enzyme catalysis. A detailed study of partial reactions of ATP hydrolysis by the modified enzyme and associated changes in the fluorescence intensity of the incorporated NBD label revealed that a predominant effect of the NBD-modification was the inhibition of Ca2+ release from the ADP-sensitive phosphoenzyme intermediate and that two major fluorescent states of the enzyme alternated during ATP hydrolysis. The latter fluorescent data are consistent with the E1-E2 model of Ca2+-ATPase reaction.View full abstractDownload PDF (1219K) -
Kunihiko Gekko, Hiroharu Ito1990 Volume 107 Issue 4 Pages 572-577
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe denaturation of lysozyme and ribonuclease A by guanidine hydrochloride was followed in the presence and absence of glycerol and sorbitol by means of circular dichroism measurements at 25°C. The protein-solvent interactions in the presence of these polyols were also studied by means of density measurements, for discussion of the mechanism of protein stabilization by polyols in terms of the multicomponent thermodynamic theory. The free energy of denaturation depends linearly on the molarity of guanidine hydrochloride at a given polyol concentration, without modification of the cooperativity of the transition. The free energy of denaturation at an infinite dilution of guanidine hydrochloride increases in proportion to the polyol concentration. The results indicate the competing solvent effects of polyols and guanidine hydrochloride on the structures of proteins. In water-protein-polyol systems, protein is preferentially hydrated to elevate its chemical potential, predominantly due to the unfavorable interaction of polyols with the exposed nonpolar amino acid residues. By linkage with the free energy of denaturation, it was quantitatively determined that the chemical potential of denatured protein is more extensively elevated by addition of polyols than that of native protein. These results demonstrate that polyols stabilize the protein structure through strengthening of the hydrophobic interaction, competing with the effect of guanidine hydrochloride.View full abstractDownload PDF (658K) -
Atsushi Irie, Hideo Kubo, Motonori Hoshi1990 Volume 107 Issue 4 Pages 578-586
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlucosylceramide (G1cβ1-1Cer) was isolated from the spermatozoa of the starfish, Asterias amurensis. The long-chain bases of the glycolipid consisted of dihydroxy (d18:2, d18:3, d19:3, and d22:2), and trihydroxy (t22:1) types. Long-chain aldehydes derived from them were analyzed mainly by proton nuclear-magnetic resonance to determine the detailed structures. Two of the tri-unsaturated bases were identified as (4E, 8E, 10E)-2-amino-4, 8, 10-octadecatriene-1, 3-diol (d18:3) and (4E, 8E, 10E)-2-amino-9-methyl-4, 8, 10-octa-decatriene-1, 3-diol (d19:3), which is a novel base. Both d22:2 and t22:1 had a cis double bond at the C9 or C13 position. All fatty acids were 2-hydroxylated (C14-C25):Most of themwere saturated and unbranched. About 10% was mono-unsaturated and unbranched (C22-C25), while saturated but branched (iso- and anteiso-types) C15-C18 acids were found as mi-nor components. The main fatty acids, which summed up to more than 93% of the fatty acids in the glucosylceramide, were n-14h: 0, n-15h:0, n-16h : 0, n-17h : 0, n-18h : 0, and n-24h:1.View full abstractDownload PDF (2115K) -
Masato Kinoshita, Haruhiko Toyohara, Yutaka Shimizu1990 Volume 107 Issue 4 Pages 587-591
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel latent proteinase of which activity was induced by heating in the presence of NaC1 was purified to homogeneity from threadfin-bream muscle by a combination of DEAE-cellulose, Con A-Sepharose, Arg-Sepharose, and Shim-pack HAC chromatographies. This proteinase was a glycoprotein having a monomeric subunit structure; Mr was estimated to be 77, 000 on SDS-PAGE analysis. The proteinase hydrolyzed Boc-Leu-Thr-Arg-MCA as well as myosin heavy chain in the presence of 2-4% NaCl at pH 7.0 and at 60°C, optimally. The proteinase was classified as serine proteinase based on the effects of soybean trypsin inhibitor, leupeptin, and antipain.View full abstractDownload PDF (2992K) -
Yukio Takishita, Kiyotaka Hiraiwa, Masaru Nagayama1990 Volume 107 Issue 4 Pages 592-596
