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Volume 119, Issue 1
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Ken-ichi Kasai, Jun Hirabayashi1996 Volume 119 Issue 1 Pages 1-8
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGalectins, animal lectins exhibiting specificity for galactosides, are now known to be widely distributed from lower invertebrates, such as sponges and nematodes, to higher vertebrates. The origin of the family can be traced back to the Precambrian era. They are classified into proto-, chimera-, and tandem-repeat types on the basis of protein architecture. The molecular functions of these types should be different because they can cross-link pairs of biomolecules of different combinations. Their biological significance, however, is not yet fully understood because they are involved in too many phenomena, such as differentiation, morphogenesis, metastasis, etc., and too many problems remain unsolved, such as those regarding their controversial cellular localization, mechanism of externalization, etc. Nevertheless, such difficulties seem to indicate their importance as household equipment and their common roles throughout the animal kingdom. They are likely to be responsible for recognizing the N-acetyllactosamine (LacNAc) structure, which is included in various glycoconjugates and considered to be an important glycocode, and then carry out appropriate tasks under given circumstances. Recently, crystallographic studies revealed that galectins and legume lectins such as concanavalin A have a common topology in spite of the absence of sequence homology. This suggests a possible relationship between animal and plant lectins, and the existence of a lectin super family. Studies on the galectin family are becoming increasingly important for glycobiology.View full abstractDownload PDF (1168K) -
Kazuko Ohgi, Mitsuaki Takeuchi, Masanori Iwama, Masachika Irie1996 Volume 119 Issue 1 Pages 9-15
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to establish the role of Tyr57 and Trp49 in the enzymatic reaction of RNase Rh, several mutant enzymes at Tyr57 and Trp49 were prepared by protein engineering and their enzymatic properties were investigated. Among the four mutant enzymes at Trp49 (W49F, W49Y, W49A, and W49I), W49F showed 16% of the activity of the native enzyme, but the others (W49Y, W49A, and W49I) showed greatly decreased activity. The data showed that Trp49 is very important for the enzyme activity. Among 8 mutant enzymes at the 57th position, Y57F and Y57W showed similar enzymatic activity toward RNA to that of the wild-type enzyme, but the others (Y57G, Y57A, Y57V, Y57M, and Y57K) are more active toward RNA and less active toward XpGs. The reason for the apparent increase for RNA activity is discussed from the view point of substrate inhibition. It is noteworthy that W49F and Y57W became more pyrimidine base- and purine base-preferential, respectively.View full abstractDownload PDF (1923K) -
Yasuhiko Yamamoto1996 Volume 119 Issue 1 Pages 16-22
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe molecular structure of the active site of horse heart met-cyano cytochrome c, as a function of temperature, has been investigated using 1H-NMR. A temperature dependence study of the NMR spectra revealed that one heme methyl proton resonance exhibits antiCurie behavior, i.e., the hyperfine shift increases with increasing temperature. Analyses of the average heme methyl proton hyperfine shift and the proximal His imidazole proton resonances indicated that the iron-His bonding interaction in this protein is essentially independent of temperature. Since such an anomalous temperature dependence of the heme methyl proton resonance disappears in met-cyano complex of a heme peptide prepared by enzymatic degradation of the protein [Smith, M. and McLendon, G. (1981) J. Am. Chem. Soc. 103, 4912-4921], the anti-Curie behavior observed for the heme methyl proton resonance in met-cyano cytochrome c is attributed to a rotational displacement of the heme about the iron-His bond relative to the protein moiety due to a temperature-dependent conformational alteration of the heme-protein linkage. Such rotational mobility of heme at the active site of a protein may be responsible for the anomalous temperature dependence of heme methyl proton hyperfine shifts reported for many c-type ferri cytochromes.View full abstractDownload PDF (945K) -
D. Yu, S. Imajoh-Ohmi, K. Akagawa, S. Kanegasaki1996 Volume 119 Issue 1 Pages 23-28
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman peripheral monocytes cultured with GM-CSF and IL-4 differentiated to dendritic cells (DCs) and with GM-CSF alone to macrophages. Superoxide-generating ability in such DCs was found to be suppressed whereas that in macrophages remained constant. To examine the reason for the suppression in DCs, we evaluated by immunoblotting the levels of essential components of the superoxide generating system in the cells during the differentiation. In contrast to the levels of cytosolic 47- and 65-kDa components and Rac-p21, which remained constant throughout cultivation, those of the large and the small subunits of cytochrome b558 were found to decrease quickly by day 2 during cultivation of monocytes with GM-CSF and IL-4. DCs obtained after 7 days of cultivation had lost the large subunit almost completely and most of the small subunit. A cell surface epitope of the cytochrome detected by a monoclonal antibody also decreased during the differentiation. On the other hand, these components, including both subunits of cytochrome b558, were maintained in the cells during differentiation of monocytes to macrophages. These results indicate that the decreased levels of cytochrome b558, especially that of the large subunit, is responsible for the low level of superoxide-generating ability of DCs and that the suppression is caused by IL-4.View full abstractDownload PDF (2476K) -
