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Volume 115 (1994)
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- Issue 5 Pages 809-
- Issue 4 Pages 623-
- Issue 3 Pages 369-
- Issue 2 Pages 175-
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Volume 115, Issue 5
Displaying 1-38 of 38 articles from this issue
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Makoto Komiyama, Teruyuki Kodama, Naoya Takeda, Jun Sumaoka, Tetsuro S ...1994 Volume 115 Issue 5 Pages 809-810
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCerium (III) chloride efficiently hydrolyzes DNA at pH 7 under aerobic conditions. A titration study showed that Ce (III) is oxidized to Ce (IV) in the reaction mixture and the resultant Ce (IV) is responsible for the DNA hydrolysis.View full abstractDownload PDF (202K) -
Junzo Hirose, Takeshi Sakurai, Kaori Imamura, Hideomi Watanabe, Hiroyu ...1994 Volume 115 Issue 5 Pages 811-813
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe ascorbate oxidase obtained from a microorganism, Acremonium sp. HI-25 (molecular weight, 80 kDa; monomeric protein), was studied with respect to atomic absorption, EPR, absorption spectra, circular dichroism (CD) spectra, and steady-state kinetics. The enzyme was found to be a multicopper protein, containing fourr copper atoms of three kinds, types 1, 2, and 3 copper, in the ratio of 1:1:2. The EPR parameters of the type 1 and 2 copper atoms in the ascorbate oxidase are very similar to those in the case of the ascorbate oxidase obtained from cucumber, which is a dimeric protein. The apparent Km and kcat values for ascorbic acid of the ascorbate oxidase from Acremonium sp. HI-25 are almost the same as those of the monomeric unit of the ascorbate oxidase from cucumber.View full abstractDownload PDF (249K) -
Motoo Yamasaki, Makoto Asano, Masami Okabe, Makoto Morimoto, Yoshiharu ...1994 Volume 115 Issue 5 Pages 814-819
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRecombinant human granulocyte colony-stimulating factor (rhG-CSF) has been obtained from genetically engineered Escherichia coli as an unglycosylated protein. Both native glycosylated hG-CSF and rhG-CSF are rapidly cleared from the circulation, which may limit their effectiveness for clinical use. To improve this biological property, rhG-CSF and its derivative ND 28, which has a higher specific activity than does rhG-CSF, were modified with polyethylene glycol (PEG). Modified rhG-CSF and ND 28 in which 1 to 3 mol of PEG were bound, were purified by two-step chromatography and characterized by several methods. The results of their physicochemical characterization suggest that PEG-modification does not appreciably change the conformation of rhG-CSF and ND 28. As a result of the whole characterization, the PEG-modification of rhG-CSF and ND 28 enhanced the stability of rhG-CSF and ND 28 and decreased the plasma clearance rate, which led to more effective hemopoiesis.View full abstractDownload PDF (617K) -
Masahiro Uritani, Kyoko Nakano, Yuhko Aoki, Hisao Shimada, Mikio Arisa ...1994 Volume 115 Issue 5 Pages 820-824
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEF-3 is a translational elongation factor specific to yeasts and fungi. Its carboxy-terminal region contains three lysine-clusters and is very basic. The region has been reported to be responsible for the interaction with ribosomes [Ishiyama, A., Ogawa, K., & Miyazaki, M. (1992) in Abstracts of the 15 th Annual Meeting of the Molecular Biology Society of Japan, p.190]. To find specific inhibitors for the interaction of EF-3 with ribosomes, the effects of two basic polyamino acids, poly-L-(Lys) and poly-L-(Arg), and two acidic polyamino acids, poly-L-(Asp) and poly-L-(Glu), were examined using two assay systems for ATPase of EF-3. One was for the ribosome-activated ATPase and the other for the intrinsic (ribosome-independent) ATPase of EF-3. Basic polyamino acids were expected to act as analogues of the carboxy-terminal region of EF-3, and acidic ones to interact with EF-3. The basic polyamino acids inhibited the ribosome-activated ATPase, but they also inhibited the intrinsic one more effectively. Acidic polyamino acids, poly-L-(Asp) and poly-L-(Glu), inhibited the ribosome-activated ATPase but not the intrinsic one. Thus, acidic polyamino acids could be specific inhibitors of the interaction between EF-3 and ribosomes. Furthermore, a system for detecting the binding of EF-3 to ribosomes was constructed. That is, ribosome-bound EF-3 was detected by measuring the ATPase on precipitated ribosomes after a mixture of EF-3 and ribosomes had been ultracentrifuged. Using this system, poly-L-(Asp) was shown to inhibit the binding of EF-3 to ribosomes directly.View full abstractDownload PDF (527K) -
