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Volume 127 (2000)
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Volume 127, Issue 6
Displaying 1-26 of 26 articles from this issue
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Yoshikazu Tomisugi, Masaki Unno, Tsunehiro Mizushima, Yukio Morimoto, ...2000 Volume 127 Issue 6 Pages 941-943
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS20 S proteasomes from higher eukaryotes have immunological functions rather than those from archibacteria or yeast. To clarify the mechanism of the sorting and production of antigen-presenting peptides, it is important and worthwhile to determine the structure of mammalian proteasomes using a third generation synchrotron radiation source. Here we report new crystal forms of 20 S proteasomes from bovine liver and preliminary structure analysis of them. The crystals belong to the same space group but have different cell dimensions. One crystal (form I) belongs to space group P 212121 with unit cell dimensions of a=124.8, b=197.4, c=323.8 Å, and diffracts to 3.0 Å resolution. The other crystal (form II) belongs to the same space group with a=115.1, b=205.6, c=316.0 Å, and diffracts to 4.0 Å resolution. The diffraction data for the form I crystal provided an interpretable electron density map for presenting the structural differences from yeast proteasomes.View full abstractDownload PDF (1831K) -
Motoko Sasaki, Kazuhiro Ogata, Hideki Hatanaka, Yoshifumi Nishimura2000 Volume 127 Issue 6 Pages 945-953
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe DNA-binding domain of c-Myb consists of three imperfect tandem repeats, R1, R2, and R3. Each repeat contains three helices. The minimal DNA-binding domain is an R2 R3 fragment. Here, we have examined the backbone dynamics of R2 R3 in its DNA-bound form by NMR. Upon binding to DNA, the N- and C-termini, and the linker between R2 and R3 become less flexible. In the free form the third helix of R2 exhibits slow conformational exchange fluctuations owing to a cavity in the hydrophobic core of R2. Upon binding to DNA, the conformational exchange contributions in R2 are reduced but remain significant in NMR relaxation measurements. Upon binding to DNA, the third helix of R3 comes to exhibit significant chemical exchange contributions. These findings suggest that the orientations of the third helices of both R2 and R3 as to DNA are being chemically exchanged. In the DNA-bound form both R2 and R3 exhibit similar dynamical characters, except for amino acids Trp 95, Thr 96, and Val 103 of R2, which are located around the cavity of the unbound form. Upon binding to DNA, since Trp 95 moves into the cavity to fill it up, the local conformational exchange contributions seem to be still observable around the filled cavity.View full abstractDownload PDF (689K) -
Makoto Kimura, Shigeko Sekido, Yasuhiro Isogai, Isamu Yamaguchi2000 Volume 127 Issue 6 Pages 955-963
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe established an efficient overproduction-purification system for blasticidin S deaminase (BSD) using the cDNA cloned from Aspergillus terreus. The estimated molecular mass of the purified enzyme indicated BSD was a tetramer. This tetrameric form was very resistant to denaturation by SDS and showed heat-modifiable behavior on SDS-PAGE; i.e., BSD migrated much slower (as a single band of 36 kDa) in its active conformation than its completely denatured polypeptide (13 kDa) if heat treatment in 2% SDS was not performed before electrophoresis. As predicted from the presence of the catalytic zinc-coordinating sequence motif conserved in the cytosine nucleoside/nucleotide deaminase family, BSD also contained one zinc per deaminase subunit. However, the predicted catalytic function appeared not to be the only role of this zinc in the enzyme. First, titration of the zinc-chelating -SH groups with p-hydroxymercuriphenylsulfonate led to dissociation of the BSD tetramer into unstable monomers or dimers. Second, depletion of zinc on reconstitution of chemically denatured BSD (with either guanidine-HCl or acidic pH) resulted in improper folding of the polypeptide. These results suggest that zinc also plays a structural role in maintenance of the protein structure. When we introduced mutations at Glu-56 (the proposed active site) and Cys-91 (a proposed catalytic zinc-binding Cys) in BSD, none of the resulting mutants (E 56D, E 56Q, C 91A, C 91S, and C 91H) showed any detectable activity, as judged with the spectrophotometric assay. Replacements of Cys-91 resulted in gross perturbation of the enzyme structure although the catalytically essential Glu-56 was not necessarily required for proper folding of the enzyme. These results further support our proposal that the catalytic zinc coordinated by the conserved sequence motif is also structural in BSD.View full abstractDownload PDF (2305K) -
