All issues
Volume 102 (1987)
- Issue 6 Pages 1329-
- Issue 5 Pages 963-
- Issue 4 Pages 673-
- Issue 3 Pages 445-
- Issue 2 Pages 243-
- Issue 1 Pages 1-
Volume 102, Issue 2
Displaying 1-27 of 27 articles from this issue
- |<
- <
- 1
- >
- >|
-
Masaharu KAMO, Akira TSUGITA1987 Volume 102 Issue 2 Pages 243-246
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe detergents, sodium dodecyl sulfate (SDS), lauryl glutamate (LG), and octyl-(polydisperse) oligooxyethylene (Octyl-POE), were tested as to their effects on theactivities of carboxypeptidases A, B, and P. In general, Octyl-POE showed little inhibition and SDS showed the strongest inhibition. Carboxypeptidase B was only slightly inhibited by SDS. The inhibitory effect of SDS on these enzyme activities depended not on its concentration but on its absolute amount. For a constant amount of SDS, the activities of carboxypeptidases A and B remained almost constant with increasing reaction volume. Commercial carboxypeptidase B is usually contaminated by carboxypeptidase A. With the addition of SDS, almost only carboxypeptidase B activity was detected.View full abstractDownload PDF (291K) -
Fumio SAKANE, Kazuhiko TAKAHASHI, Hajime TAKAYAMA, Jiro KOYAMA1987 Volume 102 Issue 2 Pages 247-253
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe membrane fraction of guinea pig polymorphonuclear leukocytes stimulated with phorbol myristate acetate exhibits the respiratory burst NADPH oxidase activity. This activity is markedly unstable at 37°C, disappearing with a half-life of 11.0 min. When the membrane fraction was pretreated with 0.1% glutaraldehyde, the NADPH oxidase was found to become more stable; its half-life increased about sixfold without any enhancement of the initial activity. The glutaraldehyde treat-ment of the membrane fraction also protected the NADPH oxidase against inactiva-tion with 0.1-0.2 % Triton X-100. These stabilizing effects of glutaraldehyde on the NADPH oxidase seem to be due to its protein cross-linking ability, since its monovalent analogue, butyraldehyde, did not show any effect on the NADPH oxidase activity. In fact, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that glutaraldehyde cross-linked many proteins constituting the membrane.View full abstractDownload PDF (539K) -
Hiroshi WAKAO, Takayoshi WAKAGI, Tairo OSHIMA1987 Volume 102 Issue 2 Pages 255-262
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn NADH dehydrogenase was purified to electrophoretical homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium optimally growing at pH 2-3 and 75°C. A 2, 100-fold purification was achieved. The purified enzyme is an acidic protein with an isoelectric point of 5.6 and a molecular weight of 95, 000, consisting of two 50, 000-dalton subunits. The enzyme showed an absorption spectrum characteristic of flavoproteins, with maxima at 272, 372, and 448 nm. The enzyme is highly thermostable, is specific for NADH as an electron donor, and is capable of using 2, 6-dichlorophenolindophenol, ferricyanide, benzoquinone, and naphthoquinone as electron acceptors. Though at a low rate, caldariellaquinone, a unique and sole benzothiophenequinone in the genus Sulfolobus, was also reduced by the enzyme, suggesting that the enzyme is a possible member of the respiratory chain of the thermoacidophilic archaebacterium.View full abstractDownload PDF (1044K) -
Hiroshi MORII, Haruhiko TAKISAWA, Taibo YAMAMOTO1987 Volume 102 Issue 2 Pages 263-271
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Ca2+-induced Ca2+ release channel in the heavy fraction of the sarcoplasmic reticulum (SR) from rabbit skeletal muscle is inactivated during ATP-dependent Ca2+ uptake (Morii, H., Takisawa, H., & Yamamoto, T. (1985) J. Biol. Chem. 260, 11536-11541). AMP, one of the adenine nucleotides which activate the Ca2+ release, delayed the onset of the channel inactivation when added early during the course of the Ca2+ uptake. However, AMP could no longer activate the channel but accel-erated the inactivation when added during the later phase of the Ca2+ uptake. In SR passively loaded with Ca2+, the Ca2+ channel which had been activated by AMP and Ca2+ was not spontaneously inactivated. Similarly, during GTP-dependent Ca2+ uptake, the channel activated by AMP was not inactivated. In addition acid phosphatase markedly delayed