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Volume 110 (1991)
- Issue 6 Pages 847-
- Issue 5 Pages 677-
- Issue 4 Pages 469-
- Issue 3 Pages 315-
- Issue 2 Pages 163-
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Volume 110, Issue 4
Displaying 1-35 of 35 articles from this issue
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Mikihiko Naito, Ichiro Kudo, Yasuhito Nakagawa, Keizo Waku, Hisao Noji ...1991 Volume 110 Issue 4 Pages 469-471
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSynthesis and degradation of glycerophospholipids in HL-60 cells and retinoic acid (RA)-treated HL-60 cells were examined. The synthesis of each subclass of ethanolamine-con-taining glycerophospholipids was extremely suppressed in RA-treated HL-60 cells, while that of other glycerophospholipids was not seriously affected. A pulse-chase experiment revealed that about 88% of 1, 2-diacyl and 28% of 1-alkenyl-2-acyl glycerophosphoethanolamine were degraded during 4 days in RA-treated HL-60 cells. These characteristics of metabolism observed in RA-treated HL-60 cells might be responsible for the change of subclass composition of ethanolamine-containing glycerophospholipids in HL-60 cells during differentiation to granulocytes.View full abstractDownload PDF (300K) -
Tomomi Fujii, Hideaki Moriyama, Akio Takenaka, Nobuo Tanaka, Takayoshi ...1991 Volume 110 Issue 4 Pages 472-473
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFerredoxin from a thermoacidophilic archaebacterium, Sulfolobus acidocaldarius, has been crystallized. The space group is P43212 or P41212 and the cell dimensions are a=b=50.12Å and c=69.52Å. The Vm value is calculated to be 1.88 Å3/Da, assuming one molecule per asymmetric unit. The crystal diffracts X-rays beyond 2.0Å resolution.View full abstractDownload PDF (507K) -
Tetsu Matsuura, Sumiko Kimura, Satoshi Ohtsuka, Koscak Maruyama1991 Volume 110 Issue 4 Pages 474-478
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen rabbit skeletal muscle myofibrils were kept for 12h at 4°C, α-connectin was partially degraded and 1, 200 kDa peptide was newly formed [Takahashi, K. & Takai, H. (1988) Abst. 80th Jpn. Soc. Zootech. Sci. p-102]. The latter was isolated together with remaining α-connectin. Ultracentrifugation of the mixture at low ionic strength resulted in sedimentation of α-connectin, leaving the 1, 200 kDa peptide in the supernatant. Physicochemical properties of the isolated 1, 200 kDa peptide were investigated: UV absorption spectra, circular dichroism spectra, amino acid composition, and molecular size and shape. Polyclonal antibodies against the 1, 200 kDa peptide [PcAb(1200)] bound the Z line and I-band. The position of the stripe in the I band near the N1 line due to the binding of PcAb(1200) moved both away from the Z lines and from the A band as sarcomeres were elongated. Therefore, it is considered that the 1, 200 kDa portion of α-connectin is elastic.View full abstractDownload PDF (920K) -
Saki Itonori, Miwako Nishizawa, Minoru Suzuki, Fuyuhiko Inagaki, Taro ...1991 Volume 110 Issue 4 Pages 479-485
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA series of glycosphingolipids containing 2'-caminoethylphosphoryl(→6)-N-acetylglucosamine as a polar group has been demonstrated in larvae of the green-bottle fly, Lucilia caesar. The thin-layer chromatographic pattern of the total polar glycolipid revealed the presence of more than eight components, of which five major components were purified by the use of successive column chromatography on QAE- and DEAE-Sephadex and silicic acid (Iatrobeads). From structural studies including compositional sugar analysis, hydrogen fluoride degradation, proton magnetic resonance spectroscopy, methylation analysis, and fast atom bombardment mass spectrometry, their structures were deduced to be as follows: 2'-aminoethylphosphoryl→6G1cNAcβ1-3Manβ1-4G1cβ1-Cer, GaINAcβ1-4(2'-aminoethylphosphoryl→6)G1cNAcβ1-3Man β1-4G1cβ1-Cer, GalNAcαal-4 GalNAcβ1-4(2'-aminoethylphosphoryl→6)G1cNAcβ1-3Manβ1-4G1cβ1Cer, Galβ1-3GalNAcα 1-4GalNAc-β1-4(2'-aminoethylphosphoryl→6)G1cNAcβ1-3Manβ1-4G1cβ1-Cer, and GleNAcβ1-3Gal-β1-3GaINAcα1-4Ga1NAcβ1-4(2'-aminoethylphosphoryl→6)G1cNAcβ1-3Manβ1-4G1c-β1-Cer. The main molecular species of the ceramide moiety was arachidinyltetradecasphingenine in all of the major glycolipids.View full abstractDownload PDF (812K) -
Seiichi Hashida, Koichiro Tanaka, Naoko Yamamoto, Takeshi Uno, Ken'ich ...1991 Volume 110 Issue 4 Pages 486-492
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOne attomole of [Arg8]-vasopressin (AVP) was detected by a novel noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay). AVP was indirectly biotinylated using N-hydroxysuccinimidobiotin and trapped onto an anti-AVP IgG-coated polystyrene ball. After washing, biotinylated AVP was eluted from the polystyrene ball with HC1 and was reacted with 2, 4-dinitrophenyl-fluorescein disulfidebovine serum albumin-rabbit anti-AVP IgG conjugate. The complex formed was trapped on [anti-2, 4-dinitrophenyl group] IgG-coated polystyrene balls and, after washing, reacted with avidin-β-D-galactosidase conjugate. The polystyrene balls were washed, and the complex of the three components was eluted with 2, 4-dinitrophenyl-L-lysine and trans-ferred to anti-fluorescein IgG-coated polystyrene balls. After washing, the complex was released from the polystyrene balls by reduction with 2-mercaptoethylamine and transferred to [anti-rabbit IgG] IgG-coated polystyrene balls. β-D-Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. The detection limit of AVP was 1.1fg (1amol) /tube. Interference by proteins in biological fluids was eliminated by separation of peptides from proteins using a molecular sieve. The principle of the present method may be applicable to the measurement of haptens, including peptides, that can be derivatized so as to be bound simultaneously by both anti-hapten antibody and avidin molecules.View full abstractDownload PDF (763K) -