Peter Borowski, Sven Medem, Rainer Laufs, Wolfgang Weber1994 Volume 115 Issue 5 Pages 825-829
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe investigated polyionic agents with regard to their effects as modulators of epidermal growth factor receptor (EGF-R) kinase activity. Many synthetic polypeptides containing glutamine as well as casein were phosphorylated, while polycationic compounds with tyrosine residues were not phosphorylatable and thus inhibited the EGF-R activity. Polyarginine, protamine sulfate, spermidine, heparin, and poly-L-lysine with a chain length of<20.5 kDa triggered the phosphorylation of poly(Tyr1, Glu4). On the other hand, dextran sulfate and poly-L-lysine with chain lengths of<20.5 kDa inhibited the EGF-R kinase activity. Alteration of the state of autophosphorylation of EGF-R is not in agreement with the activity of EGF-R kinase towards poly(Tyr1, Glu4). Casein and histone H1 both increased the autophosphorylation of EGF-R in a concentration-dependent manner, but only casein increased the activity of the enzyme towards an exogenous substrate. The compounds enhancing the EGF-R activity, such as poly-L-lysine, protamine, and poly-L-arginine, down-modulated the autophosphorylation reaction. We discuss the conse-quences of these effects as to in vivo conditions.View full abstractDownload PDF (1655K) -
Taketoshi Uzawa, Akihiko Yamagishi, Kazuya Nishikawa, Tairo Oshima1994 Volume 115 Issue 5 Pages 830-832
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of novel polyamines on aminoacyl-tRNA formation catalyzed by Escherichia coli, Sulfolobus acidocaldarius, and Thermus thermophilus HB8 S-100 extracts were investigated. These effects were diverse and differed depending on the amino acid and the enzyme used. A quaternary polyamine, tetrakis (3-aminopropyl) ammonium, inhibited phenylalanyl-tRNA synthesis catalyzed by the T. thermophilus extract, but not inhibit the other aminoacyl-tRNA formations tested. The inhibition was observed in hybrid reactions where the thermophile tRNA or extract was replaced by its E. coli counterpart, although the quaternary amine did not inhibit Phe-tRNA formation by the E. coli homologous system. Spermine relieved the inhibition of the reaction of thermophile enzyme and tRNA, but not the inhibition of the hybrid reactions. These results suggest that the branched polyamine interacts with both the thermophile enzyme and tRNAphe.View full abstractDownload PDF (344K) -
Elena Bravo, Elena Pignatelli, Roberta Masella, Roberto Verna, Alfredo ...1994 Volume 115 Issue 5 Pages 833-836
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have shown previously that the age-dependent increase in plasma cholesterol levels observed in male Wistar rats is associated with relevant changes in the lipoprotein pattern (in particular, with a much higher proportion of the HDL1 class) that are evident in animals from the age of 9 months. In this study, the possibility that a decreased catabolism of HDL1 cholesterol may cause this is evaluated by infusing this lipoprotein fraction labeled with [14C] cholesterol into both young (3.5±0.5 months) and adult (13.0±1.0 months) male Wistar rats with a permanent biliary drainage. The clearance of radioactivity from the blood compartment was slower in the older animals than in the younger ones. Conversely, the incorporation of radioactivity into plasma cholesteryl esters and the secretion of radioactivity into bile was higher in the younger animals. These results support the hypothesis that the age-related increase in HDL1 proportion is due, at least in part, to a slower liver catabolism of HDL1-cholesterol.View full abstractDownload PDF (476K) -
Eiichi Hoshino, Yuichi Kubota, Mitsuo Okazaki, Kazutosi Nisizawa, Taka ...1994 Volume 115 Issue 5 Pages 837-842