the onset of the inactivation during ATP-dependent Ca2+ uptake, without affecting Ca2+ ATPase activity or GTP-dependent Ca2+ uptake by heavy SR. The effect of the phosphatase was completely blocked by ruthenium red, a potent inhibitor of the channel. These results suggest that the channel is inactivated through an ATP-dependent process, presumably phosphorylation of proteins in the SR membrane. This was supported by the findings that the reactiva-tion of the inactivated channel by added Ca2+ was markedly accelerated by the addi-tion of acid phosphatase and that several proteins of heavy SR were phosphorylated during ATP-dependent Ca2+ uptake.View full abstractDownload PDF (1488K) -
Jin KONISHI, Masafumi YOHDA, Tadao HASHIMOTO, Masasuke YOSHIDA1987 Volume 102 Issue 2 Pages 273-279
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kinetical characteristics of ATP hydrolysis by mitochondrial F1-ATPase from Saccharomyces cerevisiae (yeast) have been studied under conditions where only a single catalytic site per enzyme molecule bound ATP. Four major features were observed, that is, fast ATP binding to the enzyme, slow product release from the enzyme, an equilibrium close to unity between ATP and products on the enzyme, and promotion of ATP hydrolysis on the second addition of a large excess of ATP (cold chase). These are essentially the same as the kinetical characteristics observed for beef heart mitochondrial F1-ATPase, which were called as unisite catalysis by Grubmeyer et al. (Grubmeyer, C. et al. (1982) J. Biol. Chem. 257, 12092-12100), although the release of a hydrolysis product, P1, from the yeast enzyme appeared to occur significantly faster than that from the beef enzyme, which resulted in a decreased extent of cold chase promotion of ATP hydrolysis of the yeast enzyme. The yeast F1-ATPase showed unisite catalysis even in the absence of P1 in the reaction mixtures, while it was reported for the beef F1-ATPase that the presence of P1 in the reaction mixture was essential for unisite catalysis (Penefsky, H. S. & Grub-meyer, C. (1984) in H+-ATPase (ATP Synthase) (Papa, S. et al., eds.) pp. 195-204, The ICSU Press). Another difference in the P1 effect on the kinetics was that ATP hydrolysis was initiated without a lag time in the absence of P1 in the case of the yeast enzyme when a 1, 000-fold molar excess of ATP per enzyme molecular was mixed with the enzyme. Thus the “P1-activation” observed for the beef enzyme may not be common to all F1ATPases.View full abstractDownload PDF (577K) -
Takao SHINOZAWA, Masahiro SOKABE, Shoichi TERADA, Hiroshi MATSUSAKA, T ...1987 Volume 102 Issue 2 Pages 281-290
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to identify the cGMP-sensitive ion channel protein in frog rod outer segments (ROS), we analyzed cGMP binding proteins in the ROS by means of photoaffinity labeling with [3H]cGMP. We found four cGMP binding proteins with molecular weights (Mws) of 250K, 100K, 92K, and 53K. The 250K protein was an integral-membrane protein, which we named cG-Protein, (cG stands for cGMP). The cGMP-binding to cG-Protein was slightly increased by CaCl2. cG-Protein has a carbohydrate moiety. The amount of cG-Protein per single rod outer segment was estimated to be 9.0 × 106 molecules. Light-dependent phosphorylation of cG-Protein with [γ-32] ATP was observed. The 100K and 92K proteins were peripheral-membrane proteins, corresponding to cGMP phosphodiesterase. The 53K protein was a soluble protein. Incorporation of a membrane protein fraction of frog ROS into a planar lipid bilayer resulted in the appearance of at least three kinds of ion channel activities; two of them were related to cGMP. The possibility that cG-Protein is the cGMP-sensitive ion channel in vivo is discussed.View full abstractDownload PDF (2895K) -
Hideyoshi HIGASHI, Yoshio HIRABAYASHI, Makoto ITO, Tatsuya YAMAGATA, M ...1987 Volume 102 Issue 2 Pages 291-296