Sachiko Okuno, Takako Kitani, Hidenori Matsuzaki, Hiroaki Konishi, Ush ...2000 Volume 127 Issue 6 Pages 965-970
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProtein kinase B (PKB) was recently reported to be activated on the phosphorylation of Thr308 by Ca2+/calmodulin-dependent protein kinase kinase α (CaM-kinase kinase α), suggesting that PKB was regulated through not only the phosphoinositide 3-kinase pathway but also the Ca2+/cahnodulin protein kinase pathway. The activation of PKB by CaM-kinase kinase α was as high as 300-fold after incubation for 30min under the phosphorylation conditions, and still increased thereafter, suggesting that the maximal activation of PKB on phosphorylation of the Thr308 residue is several hundred fold. On the other hand, the Vmax value of CaM-kinase kinase α for the phosphorylation of PKB was more than two orders of magnitude lower than that for CaM-kinase IV, although the Km values for PKB and CaM-kinase IV were not significantly different, raising the question of whether or not PKB is a physiological substrate of CaM-kinase kinase α. Besides CaM-kinase kinase α, CaM-kinase H also remarkably activated PKB. However, the specific activities of CaM-kinase kinase α and CaM-kinase II as to the activation of PEB were more than three orders of magnitude lower than that of 3-phosphoinositide-dependent protein kinase 1 (PDK1).View full abstractDownload PDF (1196K) -
Noriko Noguchi, Kazumi Nakano, Yasuaki Aratani, Hideki Koyama, Tatsuhi ...2000 Volume 127 Issue 6 Pages 971-976
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLow density lipoprotein was oxidized by neutrophils derived from either C57BL/6 mice or myeloperoxidase (MPO)-knockout mice. The generation of superoxide from neutrophils of MPO-knockout mice was about 70% of that from wild-type mice. The extent of the oxidation of human low density lipoprotein (LDL) by phorbol myristate acetate (PMA)-activated neutrophils of wild-type and MPO-knockout mice was assessed by measuring consumption of α-tocopherol and formation of phosphatidylcholine hydroperoxide (PCOOH) and cholesteryl ester hydroperoxide (CEOOH). Little consumption of α-tocopherol was observed in both oxidations. It was found, however, that lipid hydroperoxides were accumulated with time in both oxidations and that the rates of formation of PCOOH and CEOOH in the oxidation by MPO-knockout neutrophils were about 66 and 44% of those by wild-type neutrophils, respectively. The lipid peroxidation was completely inhibited by adding superoxide dismutase (SOD) in both cases. The addition of L-tyrosine and SOD enhanced lipid peroxidation of LDL induced by wild-type neutrophils but not by MPO-knockout ones. These results suggest that, regardless of their MPO activity, neutrophils induce lipid peroxidation of LDL by a superoxide-dependent pathway, and that MPO-catalyzed lipid peroxidation is enhanced by the presence of an appropriate amount of free tyrosine and further enhanced by SOD.View full abstractDownload PDF (443K) -
Ryuta Koishi, Takemichi Nakamura, Tomoko Takazawa, Chigusa Yoshimura, ...2000 Volume 127 Issue 6 Pages 977-983
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn a previous study, we reported the isolation of a cDNA encoding KDRF (K-M-102-d-erived r-eductase like f-actor) from the human bone marrow-derived stromal cell line KM-102. Analysis of the sequence of this cDNA revealed it to be the previously reported human thioredoxin reductase cDNA. Human thioredoxin reductase, which was recently isolated from human lung adenocarcinoma NCI-H 441 cells as a selenocysteine-containing selenoprotein, and its substrate thioredoxin are thought to be essential for protecting cells from the damage caused by reactive oxygen species. To obtain the selenocysteine-containing recombinant KDRF/thioredoxin reductase, we introduced a secondary structure, which is identical to the selenocysteine insertion signal of Escherichia coli formate dehydrogenase H mRNA, downstream of the TGA in the KDRF/thioredoxin reductase cDNA and expressed it in E. coli. As a result, a significant amount of selenocysteine was incorporated into the C-terminus of the KDRF/thioredoxin reductase protein. The selenocysteine-containing KDRF/thioredoxin reductase showed reducing activities toward human and E. coli thioredoxin, whereas non-selenocysteine-containing KDRF/thioredoxin reductase showed no enzyme activity. Our results suggest that this strategy will be applicable to the production of other mammalian selenocysteine-containing selenoproteins in E. coli.View full abstractDownload PDF (1273K) -
Naruhiro Tanaka, Tetsuo Ishida, Sinsuke Hukuda, Kihachiro Horiike2000 Volume 127 Issue 6 Pages 985-991