Hiroyasu Inoue, Saori Takahashi, Kiyoshi Fukui, Yoshihiro Miyake1991 Volume 110 Issue 4 Pages 493-500
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe presence of a leucine zipper motif was recognized in the deduced amino acid sequences of human and rat renin-binding proteins (RnBPs) on cloning and sequence analysis of the RnBP cDNAs. The in vitro synthesized RnBPs, with the respective cDNAs, formed heterodimers with porcine renin and homodimers. On comparison of these properties with those of porcine RnBP, the leucine zipper motif was suggested to be a functional domain common to animal RnBPs. In addition to the motif, a hydrophobic domain adjacent to the motif and 10 cysteine residues were also well conserved in the three RnBPs. Moreover, about 85% of their amino acid sequences were identical. The RnBP mRNAs were expressed in the kidneys as the same size of 1. 5-kb and the genes are suggested to exist as single copies in the genomes. Despite the high similarities in genetic and molecular properties, the molecular weights of human and rat RnBPs were 43, 000, which is 1, 000 larger than that of porcine RnBP. The immunoreactivities of human and rat RnBPs toward anti-porcineRnBP antiserum were 88 and 8% that of porcine RnBP, respectively, and the affinities of the two RnBPs for porcine renin were remarkably less than that of porcine RnBP. Moreover, the human and rat RnBP homodimers were partly dissociated under the conditions under which porcine RnBP existed as a dimer. These results indicate distinct differences in the molecular properties among the three RnBPs, in spite of their being highly similar structurally and functionally.View full abstractDownload PDF (1462K) -
Yoshinori Harada, Takeshi Sakamoto, Tomoko Shinomura, Kazunori Takamot ...1991 Volume 110 Issue 4 Pages 501-507
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo carry out systematic structure-function studies of octopus rhodopsin, photoreceptor protein of octopus visual cells, by means of specific amino-acid replacements, we have totally synthesized a DNA duplex of 1, 365 base pairs that encodes the entire octopus rhodopsin of 455 amino acids [Ovchinnikov et al. (1988) FEBS Lett. 232, 69-72] by introducing codons preferred in Escherichia coli. Total synthesis simplifies site-specific mutagenesis in all parts of the gene by replacement of short restriction fragments by their newly synthesized counterparts containing the required nucleotide alterations. Thirty unique restriction sites were introduced in the octopus rhodopsin gene, which was assembled on a plasmid in two steps. Five cartridge genes of 344, 296, 320, 212, and317 base airs capable of being expressed independently were first constructed by using 48 synthetic oligonucleotides ranging in size from 54 to 73 nucleotides. The entire gene was constructed by consecutive linkage of cartridge genes. These cartridge genes were designed to correspond to the transmembrane helical unit of octopus rhodopsin, resulting in easy construction of various chimeric rhodopsins. The nucleotide sequences were confirmed by sequencing the cartridges as well as the entire gene. These synthetic genes were cloned into an expression vector carrying the trp promoter of E. coli, and were preliminarily expressed in vitro and in vivo.View full abstractDownload PDF (1115K) -
Kazuhiro Kohama, Tadashi Kohno, Tsuyoshi Okagaki, Teruo Shimmen1991 Volume 110 Issue 4 Pages 508-513
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSActin-activated ATPase activity of myosin from Physarum polycephalum decreases when it binds Ca2+ and increases when it loses Ca2+. This Ca-inhibition is observed with phosphorylated myosin [Kohama, K. (1990) Trend. Pharmacol. Sci. 11, 433-435]. The activity of dephosphorylated myosin remained at a low level both in the presence and absence of Ca2+, although Ca2+-binding ability was much the same as that of the phosphorylated myosin. The effect of phosphorylation has been studied at a conventional actin concentration, which is comparable with that of myosin by weight. When the concentration of actin was increased by 10 times, the dephosphorylated myosin became actin-activatable in the absence of Ca2+, and Ca-inhibition was recovered. As actin exists quite abundantly in non-muscle cells of Physarum, myosin phosphorylation plays virtually no role in regulating actin-myosin-ATP interaction in vivo. Physiologically the interaction may be regulated by Ca2+ by binding to and subsequent release from myosin.