Atsushi Takeda, Dousei Higuchi, Takako Yamamoto, Yoshiko Nakamura, Yut ...1996 Volume 119 Issue 1 Pages 29-36
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBleomycin (BLM) hydrolase, which hydrolyzes the carboxyamide bond in the β-aminoalanine moiety, was purified from newborn rat skin. The enzyme was purified 2, 500-fold over the crude extract to apparent homogeneity in five steps in the presence of 2-mercaptoethanol: 45-55% ammonium sulfate fractionation, followed by chromatographies on Sephacryl S-200, DEAE-cellulofine, Phe-Superose, and Mono Q ion-exchange. The native enzyme had a molecular mass of 280 kDa according to gel filtration. The subunit molecular mass was estimated as 48 kDa by SDS-PAGE, indicating that the enzyme was comprised of six identical subunits. The amino acid sequence of its NH2, -terminus was determined to be acetyl-Met-Asn-Asn-Ala-Gly-Leu-Asn-Ser-Glu-Lys-, which was not found in the amino acid sequence database. The optimum pH of the enzyme was 7.5 with pepleomycin (PLM). The Km and Vmax values were 2.1mM and 6.8μmol•mg-1•h-1 for PLM, and 1.8mM and 7.2μmol•mg-1•h-1 for BLM-A2, respectively. The enzyme activity was inhibited by iodoacetic acid, N-ethylmaleinimide (NEM), and p-chloromercuribenzoic acid (pCMB) as well as divalent cations such as Cu2+, Cd2+, Hg2+, and Zn2+. It was effectively inhibited by a cysteine protease inhibitor E-64. However, cystatins A and C did not inhibit the activity. BLM hydrolase exhibited broad aminopeptidase substrate specificity towards aminoacyl-β-naphthylamides such as basic, neutral, and hydrophobic amino acid residues, as well as acidic residues. These results indicated that BLM hydrolase represents a new family of cysteine proteases. Western blotting and immunohistochemical analyses showed that BLM hydrolase is ubiquitous in various rat tissues but at low levels in lung and adult skin tissues, suggesting that this enzyme plays an important role in the metabolism of antibiotics.View full abstractDownload PDF (1943K) -
Sachiko Murase, Noboru Yumoto, Michael G. Petukhov, Susumu Yoshikawa1996 Volume 119 Issue 1 Pages 37-41
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCompetition assays using three series of analogs of neuropeptide Y (NPY) ([Xaa11] NPY (11-36), [Xaa12] NPY (12-36), and [Xaa13] NPY (13-36)) revealed that the binding affinity for the Y2 receptor was considerably lowered by truncation of residue 11. Upon acetylation or succinylation of the α-amino group, the binding affinity of [Xaa12] NPY (12-36) recovered to a level similar to that of [Xaa11] NPY (11-36). No significant difference was observed between the increases caused by acetylation and those caused by succinylation, suggesting that the increase in binding affinity cannot be explained by the change in the net charge at the N-terminus as a consequence of the modification. The scattered data points on a plot of the α-helix content vs. IC50 of all these analogs revealed the absence of any apparent relationship, an indication that prior formation of the α-helix is not necessary for binding to the Y2 receptor. It has been widely accepted that fewer than 12 residues from the C-terminus are directly involved in binding of NPY to the Y2 receptor, while the remaining part of NPY only assists in the adoption of a favorable conformation by the C-terminal hexapeptide for recognition by the receptor. However, the present results suggest that the region around residue 12 does not project from the Y2 receptor.View full abstractDownload PDF (635K) -
Sheng-Nan Wu, Hsin-Su Yu, Yousuke Seyama1996 Volume 119 Issue 1 Pages 42-48
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSpontaneous and vasopressin-induced Ca2+ oscillations in cultured vascular smooth muscle (A7r5) cells were further examined and characterized. Intracellular Ca2+ concentrations ([Ca2+]i) were measured by use of a high-performance laser cytometer. When the oscillatory patterns in [Ca2+]i were analyzed with a power spectrum method, about 80% of cells exhibited spontaneous Ca2+ oscillations with the frequency of 0.02-0.5Hz. Nifedipine abolished these repetitive spikes, whereas pinacidil partially attenuated their amplitude and frequency. When vasopressin (100nM) was applied to A7r5 cells, there was an initial rise in [Ca2+]i, followed by a delayed sustained increase in [Ca2+]i. The one-pool, nonoscil-latory model was employed to fit this biphasic change, and the difference between the observed response and the simulated response was then analyzed with a power spectral method. About 50% of cells were noted to display oscillatory patterns in [Ca2+]i, after sustained increase in [Ca2+]i. The present study indicates that spontaneous Ca2+, oscillations in A7r5 cells are modulated by the activity of ATP-sensitive K+ channels and are not related to pertussis toxin-sensitive GTP-binding protein (s). On the basis of the one-pool, nonoscillatory model, it is suggested that the buffering capacity of internal stores appears to be stronger in the cells with spontaneous Ca2+ oscillations than in those in a quiescent state, and the vasopressin-mediated inhibition of accumulation by internal stores was attenuated when the cells exhibited spontaneous Ca2+ oscillations. The implementation of this minimum kinetic model integrated with a power spectrum method would be an alternative to understand the oscillating behavior in [Ca2+]i.View full abstractDownload PDF (915K) -