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn intervertebral disc is a large peice of avascular cartilage rich in proteoglycans and water consisting of gelatinous nucleus pulposus and fibrous annulus fibrosus. The soluble fraction of rabbit nucleus pulposus exhibited unusually high Ca2+-dependent phospholipase A2 (PLA2) activity (about 70% of the total PTA2 activity). The soluble PLA2 activity was 6-7-fold higher than those of rabbit annulus fibrosus and spleen. The PLA2 was bound to an anion-exchange column at pH 7.4, and eluted near the void volume as a broad peak on gel-filtration on a TSKgel SuperSW 3000 column developed with a buffer containing 0.1-0.2M salt. When the gel-filtration column was developed in the presence of 1M salt, almost all the PLA2 activity was eluted near the total available volume. The soluble PLA2 was purified to near homogeneity. A Ca2+-dependent PLA2 was also purified from the fractions extracted with 1M KBr from nucleus pulposus. For comparison, we purified a Ca2+-dependent PLA2 from the KBr fraction of spleen. The splenic PLA2 was identical to a group Ha PLA2, as judged from its N-terminal amino acid sequences and mass spectra. On SDS-polyacrylamide gel electrophoresis the enzymes purified from the soluble and KBr fractions of nucleus pulposus both gave a major 15.7-kDa band at the same position as splenic group IIa PLA2. These results suggest that group IIa PLA2 is associated with soluble high-molecular-weight proteins, most likely proteoglycans, in the extracellular matrix of rabbit nucleus pulposus.View full abstractDownload PDF (2212K) -
Shinji Asano, Ryoko Furumoto, Yasuhiro Tega, Saiko Matsuda, Noriaki Ta ...2000 Volume 127 Issue 6 Pages 993-1000
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy means of a functional expression system and site-directed mutagenesis, we analyzed the role of the putative K+-binding site, Glu-345, located in the fourth transmembrane segment of the gastric H+, K+-ATPase α-subunit. In the present study, we used several mutants, with alanine, isoleucine, leucine, glutamine, valine, lysine, and aspartic acid instead of Glu-345, and analyzed the H+, K+-ATPase partial reactions of the mutants to determine the precise role of this residue. All the mutants except E 345 Q exhibited no H+, K+-ATPase activity. The E 345 Q mutant showed 3-times higher affinity for ATP. This mutation shifted the optimum pH toward a more alkaline one. The E 345 A, E 345 I, E 345 L, E 345 V as well as E 345 Q mutants were phosphorylated with ATP as in the case of the wild-type H+, K+-ATPase, whereas the E 345 K mutant was not phosphorylated. The E 345 Q mutant was dephosphorylated in the presence of K+, but its affinity for K+ was significantly lower than that of the wild type. The E 345 A, E 345 I, E 345 L, and E 345 V mutants did not exhibit sensitivity to K+ in the dephosphorylation step below 3mM K+. Therefore, Glu-345 is important for the conformational change induced by K+, especially in the dephosphorylation step in which K+ reacts with the enzyme from the luminal side with high affinity and accelerates the release of inorganic phosphate. The glutamic acid in the fourth transmembrane segment is conserved, and was found to be involved in the cation-induced conformational change in H+, K+-ATPase as well as Na+, K+-ATPase and Ca2+-ATPase, however, the precise roles of the side chain in the function were different.View full abstractDownload PDF (2828K) -
Hiroshi Manya, Yuji Sato, Naomi Eguchi, Kousuke Seiki, Hiroshi Oda, Hi ...2000 Volume 127 Issue 6 Pages 1001-1011
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLipocalin-type prostaglandin D synthase (L-PGDS) is a highly glycosylated member of the lipocalin gene family and is secreted into various human body fluids. We comparatively analyzed the structures of asparagine-linked sugar chains of human L-PGDS produced by recombinant Chinese hamster ovary cells and naturally occurring human urine and amniotic fluid. After the sugar chains were liberated by hydrazinolysis followed by N-acetylation, they were derivatized with 2-aminobenzamide. All of the sugar chains of three L-PGDSs occur as biantennary complex-type sugar chains. Most of the sugar chains of three samples were fucosylated on the inner most N-acetylglucosamine residue. Although the sugar chains of the recombinant L-PGDS do not contain any bisecting N-acetylglucosamine residues, 58% and 34% of the fucosylated-sugar chains of amniotic fluid and urine L-PGDSs, respectively, contain bisecting N-acetylglucosamine residues. The sialic acid residues occur solely as Siaα2→3 Gal groups of the recombinant L-PGDS; the sialic acid residues of other L-PGDS occur as both Siaα2→3 Gal and Siaα2→6 Gal groups. Variations in L-PGDS glycosylation may prove useful as markers to further elucidate the role of L-PGDS glycoforms in different tissues.View full abstractDownload PDF (1698K) -
Yoshinobu Kimura, Sayuri Matsuo2000 Volume 127 Issue 6 Pages 1013-1019