Latex beads coated by either phosphorylated or dephosphorylated myosin moved ATP-dependently on the actin cables of Characeae cells to the same extent in the absence of Ca2+, but the movement was abolished by increasing Ca2+. When the interaction was examined by monitoring the movement of actin filaments on myosin fixed on a coverslip, the movement and Ca-inhibition of the movement were detected with phosphorylated, not dephosphorylated, myosin [Okagaki, T., Higashi -Fujime, S., & Kohama, K. (1989) J. Biochem. 106, 955-957]. The discrepancy between the in vitro motility assays suggests that the former monitors the interaction at a high concentration of actin and the latter, that at a conventional, low concentration of actin. The former assay should be more useful as a monitor of the physiological interaction.View full abstractDownload PDF (537K) -
Naoya Suzuki, Koshin Mihashi1991 Volume 110 Issue 4 Pages 514-519
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe investigated the mode of binding of cytochalasin B (CB) to F-actin in an ADP-solution with and without inorganic phosphate (P1). In the presence of P1 (20 mM), a filament of F-actin had a single high-affinity CB binding site (Kd =1.4 nM), just like in the case of an ATP-solution [Kd=5.0 nM: Suzuki, N. & Mihashi, K. (1991) J. Biochem. 109, 19-23]. But in the absence of P1, there were two low-affinity (Kd =200 nM) CB binding sites as well as one high-affinity site (Kd=1.6 nM). We determined the concentration of CB necessary for half-maximal inhibition of growth or shortening of F-actin (K1) using of pyrene-labeled actin. We obtained K1 =80 nM for growth and K1 =800 nM for shortening in the presence of ATP. The addition of P1 to the ATP-solution reduced K1 for growth to 9 nM. We propose a model explaining these results. In the model, high-affinity CB binding to the terminal subunit dimer can inhibit subunit exchange at the B-end only when the terminal subunits bind ATP or ADP•P1. When the terminal subunits bind ADP, additional low-affinity CB bindings to the terminal subunits are needed to inhibit the subunit exchange.View full abstractDownload PDF (637K) -
Setsuo Furuyoshi, Yoshito Nawa, Nariyoshi Kawabata, Hidehiko Tanaka, K ...1991 Volume 110 Issue 4 Pages 520-525
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new enzyme, L-tartrate decarboxylase, was found in cells of Pseudomonas sp. group Ve-2. The enzyme was purified to homogeneity and characterized. The enzyme requires K+, Mg2+, and NAD+ for L-tartrate decarboxylation. The dependence of the enzymatic decarbox-ylation on NAD+ suggests that the decarboxylation involves redox reactions of the sub-strate. The enzyme catalyzes NAD+-linked oxidative decarboxylation of D-malate as well. The enzyme is composed of four subunits with identical molecular weight (Mr 40, 000). The apparent Michaelis constants for L-tartrate and NAD+ are 7.0 and 1.1 mM, respectively. The cofactor requirements and the physical properties of the enzyme were similar to those of L-tartrate dehydrogenase-D-malate dehydrogenase from Rhodopseudomanas sphaeroides, and tartrate dehydrogenase from P. putida.View full abstractDownload PDF (977K) -
Kinuko Kimura, Shigeyoshi Sugano, Akihiro Funae, Yoshio Nakano1991 Volume 110 Issue 4 Pages 526-531
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe inactivation of native glutamine synthetase (GS) from Bacillus subtilis by trypsin, chymotrypsin, or subtilisin followed pseudo-fast order kinetics. Trypsin cleaved the polypeptide chain of GS into two principal fragments, one of about 43, 000 (Mr) and the other of smaller than 10, 000. Chymotrypsin and subtilisin caused similar cleavage of GS. A large fragment (Mr 35, 000) and one smaller than 10, 000 were detected on SDS-PAGE. The nicked protein remained dodecameric, as observed on gel filtration, electrophoresis, and electron micrography. In the presence of glutamate, ATP, and Mn2+, the digestion of GS by each of the three proteases was retarded completely; however, the presence of one substrate, L-glutamate, ATP+Mn2+, or ATP+Mg2+ led to partial protection. The product, L-glutamine, did not retard but altered the susceptibility of the protease sensitive sites. Amino acid sequence analysis of the two smaller polypeptide fragments showed that the nicked region was around serine 375 and serine 311, respectively, and that both large fragments (43, 000 and 35, 000) were N-terminal polypeptides of GS. The serine 311 region was involved in the formation of the enzyme-substrate complex. Tyrosine 372 near serine 375 corresponded to tyrosine 397 which was adenylylated by adenyltransferase in Escherichia coli GS.View full abstractDownload PDF (1991K) -
Yukio Morimoto, Toshiki Tani, Hirokazu Okumura, Yoshiki Higuchi, Norit ...1991 Volume 110 Issue 4 Pages 532-540
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe three-dimensional structure of cytochrome c3 from Desulfovibrio vulgaris Hildenbor-ough has been determined by use of the molecular replacement method and refined at 2.0 Å resolution. A suitable crystal of the cytochrome c3 was obtained from buffer solution (25 mM Tris-HC1, pH 7.4), with 75% ethanol as the precipitating reagent. Crystallographic data are as follows: α=43.17 Å, b=62.91 Å, c=41.17 Å, orthorhombic, P212121 and Z 4. Constrained least-squares refinement and a molecular dynamics procedure with a simulat-ed structure annealing method yielded a crystallographic R-factor of 0.212. The similarity in the folding pattern of both cytochromes c3 is established, the mean deviation of the polypeptide backbone between the two structures being 0.367 Å. Most of the amino acids substitutions from DvMF were located on the surface of the molecule, and in particular, S27 and V86 were placed near the propionic acid of the heme group so as to hang over the heme and the cleft of the molecule.View full abstractDownload PDF (771K) -