Takashi Kumasaka, Masaki Yamamoto, Hideaki Moriyama, Nobuo Tanaka, Mam ...1996 Volume 119 Issue 1 Pages 49-57
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe crystal structure of the zinc-protease, H2-proteinase, isolated from the venom of Trimeresurus Ravoviridis has been determined. The crystallographic R factor is 0.176 for 10, 635 reflections with Fobs>2σ (Fobs) in the 8.0 to 2.2Å resolution range. The enzyme has two domains with a cleft in which a catalytic zinc atom is located. The N-terminal domain is composed of four helices around a central five-stranded β-sheet. The irregularly folded C-terminal domain contains one helix and two disulfide bridges. These two domains are linked by a disulfide bridge. In the zinc environment, the catalytic zinc atom forms a distorted tetrahedral coordination with three histidines and one catalytic water molecule, and the methionine-containing turn is structurally conserved. These are distinctive features of the metzincins, one of the zinc metalloprotease superfamilies. The entire structure shows good agreement with that of two Crotalus snake venom proteases, adamalysin II and atrolysin C. The H2-proteinase, however, contains no structural calcium ions, and differences of disulfide configurations and the coordination of the catalytic water molecule exist as compared with the other two proteases.View full abstractDownload PDF (3317K) -
Keiko Sakai, Tadahiko Fujii, Toshihiko Hayashi1996 Volume 119 Issue 1 Pages 58-62
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPlasma fibronectin incubated with a low concentration of SH reagent under physiological conditions without cells formed a multimer which retained the ability of heparin-binding and cell-binding but lost gelatin affinity [Sakai, K., Fujii, T., and Hayashi, T. (1994) J. Biochem. 115, 415-421]. The conformation of the multimeric fibronectin, as observed by ultraviolet circular dichroism and fluorescence spectroscopy was different from that of dimeric plasma fibronectin. Monitoring the change in ellipticity indicated that conformational change was mostly accomplished within the first 3h of incubation with 0.5mM dithiothreitol at 37°C. In contrast, multimers became detectable after 4 h of incubation. The results indicate that the overall reaction of multimerization of plasma fibronectin consists of two steps: the initial step of conformational change of dimeric fibronectin, and the later polymerization step of the polypeptide in an altered conformation. The initial step, involving the conformational change of fibronectin, depended on temperature: it proceeded at 37°C but not at 25°C. In contrast the second step took place at 25°C at a low, yet significant rate. Proteolytic susceptibility of the fibronectin to thermolysin or m-calpain changed within 3h of incubation with dithiothreitol at 37°C in accordance with the conformational change detected by circular dichroism. Namely, the fibronectin in an altered conformation appeared to be less susceptible to thermolysin, but more susceptible to m-calpain. The changes in enzymatic susceptibilities tended to be localized in the amino- and carboxylterminal regions, which are consistent with the implications from the spectroscopic analysis.View full abstractDownload PDF (2584K) -
Hideki Nagase, Kei-ichi Enjyoji, Midori Shima, Kenji Kitazato, Akira Y ...1996 Volume 119 Issue 1 Pages 63-69
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOur previous study has shown that depolymerized holothurian glycosaminoglycan (DHG) has two different inhibitory activities in the blood coagulation cascade: heparin cofactor II-dependent thrombin inhibition; and antithrombin III- and heparin cofactor II-independent inhibition of the intrinsic factor Xase complex [Nagase et al. (1995) Blood 85, 1527-1534]. In the present study, the effect of DHG on the activation of factor VIII and factor V by thrombin was examined with purified human components. DHG inhibited the activation of factor VIII by thrombin at concentrations exceeding 80nM, but not the activation of factor V by thrombin at concentrations of up to 8μM. On Western blot analysis, DHG inhibited the cleavage of factor VIII light chain at concentrations exceeding 0.8μM. The interaction between DHG and factors VIII and V and thrombin was examined with a DHG-cellulofine column. DHG had strong affinity for factor V and thrombin, but slight affinity for factor VIII. The interaction of DHG with thrombin was analyzed, using fluorescein isothiocyanate-labeled DHG. One mole of DHG bound 2mol of thrombin, with a dissociation constant (Kd) of 3.04×10-6M. These results suggest that DHG interferes with the interaction between thrombin and factor VIII, probably by making a binary complex through the anionic binding exosite II of thrombin.View full abstractDownload PDF (2021K) -
Yoshihiro Nakamura, Tetsuo Hashimoto, Takashi Kamaishi, Jun Adachi, Fu ...1996 Volume 119 Issue 1 Pages 70-79