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method for immobilizing oligosaccharides on a TLC plate for immunostaining has been developed. N-Glycolylneuraminic acid (NeuGc)-containing oligosaccha-rides derived from II3NeuGc-LacCer, IV3NeuGc-nLcOse4er, II3NeuGc-GgOse3e3Cer, and II3(NeuGc)2-LacCer by digestion with our newly isolated endoglycoceramidase (Ito, M. & Yamagata, T. (1986) J. Biol. Chem. 261, 14278-14282) and sialyllactose were chromatographed on polyamide 11 TLC or NH2-HPTLC plates, and covalently linked to the plates by reductive amination with sodium cyanoborohydride (NaBH3-CN). The immobilized oligosaccharides were detected by enzyme-immunostaining using NeuGc-specific chicken anti-NeuGc-LacCer and horseradish peroxidase-conjugated rabbit anti-chicken IgG. II3NeuGc-nLcOse4 showed the highest reac-tivity with the antibody, followed by II3NeuGc-GgOse3. As little as 0.8 nmol of the NeuGc-containing oligosaccharides was detected. The polyamide 11 TLC aluminum plate was found to be more suitable for the immunostaining than the NH2-HPTLC plate under the conditions used. For binding of the oligosaccharides to the NI-12-1-IPTLC plate, reductive amination was found to be superior to the heating method reported earlier.View full abstractDownload PDF (2549K) -
Misao TASHIRO, Kimikazu HASHINO, Masako SHIOZAKI, Fumio IBUKI, Zensuke ...1987 Volume 102 Issue 2 Pages 297-306
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of a double-headed trypsin inhibitor (RBTI) from rice bran was determined by a combination of limited proteolysis of the native inhibitor with Streptomyces griseus trypsin at pH 3 and conventional methods. RBTI consists of 133 amino acid residues including 18 half-cystine residues which are involved in 9 disulfide bridges in the molecule. The limited proteolysis at pH 3 produced a major split of Lys(83)-Met(84) and a minor split of Arg(107)-Val(108) together with a non-enzymatic hydrolysis of Asp(19)-Pro(20) in the molecule. The established sequence showed that RBTI is composed of 4 domains, domains I and III, and domains II and IV being homologous to the first and the second domains of soybean Bowman-Birk inhibitor, respectively, indicating that RBTI has a duplicated structure of the Bowman-Birk type inhibitor.View full abstractDownload PDF (770K) -
Tatsuhiko SEKIGUCHI, Sigenori KAWAHARA, Hiroshi SHIMIZU1987 Volume 102 Issue 2 Pages 307-312
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMembrane vesicles from sarcoplasmic reticulum of rabbit skeletal muscle were incorporated into a bilayer lipid membrane. With this system, single current fluctuation was observed in the presence of 50mM Ba-gluconate. This channel activity was observed only in vesicles from terminal cisternae. The single channel conductance was 14.1pS, and the channel state was almost wholly open. The open-close transition of the channel obeyed simple two-state kinetics and was voltage-independent. The ionic selectivity was also studied, and the channel showed no selectivity among Ba, Ca, Mn, and Mg. On the other hand, it was less permeable to Cs than to Ba. Based on these results, the relation of the Ca channel to excitation-contraction coupling is discussed.View full abstractDownload PDF (396K) -
Mohammad Yahiya KHAN, Sudhir Kumar AGARWAL, Sharika HANGLOO1987 Volume 102 Issue 2 Pages 313-317
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUrea-induced unfolding of bovine serum albumin and one of its fragments containing domain II+III has been studied by difference spectral and fluorescence emission measurements. The unfolding-refolding curves of both the proteins showed the presence of at least one stable intermediate when the transition was monitored at 288nm. The presence of the intermediate was not detectable at 293 nm where only tryptophan contributed towards the protein absorption. However, both the proteins did show the presence of intermediate when the denaturation was monitored fluorometrically. Since domain III of the albumin is devoid of tryptophan, it is concluded that the formation of intermediate in the unfolding-refolding transition of serum albumin involves (i) unfolding of domain III, (ii) minor structural transfor-mations in domain II, and/or (iii) the separation of the sub-domains of domain III from each other.View full abstractDownload PDF (427K) -
Takaomi Comings SAIDO, Satoshi TOYOSHIMA, Toshiaki OSAWA1987 Volume 102 Issue 2 Pages 319-326