Kunizo Arai, Toku Kanaseki, Shoji Ohkuma1991 Volume 110 Issue 4 Pages 541-547
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing Percoll density gradient centrifugation after treatment of the postnuclear supernatant (PNS) with 1 mM Ca2+ to swell and lighten mitochondria, we isolated highly purified lysosomes (dextranosomes) in high yield (25%) from the livers of rats to which dextran had been administered. The lysosomal fraction obtained by this method was enriched more than 100-fold in N-acetyl-β-glucosaminidase and arylsulfatase and 40-fold in acid phosphatase and β-glucosidase. Electron microscopic examination and measurement of marker enzyme activity for various subcellular organella indicated that the lysosomal fraction was essentially free from contamination by other organella. Flavins, ubiquinones, and hemochromes were found on lysosomal membranes and investigated. The FAD and ubiquinone-9 contents of the purified lysosomal membranes were 0.118 and 6.93 nmol/mg of protein, respectively. Hemochromes in lysosomes showed spectra similar to that of a b-type cytochrome, with the α-peak at 562 nm and the γ-peak at 436 nm.View full abstractDownload PDF (1311K) -
Shin-ichi Miyoshi, Sumio Shinoda1991 Volume 110 Issue 4 Pages 548-552
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe metalloprotease produced by Vibrio vulnificus (VVP) is known to be quickly inactivated by plasma proteins which belong to the class of α-macroglobulins in vitro at a molar ratio of 1:1. But the in vivo potential of the inactivators has not been studied. Macroalbumin (MA), a member of a -macroglobulins in guinea pig plasma, was found to inactivate VVP by means of physical entrapment in vitro. In vivo actions of VVP, permeability-enhancing and hemorrhagic actions, were greatly augmented by simultaneous injection of the antibody against MA, which had no effect on in vitro proteolytic action toward azocasein. The interstitial-tissue space in the normal guinea pig skin contains a negligible amount of MA. However, sufficient MA was present in the extravascular fluid collected after the intradermal injection of VVP. Besides, in the extravascular fluid, VVP formed a complex with MA and no inactivator other than MA was found. These results indicate that plasma MA leaked from the vascular system owing to the permeability-enhancing and hemorrhagic actions of VVP, resulting in inactivation of VVP in situ.View full abstractDownload PDF (490K) -
Hidenori Yoshino, Osamu Minari1991 Volume 110 Issue 4 Pages 553-558
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA technique in which the subunits of human erythrocyte spectrin were immobilized on a nitrocellulose membrane was developed to study which domains of the subunit are able to bind to the counterpart subunit. The limited tryptic digestion of the isolated α and β subunits of human erythrocyte spectrin produced eight fragments in the α subunits and nine fragments in the β subunit. Four fragments of the β (80, 60, 44, and 18 kDa) and two of the α (82 and 33 kDa) bound to α and β subunits which were immobilized on nitrocellulose membrane strips, respectively. The binding affinities of all the fragments to the subunits, however, were remarkably lower than that of the mother proteins. The titration of fluorescence anisotropy of N-(1-anilinonaphthyl-4)maleimide which was covalently attached to the subunit by the trypsin-digested fraction of the counterpart subunit also indicate weak binding of the fragments even in solution. These findings suggest that the high-affinity binding of the α subunit to the β subunit to form spectrin αβ dimer occurs only when the binding domains are arrayed along the polypeptide chains at the appropriate positions on the subunits.View full abstractDownload PDF (1551K) -
Shin-ichi Hayashi, Junko Watanabe, Kaname Kawajiri1991 Volume 110 Issue 4 Pages 559-565
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe identified genetic polymorphisms in the 5'-flanking region of the human cytochrome P45011E1 gene and investigated the effect of these polymorphisms on the transcriptional regulation of the gene. PCR direct sequencing of the two homozygous alleles [types A (c1/c1) and C (c2/c2)] revealed the existence of several point mutations in the distal 5'-flanking region of the gene, but no differences in the proximal promoter region. The DNA segment ( -1372 to -960) placed upstream of SV40 promoter and the chloramphenicol acetyltrans-ferase (CAT) gene enhanced the expression of the gene, and the enhancement of expression by type C DNA was about 10 times that by its type A counterpart. DNase I footprinting analysis showed at least one protected region in which one of the polymorphic loci (RsaI polymorphism) was located. The DNase I sensitivities and protection profiles of the two genotypes were different. The protected region had high homology to the consensus sequence of the binding region of liver specific transcription factor HNF1 (LF-B1), and this was confirmed by gel retardation assay. These results indicate that genetic polymorphisms in the 5'-flanking region of the human P45011E1 gene affect its binding of trans-acting factor and change its transcriptional regulation. This may lead to inter-individual differences of microsomal drug oxidation activity.View full abstractDownload PDF (2137K) -