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPartial regions of the mRNA encoding a major part of translation elongation factor 2 (EF-2) from a kinetoplastid protozoan, Trypanosoma cruzi, were amplified by means of polymerase chain reaction and their primary structures were analyzed. The deduced amino acid sequence was aligned with those of other eukaryotic and archaebacterial EF-2s, and the phylogenetic relationships among eukaryotes were inferred by the maximum likelihood (ML) method. ML analyses of EF-2 phylogeny using six different stochastic models of amino acid substitutions consistently suggested that the phylogenetic position of T. cruzi is likely to be closer to higher eukaryotes than that inferred from the phylogeny of small subunit ribosomal RNA (SrRNA). These results are consistent with those for the elongation factor 1α (EF-1α) phylogeny. When the EF-1α and EF-2 phylogenies were totally evaluated, it became much clearer that the divergence of T. cruzi occurred later than that of a mitochondrion-lacking protozoan, Entamoeba histolytica, although this is not conclusive.View full abstractDownload PDF (1397K) -
Kazumi Yamamoto, Yoshio Kojima, Toshiro Kikuchi, Tatsuro Shigyo, Kohji ...1996 Volume 119 Issue 1 Pages 80-84
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nucleotide sequence of the uricase gene from the thermophilic bacterium Bacillus sp. TB-90 was determined. The primary structure of the uricase deduced from the nucleotide sequence comprised 332 amino acids, with a total molecular mass of 37, 994 Da. The molecular mass of the subunit of the uricase produced by the transformant of Escherichia coli agreed well with this value. However, the molecular mass of a subunit of the uricase produced by Bacillus sp. TB-90 was found to be 34, 000 Da by SDS-PAGE. The difference between these molecular masses was attributed to processing of the C-terminal 13 amino acid residue in Bacillus sp. TB-90. Comparison of the enzymatic properties of both uricases showed that the thermostability of the uricase produced by the transformant was enhanced by about 10°C in comparison to that produced by Bacillus sp. TB-90.View full abstractDownload PDF (612K) -
Kishan Lal Agarwala, Shun-ichiro Kawabata, Michimasa Hirata, Masaru Mi ...1996 Volume 119 Issue 1 Pages 85-94
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cysteine protease inhibitor with an apparent Mr=12, 600, designated limulus (L)-cystatin, was isolated from hemocyte lysates of the Japanese horseshoe crab (Tachypleus tridentatus), using two steps of chromatography, including dextran sulfate-agarose, and carboxymethylated papain-agarose. L-cystatin inhibits amidolytic activity of papain by forming a noncovalent 1:1 complex with an equilibrium constant (Ki) of 0.08nM. It also inhibits cathepsin L (Ki=0.17nM) and ficin (Ki=0.52nM), but not argingipain (a bacterial cysteine protease) and calpains. A cDNA for L-cystatin was isolated and the open reading frame coded for a mature protein of 114 amino acids, of which 99 residues were confirmed by peptide sequencing. L-cystatin shows significant sequence identities to members of the family 2 cystatin, such as bovine colostrum cystatin (33%) and human cystatin S (31%). Northern blotting revealed expression of the mRNA in hemocytes and slightly in heart but expression was negligible in hepatopancreas, intestine, stomach, and muscle. Immunoblotting revealed the localization to be in the large granules of hemocytes. Furthermore, L-cystatin has an antimicrobial activity against Gram-negative bacteria, which is much stronger than that of chicken egg white cystatin. These data suggest that the large granule-derived L-cystatin serves synergistically to accomplish an effective defense against invading microbes, together with other defense molecules that are released in response to external stimuli.View full abstractDownload PDF (3608K) -
Takeo Yamaguchi, Misuzu Yamamoto, Eiji Kimoto1996 Volume 119 Issue 1 Pages 95-99
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMembrane vesicles, which were released from human erythrocyte ghosts by dimyristoylphosphatidylcholine (DMPC), showed a protein composition similar to that of the erythrocyte membrane, despite a reduction of in spectrin content. The spectrin content of vesicles decreased with increasing hemoglobin concentration within ghost membranes, but increased upon exposure of hemoglobin-free ghosts to a pressure of 100 MPa. The ESR spectra of spin-labeled membrane proteins showed that membrane proteins in ghosts became unfolded at high pressure. Furthermore, spectrin-poor and protein 4.1-rich vesicles were released by DMPC from diamide-treated ghosts in which spectrin was cross-linked and stabilized. Taking into account that the spectrin tetramer is stabilized by hemoglobin [Liu and Palek (1984) J. Biol. Chem. 259, 11556-11562], these results suggest that the spectrin content of DMPC-induced vesicles from erythrocyte ghosts increases with increasing instability of the cytoskeletal network in parent cells.View full abstractDownload PDF (1713K) -
Akihiko Tsuji, Keisuke Edazawa, Kohji Sakiyama, Kaya Nagata, Yuka Sasa ...1996 Volume 119 Issue 1 Pages 100-105