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mode of hydrolysis of cotton cellulose by two highly purified exo- and endo-type cellulases from Irpex lacteus was investigated by differential scanning calorimetry, to measure changes in the size of the amorphous region in cotton fibers with the enzymatic reaction. The cellulases induced entirely different changes in the size of the amorphous region, particularly at earlier stages of reaction. Exo-type cellulase gradually reduced the amorphous region with release of cellobiose from the initial stage of hydrolysis, but began to increase the amorphous region at more advanced stages of hydrolysis. By contrast, endo-type cellulase caused no liberation of reducing sugar at the initial stage of hydrolysis but caused a sharp increase in the amorphous region, and it thereafter caused a rapid decrease of the amorphous region, accompanied with the production of various kinds of cellooligosaccharides. The rate of size reduction of the amorphous region caused by endo-type cellulase was much higher than that by exo-type cellulase. Convergence of the decrease in the size of amorphous region during hydrolysis by endo-type cellulase is followed by the increase in this region being influenced by further hydrolysis of remained crystalline region. Substantial changes in the morphology of cotton occurred with the two cellulases after the hydrolysis stages at which the size of the amorphous region was minimum.View full abstractDownload PDF (3287K) -
Noriaki Sakata, Erzsébet Jakab, Torkel Wadström1994 Volume 115 Issue 5 Pages 843-848
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe existence of a second binding site to Staphylococcus aureus on fibronectin was explored by using a recombinant protein, alb-FnBPA, which was obtained from an Escherichia coli clone containing a part of the gene for fibronectin-binding protein of S. aureus. Limited digestion with trypsin produced a 215K fragment from the B chain of fibronectin by releasing an N-terminal 32 kDa region. This C-terminal large fragment captured alb-FnBPA in spite of lacking the N-terminal binding region to S. aureus. This treatment also produced a 185K fragment from A chain by releasing N-terminal 32 kDa and C-terminal 37 kDa regions, and the 185K fragment possessed no binding ability. Thermolysin digestion was also performed to investigate the effect on the binding of releasing the C-terminal region from both of the chains and to locate the binding region to alb-FnBPA on the tryptic 215K fragment. But this digestion produced no fragment binding to alb-FnBPA except for the N-terminal 24K and its transient precursory fragments. These results indicate that both the N-terminal and the C-terminal regions of fibronectin bind to S. aureus, and suggest that the binding appears to involve cooperation between type III repeats and C-terminal type I repeats.View full abstractDownload PDF (3062K) -
Satoru Oshiro, Yuichi Nakamura, Ryuta Ishige, Makoto Hori, Hiroshi Nak ...1994 Volume 115 Issue 5 Pages 849-852
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMammalian cells internalize iron as diferric transferrin (Fe2Tf) iron via receptor-mediated endocytosis (RME) and a redox mechanism under physiological condition and as an iron salt through the Tf-independent iron uptake (Tf-IU) system under morbid conditions. We have previously shown that Tf iron is reduced at the Tf molecule through a redox system on the plasma membrane prior to uptake [Oshiro, S., Nakajima, H., Markello, T., Krasnewich, D., Bernadini, I., & Gahl, W. (1993) J. Biol. Chem. 268, 21586-21591]. In the present study, the reduction site for Tf iron uptake via RME and for Tf-independent iron uptake via the Tf-IU system were examined using specific iron sources and well-characterized ferric or ferrous iron chelators in cultured human fibroblasts. At 4°C for 1 h, although [55Fe]2Tf was not internalized into the cells, 55Fe from [55Fe]2-Sepharose was taken up. However, under the same conditions, EDTA or dipyridyl removed the radioactive iron as a ferric or ferrous iron-chelator complex from [55Fe]2 bound to the Tf receptor on the cell surface. Moreover, after 55Fe-citrate was loaded into the cells at 4°C for 1 h, 55Fe was removed from the cell surface by dipyridyl just as observed for [55Fe]2-Sepharose. In inhibition experiments, ferric citrate showed a dose-dependent inhibition of the iron uptake of both Tf iron and Tf-independent iron. Conversely, Fe2Tf dose-dependently inhibited the uptake of 55Fe-citrate. These results suggest that Tf-independent iron as well as Fe2Tf is reduced at the plasma membrane prior to uptake, as the ferric reduction of Fe2Tf taken up by the redox mechanism occurs at the same site.View full abstractDownload PDF (472K) -