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAs a part of our studies to elucidate the physiological significance of free N-glycans in differentiating or growing plant cells, we first demonstrate that two kinds of free N-glycans already occur at an early stage of seed development. In this report, we used the developing Ginkgo biloba seeds as a model plant, since we have already revealed a functional feature of the Ginkgo endo-β-N-acetylglucosaminidase and structural features of N-glycans linked to storage glycoproteins in the developing seeds [Kimura, Y. et al. (1998) Biosci. Biotechnol. Biochem. 62, 253-261; Kimura, Y. and Matsuo, S. (2000) Biosci. Biotechnol. Biochem. 64, 562-568]. The structures of free N-glycans, which were determined by a combination of ESI-MS, sequential α-mannosidase digestions, partial acetolysis, and two dimensional sugar chain map, fell into two categories. One dominant species is a high-mannose type structure having one GlcNAc residue at the reducing end (Man9-5GlcNAc1). The concentration of this type of free glycan (as the pyridylaminated derivatives) is about 2.2 nmol in 1g fresh weight. The detailed structural analysis revealed that the high-mannose type structures have a common core unit; Manα1-6 (Manl-3) Manα1-6 (Manα1-3) Manβ1-4 GlcNAc. The other minor species of free N-glycans is the plant complex type structure having an N-acetylchitobiose unit at the reducing end (Man3Xyl1Fuc1GlcNAc2). The concentration of this type of free glycan (as the pyridylaminated derivative) was about 75 pmol in 1g fresh weight.View full abstractDownload PDF (496K) -
Ulrike Hubl, Hideharu Ishida, Makoto Kiso, Akira Hasegawa, Roland Scha ...2000 Volume 127 Issue 6 Pages 1021-1031
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe sensitivity and specificity of two influenza C virus assays, solid-phase and overlay assays, were investigated using naturally occurring 9-O-acetylated GD3, rat serum glycoproteins containing 60% of N-acetyl-9-O-acetylneuraminic acid, and synthetically O-acetylated sialylated compounds. The sensitivity of the solid-phase assay was higher for glycoproteins containing N-acetyl-9-O-acetylneuraminic acid than for gangliosides, and also differed for various 9-O-acetylated gangliosides. The overlay assay was less sensitive for all glycoconjugates tested. For virus recognition the presentation of the sialic acid within the molecule and the structure of the sialic acid are essential. Investigation of gangliosides from human melanomas and normal skin with the influenza C virus assay showed an increase of O-acetylation of sialic acids in most tumour samples and the occurrence of several O-acetylated gangliosides.View full abstractDownload PDF (3383K) -
Shaoliang Li, Shigemi Norioka, Fumio Sakiyama2000 Volume 127 Issue 6 Pages 1033-1039
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel bacteriolytic enzyme CwhA (c-ell w-all h-ydrolytic a-midase) was purified by ion exchange and gel-filtration chromatographies from a commercial bacteriolytic preparation from Achromobacter lyticus. CwhA exhibited optimal pH at 8.5 and lysed CHCl3-treated Escherichia coli more efficiently than Micrococcus luteus, Staphylococcus aureus, Enterococcus faecalis, and Pediococcus acidilactici. The enzyme was inhibited by 1, 10-phenanthroline strongly and by EDTA to a lesser extent, suggesting that it is probably a metalloenzyme. Amino acid composition and mass spectrometric analyses for the CwhA-derived M. luteus muropeptides revealed that CwhA is N-acetyhnuramoyl-L-alanine amidase [EC 3. 5. 1. 28]. The complete amino acid sequence of CwhA was established by a combination of Edman degradation and mass spectrometry for peptides obtained by Achromobacter protease I (API) digestion and cyanogen bromide (CNBr) cleavage. The enzyme consists of a single polypeptide chain of 177 amino acid residues with one disulfide bond, Cys 114-Cys 121. CwhA was found to be homologous to N-acetylmuramoyl-L-alanine amidase from bacteriophage T 7 (BPT 7). Its sequence identity with BPT 7 is 35%, but the amino acid residues functioning as zinc ligands in BPT 7 are absent in CwhA. These results suggest that CwhA is a new type of N-acetylmuramoyl-L-alanine amidase.View full abstractDownload PDF (527K) -
Kuniyo Inouye, Akitoshi Saito, Manabu Orita, Ben'ichiro Tonomura, Hiro ...2000 Volume 127 Issue 6 Pages 1041-1046