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe obtained terminally differentiated chondrocytes in monolayer culture from chick embryonal growth plates, and examined the effect of retinoic acid on these cells. The cells treated with retinoic acid ceased type X collagen synthesis and showed decreased calcium incorporation into cell layers. Retinoic acid tended to stimulate proliferation of the cultured chondrocytes. It also increased DNA accumulation dose-dependently in the range from 1 nM to 1 μM. DNA synthesis in the growth phase and continency was stimulated within 10 h after addition of 0.1 μM retinoic acid. [3H]Retinoic acid binding, which was inhibited by simultaneous addition of excess unlabeled retinoic acid, was detected in both the cytosolic and nuclear fractions of the chondrocytes. The retinoic acid binding capacity of the nuclear fraction was increased by pretreating the cells with retinoic acid. These results indicate that retinoic acid binds to both the cytosolic and nuclear fractions of cultured chondrocytes, and induces their proliferation and dedifferentiation.View full abstractDownload PDF (4071K) -
Nobuhito Sone, Eiji Kutoh, Ko Sato1990 Volume 107 Issue 4 Pages 597-602
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cytochrome o-type oxidase from the thermophilic bacterium PS3 grown under airlimited conditions was purified by ion-exchange chromatography in the presence of a non-ionic detergent. The enzyme was composed of three subunits (60, 30, and 16 kDa) and seemed to contain two molecules of heme b as prosthetic groups. It contained no detectable copper. The reduced enzyme showed absorption bands at 426 and 558.5 nm, and a character-istic spectral change upon binding CO. It oxidized several cytochromes c and artificial dyes such as N, N, N', N'-tetramethyl-p-phenylenediamine and phenazonium methosulfate at appreciable rates. Its Km for O2 was low (0.09 μM). It was capable of transmembrane electron transfer, because when reconstituted into liposomes, it generated a membrane potential upon oxidation without pumping protons.View full abstractDownload PDF (1766K) -
Kouji Nakamura, Akira Nakamura, Hiromu Takamatsu, Hirofumi Yoshikawa, ...1990 Volume 107 Issue 4 Pages 603-607
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA 3.5-kb HindIII DNA fragment containing the sec Y gene of Bacillus subtilis has been cloned into plasmid pUC13 using the Escherichia coli sec Y gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained five open reading frames, and their order in the region, given by the gene product, was suggested to be L30-L15-SecY-Adk-Map by their similarity to the products of the E. coli genes. The region was similar to a part of the spc operon of the E. coli chromosome, although the genes for Adk and Map were not included. The gene product of the B. subtilis sect homologue was composed of 423 amino acids and its molecular weight was calculated to be 46, 300. The distribution of hydrophobic amino acids in the gene product suggested that the protein is a membrane integrated protein with ten transmembrane segments. The total deduced amino acid sequence of the B. subtilis Sec Y homologue shows 41.3% homology with that of E. coli SecY, but remarkably higher homologous regions (more than 80% identity) are present in the four cytoplasmic domains.View full abstractDownload PDF (1700K) -
Akira Yoshida, Masami Takahashi, Yoshiko Fujimoto, Haruhiko Takisawa, ...1990 Volume 107 Issue 4 Pages 608-612
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA monoclonal antibody designated as MAC-L1 immunoprecipitated [3H]PN200-110- labeled calcium channels of chick cardiac and skeletal muscle. On specific immuno-precipitation of 125I-labeled proteins, two large polypeptides (Mr 197, 000 and 139, 000 for heart, and 172, 000 and 135, 000 for skeletal muscle, under reducing conditions) were identified as the major components of these channels. Both polypeptides were found to exist together as a complex in 1% digitonin, but to become separated from each other in 1% Triton X-100. The 197 and 172 kDa peptides of cardiac and skeletal muscles, respectively, were photolabeled with [3H]azidopine. Under nonreducing conditions, the 139 kDa polypeptide of heart and the 135 kDa polypeptide of skeletal muscle took on larger molecular weights of 192, 000 and 190, 000, respectively. The 139 kDa but not the 197 kDa component of the heart was capable of binding to wheat germ agglutinin-Sepharose. Among the polypeptides specifically precipitated by MAC-L1, a 165 kDa peptide of skeletal muscle was phospho-rylated by cAMP-dependent protein kinase. In contrast, a minor 99 kDa polypeptide, but not the major 197 kDa polypeptide, of the heart was phosphorylated by this kinase. These results suggest that the dihydropyridine-sensitive cardiac calcium channel has αl and α2 subunits that are homologous but not identical to those of the skeletal muscle calcium channel.View full abstractDownload PDF (3052K) -
Kenji Hosoya, Yasuo Nagareda, Shuichi Hasemi, Akihiro Sanda, Yoshio Ta ...1990 Volume 107 Issue 4 Pages 613-618