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytosolic protein-O-carboxylmethyltransferase was purified more than 4, 000-fold in specific activity and membrane-associated protein-O-carboxylmethyltransferase carboxymethylase about 900-fold from chicken erythrocytes by use of a combination of affinity chromatography on immobilized S-adenosyl-L-homocysteine and gel filtration on Sephacryl S-200 (Pharmacia), together with 3-((3-cholamidopropyl)-dimethylammonio)-1-propane-sulfonate as a detergent to solubilize the membrane associated enzyme. The two enzymes were characterized by examining the dependence of their activity on pH and on concentration of S-adenosyl-L-methionine using fetuin as an exogenous methyl-acceptor substrate, and were found to differ somewhat. The cytosolic enzyme had a pH optimum of 6.0 with an apparent Km value of 2.1μM for S-adenosyl-L-methionine, whereas corresponding values for the membrane-associated enzyme were 6.5 and 0.71μM. This report deals with the biochemical differences between purified cytosolic and membrane-associated protein carboxymethylase from the same cell source.View full abstractDownload PDF (1139K) -
Yasuzo NISHINA, Hiromasa TOJO, Hidetaka USHIJIMA, Kiyoshi SHIGA1987 Volume 102 Issue 2 Pages 327-332
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe equilibrium constants and the rate constants (binding and dissociation constants) between reduced D-amino acid oxidase and pyridine carboxylates were obtained at various pH values (from pH 6.0 to 8.3). The pH dependence of the constants is consistent with the previous conclusion from a resonance Raman study that pyridine carboxylates in the form of a cation protonated at the N atom can bind to the reduced enzyme, but those in the neutral form cannot bind, showing that the positive charge of cationic pyridine carboxylates interacts with the negative charge of the anionic reduced flavin in the reduced enzyme. The binding rate constants of picolinate and nicotinate in the cationic form for the reduced enzyme were quite similar to each other, but the dissociation rate constant of picolinate is several times smaller than that of nicotinate. Thus, it is concluded that the difference in affinity of picolinate and nicotinate for the reduced enzyme is derived from the difference of the dissociation rate constants.View full abstractDownload PDF (418K) -
Keiichi NOMURA, Bunzo MIKAMI, Yoshimitsu NAGAO, Yuhei MORITA1987 Volume 102 Issue 2 Pages 333-340
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMethyl 2, 4-dinitrophenyl disulfide (MDPS) is shown to be an effective methanethiolating reagent for sulfhydryl groups in proteins via thiol-disulfide exchange reaction. It reacts with the two reactive sulfhydryl groups (SH1 and SH2) in soybean Β -amylase. A decrease of the enzymatic activity accompanies the methanethiolation of SH2. After complete methanethiolation of SH2, the modified enzyme still has 9% of the initial activity. Modification of SH2 with cyanide and iodoacetamide reduces the enzymatic activity to 65 and 2% of the initial activity, respectively. Apparently, the residual activity depends upon the size of the substituent at SH2. The modified enzymes still have the almost same Km values for amylopectin and Kd values for enzyme-maltose and enzyme-cyclohexaamylose complexes as the native enzyme. In contrast to maltose and cyclohexaamylose, the Kd value of the enzymeglucose complex increases in the order of cyanide-, MDPS-, and iodoacetamide-modified enzymes, indicating that SH2 is located near the binding site of glucose. It is proposed from the subsite structure of soybean β -amylase that the position of SH2 and the glucose binding site is around subsite 1, where the nonreducing ends of the substrate bind productively.View full abstractDownload PDF (654K) -
Keiichi NOMURA, Bunzo MIKAMI, Yuhei MORITA1987 Volume 102 Issue 2 Pages 341-349
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSulfhydryl (SH) groups of soybean β-amylase were modified with 5-(iodoacetoamidoethyl)aminonaphthalene-1-sulfonate (IAEDANS) and the SH-containing peptides exhibiting fluorescence were purified after chymotryptic digestion of the modified enzyme. The sequence analysis of the peptides derived from the modification of all SH groups in the denatured enzyme revealed the existence of six SH groups, in contrast to five reported previously. One of them was found to have extremely low reactivity toward SH-reagents without reduction. In the native state, IAEDANS reacted with 2mol of SH groups per mol of the enzyme (SH1 and SH2) accompanied with inactivation of the enzyme owing to the modification of SH2 located near the active site of this enzyme. The selective modification of SH2 with IAEDANS was attained after the blocking of SH1 with 5, 5'-dithiobis-(2-nitrobenzoicacid). The amino acid sequences of the peptides containing SH1 and SH2 were determined to be Cys-Ala-Asn-Pro-Gln and His-Gln-Cys-Gly-Gly-Asn-Val-Gly-Asp-Ile-Val-Asn-Ile-Pro-Ile-Pro-Gln-Trp, respectively.View full abstractDownload PDF (636K) -