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe reported previously that various naphthoquinone derivatives inhibited cytochrome P 450-dependent monooxygenase of liver and placenta microsomes [Muto, N. et al. (1987) Biochem. Biophys. Res. Commun. 146, 487-494]. To understand the complex inhibitory behaviors that were observed, it is desirable to study the relationship between structure and inhibitory activity of naphthoquinones in a simplified system containing a single P 450 species. In the present study, the inhibitory effects of six derivatives of 1, 4-naphthoquinone (hereafter referred to as NQ) on rat cytochrome P 4501 A 1-dependent 7-ethoxycoumarin O-deethylation were examined using yeast microsomes containing overexpressed rat P 4501 A 1. Of these, 2-methyl-5-hydroxy-NQ, 2-methyl-NQ, 2-hydroxy-NQ, and NQ showed competitive inhibition, whereas 5, 8-dihydroxy-NQ and 5-hydroxy-NQ showed noncompetitive inhibition. Judging from the inhibitor constant (Ki), the binding affinity of the four competitive inhibitors for the substrate-binding pocket of P 4501 A 1 is in the order: 2-CH3-5-OH-NQ>2-CH3-NQ>NQ>>2-OH-NQ. On binding with P 4501 A 1, 2-CH3-5-OH-NQ, 2-CH3-NQ, and NQ induced distinct Type II, Type I, and reverse Type I spectra, respectively. These results indicate that methyl and hydroxyl groups introduced into NQ have unique effects on their binding mode and binding affinity.View full abstractDownload PDF (513K) -
Yasushi Kikuta, Emi Kusunose, Masamichi Kusunose2000 Volume 127 Issue 6 Pages 1047-1052
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously reported the cloning of a human liver leukotriene B4 (LTB4) ω-hydroxylase P 450 designated CYP 4 F 2 [Kikuta et al. (1994) FEBS Lett. 348, 70-74]. However, the properties of CYP 4 F 2 remain poorly defined. The preparation solubilized using n-octyl-β-D-glucopyranoside from microsomes of CYP 4 F 2-expressing yeast cells catalyzes ω-hydroxylation of LTB4, 6-traps-LTB4, lipoxin A4, 8-hydroxyeicosatetraenoate, 12-hydroxyeicosatetraenoate, and 12-hydroxystearate in the presence of rabbit liver NADPH-P 450 reductase. In addition, the enzyme shows ethoxycoumarin O-deethylase and p-nitroanisole O-demethylase activities. The enzyme was purified to apparent electrophoretic homogeneity from yeast cells by sequential chromatography of solubilized microsomes through amino-n-hexyl-Sepharose 4 B, DEAE-HPLC, and hydroxylapatite HPLC columns. The final preparation showed a specific content of 11.1 nmol of P 450/mg of protein, with an apparent molecular mass of 56.3 kDa. CYP 4 F 2 was distinguished from the closely homologous CYP 4 F 3 (human neutrophil LTB4 ω-hydroxylase) by its much higher Km for LTB4 inability to ω-hydroxylate lipoxin B4, and extreme instability.View full abstractDownload PDF (983K) -
Hyean-Woo Lee, Joon Hyung Sohn, Byung-II Yeh, Jong-Whan Choi, Seunho J ...2000 Volume 127 Issue 6 Pages 1053-1056
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGrowth of Escherichia coli in the presence of glyphosate, an inhibitor of aromatic amino acid biosynthesis, has permitted the production of proton-dislocating ATPase that is specifically labeled with 5-fluorotryptophan. Five sets of 19F resonances could be assigned to each tryptophan residue by lauryldimethylamine oxide and carboxypeptidase treatment. On labeling with 4-chloro-7-nitro-benzofurazan, the label attached to β155 Lys, which is known to be in the catalytic site, which caused one of the residues, β108 Trp, to become nonequivalent. 19F NMR spectroscopic investigation of internally fluorotryptophan-labeled F1-ATPase will provide valuable information about the asymmetric nature of F1-ATPase and the conformational changes induced by ligand binding.View full abstractDownload PDF (1548K) -
Yan Zhang, Jeong Won Pak, Ichiro N. Maruyama, Masayuki Machida2000 Volume 127 Issue 6 Pages 1057-1063
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo transcription factors, human ATF 1, its DNA-binding domain (ATFIBD), and the DNA-binding domain (GAL 4 BD) of the yeast GAL 4 protein, were displayed on the surface of bacteriophage λ vectors and efficiently selected by DNA fragments immobilized in microtiter wells. The DNA-binding proteins are fused to the carboxy terminus of the tail protein gpV and head protein gpD of the vectors, λfoo and λfooDc, respectively. After a single round of affinity selection, the fusion phages were successfully enriched 60- to 4, 000-fold over the vector phages. Further, the GAL 4 BD fusion phages were enriched 5- and 15-fold by affinity selection using specific DNA as probes over nonspecific DNA when expressed on AfooDc and λfoo, respectively. The ATF 1 BD fusion phages were also sequence-specifically enriched greater than 4-fold when displayed on λfoo. These results suggest that the λfoo display system is useful for in vitro studying of protein-DNA interactions and may be applied to screening of DNA-binding protein from complex cDNA libraries through DNA-binding affinity.View full abstractDownload PDF (2974K) -
Cloning and Characterization of a Myosin from Characean Alga, the Fastest Motor Protein in the WorldTaku Kashiyama, Naohiro Kimura, Tetsuro Mimura, Keiichi Yamamoto2000 Volume 127 Issue 6 Pages 1065-1070