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA pyrimidine base specific and most basic alkaline RNase named RNase BL4 was isolated from bovine liver as a protein showing a single band on slab gel-electrophoresis. The enzyme is most active at pH 7.5. The enzyme was immunologically distinguishable from the known bovine RNases such as pancreatic RNase (RNase A), seminal RNase, kidney non-secretory RNase (RNase K2), and brain RNase (RNase BRb). The primary structure of this pyrimidine base-specific RNase was determined to be < EDRMYQRFLRQHVDPDETG-GNDSYCNLMMQRRKMTSHQCKRFNTFIHEDLWNIRSICSTTNIQCKNGQMNCHEGVVRV-TDCRETGSSRAPNCRYRAKASTRRVVIACEGNPEVPVHFDK. It consists of 119 amino acid residues, and is 5 amino acid residues shorter than RNase A. The sequence homology of RNase BL4 with RNase A is 46.2%, and optimal alignment of RNase A and RNase BL4 requires five deletions, one at the 24th position, two at the 75th and 76th positions, and two at the C-terminus in RNase BL4. The RNase BL4 was highly homologous with a porcine liver RNase (RNase PL3, 94.1% homology) studied by Hofsteenge et al. (personal communi-cation from Hofsteenge, J., Matthies, R., and Stones, S. R.).View full abstractDownload PDF (668K) -
Marco Trinchera, Riccardo Ghidoni1990 Volume 107 Issue 4 Pages 619-623
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe demonstration of a precursor-product relationship in the course of GM1 and GD1a biosynthesis is described in the present paper. We injected rats with GM2 gangliosides [GalNAβ1-4(NeuAcα2→3)Galβ1-4Glcβ1-1'Cer] of brain origin, which were iso-topically radiolabeled on the Ga1NAc ([GalNAc-3H]GM2) or sphingosine ([Sph-3H]GM2) residue. We then compared the time-courses of GM1 and GD1a biosynthesis in the liver after the administration of each radiolabeled GM2 derivative. After the administration of [GalNAc-3H]GM2, GM1, and GD1a were both present as doublets, that could be easily resolved on TLC. The lower spot of each doublet was identified as a species having the typical rat brain ceramide moiety and represented gangliosides formed through direct glycosylation of the injected GM2. The upper spot of each doublet was identified as a species having the typical rat liver ceramide moiety and represented gangliosides formed through recycling of the [3H]GalNAc residue, released during ganglioside catabolism. After the administration of [Sph-3H]GM2, only ganglioside with the rat brain ceramide moiety were found, that represented the sum of ganglioside formed through direct glycosylation and those formed through recycling of some sphingosine-containing fragments. In each case, the time-course of GM1 and GD1a biosynthesis exhibited a precursor-product relationship. The curve obtained from the direct glycosylation showed a timing delay with respect to those obtained from recycling of GM2 fragments. These results are consistent with the hypothesis that the sequential addition of activated sugars to a sphingolipid precursor is a dissociative process, catalyzed by physically independent enzymatic activities.View full abstractDownload PDF (1572K) -
Ayae Honda, Jun Mukaigawa, Akiko Yokoiyama, Atsushi Kato, Susumu Ueda, ...1990 Volume 107 Issue 4 Pages 624-628
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe RNA-dependent RNA polymerase of influenza virus A/PR/8 was isolated from virus particles by stepwise centrifugation in cesium salts. First, RNP (viral RNA-NP-P proteins) complexes were isolated by glycerol gradient centrifugation of detergent-treated viruses and subsequently NP was dissociated from RNP by cesium chloride gradient centrifugation. The P-RNA (P proteins-viral RNA) complexes were further dissociated into P proteins and viral RNA by cesium trifluoroacetate (CsTFA) gradient centrifugation. The nature of P proteins was further analyzed by glycerol gradient centrifugation and immunoblotting using monospecific antibodies against each P protein. The three P proteins, PB1, PB2, and PA, sedimented altogether as fast as the marker protein with the molecular weight of about 250, 000 Da. Upon addition of the template vRNA, the RNA-free P protein complex exhibited the activities of capped RNA cleavage and limited RNA synthesis. When a cell line stably expressing cDNAs for three P proteins and NP protein was examined, the three P proteins were found to be co-precipitated by antibodies against the individual P proteins. These results indicate that the influenza virus RNA-dependent RNA polymerase is a heterocomplex composed of one each of the three P proteins and that the RNA-free RNA polymerase can be isolated in an active form from virus particles. Furthermore, the three P proteins form a complex in the absence of vRNA.View full abstractDownload PDF (2990K) -
Hisako Muramatsu, Takashi Muramatsu1990 Volume 107 Issue 4 Pages 629-634