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel trypsin-like serine proteinase was purified to homogeneity from the bovine pancreas microsome fraction. The enzyme was solubilized with 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), and purified by a series of column chromatographic steps on Ultrogel AcA-34, trypsin inhibitor-Sepharose 4B, and arginineSepharose 4B. The molecular mass of this pancreas trypsin-like proteinase (bPTLP) was estimated to be 29.5 kDa by SDS-PAGE under reducing conditions. The NH2-terminal sequence of bPTLP is very homologous, but not identical to those of other serine proteinases, especially such as elastases IV, II, and III. Substrate specificity studies involving a synthetic substrate and glucagon indicated that the enzyme hydrolyzes Arg-X, Lys-X, and Leu-X bonds. The best synthetic substrate for bPTLP was t-butyloxycarbonyl Gln-Arg-Arg-4-methylcoumaryl 7-amide. The enzyme failed to hydrolyze the substrate for chymotrypsin and elastase. The enzyme activity was inhibited by diisopropyl fluorophosphate, p-amidinophenylmethane sulfonylfluoride, and leupeptin, indicating that it is a serineproteinase. These findings show that bPTLP is a novel serine-proteinase which differs from all known proteinases. The physiological function of the enzyme has yet to be determined.View full abstractDownload PDF (1655K) -
Osamu Okamoto, Yutaka Suzuki, Sadao Kimura, Hiroshi Shinkai1996 Volume 119 Issue 1 Pages 106-114
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA protein with an Mr of 22, 000 was purified from bovine dermis. The amino acid sequence of the protein was identical to 22-kDa protein [Neame, P. J. et al. (1989) J. Biol. Chem. 264, 5474-5479] and showed highly homologous sequences to TRAMP (tyrosine rich acidic matrix protein) [Cronshaw, A. D. et al. (1993) Matrix 13, 255-266] and dermatopontin [Superti-Furga, A. et al. (1993) Cenomics 17, 463-467]. The protein was proved to associate with decorin and a modified decorin with carboxymethylated cysteinyl residues, but not to assemble to hyaluronate or dermatan sulfate chains. The pyridylethylation of cysteinyl residues in the 22-kDa protein did not affect its binding activity to decorin or modified decorin. Immunohistochemical analyses revealed positive stains in endothelial cells and the periphery of collagen fibers in normal dermis but not in the fibroblasts in tissue. Collagen fibers in sclerotic regions of progressive systemic sclerosis were stained diffusely, suggesting that the 22-kDa protein increases in parallel to the accumulation of collagen in the disease. Western blotting analyses of extracts of cultured endothelial cells revealed a lower Mr protein than that from cultured fibroblasts, suggesting the presence of a molecule related to the 22-kDa protein.View full abstractDownload PDF (3605K) -
Kiyoshi Yamauchi, Katsuya Togawa, Masayoshi Kinoshita1996 Volume 119 Issue 1 Pages 115-119
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS2, 2'-Dotriacontanedioylbis (1-palmitoyl-sn-glycero-3-phosphocholine) was synthesized chemically as a dimeric version of 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine. Upon sonication in water, the bipolar lipid furnished a planar membrane of about 50 Å thickness with gel-to-liquid crystalline phase transition constants of Tm=53.4°C, ΔH=20.9kcal/mol, and ΔS=64.0 e. u. In an air/water interface, the lipid displayed unique surface pressuresurface area isotherms with a limiting area of 105-190 Å/molecule at 10-20°C. The lipid was a substrate of phospholipase A2 (Naja mocambique), which afforded 1-palmitoyl-sn-glycero-3-phosphocholine and dotriacontanedioic acid as final products.View full abstractDownload PDF (3462K) -
Hideki Nikami, Yasutake Shimizu, Michihiro Sumida, Yasuhiko Minokoshi, ...1996 Volume 119 Issue 1 Pages 120-125
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPrecursor cells of brown adipocytes were isolated from the interscapular brown fat of newborn rats and cultured on collagen-coated dishes. When confluent cells were treated with dexamethasone, mRNAs for muscle/adipocyte type of glucose transporter, hormonesensitive lipase, and CCAAT/enhancer binding protein α were increased remarkably, confirming a predominant effect of dexamethasone on the terminal differentiation of the cultured cells. Effects of dexamethasone on the expression of three subtypes of β-adrenoceptor were also examined., β1- and β2-adrenoceptor mRNAs remained constant regardless of dexamethasone-treatment, while β3-adrenoceptor mRNA was present only in dexamethasone-treated differentiated cells. To assess the metabolic response mediated by β3-adrenoceptor, glucose transport into the cells was estimated. Norepinephrine enhanced glucose transport in dexamethasone-treated differentiated cells, but not in undifferentiated cells. β3-Adrenergic agonists mimicked completely the stimulatory effect of norepinephrine at concentrations lower by two orders of magnitude. These results suggest that the β3-adrenoceptor is expressed during the course of differentiation in brown adipocytes and plays a significant role in the response of glucose transport to adrenergic stimulation.View full abstractDownload PDF (3103K) -
Takayuki Tsukuba, Hideaki Sakai, Masayuki Yamada, Hidefumi Maeda, Hito ...1996 Volume 119 Issue 1 Pages 126-134