Mitsuhiro SHIMIDZU, Heisaburo SHINDO, Kojiro TAKAHASHI, Shigehiko TANI ...1987 Volume 102 Issue 2 Pages 351-358
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe 31P NMR method was first applied to characterize in vivo phosphorylation of H1 and H5 in calf thymus and chicken erythrocytes as well as in vitro phospho-rylation of H1 and H5 by cAMP-dependent protein kinase. The amino acid resi-dues phosphorylated in vivo in the histones were exclusively serine residues, and the mole fraction of phosphoserine was estimated to be 0.34 and 0.27 per molecule of calf thymus H1 and chicken erythrocyte H5, respectively. Interestingly, chickenerythrocyte H1 was not phosphorylated in vivo. Three H1 subtypes from calf thymus H1 varied in the 31P NMR spectra, and the bisected fragments of calf thymus H1 and chicken erythrocyte H5 exhibited characteristic spectral patterns, indicating that there are considerable diversities of the degree of phosphorylation and phospho-rylation sites in very-lysine-rich histones. Furthermore, it was foundthat the microenvironment of phosphoserine residues phosphorylated in vivo in calf thymus H1 and chicken erythrocyte H5 is quite distinct from that of phosphoserine residues phosphorylated in vitro by bovine heart cAMP-dependent protein kinase.View full abstractDownload PDF (1437K) -
Kazuo ITO, Takehiko YAMAMOTO, Noshi MINAMIURA1987 Volume 102 Issue 2 Pages 359-367
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhosphodiesterase I [EC 3. 1. 4. 1] was purified from normal human urine in a highly purified state free from phosphodiesterase II, RNase, DNase I, DNase II, and phosphatase by column chromatographies of DEAE-Toyopearl, butyl-Toyopearl, Affi-Gel blue, and Sephadex G-150. The molecular weight of the enzyme was 1.9×105 and the pH optimum around 9.0 with p-nitrophenyl deoxythymidine 5'-phosphate asthe substrate. The enzyme hydrolyzed the 3'-5' linkage of various dinucleoside monophosphates at approximately the same rate and the phosphodiester bonds of cyclic 3', 5'-mononucleotides to produce mononucleoside 5'-phos-phate. The enzyme also hydrolyzed ADP to 5'-AMP and P1, ATP to 5'-AMP and PP1, and NAD+ to 5'-AMP and NMN. The enzyme activity was abolished by removal of metal ions with EDTA, and the metal-free enzyme was reactivated on the addition of Zn2+. The enzyme activity was also abolished by some reducing agents and the inhibition was reversed by Zn2+. The metal-free enzyme was less stable than the native enzyme, and Zn2+ and Co2+ restored the stability of the metal-free enzyme to the level of the native enzyme. The enzyme degraded oligonucleo-tides and high molecular nucleotides stepwise from the 3'-termini to give 5'-mono-nucleotides. The enzyme hydrolyzed single-stranded DNA more preferentially than double-stranded DNA. The enzyme also nicked superhelical covalently closed circular φX174 DNA to yield first open circular DNA and then linear DNA.View full abstractDownload PDF (2408K) -
Tomoko HAYASHI, Hiroaki HAYASHI, Koichi IWAI1987 Volume 102 Issue 2 Pages 369-376
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of a single H1 histone of the protozoan Tetrahymena pyriformis was determined, following previous determinations of the sequences of histones H2B, H2A, H3, and H4. Only a single H1 species was obtained by fractionation of a 0.5M HClO4-soluble fraction from the whole histone extract and further purification. This starting material for sequencing contained 1.1mol/mol phosphate and showed a single electrophoretic band after dephosphorylation. The sequence determination was performed by Edman degradation of BrCN fragments, staphylococcal protease peptides, and tryptic peptides, as well as secondary peptides from one BrCN fragment and one staphylococcal protease peptide. Phosphorus analysis of the tryptic peptides, containing serine or threonine, showed that five sites of the sequence were phosphorylated to various extents (5-30%). Thus, the