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEmbryoglycan is the high-molecular-weight poly-N-acetyllactosamine characteristically and abundantly present in early embryonic cells. Among lectins reacting with poly-N-acetyllactosamines pokeweed agglutinin (PWA) was found to be most useful in analyzing glycoproteins from HM-1 pluripotent embryonal carcinoma (EC) cells, since virtually all glycoproteins carrying embryoglycan in these cells could be isolated by affinity chromatog-raphy on PWA-agarose. Furthermore almost all of embryoglycan from HM-1 cells hound to PWA-agarose. Since PWA-agarose used for the present study was confirmed to bind branched but not linear poly-N-acetyllactosamines, the above result indicated that em-bryoglycan lacking branched poly-N-acetyllactosaminyl chain was scarcely present in these cells. The same approach was used as a mean to show that embryoglycan with Lotus tetragonolobus agglutinin receptor activity also usually has the branched poly-N-acetyllactosamine structure in EC cells. Glycoprotein fractions from PYS-2 parietal endoderm cells and from STO fibroblasts also bound to PWA-agarose. However, the ratio of PWA binding fraction to the total [14C]galactose-labeled glycoproteins was less in these cells as compared to the value in EC cells, and the poly-N-acetyllactosamines from these cells exhibited lower molecular weights than embryoglycan. These results are consistent with our proposal that the complexity and abundance of poly-N-acetyllactosamines distinguishes EC cells from most other cells.View full abstractDownload PDF (707K) -
Ken-ichirou Morohashi, Yasuki Nonaka, Shirou Kirita, Osamu Hatano, Aki ...1990 Volume 107 Issue 4 Pages 635-640
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExpression plasmids were constructed using two cDNA clones of P-450(11β), pcP-450-(11β)-2, and pcP-450(11β)-3 (Morohashi et al. (1987) J. Biochem. 102, 559-568 and Kirita et al. (1988) J. Biochem. 104, 683-686), and introduced into COS-7 cells by electroporation. The expression of P-450(11β) proteins and their localization in the mitochondria were demonstrated by immunoblotting, immunofluorescence microscopy, and immunoelectron microscopy. The enzymatic activities of the expressed P-450(11β)s were determined using deoxycorticosterone (DOC), deoxycortisol, and corticosterone as substrates. Though the activities of the two P-450(11β)s for 11-, 18-, and 19-hydroxylation of DOC were almost equal, the production of 18-hydroxycorticosterone and aldosterone from corticosterone by P-450(11β)-3 was greater than that by P-450(11β)-2.View full abstractDownload PDF (2620K) -
Tomohiro Mega, Tokihiko Nishijima, Tokuji Ikenaka1990 Volume 107 Issue 4 Pages 641-644
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSβ-D-Galactopyranosylmethyl-p-nitrophenyltriazene (β-GalMNT), a specific inhibitor of β-galactosidase, was isolated as crystals by HPLC and its chemical and physicochemical characteristics were examined. Aspergillus oryzae β-galactosidase was inactivated by the compound. We studied the inhibition mechanism in detail. The inhibitor was hydrolyzed by the enzyme to p-nitroaniline and an active intermediate (β-galactopyranosylmethyl carbonium or β-galactopyranosylmethyldiazonium), which inactivated the enzyme. The efficiency of inactivation of the enzyme (the ratio of moles of inactivated enzyme to moles of β-GalMNT hydrolyzed by the enzyme) was 3%; the efficiency of Escherichia coli β-galactosidase was 49%. In spite of the low efficiency, the rate of inactivation of A. oryzae enzyme was not very different from that of the E. coli enzyme, because the former hydrolyzed β-GalMNT faster than the latter did. A. oryzae β-galactosidase was also inactivated by p-chlorophenyl, p-tolyl, and m-nitrophenyl derivatives of β-galacto-pyranosylmethyltriazene. However, E. coli β-galactosidase was not inactivated by these triazene derivatives. The results showed that the inactivation of A. oryzae and E. coli β-galactosidases by β-GalMNT was an enzyme-activated and active-site-directed irrevers-ible inactivation. The possibility of inactivation by intermediates produced nonenzymatically was ruled out for E. coli, but not for the A. oryzae enzyme.View full abstractDownload PDF (482K) -
Yoshihiko Maekawa, Hiroo Yasukawa, Bunsei Kawakami1990 Volume 107 Issue 4 Pages 645-649
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe genes of the BanI restriction-modification system specific for GGPyPuCC were cloned from the chromosomal DNA of Bacillus aneurinolyticus IAM1077, and the coding regions were assigned on the nucloetide sequence on the basis of the N-terminal amino acid sequences and molecular weights of the enzymes. The restriction and modification genes coded for polypeptides with calculated molecular weights of 39, 841 and 42, 637, respectively. Both the enzymes were coded by the same DNA strand. The restriction gene was located upstream of the methylase gene, separated by 21bp. The cloned genes were significantly expressed in E. coli cells, so that the respective enzymes could be purified to homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration indicated that the catalytically active form of the endonuclease was dimeric and that of the methylase was monomeric. Comparison of the amino acid sequences revealed no significant homology between the endonuclease and methylase, though both enzymes recognize the same target sequence. Sequence comparison with other related enzymes indicated that BanI methylase contains sequences common to cytosine-specific methylases.View full abstractDownload PDF (1532K) -