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCathepsin E (CE) is the only known aspartic proteinase that exists as a homodimer consisting of two fully catalytically active monomers, which are covalently bound by a disulfide bond between two cysteine residues at the NH2-terminal region (Cys43 in human pro-CE). To understand the physiological significance of the dimer formation, the monomeric mutant of human CE was constructed by site-directed mutagenesis (Cys43→Ser43) and expressed in Chinese hamster ovary (CHO) cells. Immunolocalization of the mutant protein at both the light and electron microscopic levels revealed the monomeric CE to be associated predominantly with the endoplasmic reticulum and the non-lysosomal endocytic organelles. The cellular localization of the monomeric protein was compatible with that of the wild-type (dimeric form) of recombinant human CE expressed in the same cells. The monomeric protein was generated primarily as the 46-kDa pro-CE with a high-mannosetype oligosaccharide chain in the cells. In addition to the maximal activation at around pH 3.5, a substantial proportion of the monomeric pro-CE was converted to the mature form by incubation at pH 7 and 37°C for 5min. In contrast, the dimeric pro-CE was scarcely activated by treatment at pH 7. Although catalytic properties of the in vitro-activated monomeric CE appeared to be indistinguishable from those of the dimeric forms of natural and recombinant CE, the monomeric form was more unstable to pH and temperature changes than these dimeric forms. These results indicate that the dimerization of CE is not necessarily required for proper folding to express activity, correct intracellular localization and carbohydrate modification, but that it may be essential to structurally stabilize the molecule in vivo.View full abstractDownload PDF (4624K) -
Akihiro Okamoto, Ryuichi Kato, Ryoji Masui, Akihiko Yamagishi, Tairo O ...1996 Volume 119 Issue 1 Pages 135-144
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe aspartate aminotransferase gene (AspAT, EC 2. 6. 1. 1) of an extremely thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced, and its gene product was overproduced. The purified T. thermophilus AspAT was stable up to about 80°C at neutral pH. T. thermophilus AspAT was strictly specific for acidic amino acid substrates, such as aspartate, glutamate, and the respective keto acids. The gene coding for T. thermophilus AspAT showed that it comprised 1, 155bp with a high G+C content (70mol%), and encoded a 385-residue protein with a molecular weight of 42, 050. The amino acid sequence of T. thermophilus AspAT deduced from its gene showed about 15, 46, and 29% homology with those from Escherichia coli, Bacillus sp. YM-2, and Sulfolobus solfataricus, respectively. When the amino acid sequence of T. thermophilus AspAT was compared with that of E. coli AspAT, the number of Cys was found to have decreased from 5 to 1, that of Asn from 23 to 9, that of Gln from 16 to 8, and that of Asp from 20 to 13, all of which are known to be relatively labile at high temperatures. Conversely, the number of Pro was increased from 15 to 25, Arg from 22 to 32, and Glu 27 to 37. As shown by the E. coli AspAT structure, there was a marked tendency for the extra prolyl residues to be located around the surface of the molecule. This was quite different from that in the case of RecA protein, which shows an increased number of prolyl residues in the interior of its molecule. Different strategies of different proteins as to prolyl contribution to thermostability have been suggested. Despite the high degree of conservation of active-site residues, Arg292 in E. coli AspAT, which interacts with the distal carboxylate of the substrate, was not found in T. thermophilus AspAT. Arg89 may complement the function of Arg292.View full abstractDownload PDF (4201K) -
Yoshio Hashimoto, Keiko Yamada, Hiroyuki Motoshima, Tadahiro Omura, Hi ...1996 Volume 119 Issue 1 Pages 145-150
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe constructed a system for the expression and secretion of mature hen lysozyme by yeast using an intermediate “secretion-signal cassette” vector, pKP1700, containing the yeast invertase signal sequence and an expression vector, pAM82, for secretion and maturation of the enzyme. Using this system, mutants of hen lysozyme were produced and the catalytic mechanism in hen lysozyme was definitely confirmed. The hydrolytic activity of D52A as to substrate (NAG)6 at pH 5.0 was obviously decreased to one-four hundredth of that of the wild type. The acidic limb of the pH-activity profile observed for the wild-type was not observed for D52A, and the pKa of Glu 35 on the alkaline limb was seen for both enzymes. Moreover, no structural change was detected on X-ray analysis of D52A. Therefore, we confirmed that dissociated Asp 52 assists catalysis by producing an electrostatic field and by stabilizing the oxocarbonium ion intermediate in the dissociated form.View full abstractDownload PDF (738K) -
Tetsu Hozumi, Masao Miki, Sugie Higashi-Fujime1996 Volume 119 Issue 1 Pages 151-156
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMuscle G-actin treated with the hetero-bifunctional cross-linking reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), denoted as MBS-G-actin, is not induced to polymerize into F-actin by salt and myosin subfragment 1 [Bettache, N., Bertrand, R., and Kassab, R. (1989) Proc. Natl. Acad. Sci. USA 86, 6028-6032]. However, the addition of salt and phalloidin together allowed MBS-G-actin to polymerize and the resulting polymer (P-MBS-G-actin) could activate the Mg2+-ATPase of S-1 [Miki, M. and Hozumi, T. (1991) Biochemistry 30, 5625-5630]. When F-actin was treated with MBS (MBS-F-actin), unlike MBS-G-actin, intercross-links between monomers in F-actin occurred. The MBS-F-actin could activate the Mg2+ -ATPase of heavy meromyosin (HMM): its maximum turnover rate, Vmax, was almost the same as that of native F-actin, but the affinity of HMM for MBS-F-actin in the presence of ATP, Km, was about 3 times higher. Electron microscopy showed that both P-MBS-G-actin and MBS-F-actin had the double stranded structures of F-actin and formed the arrowhead structures when combined with HMM. By in vitro motility assay, the sliding velocities of P-MBS-G-actin and MBS-F-actin were found to be slightly slower than that of native F-actin. But the critical concentration of KCl, over which the sliding movement was not observed, for MBS-modified actins was considerably higher than for native F-actin. When MBS-modified actins were regulated by tropomyosin-troponin complex, they were less sensitive to the Ca2+ concentration for HMM ATPase activation and sliding movement. These results showed that the modification of some of the lysine residues in the actin molecule leads to change in the biochemical properties of F-actin.View full abstractDownload PDF (4019K) -