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn characean algae, very rapid cytoplasmic streaming is generated by sliding movement of an unconventional myosin on fixed actin cables. The speed of this sliding movement is the fastest among many molecular motors known so far. We have cloned a set of overlapping cDNAs encoding the heavy chain of this myosin by immunoscreening with antibody raised against characean myosin. The molecular mass of this heavy chain is 248 kDa, and the protein has a conserved motor domain, six IQ motifs, an extensive α-helical coiled-coil domain, and a C-terminal globular domain. Phylogenetic analysis suggested that this myosin belongs to class XI.View full abstractDownload PDF (1728K) -
Hitoshi Nakatogawa, Hiroyuki Mori, Gen Matsumoto, Koreaki Ito2000 Volume 127 Issue 6 Pages 1071-1079
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe secY205 mutant is cold-sensitive for protein export, with an in vitro defect in supporting ATP- and preprotein-dependent insertion of SecA into the membrane. We characterized SecA81 with a Gly516 to Asp substitution near the minor ATP-binding region, which suppresses the secY205 defect at low temperature and exhibits an allele-specific synthetic defect with the same SecY alteration at 42°C. The overproduced SecA81 aggregated in vivo at temperatures above 37°C. Purified SecA81 exhibited markedly enhanced intrinsic and membrane ATPase activities at 30°C, while it was totally inactive at 42°C. The trypsin digestion patterns indicated that SecA81 has some disorder in the central region of SecA, which encompasses residues 421-575. This conformational abnormality may result in unregulated ATPase at low temperature as well as the thermosensitivity of the mutant protein. In the presence of both proOmpA and the wild-type membrane vesicles, however, the thermosensitivity was alleviated, and SecA81 was able to catalyze significant levels of proOmpA-stimulated ATP hydrolysis as well as proOmpA translocation at 42°C. While SecA81 was able to overcome the SecY205 defect at low temperature, the SecY205 membrane vesicles could not significantly support the translocation ATPase or the proOmpA translocation activity of SecA81 at 42°C. The inactivated SecA81 molecules seemed to jam the translocase since it interfered with translocase functions at 42°C. Based on these results, we propose that under preprotein-translocating conditions, the SecYEG channel can stabilize and activate SecA, and that this aspect is defective for the SecA81-SecY205 combination. The data also suggest that the conformation of the central region of SecA is important for the regulation of ATP hydrolysis and for the productive interaction of SecA with SecY.View full abstractDownload PDF (2579K) -
Tomofumi Fujino, Ken Ando, Masatoshi Beppu, Kiyomi Kikugawa2000 Volume 127 Issue 6 Pages 1081-1086
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSErythrocytes oxidized or aged in the circulation undergo membrane protein aggregation and anti-band 3 autoantibody binding to the cell surface. When human erythrocytes were mildly oxidized in vitro with 0.1mM Fe(III) at 37°C for 3 h, the aggregation of nonionic detergent C12E8-insoluble membrane protein and the binding of anti-band 3 IgG to the cell surface were increased. Incubation of membranes isolated from the oxidized cells increased the amount of protein aggregates by 5-fold after 6 h, while incubation for a further 12 h sharply decreased the amount of aggregates. In the presence of diisopropyl fluorophosphate (DFP), however, the increased amount of aggregates was maintained in the subsequent incubation. Western blot analysis of the aggregates using rabbit anti-band 3 showed that band 3 protein aggregates increased in the initial stage of incubation and decreased upon subsequent incubation, whereas the increased band 3 protein aggregates did not subsequently decrease when membranes were incubated in the presence of DFP. Incubation of the oxidized cells at 37°C for 18 h caused reduction of the membrane protein aggregates and the 125I-anti-band 3 IgG binding to the cell surface, while incubation in the presence of DFP did not cause these reductions. The results suggest that the oxidation-induced cell membrane protein aggregates were probably removed by 80-kDa serine protease, namely, oxidized protein hydrolase (OPH), in the oxidized cell membranes [Fujino et al. (1998) Biochim. Biophys. Acta 1374, 47-54; (1998) J. Biochem. 124, 1077-1085; (2000) Biochim. Biophys. Acta 1478, 102-112], and as a result the increased anti-band 3 binding to the cell surface was reduced.View full abstractDownload PDF (1174K) -
Tomofumi Fujino, Masaki Kojima, Masatoshi Beppu, Kiyomi Kikugawa, Hide ...2000 Volume 127 Issue 6 Pages 1087-1093