total sequence, consisting of 165 amino acid residues and having a molecular weight of 17, 942 in the unmodified form, was completely determined. This unusually small H1 sequence differs substantially from the human spleen H1 sequence of 218 residues, having larger proportions of hydrophilic residues and smaller proportions of hydrophobic residues. Comparison of the distribution pattern of hydrophilic and hydrophobic residues, between the protozoan and human sequences, showed that the protozoan sequence lacks the central hydrophobic domainthat is conserved in the known vertebrate and other H1 histones. The implications for the function of H1 are discussed from the evolutionary viewpoint.View full abstractDownload PDF (559K) -
Keiko SAKAI, Seiichi KAWASHIMA, Koichi SUZUKI, Kazutomo IMAHORI1987 Volume 102 Issue 2 Pages 377-384
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSD-Fructose 1, 6-bisphosphatase [EC 3.1.3.11, FBPase] is one of the key enzymes in glyconeogenesis and its activity is controlled by various effectors such assubstrate, AMP and ATP. To analyze this complex regulation system, we tried an affinity labeling of FBPase with an AMP derivative, since AMP is a potent allosteric inhibitor of this enzyme. The results obtained are as follows. 1. To determine the func-tional groups which are essential for AMP as an inhibitor, inhibitory activities of some AMP derivatives were examined. These derivatives modified at the purine ring or phosphate group lost the activity while one modified at theribose ring retained the ability to inhibit FBPase. This shows that an affinity labeling reagent should be an AMP derivative in which the ribose ring is modified. 2. 2', 3'-Dialdehyde AMP (dial-AMP) was prepared by periodate oxidation of AMP and was reacted with FBPase. Under appropriate conditions, 1 mol of the reagent was incorporated per mol of enzyme subunit with a concomitant loss of enzyme activity. The reaction was prevented by the presence of AMP but not of ATP. The heat-stability, the kinetic parameters and the UV-absorption spectrum of the modified enzyme were all the same as those of native FBPase in the presence of AMP. Thus it was con-cluded that the allosteric AMP site in FBPase was modified specifically. Department of Chemistry, College of Arts and Sciences, The University of Tokyo.View full abstractDownload PDF (602K) -
Kiyoshi MIKI, Etsuro SUGIMOTO, Yasuo KITAGAWA1987 Volume 102 Issue 2 Pages 385-392
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe differentiation of retinoic acid-treated F9 cells (primitive endoderm-like F9 cells) into parietal endoderm-like F9 cells induced by dibutyryl cAMP was studied as a culture model of the morphogenesis of early mouse embryo. For this purpose, 6 cDNA clones coding for mRNAs specifically expressed in parietal endoderm-like F9 cells were selected. Northern hybridization of RNA extracted from variously treated F9 cells to nick-translated plasmid DNA of these clones demonstratedthe reversible expression of many mRNAs depending on the presence of dibutyryl cAMP in the culture medium. This result suggested that the differentiated state of parietal endoderm, which is formed from primitive endoderm at a position ad-jacent to the trophectoderm in mouse embryo, can be reversed if the local signal isremoved. One of the selected clones, pLAM, hybridized to an mRNA of 6.3kb andselected mRNA producing a laminin B subunit in an in vitro translation system. This clone has an inserted sequence of 3.1kb. Among the restriction sites in this sequence, six were consistent with those in a 1.7kb inserted sequence of pPE 49 and pPE 386, which were isolated by Barlow et al. as laminin B1 clones. An XbaI site found in both pPE 49 and pPE 386 was, however, not found at the corresponding position of pLAM. Dot hybridization of RNA with pLAM showed that expression of laminin B in F9 cells is stimulated more than 100-fold during differentiation of F9 stem cells into parietal endoderm-like F9 cells.View full abstractDownload PDF (2511K) -
Hiroshi KAWASAKI, Shinobu IMAJOH, Koichi SUZUKI1987 Volume 102 Issue 2 Pages 393-400