Tadashi Ueda, Hiroki Iwashita, Yoshio Hashimoto, Taiji Imoto1996 Volume 119 Issue 1 Pages 157-161
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe designed mutant lysozymes with N-glycosylation signal sequences (Asn48 -G1y49-Thr-50 and Asn87-Ile88-Thr89) by substituting Asp to Asn at positions 48 and 87. When these mutant lysozymes were expressed by using yeast (Saccharomyces cerevisiae) in Burkholder minimum medium, N-glycosylation occurred in both lysozymes. The mutant lysozyme with the oligosaccharide at Asn87 showed a similar character to a reported polymannosyl lysozyme [Nakamura, Takasaki, Kobayashi, and Kato (1993) J. Biol. Chem. 268, 12706-12712; Kato, Takasaki, and Ban (1994) FEBS Lett. 355, 76-80]. As judged from the thermodynamic stabilities of the lysozymes obtained by the guanidine hydrochloride denaturation method, the oligosaccharide-bearing mutant lysozymes were more stable by 0.4-1.6kcal/mol than the corresponding unglycosylated lysozymes. Therefore, it is suggested that the introduction of an N-glycosylation signal sequence into a protein is an effective means to increase the stability of the protein.View full abstractDownload PDF (3903K) -
Rujun Kang, Kazuya Yamada, Takashi Tanaka, Ting Lu, Tamio Noguchi1996 Volume 119 Issue 1 Pages 162-166
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPrevious studies have suggested that some glycolytic intermediates are involved in the regulation of L-type pyruvate kinase gene expression by carbohydrates such as glucose and fructose. To find such intermediates, we examined the relationship between the levels of L-type pyruvate kinase mRNA and glycolytic metabolites in hepatocytes cultured under various conditions. Of the metabolites, the levels of 3-phosphoglycerate and phosphoenolpyruvate only increased significantly under conditions under which the expression of the L-type pyruvate kinase gene was stimulated. The level of glucose 6-phosphate, which was reported to be involved in dietary stimulation of this gene expression, was not correlated with the mRNA level since marked accumulation of deoxyglucose 6-phosphate occurring on the addition of deoxyglucose, a nonmetabolizable glucose analog, was not accompanied by an increase in the L-type pyruvate kinase mRNA level. In addition, we found that fructose at a low concentration in the presence of glucose failed to increase the mRNA and metabolite levels in contrast to other reports that the promoter activity of the L-type pyruvate kinase gene is stimulated by this treatment. Thus we propose that 3-phosphoglycerate and/or phosphoenolpyruvate are involved in the carbohydrate regulation of L-type pyruvate kinase gene expression.View full abstractDownload PDF (1821K) -
Masatoshi Muraoka, Koichi Nakazato, Toshihiko Hayashi1996 Volume 119 Issue 1 Pages 167-172
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSType IV collagen was prepared from bovine lens capsule by acetic acid extraction, followed by purification including DEAE-Sephacel chromatography and dialysis. The type IV collagen solution became viscous and eventually gelated upon dialysis against 2M guanidine-HCl and 10mM dithiothreitol. Gelation was not observed for heat-denatured type IV collagen, suggesting that collagenous conformation may be required for the gelation. A reducing agent, dithiothreitol, was essential for the gelation of type IV collagen in 2M guanidine-HCl. An optimal concentration of guanidine-HCl for the gelation lays between 1.5 and 2.5M: gelation did not occur at 1M or lower and at 3M or higher, although the circular dichroism spectrum characteristic of the collagenous triple-helix was not changed in 3M guanidine-HCl. This suggests that an appropriate change in conformation of the type IV collagen at a region other than the triple-helical region or/and partial dissociation of complexed type IV collagen aggregates may drive intermolecular interactions of the type IV collagen leading to polymerization and eventually to gelation. To our knowledge, this is the first report that the type IV collagen alone has the ability to form a rigid gel. The assembled structure of the type IV collagen in gel form might be related to the skeletal architecture of basement membrane (s).View full abstractDownload PDF (1792K) -
Yoko Shiga, Shunichi Kametani, Hideaki Mizuno, Hiroshi Akanuma1996 Volume 119 Issue 1 Pages 173-179
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe cyclic polyol 1, 5-anhydro-D-glucitol (AG) is detected in most organisms, but little is known about its metabolism and physiological roles. Our previous study demonstrated that Escherichia coli C600 synthesizes AG when glucose is exhausted in the medium and that it temporarily releases AG into and then takes it back from the medium, thus forming a sharp peak in AG concentration in the medium a few hours after reaching stationary growth phase. The present study demonstrates that when glucose is absent in the culture medium, E. coli C600 takes up and phosphorylates AG and releases a large portion of it back into the medium in the form of a phosphate ester. [U-13C] AG was added to the medium after the exhaustion of glucose and the resulting [U-13C]AG phosphate was partially purified by several steps of anion exchange chromatography and identified as AG 6-phosphate by 13C-NMR. The identity of the phosphate ester was also confirmed by GC-MS analysis after further purification.View full abstractDownload PDF (1001K) -