Yutaka Kikuchi, Masachika Irie, Hideharu Ikebuchi, Jun-ichi Sawada, Ta ...1990 Volume 107 Issue 4 Pages 650-654
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to determine the epitope of metallothionein (MT) to a murine monoclonal antibody (MT 189-14-7) which had been produced by immunization with rat MT 2 (Kikuchi et al. (1988) Mol. Immunol. 25, 1033-1036), various lengths of synthetic oligopeptides were tested for their inhibitory activities in competitive radioimmunoassay (RIA). The amino-terminal acetylated pentapeptide, AcMDPNC, exhibited an inhibitory activity comparable to that of native MTs, whereas the acetylated tetrapeptide, AcMDPN, and the deacetylated heptapeptide, MDPNCSC, were much less inhibitory. The results suggest that the major part of the epitope structure of MT to the MT 189-14-7 monoclonal antibody is located within the amino-terminal acetylated pentapeptide, AcMDPNC. The specificities of polyclonal rabbit anti-MT antisera raised against the same immunogen were also determined by using various animal MTs and synthetic peptides as inhibitors in the RIA. Among three antisera tested, two reacted with several amino-terminal oligopeptides similarly to the MT 189-14-7 antibody. The major epitope structures to these polyclonal antibodies were shown to be located within the acetylated tetrapeptide, AcMDPN. Another antiserum contained at least two different populations of antibodies: one consisted of antibodies reactive with the amino-terminal synthetic peptides, while the other was not reactive with them. These results suggest that, in the rabbit also, the amino-terminal region common to various animal MTs can be an epitope to antibodies raised against rat MT, as shown in the mouse. Moreover, the results indicate that the synthetic amino-terminal peptides are useful for determination of the specificity of polyclonal rabbit anti-MT antibodies, which have been widely used for the quantification of MTs.View full abstractDownload PDF (633K) -
Regulation of L-Type Pyruvate Kinase Gene Expression by Dietary Fructose in Normal and Diabetic RatsTamiko Matsuda, Tamio Noguchi, Masaru Takenaka, Kazuya Yamada, Takehik ...1990 Volume 107 Issue 4 Pages 655-660
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPrevious stuides have shown that dietary fructose stimulates expression of the L-type pyruvate kinase gene mainly at the post-transcriptional level, in diabetic liver, and that this effect may be mediated by a metabolite common to both fructose and glycerol. In the present work, we carried out further studies on the mechanism of fructose induction of L-type isozyme mRNA in the liver, kidney and small intestine of rats. The L-type isozyme mRNA in the kidney of normal and diabetic rats was increased by dietary fructose, the time course of the increase being similar to that observed in the small intestine and liver. The mRNA was not induced in the kidney by dietary glucose or insulin. Glycerol was also a potent inducer of the mRNA in the kidney and liver, but not in the small intestine. These results show that fructose and glycerol induced increase in the mRNA only in organs in which they were metabolized, and thus support the metabolite hypothesis. No other carbohydrates tested increased the level of mRNA in these tissues, except glucose, which increased the level in the small intestine. Thus a molecule that increase the mRNA level may accumulate significantly during metabolism of only certain carbohydrates. Dietary fructose or glycerol slightly stimulated transcription of the gene for the L-type isozyme in diabetic liver, and also in normal and diabetic kidney, but the magnitudes of increase were much lower than those of the mRNA, confirming our previous findings described above. On the other hand, dietary fructose caused marked stimulation of gene transcription in normal rat liver, although the magnitude of its induction of the mRNA was similar to that in diabetic liver. Insulin treatment of fructose-fed diabetic rats also caused a marked increase in the gene transcription without any concomitant change in the mRNA level. Thus, the mechanism of fructose induction of the L-type pyruvate kinase in diabetic liver, which is similar to that found in the kidney, is different from that in normal liver, and this difference is attributable to the difference in the level of insulin.View full abstractDownload PDF (4139K)
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