Kazuhiro Kohama, Tsuyoshi Okagaki, Hiromi Takano-Ohmuro, Ryoki Ishikaw ...1991 Volume 110 Issue 4 Pages 566-570
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhysarum myosin is uniquely under an inhibitory Ca2+ -regulation in the ATP-dependent interaction with actin [Kohama (1990) Trends Pharmacol. Sci. 11, 433-435, for review]. Calcium-binding light chain (CaLc) has been suggested to be of primary importance to the control from its amino acid sequence [Kobayashi et al. (1988) J. Biol. Chem. 263, 305-313]. To provide a biochemical basis for this suggestion, the Ca-binding capacity of CaLc and its Kd for Ca2+ were measured. The Ca-binding properties of CaLc allowed those of Physarum myosin to be explained in terms of CaLc. However, the mode of Ca2+ -regulation by CaLc differs according to the enzyme upon which Ca-sensitivity is confered by CaLc, i.e., CaLc activated bovine phosphodiesterase activity and inhibited Physarum myosin ATPase activity, with the same Kd in μM levels. Thus, CaLc appears to work as a mere Ca-receptive subunit in Physarum myosin, with the secret of the inhibition lying in other subunits. CaLc was also shown to belong to a family of alkali light chains (AlLc) by allowing it to bind skeletal myosin as a substitute for its A1Lc. Therefore, present study is the first biochemical indication that the A1Lc family is involved in regulating the myosin function.View full abstractDownload PDF (1559K) -
Tomiko Asano, Rika Morishita, Kanefusa Kato1991 Volume 110 Issue 4 Pages 571-574
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPolyclonal antibodies to the α subunits of Go type G proteins (Goα) were coupled to agarose gel and used to isolate Goα from solubilized membranes of various bovine tissues. The cholate extract of membranes was applied to the anti-Goα-agarose gel column. The column was washed extensively, then bound proteins were eluted at a neutral pH using a commer-cial ActiSep Elution Medium. The proteins in the eluate displayed a single band of 39 kDa on SDS-polyacrylamide gel electrophoresis. They bound to GTPγS and were ADP-ribosylated by pertussis toxin. The yield of the immunoreactive Goα from the extract was about 40%. Isoelectric focusing, immunoassay and peptide mapping analysis of the Goα-like proteins purified from the heart and adrenal medulla indicated that these proteins were very similar to the α subunit of a minor subtype of Go in the brain which was previously referred to as Go*αView full abstractDownload PDF (1250K) -
Ryoji Masui, Sadao Wakabayashi, Hiroshi Matsubara, Youssef Hatefi1991 Volume 110 Issue 4 Pages 575-582
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMitochondrial NADH: ubiquinone oxidoreductase (complex I) is the most complicated enzyme in the respiratory chain and is composed of at least 26 distinct polypeptides. Two hydrophilic subfractions of bovine heart complex I were systematically resolved into individual polypeptides by chromatography. Three polypeptides (51, 24, and 9 kDa) were isolated from the flavoprotein fraction (FP) of complex I, and the complete amino acid sequence of the 9 kDa polypeptide was determined. The 9 kDa polypeptide is composed of 75 amino acids with a molecular weight of 8, 437. This protein exhibits no obvious sequence similarity to other proteins. The iron-sulfur protein fraction (IP) of complex I was separated into eight polypeptides, 75, 49, 30, 20, 18, 15, 13 kDa-A, and 13 kDa-B. The 20 kDa polypeptide was recognized as a novel component of IP for the first time. The N-terminal and several peptide sequences of the 20 kDa polypeptide were determined. Comparison of the sequences revealed significant sequence similarities of the 20 kDa polypeptide to the psbG gene products encoded in the chloroplast genome. The conserved sequence in these proteins was also found in the small subunit of the nickel-containing hydrogenases. These results suggest that complex I is related to other redox enzyme complexes.View full abstractDownload PDF (1140K) -
Keiko Kakeda, Hajime Ishikawa1991 Volume 110 Issue 4 Pages 583-587
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSymbionin, that is selectively produced by an intracellular symbiont harbored by the aphid bacteriocyte, is structurally homologous to the Escherichia coli groEL protein, a heat shock protein functioning as a molecular chaperon. It was shown that symbionin has ATPase activity and, in the presence of Mg-ATP, is converted into lower molecular mass species. Like the groEL protein, symbionin was able to reconstitute dimeric ribulose 1, 5-bisphosphate carboxylase/oxygenase holoenzyme from its unfolded subunits in vitro, suggesting that this protein functions as a molecular chaperon in the endosymbiont. The groES-homologous protein did exist in the endosymbiont, but its amount was small relative to that of symbionin.View full abstractDownload PDF (1459K) -
Tsunenori Nozawa, Michael T. Madigan1991 Volume 110 Issue 4 Pages 588-594
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTemperature and solvent effects on reaction center structures were examined in two thermophilic photosynthetic bacteria, Chlorollexus aurantiacus and Chromatium tepi-dum, in order to gain insight into the interactions among the reaction center proteins and pigment systems. Thermal stability of the reaction centers was found to be proportional to the optimum growth temperature. Circular dichroism (CD) spectra in the 250-300 nm region indicated that thermal denaturation destroyed tertiary structures (helix-to-helix interactions or amino acid residue conformation) in the native reaction center, keeping helical structures intact. Absorption and circular dichroism spectral changes showed that alcohol denatured the so-called special pair and the accessory BCh1 a independently. The alcohol denaturation further indicates that the coordination between BCh1 a and amino acid residue in the protein is one of the important interactions maintaining the pigment organization of the reaction centers.View full abstractDownload PDF (546K) -