Shunichi Kametani, Hideaki Mizuno, Yoko Shiga, Hiroshi Akanuma1996 Volume 119 Issue 1 Pages 180-185
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOf the all-carbon-13 compounds, glucose is one of the most easily accessible, and therefore we applied 13C-NMR technique to the metabolic study of glucose-related compounds, 1, 5-anhydro-D-glucitol and 1, 5-anhydro-D-fructose (AF). Applying an INADEQUATE method to the substitutes of these novel sugars fully labeled with carbon-13, we could trace out the entire carbon skeleton with high sensitivity and confirm the chemical structures of these sugars. The method also provided a much easier way to optimize the enzymatic oxidation for AF preparation: we selectively and continuously monitored the quantities, as well as their structures in aqueous solution, of the substrate and products in a noninvasive manner. Similarly relying upon information from the 13C-NMR, we developed a valuable derivatization method of AF for its GC-MS application, which was so sensitive that we were able to demonstrate the natural occurrence of AF in rat liver.View full abstractDownload PDF (689K) -
Toyofumi Yamaguchi, Shigeko Nishimura, Keiko Takahashi, Michiyasu Yosh ...1996 Volume 119 Issue 1 Pages 186-192
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDNA polymerase βs from cherry salmon, Oncorhynchus masou, liver and testes were purified to near homogeneity, and no substantial differences between the enzymes were observed. The molecular weight of both enzymes, determined by SDS-polyacrylamide gel electrophoresis, was 39, 000. The amino acid sequences of the N-terminus of the liver and testes enzymes were determined and compared with that of the rat enzyme. Of the N-terminal 30 amino acid residues of salmon liver DNA polymerase β, 21 (70%) were identical to those of the rat enzyme sequence. However, unlike most eukaryotic DNA polymerase βs,
the isoelectric points (pIs) of the DNA polymerase βs from salmon liver and testes were both estimated to be 6.2, which is significantly different from the alkaline isoelectric points (pI=8.5-9.5) established for other highly purified vertebrate DNA polymerase βs. The cherry salmon DNA polymerase βs were still active at below 10°C, compared with the rat enzyme.View full abstractDownload PDF (1844K) -
Naoki Ichikawa, Yukuo Yoshida, Tadao Hashimoto, Kunio Tagawa1996 Volume 119 Issue 1 Pages 193-199
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn ATPase inhibitor and its stabilizing factor, the 9K protein, are regulatory factors of F1F0-ATPase. The binding sites for these factors on F1 were examined using the zero length cross-linkers, N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline, and 1-ethyl-3-[3-dimethylamino) propyl] carbodiimide. The cross-linked products were analyzed by immunoblotting after SDS-polyacrylamide gel electrophoresis. The inhibitor and the 9K protein cross-linked to the α and β subunits of F1 indicating that they interacted with both subunits. Peptide mapping and amino acid sequence analysis of the cross-linked products after weak acid hydrolysis showed that the inhibitor cross-linked to the Pro334-Asp363 region of the β subunit. Amino acid sequence analysis of the cross-linked peptide showed that the inhibitor binds to Asp363 of the β subunit. As this region contains the amino acid residues, including Tyr359, that are modified by nucleotide analogs and form the active site, the inhibitor probably binds to the catalytic site of F1.View full abstractDownload PDF (3428K) -
Tsuyoshi Miyaoka, Mikako Tsuchiya, Nobumasa Hara, Hiroshi Ishino, Mako ...1996 Volume 119 Issue 1 Pages 200-207
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of KCl on ADP-ribosylation of the recombinant RhoA protein catalyzed by the Clostridium botulinum C3 enzyme was studied. When the recombinant glutathione S-transferase-RhoA fusion protein (GST-RhoA) was incubated with C3 and [adenylate-32P] NAD, incorporation of radioactivity into the recombinant RhoA increased in the presence of KCl. The increase in ADP-ribose incorporation into RhoA due to KCl appeared in the presence of MgCl, and was abolished by EDTA. C3 was stabilized by KCl, but the stabilization was also seen with BSA. The KCl-induced increase in the ADP-ribosylation was observed even in the presence of BSA during the modification reaction, thus the effect of KCl was not due to the stabilization of C3. While the initial rate of the reaction was increased by KCl, maximum incorporation of ADP-ribose per GST-RhoA molecule did not increase in the presence of KCl. Kinetic analysis revealed that KCl increased Vmax but did not alter Km for either NAD or RhoA. The NAD glycohydrolase activity of C3 was also increased by KCl. These results indicate that KCl directly activates the C3 enzyme.View full abstractDownload PDF (2238K) -
1996 Volume 119 Issue 1 Pages 208
Published: 1996
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (28K)
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