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe microscale separation of peptides based on the difference in positive chargewas examined with tryptic digests of apomyoglobin and calmodulin. By this separation method, C-terminal and blocked N-terminal peptides could be selectively isolated in the same fraction without any chemical modifications. Separated peptides, including internal peptides, were further purified by reversed phase high performance liquid chromatography, and the purified peptides could be directly subjected to sequence and amino acid analyses. The N-terminal peptides of calcium-activated neutral protease were successfully isolated by this method.View full abstractDownload PDF (601K) -
Toshiharu HASE, Masayuki HARABAYASHI, Kiyozo KAWAI, Hiroshi MATSUBARA1987 Volume 102 Issue 2 Pages 401-410
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSYeast cytochrome c1 is a component of complex III, an oligomeric enzyme of the mitochondrial respiratory chain. In order to investigate the structural requirement of cytochrome c1 for the function and assembly of the enzyme, we used an in vivo complementation assay to determine whether or not an in vitro mutated cytochrome c1 is functional. A yeast mutant whose nuclear cytochrome c1 gene was specifically inactivated was constructed by means of a gene disruption technique. The mutant was unable to respire, and lacked spectrally and immunochemically detectable cytochrome c1. These defects disappeared on the introduction of a plasmid carrying the cytochrome c1, gene coding the wild-type molecule or one coding a mutant molecule lacking the carboxyl (C)-terminal 17 amino acid residues. On theother hand, another mutant gene with a deletion corresponding to the C-terminal 71 residues showed no such ability. These results suggest that the region between the C-terminal 17 and 71 residues is necessary for the function of cytochrome c1.View full abstractDownload PDF (2124K) -
Toshiharu HASE, Masayuki HARABAYASHI, Kiyozo KAWAI, Hiroshi MATSUBARA1987 Volume 102 Issue 2 Pages 411-419
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome c1 is an amphiphilic protein which binds to the mitochondrial inner membrane, presumably through a hydrophobic region near the carboxyl (C)-ter-minus. In the preceding study (Hase, T., et al. (1987) J. Biochem. 102, 401-410), two cytochrome c, mutations were constructed: Δ1 and Δ2 cytochromes c1, in whichthe C-terminal segments of 17 and 71 residues were replaced by foreign sequencesof 20 and 15 residues, respectively. Δ2 cytochrome c1 had lost the putative mem-brane anchor. The two cytochrome c1 mutants were localized in mitochondria, butsuccinate-cytochrome c1 reductase activity was detected only in the mitochon-dria containing Δ1 cytochrome c1. The membrane association of the two mutant molecules as well as that of authentic cytochrome c1 was investigated. These three molecules were firmly attached to mitochondrial membranes and not solubilized on either sonication or sodium carbonate (pH 11) treatment. However, when the membranes were solubilized with Triton X-100, both the Δ1 and authentic cyto-chromesc1were extracted from the membranes more easily than Δ2 cytochrome c1. By fractionating cholate extracts of mitochondrial membranes with ammonium sul-fate, Δ1 cytochrome c1 was cofractionated with the enzymatic activity of complex III, butΔ2cytochrome c1 was clearly separated from the complex III fraction. Trypsin treatment of mitochondria and mitoplasts showed that Δ2 cytochrome c1 was exposed to the intermembrane space, with such a topology that its trypsin susceptibility be-came much higher than that of the authentic molecule. These results together suggest that Δ1 cytochrome c1 is assembled into the complex III in a fairly similar manner to the authentic cytochrome c1, whereas Δ2 cytochrome c1 is attached to the cytosolic side of the inner membrane quite differently. The C-terminal region containing the putative membrane anchor is thus indispensable for functional as-sembly into the mitochondrial respiratory chain.View full abstractDownload PDF (2412K) -
Akio AMETANI, Shuichi KAMINOGAWA, Makoto SHIMIZU, Kunio YAMAUCHI1987 Volume 102 Issue 2 Pages 421-425