Daitaro Kurosaka, Shunji Hattori, Hisae Hori, Noriko Yamaguchi, Tomoko ...1994 Volume 115 Issue 5 Pages 853-857
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProcollagen synthesized by skin fibroblasts from a patient with a lethal variant of osteogenesis imperfecta has been characterized. After pepsin digestion of the type I procollagen, a portion of the α1 (I) chains was recovered as a disulfide-bonded dimer. Cyanogen bromide peptide mapping suggested that a new cysteine residue was present in the α1 (I) CB6 fragment. Sequencing of cloned cDNAs prepared using mRNA from the proband's fibroblasts demonstrated that some of the clones contained a single base mutation that converted the glycine codon in amino acid position 946 of the α1 (I) chain to a cysteine codon. The thermal stability of the molecules was markedly lower than that in the case of the normal control.View full abstractDownload PDF (2926K) -
Akira Nagayoshi, Norio Matsuki, Hiroshi Saito, Kazuhisa Tsukamoto, Mas ...1994 Volume 115 Issue 5 Pages 858-861
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe reported previously that apolipoprotein B is not actively secreted from suncus liver. In the present study we have investigated the hepatic cholesterol metabolism, which plays a critical role in the secretion of apo B. We found that the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in suncus liver is high and that acyl-coenzyme A: cholesterol acyltransferase (ACAT) activity is almost absent in contrast to rats. As a result, the hepatic content of cholesterol ester, upon which apoprotein B secretion partly depends, is very low in suncus. The deficiency of ACAT activity may cause the defect in active secretion of apoprotein B-containing lipoproteins in suncus.View full abstractDownload PDF (1359K) -
Kazuki Okamoto, Gang Liu, Wen Gong Yu, Fumihide Isohashi1994 Volume 115 Issue 5 Pages 862-867
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously found a novel endogenous factor in rat liver cytosol, named ATP-stimulated glucocorticoid-receptor-translocation promoter (ASTP), that increased the binding of activated glucocorticoid-receptor to nuclei in the presence of ATP. In this work, we immunized rabbits with the purified ASTP protein and characterized the antibodies with regard to titer, cross-reactivity and specificity. An IgG fraction from sera of the immunized rabbits contained specific antibodies to ASTP. The anti-ASTP IgG could precipitate the ASTP protein without the activity. Immunoblot analysis revealed a major band of 48 kDa in rat liver cytosol that migrated to the same position as the purified ASTP protein by SDS-PAGE, and an additional minor band of about 50 kDa. Monospecific antibodies purified from the IgG fraction using the antigen (the purified 48-kDa ASTP protein) immobilized on a polyvinylidene difluoride membrane also reacted with both the 48-kDa ASTP protein and the 50-kDa protein in rat liver cytosol, suggesting that this 50-kDa protein is immunologically related to the 48-kDa ASTP protein. Densitometric quantification of immunoblots demonstrated that the rat kidney cytosol contained ASTP protein at a concentration of about 20% of that of liver cytosol. Other tissues such as brain, skeletal muscle, heart, and lung, contained neither the ASTP protein nor the activity.View full abstractDownload PDF (2702K) -
Shigeo Yamamoto, Noriyuki Okujo, Takashi Yoshida, Shiro Matsuura, Sumi ...1994 Volume 115 Issue 5 Pages 868-874
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe structure of vibrioferrin, a siderophore from Vibrio parahaemolyticus, was elucidated based on a combination of partial hydrolysis and spectroscopic techniques. HPLC of purified vibrioferrin showed two peaks with an area ratio of approximately 2:1. However, upon reinjection of each of those isolated compounds, the original chromatographic pattern was obtained, indicating an equilibrium between two compounds in aqueous solution. Consistent with this finding, most of the NMR signals of vibrioferrin were duplicated. The structure was determined as 1-{2-[2-(5-carboxy-5-hydroxy-2-oxo-l-pyrrolidinyl)propionamide] ethyl} citrate, which exists in two epimeric forms resulting from cyclization between an amidic nitrogen of the alanine residue and a keto group of the 2-ketoglutaric acid residue. Transport experiments with 55Fe-labeled vibrioferrin demonstrated the function of vibrioferrin as a siderophore in V. parahaemolyticus. Kinetic studies with mid-log phase cells revealed that the iron uptake system was receptor-mediated, with Km and Vmax values of 67 nM and 54 pmol Fe/mg cell protein/min, respectively. Moreover, iron uptake mediated by vibrioferrin was blocked both by uncouplers and by ATPase inhibitors.View full abstractDownload PDF (848K) -