Masao Nakamura1991 Volume 110 Issue 4 Pages 595-597
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDirect reactions of peroxidases with Trolox C (a vitamin E analogue) and vitamin E were observed in 50% (v/v) methanol. The kinetic results revealed that the reaction of horseradish peroxidase intermediate Compound II with Trolox C and vitamin E was the rate-determining step, and the rate constants were estimated to be 1.7×103 and 5.1×102 M-1•s-1, respectively. Peroxidases catalyzed the one-electron oxidation of Trolox C and vitamin E, and the vitamin E phenoxyl radicals resulting from the peroxidase reactions were detected by continuous-flow ESR spectroscopy.View full abstractDownload PDF (250K) -
Hiroshi Kitagawa, Mikiyo Takaoka, Hiroshi Nakada, Shigeyuki Fukui, Iku ...1991 Volume 110 Issue 4 Pages 598-604
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have determined the structures of six oligosaccharides isolated from human milk using a monoclonal antibody, MSW 113. The isolation involved affinity chromatography on a column of the immobilized monoclonal antibody and high-performance liquid chromatography. From the results of 500 and 600 MHZ 1H NMR spectroscopy and fast atom bombard-ment-mass spectrometry their structures were deduced to be:
NeuAcα2→6
1. NeuAcα2-3Galβ1-3GlcNAcβ1-4Gle 2. NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc
NeuAcα2→6
NeuAcα2→6
3. NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glec 4. NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc
Fucα1↑4 Galβ1-4GlcNAcβ1↑6
NeuAcα2↓6
5. NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc 6. NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc
NeuAcα2-6Galβ1-4GlcNAcβ1↑6 Galβ1-4GlcNAcβ1↓6
Fucα1↑3
Two of these oligosaccharides, numbers 4 and 5, have not previously been described. All of them bound to MSW 113, but their reactivities are weaker than those of sialyl-Le8 oligosaccharides. The results indicate that MSW 113 reacts with oligosaccharides with the mono- and disialyl-Le8, and other sialyl type 1 structures.View full abstractDownload PDF (546K) -
Hiroshi Yamashita, Hiroshi Nakatani, Ben'ichiro Tonomura1991 Volume 110 Issue 4 Pages 605-607
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPorcine pancreatic α-amylase (1, 4-α-D-glucan glucanohydrolase) [EC 3.2.1.1] has both amylase activity (hydrolysis of α-1, 4-D-glucoside bond of starch) and maltosidase activity (hydrolysis of p-nitrophenyl-α-D-maltoside to p-nitrophenol and maltose). By the modification of histidine residues of porcine pancreatic α-amylase with diethylpyrocarbonate (DEP), both amylase and maltosidase activities were decreased in the absence of chloride ion. In the presence of chloride ion, however, maltosidase activity of the modified enzyme was increased to more than 260% of that of the native enzyme, whereas amylase activity was decreased to less than 15% of the native enzyme. Since the chloride ion binding site is part of the active site loop [Buisson et al. (1987) Food Hydrocolloids 1, 399-406 and Buisson et al. (1987) EMBO J. 6, 3909-3916], the special arrangements of both catalytic and modified histidine residues induced by the chloride ion binding would enhance only the maltosidase activity of the histidine-modified enzyme.View full abstractDownload PDF (266K) -
Hitoshi Shimoi, Makoto Tadenuma1991 Volume 110 Issue 4 Pages 608-613
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRarobacter faecitabidus protease I, a yeast-lytic serine protease, was characterized in order to elucidate the mechanism of lysis of yeast cells by this enzyme. The N-terminal amino acid sequence of the enzyme was found to be homologous to those of Lysobacter enzymogenes α-lytic protease and Streptomyces griseus proteases A and B around the catalytic His residue, showing that it is a mammalian type serine protease. In a study of its substrate specificity, it preferentially hydrolyzed the ester of alanine among amino acid p-nitrophenylesters. It also efficiently hydrolyzed succinyl Ala-Pro-Ala p-nitroanilide, the specific synthetic substrate for pancreatic elastase. With oxidized insulin B-chain, it hydrolyzed almost exclusively the peptide bond between valine 18 and cysteic acid 19 in the early step of the reaction, and thereafter it partially hydrolyzed Va112-G1u13, A1a14-Leu15, and Leu15-Tyr16. These results indicate that Rarobacter protease I is elastase-like in its substrate specificity, preferentially hydrolyzing the peptide bond of aliphatic amino acids. Its affinity for yeast cells was also investigated, and while Rarobacter protease I was adsorbed by yeast cells, pancreatic elastase was not. This difference was thought to account for the failure of pancreatic elastase to lyse yeast cells, even though its specificity is similar to that of the yeast-lytic enzyme. Rarobacter protease I was adsorbed by a mannose-agarose column and specifically eluted from the column with a buffer containing D-mannose or n-glucose. These monosaccharides also inhibited its yeast-lytic activity. These results indicate that Rarobacter protease I binds to mannan on the yeast cell surface in a lectin-like manner. We believe that the elastase-like substrate specificity and the affinity for mannose are the essential characteristics of the yeast-lytic protease.View full abstractDownload PDF (541K) -