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAmino acid sequences in H2O2-oxidized bovine serum albumin (BSA) that are susceptible to proteolytic cleavage by oxidized protein hydrolase (OPH) were investigated. When oxidized BSA was treated with OPH, low-molecular-weight fragments (54, 46, 24, 22, 20, and 8 kDa) were produced as analyzed by SDS-PAGE. N-Terminal amino acid sequence analysis of these fragments indicated that oxidized BSA was cleaved by OPH at three major sites, Leu218-Ser219, Tyr410-Thr411, and Phe506-Thr507, at an early stage of the proteolytic degradation. In the three-dimensional structure of BSA deduced by computer modeling, these cleavage sites were found to be located slightly inside the BSA molecule, in positions not easily accessible by OPH. The influence of oxidation on the tertiary structure of BSA was then investigated by hypothetically replacing all the four methionine and two tryptophan residues with their oxidized forms, methionine sulfoxide and N'-formyl-kynurenine, respectively. The three-dimensional structure of the hypothetically oxidized BSA indicated that all the three cleavage sites in the protein could become more exposed to the solvent than in unoxidized BSA. These results suggest that, upon oxidation of BSA, the amino acid sequences that are potentially cleavable by OPH but present inside the molecule become exposed on the surface and susceptible to proteolysis by OPH. This is the first report demonstrating the cleavage sites of oxidized protein by oxidized protein-selective protease, suggesting the possible mechanism of oxidized protein-selective degradation by the enzyme.View full abstractDownload PDF (1098K) -
Ken-Ichi Sano, Kayo Maeda, Toshiro Oda, Yuichiro Maéda2000 Volume 127 Issue 6 Pages 1095-1102
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVertebrate skeletal muscle α-tropomyosin polymerizes in a head-to-tail manner and binds cooperatively to actin. It has been postulated that the cooperative actin binding is governed by the strength of the head-to-tail interaction. In order to know the relationship between the head-to-tail affinity and actin binding, we studied the properties of tropomyosin variants with single residue substitutions at serine-283, the penultimate residue at the carboxyl terminus that is involved in the head-to-tail interaction. It has been shown that the phosphorylation of serine-283 strengthens the head-to-tail interaction. Viscometry was employed to compare the head-to-tail affinity of tropomyosin variants. Variant S283E showed higher viscosity whereas variant S283K showed lower viscosity compared with the wild type non-phosphorylated α-tropomyosin. The results confirm the idea that the interaction is sensitive to the ionic properties of residue 283. The strength of the head-to-tail interaction was assessed directly by sedimentation equilibrium using two pairs of tropomyosin variants designed so that only dimeric interactions were allowed within each pair. From one pair of variants with serine-283, the association constant was determined to be 2.6×104M-1 (SD=1.0×104), whereas for the second pair with glutamate-283, the affinity was 3.9×104M-1 (SD=1.6×104), slightly stronger than the former, consistent with the results of viscometry. The results indicate that the head-to-tail association is weak as previously implicated from light scattering measurements. Cosedimentation was employed to measure the cooperative actin binding of tropomyosin variants. Although previous results indicated the phosphorylation has no significant influence on the actin affinity, variant S283E shows a lower affinity compared with the control. Variants S283K and S283A show even lower affinities to actin, although these species bind to actin more cooperatively than does variant S283E. The results indicate that the affinity of the head-to-tail interaction between adjacent tropomyosin molecules is weak, and is substantially influenced by an extra charge at residue 283. On the other hand, the interaction with actin, the affinity and the cooperativity in actin binding, is dependent on amino acid residues at 283 and is not simply correlated with the strength of the head-to-tail interaction between Tm molecules in solution.View full abstractDownload PDF (1368K) -
Takanori Matsui, Nobuyuki Kurosawa, Kenji Hibi, Seiji Akiyama, Yasushi ...2000 Volume 127 Issue 6 Pages 1103-1107
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMGC-24/CD164 is a sialomucin expressed in many normal and cancerous tissues. In humans, soluble and transmembrane forms of MGC-24 are produced by alternative splicing. The total MGC-24 RNA level was found to be lower in human colorectal carcinomas as compared with the adjacent normal mucosal tissues. Lower MGC-24 mRNA levels in colon carcinomas and in the adjacent normal mucosa epithelium correlate with lymphatic vessel invasion by the carcinoma. The ratio of the soluble form to the transmembrane form of the mRNA in colorectal carcinomas was determined by ribonuclease protection assay. Higher ratios were correlated with less venous invasion and less remote metastasis, which became evident during postoperative observation.View full abstractDownload PDF (1716K) -