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSScreening of antigenically reactive fragments of αs1-casein (αs1-CN), the major casein in bovine milk, was done by using HPLC and enzyme-linked immunosorbent assay (ELISA). BALB/c mice (6-week-old) were injected intraperitoneally with αs1-CN and complete Freund's adjuvant, and 14 days later, all the mice were boosted with αs1-CN and incomplete Freund's adjuvant. Twenty-one days after the 1st immunization, the mice were bled and antiserum was separated. Anti αs1-CN antibody fraction was obtained by precipitation from the antiserum with 50% saturated ammonium sulfate. αs1-CN was digested with trypsin and chymotrypsin, and 35 peptides were purified from the digests by reversed-phase HPLC with ODS (octadecylsilica) columns. Reactivity of peptides with the antibody were examined by ELISA. The solid phase in the wells of the polystyrene microtiter plate was coated with peptides, and the plate was successively incubated with anti αs1-CN antibody, conjugate of anti mouse immunoglobulin with alkaline phosphatase (ALP) and substrate of ALP. Two tryptic fragments (the residues 104-119 and 133-151) and three chymotryptic fragments (33-54, 105-121, and 174-199) were positive in an ELISA test. These five fragments would correspond to four antigenic sites. We could thus find antigenically reactive fragments of αs1-CN by the direct and simple detection of specific antigen-antibody interaction.View full abstractDownload PDF (422K) -
Tomoko NISHIMAKI-MOGAMI, Hiroshi YAMANAKA, Michinao MIZUGAKI1987 Volume 102 Issue 2 Pages 427-432
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe activity of acetyl-CoA-dependent chain elongation of fatty acids in Escherichia coli was enhanced when the organism was grown on oleic acid as the sole carbon source, but not detected when grown on glucose. Antibodies raised against fatty acid oxidation complex of E. coli inhibited both the reaction catalyzed by crotonase and the chain elongation in a similar manner, showing that the oxidationcomplex participates in the chain elongation. The activities of condensation andthe ac-tivities of NADH- and NADPH-dependent 3-ketoacyl reduction in the cell-free extract were precipitated by antibodies to the complex in parallel with thoseof 3-ketoacyl-CoA thiolase and crotonase. These results together with the presence of NADPH-dependent trans-2-enoyl-CoA reductase in E. coli (Mizugaki, et al. (1982) Chem. Pharm. Bull. 30, 2503-2511) indicate that the acetyl-CoA-dependent chain elongation of fatty acids in E. coli occurs by the reversal of fatty acid oxidation other than the step of enoyl reduction.View full abstractDownload PDF (461K) -
Takuji SASAKI, Kazuto KOBAYASHI, Tomochika OZEKI1987 Volume 102 Issue 2 Pages 433-441
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAntitrypsin isolated from silkworm larval hemolymph can inhibit bovine trypsin. The apparent molar ratio of silkworm antitrypsin (sw-AT) to trypsin at extrapolated null trypsin activity was determined to be 1.3 by titration of trypsin with sw-AT. The undissociability of the complex between sw-AT and trypsin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was confirmed by immunoblottingand fluorescence labeling techniques. Chemical analysis of the complex elucidated that it contained equimolar sw-AT and trypsin. Densitometric analysis of electrophoretic patterns obtained during the titration of trypsin by sw-AT suggestedthe presence of a suicide product formed from sw-AT. This was the reason why excess sw-AT was needed for complete inhibition of trypsin. In the complex, the sw-AT molecule was cut at one site but the fragments produced were still joined together. Trypsin in the complex was released by treatment at pH 10.0, and it was deduced that the complex formation involved acyl-bond formation between sw-AT andtrypsin. The sw-AT component obtained from the alkali-treated complex possessed two kinds of NH2-terminal amino acid sequences.Non-covalent forces may bind the two fragments of sw-AT, which could be separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino-terminal amino acid sequence analysis of the large fragment gave a sequence identical with that of intact sw-AT. This indicated that the reactive site of sw-AT with trypsin was located at the COOH-terminal region of the molecule. These characteristics resemble those of inhibitors belonging to the serpin family.View full abstractDownload PDF (1775K) -
1987 Volume 102 Issue 2 Pages 443a
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (20K) -
1987 Volume 102 Issue 2 Pages 443b
Published: 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (20K)
- |<
- <
- 1
- >
- >|