Nobuya Itoh, Yoshinao Koide, Haruyo Fukuzawa, Susumu Hirose, Tadahiko ...1991 Volume 110 Issue 4 Pages 614-621
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNovel host-vector systems have been developed for gene cloning in the metabolically versatile bacterial genus Pseudomonas. We found that a new Pseudomonas strain, Pseudomonas flavida IF-4, isolated from soil, carried two small cryptic plasmids, named pNI10 and pNI20. They were multi-copy, but not self-transmissible, and the genome size was 3. 7 kb for pNI10 and 2. 9 kb for pNI20. Several types of cloning vectors containing a kanamycin or streptomycin resistance (Kmr or Smr) gene were constructed from pNI10 and pNI20. These plasmid vectors were efficiently transformed into several strains of Pseudomonas at a frequency up to 4×105 transformants per 1 μg plasmid DNA by the usual competent cell method. The vectors derived from pNI10 replicated not only in Pseudomonas but also in some other Gram-negative enteric bacteria such as Escherichia coli, Enterobacter aerogenes, and Proteus mirabilis.View full abstractDownload PDF (1273K) -
Teruhiro Takabe, Yukimoto Iwasaki, Takashi Hibino, Takayuki Ando1991 Volume 110 Issue 4 Pages 622-627
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe PSI core complex prepared from cucumber cotyledons, which contains 80 chlorophylls per reaction center (P700) and eight polypeptides with apparent molecular masses of 65/63, 20, 19.5, 18.5, 17.5, 7.6, and 5.8 kDa, has been shown to catalyze the light-dependent transfer of electrons from plastocyanin to ferredoxin. The “native” PSI complex, which contains more than fifteen polypeptides and 120 chlorophylls per P700, did not show higher activity. Any attempt to deplete subunit(s) of the core complex decreased its activity. These results suggest that in addition to light-harvesting chlorophyll a/b protein complexes, several genes of psaA-psaK, which have been proposed as components of PSI complex, are not involved in the activity of PSI complex. It was also found that the amount of 18.5-kDa polypeptide in the PSI complex affects the activity: when this polypeptide was largely depleted, the complex was almost inactive. The inactivation was due to inhibition of electron transfer from plastocyanin to photooxidized P700. Chemical cross-linking and N-terminal amino acid sequencing experiments indicated that the 18.5-kDa polypeptide is the plastocyanin-docking protein and the psaF gene product. The function of the psaF gene product was discussed.View full abstractDownload PDF (1239K) -
Hiroshi Ohashi, Yumiko Katsuta-Enomoto, Katsuki Yasufuku, Kenji Okada, ...1991 Volume 110 Issue 4 Pages 628-634
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree DNA constructs, pETB-40, 41, and 42, encoding human big endothelin-1 (ET-1) preceded by the specific recognition sequence (Ile-Glu-Gly-Arg) for the activated blood coagulation factor Xa (FXa), fused in frame to the N-terminal portion of β Gal, were expressed in Escherichia coli. The fusion proteins, pETB-40P, 41P, or 42P, consisted of the 55-, 51-, or 42-aa N-terminal peptide of β Gal and the 38-aa of big ET-1, and had 1, 0, or 0 Cys residues and 5, 5, or 1 Arg residues in the N-terminal peptide of β Gal, respectively. Enzymatic cleavage of the purified fusion proteins by FXa or trypsin allowed the recovery of authentic human big ET-1. The rates of conversion of pETB-40P, 41P, and 42P to big ET-1 by FXa digestion were 5.6, 11.2, and 30.0%, respectively. pETB-40P with a deletion of one Cys residue and four Arg residues in the N-terminal part was a better substrate than the other two for FXa or trypsin in the production of big ET-1.View full abstractDownload PDF (1229K) -
Kwang-Ho Hahn, Hyoungman Kim1991 Volume 110 Issue 4 Pages 635-640
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman apohemoglobin in acidic media was found to induce fusion of phosphatidylcholine/phosphatidylserine (1:1) vesicles at low protein concentration but to fragment the same vesicles to form micellar complex at high protein concentration. The fusion was demonstrated by size increase, vesicle content mixing, lipid mixing, and electron microscopy. The micellization of phospholipid vesicles was observed by light scattering, gel filtration, and electron microscopy. The hydrophobic labeling of the apohemoglobin/vesicle complex followed by CNBr cleavage of apohemoglobin showed that an N-terminal segment of the βsubunit with a molecular weight of approximately 6, 000 seems to be mainly involved in the fusion process, but the whole sequences of both α and β chains participate in the micellization process.View full abstractDownload PDF (1234K) -
Ikuko Sagami, Tetsuo Ohmachi, Hiroshi Fujii, Hideaki Kikuchi, Minro Wa ...1991 Volume 110 Issue 4 Pages 641-647
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methyl-cholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2, 916 by long and contained an entire coding region for 524 amino acids with a molecular weight of 59, 408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1, 876 by long and encoded a polypeptide of 513 amino acids with a molecular weight of 58, 079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between 1128 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2. 9 and 1. 9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.View full abstractDownload PDF (1040K) -