Nika Hotoda, Ryo Hanai2000 Volume 127 Issue 6 Pages 1109-1113
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRecombinant human DNA topoisomerase IIIα was expressed in mutant yeast cells devoid of both topoisomerases I and III, and the gene product was partially purified. The activity of the protein in supercoil removal was found to be limited and also biphasic: in the first phase it processively changed the linking-number of hypernegatively supercoiled DNA, but only to the superhelicity of a regular negative supercoil; in the second phase the enzyme relaxed the DNA further, but only slightly and slowly. The optimal solution conditions for the enzyme activity were found to be physiological. The assay results with a truncation mutant showed that the C-terminal 334 amino acids are unnecessary for the activity, suggesting that this region, and perhaps the entire protein, is involved in a function other than supercoil removal.View full abstractDownload PDF (2917K) -
Kazuo Inaba2000 Volume 127 Issue 6 Pages 1115-1120
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSConformational changes of dynein during ATP hydrolysis are demonstrated by the difference in the tryptic fragments of the dynein heavy chain between in the absence and presence of ATP and vanadate. Here tryptic sites in the presence of ATP and vanadate (Tav sites) have been mapped on the β heavy chain of outer arm dynein from sea urchin sperm flagella. Tav sites are located not only near the central catalytic domain which includes four P-loops, but also near the carboxyl-terminal coiled-coil region. The Tav2 site is located in the most carboxyl-terminal region, which is nearly 850 amino acid residues apart from the the fourth P-loop (P4 site). The region from the most amino-terminal Tav site (Tav1 site) to the Tav2 site covers approximately 2, 100 amino acid residues, which is almost half the whole β heavy chain. Comparison of the sequences around the tryptic sites of the sea urchin β chain and those of the dynein heavy chains from other organisms reveals that the sequence around the Tav1 site is highly conserved in both cytoplasmic and axonemal dyneins but that around Tav2 sites is only conserved in axonemal dyneins, suggesting functional differences in the Tav2 region between the two subfamilies of dynein.View full abstractDownload PDF (1681K) -
Yasushi Makino, Atsuyuki Shimazaki, Kaoru Omichi, Shoji Odani, Sundhir ...2000 Volume 127 Issue 6 Pages 1121-1126
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA processing The processing pathway of N-glycans in Carica papaya was deduced from the structures of N-glycans. The N-glycans were liberated by hydrazinolysis followed by N-acetylation. Their reducing-end sugar residues were tagged with 2-aminopyridine and the pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Eleven PAsugar chains were found, and their structures were analyzed by two-dimensional sugar mapping combined with partial acid hydrolysis and exoglycosidase digestion. The structures of the N-glycans were of the highmannose types with xylose and fucose; however, among them two new N-glycans, Manα1-6(Manα1-3)Manal-6(Xy1β1-2) Manβ1-4G1cNAcβ1-4 (Fucα1-3)G1cNAc and Manα1-3Martα1-6(Xylβ1-2) Manβ1-4G1cNAcα1-4 (Fucα1-3) GlcNAc, were found. Judging from these structures together with Manα1-6(Manα1-3) Manα1-6 (Manα1-3) (Xy1β1-2) Manβ1-4G1cNAcβ1-4(Fucαl-3)G1cNAc reported previously [Shimazaki, A., Makino, Y., Omichi, K., Odani, S., and Hase, S. (1999) J. Biochem, 125, 560-565], a processing pathway for N-glycans in C. papaya is inferred in which the activity of Golgi α-mannosidase II is incomplete.View full abstractDownload PDF (385K) -
Hirohisa Shiraishi, Ayako Kobayashi, Yastunitsu Sakamoto, Takamasa Non ...2000 Volume 127 Issue 6 Pages 1127-1134
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreviously, we purified a 59-kDa protein that binds to the κB motif of the Sarcophaga lectin gene. Here we report its cDNA cloning and some of its characteristics as a novel member of the Rel/Ankyrin-family. This protein, named SRAM, contained a Rel homology domain, a nuclear localization signal and 4 ankyrin repeats, but lacked the Ser-rich domain and PEST sequence that Relish contained. We found that SRAM was localized in the nuclei of NIH-Sape-4 cells, which are an embryonic cell line of Sarcophaga. The Sarcophaga lectin gene promoter containing tandem repeats of the κB motifs was activated in NIH-Sape-4 cells. In Drosophila mbn-2 cells, Dif alone activated this reporter gene and a cooperative effect was detected when SRAM and Dif were co-transfected, although SRAM alone did not activate it. This is the first report of a Rel/Ankyrin molecule that exists in the nuclei.View full abstractDownload PDF (2922K)
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