Satoko Isemura, Eiichi Saitoh, Kazuo Sanada, Kayoko Minakata1991 Volume 110 Issue 4 Pages 648-654
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOur recent work on the gene structures for human salivary (S-type) cystatins [ Saitoh, E. et al. (1987) Gene 61, 329-338] has suggested that the structures of cystatins which we determined previously at the protein level lack N-terminal peptide portions of the fullsized intact forms. In the present study, attempts were made to isolate full-sized S-type cystatins by introducing methanol fractionation into the purification steps to suppress the enzymatic activity present in saliva. Full-sized cystatin SN and two phosphorylated forms of full-sized cystatin S were thus isolated. Analysis of one fraction indicated that this was a mixture of full-sized cystatin SA and non-phosphorylated cystatin S. The phosphorylation sites of cystatin S were determined to be Ser-Ser-Ser1(P)-Lys-Glu-Glu- for monophos-phorylated cystatin S and Ser1 (P)-Ser-Ser3(P)-Lys-Glu-Glu- for diphosphorylated cystatin S. Immunoblotting analysis with anti-cystatin S antiserum revealed that tears and seminal plasma also contained S-type cystatins, but diphosphorylated cystatin S was detected neither in tears nor in seminal plasma and no cystatin SN was found in seminal plasma. These data indicate that S-type cystatins are secreted into the oral cavity without significant degradation in salivary glands or ducts and that they are expressed tissue specifically.View full abstractDownload PDF (1010K) -
Masahiro Tominaga, Yasuo Kitagawa, Shin-ichiro Tanaka, Akira Kishimoto1991 Volume 110 Issue 4 Pages 655-660
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat brain type II (β) protein kinase C (PKC) was phosphorylated by rat lung casein kinase II (CK-II). Neither type I (γ) nor type III (α) PKC was significantly phosphorylated by CK-II. CK-II incorporated 0. 2-0. 3 mol of phosphate into 1 mol of type II PKC. This phosphate was located at the single seryl residue (Ser-11) in the V1-variable region of the regulatory domain of the PKC molecule. A glutamic acid cluster was located at the carboxyl-terminal side of Ser-11, showing the consensus sequence for phosphorylation by CK-II. The velocity of this phosphorylation was enhanced by the addition of Ca2+, diolein, and phosphatidylserine, which are all required for the activation of PKC. Phosphorylation of casein or synthetic oligopeptides by CK-II was not affected by Ca2+, diolein, or phos-phatidylserine. Available evidence suggests that CK-II phosphorylates preferentially the activated form of type II PKC. It remains unknown, however, whether this reaction has a physiological significance.View full abstractDownload PDF (1070K) -
Hiroshi Terakawa, Hidenari Takahara, Kiyoshi Sugawara1991 Volume 110 Issue 4 Pages 661-666
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree types of mouse peptidylarginine deiminase were separated by DEAE-Sephacel ion-exchange column chromatography, and we propose designating them peptidylarginine deiminase type I, II, and III according to the order of elution. The type II enzyme was widely distributed in various tissues including the skeletal muscle, whereas the type I enzyme was localized in the epidermis and uterus, and the type III enzyme was detected in the epidermis and hair follicles. These enzymes were distinguished by their molecular weights and substrate specificity. The molecular weights were estimated to be approximately 54, 000 (type I) and 100, 000 (type II and III) by Sephacryl S-200 gel filtration column chromatography. On SDS-PAGE the type II and III enzymes gave Mr=81, 000 and Mr=76, 000, respectively. Among the substrates tested, the type I enzyme showed highest activity toward Bz-L-Arg-NH2, type II toward Bz-L-Arg-O-Et, and type III toward protamine. Western blot analysis showed that antibodies against the type II enzyme were immuno-crossreactive to the type III enzyme.View full abstractDownload PDF (710K) -
Tatsuo Sugano, Tsuyoshi Uchida, Masaru Yamaizumi1991 Volume 110 Issue 4 Pages 667-674
Published: October 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA protein factor which corrects the defect in xeroderma pigmentosum cells belonging to complementation group A (XP-A cells) was detected in a cell extract prepared from calf thymus. The activity of this factor was measured as the amount of unscheduled DNA synthesis (UDS) reappearing in UV-irradiated XP-A cells after microinjection of the extract. The native molecular mass of this factor was estimated to be 80 kDa by gel-filtration and 25 kDa by glycerol gradient centrifugation. The activity was, however, recovered at a position corresponding to 43 kDa after renaturation on an SDS-PAGE gel. The isoelectric point was determined to be approximately 7. 5 by measuring the activity after renaturation on an IEF gel. These values were obtained with a partially purified sample. A spot corresponding to these values was detected on two-dimensional gel electrophoresis with a highly purified sample recovered from an SDS-PAGE gel. The purified protein stimulated UDS specifically in the XP-A cells and endowed the cells with a normal level of UV-resistance. The XP-A cells injected with the factor also showed a normal level of UDS after treatment with either 4HAQO or psoralen plus UV-A. This factor (XP-A complement-ing factor; XP-ACF) may be involved in the repair of DNA damage induced by various agents.View full abstractDownload PDF (1039K) -
1991 Volume 110 Issue 4 Pages 675
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (14K)
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