Tetsuro Fujisawa, Tomoya Uruga, Ziro Yamaizumi, Yoji Inoko, Susumu Nis ...1994 Volume 115 Issue 5 Pages 875-880
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe small-angle X-ray scattering technique was used to characterize the structure in solution of wild type ras p21 as well as the oncogenic proteins mutated at residue 12, 59, or 61. In the presence of GDP, the radius of gyration, Rg, determined for wild type ras p21 was 16.89±0.01 Å, while the wild type ras p21 bound to the GTP analogue GDPNHP (5'-guanyl imido drphosphate β-γ-imidoguanosine 5'-triphosphate) showed an Rg value of 17.46±0.01 Å, which is 3.3% larger. The result shows that ras p21 expands upon GTP binding. The Rgs of mutated proteins were 17.04±0.01, 16.98±0.01, and 17.03±0.01 Å for the Gly-12 to Val, Ala-59 to Thr, and Gln-61 to Leu mutants, respectively. The scattering profiles were analyzed by simulation of hydrated ras p21, based on the crystal atomic coordinates, and it was concluded that the ras p21 molecule incorporates 20% more bulk water upon GTP binding. The increase of bulk water is especially conspicuous around the interface between switch I (residues 32-40) and switch II (residues 60-66) regions. This suggests that hydration plays an important role in the interaction with GAP.View full abstractDownload PDF (837K) -
Amino Acid Sequence of an Inhibitor from the Silkworm (Bombyxmori) Hemolymph against Fungal ProteaseMasaharu Eguchi, Masanobu Itoh, Kaori Nishino, Hiroshi Shibata, Takaha ...1994 Volume 115 Issue 5 Pages 881-884
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel protein protease inhibitor (FPI-F) which is highly specific for fungal proteases and subtilisin was isolated from the silkworm hemolymph, and its amino acid sequence was determined by conventional methods. The inhibitor consisted of 55 amino acid residues and had a molecular weight of 6, 100. The inhibitor included eight cysteine residues and relatively large amounts of acidic amino acids, but neither alanine, methionine nor tryptophan. The amino acid sequence of FPI-F was not homologous with those of other known protease inhibitors of microbe, plant or animal origin.View full abstractDownload PDF (426K) -
Zong Xuan Jin, Kazuo Inaba, Ken-ich Manaka, Masaaki Morisawa, Hiroshi ...1994 Volume 115 Issue 5 Pages 885-890
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe isolated and characterized several monoclonal antibodies against a protein complex containing the flagellar movement-initiating phosphoprotein (MIPP) that appears to play a crucial role in the initiation of flagellar movement in quiescent spermatozoa of Salmonid fish. The effects of the antibodies on the phosphorylation of MIPP, as well as on the initiation of movement, in model sperm cells were studied. Three monoclonal antibodies, namely, FMI7, FMI18, and FM127, were found specifically to inhibit both the initiation of flagellar movement and the phosphorylation of MIPP. These antibodies did not recognize denatured MIPP; they only recognized the native antigen. FMI7 exclusively recognized the denatured form of a 38-kDa protein, which may possibly be a protein kinase responsible for the phosphorylation of MIPP. Immunofluorescence analysis in situ of model sperm cells with the antibodies showed that the antigen was localized predominantly in the basal structure of the spermatozoon. Thus, the results clearly demonstrate the involvement of MIPP in the initiation of flagellar movement and the control of flagellar motility.View full abstractDownload PDF (2726K) -
Hiroyuki Nakagawa, Yasushi Koyama, Takashi Okada1994 Volume 115 Issue 5 Pages 891-897
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeventy-four kinds of cytochrome c sequences have been compared in order to determine the conserved residues and residues of which the characters are conserved. Twenty-three residues are invariant throughout all the aligned sequences, while the residues at 17 other positions share common characters. The prosthetic group as well as these conserved and character-conserved residues are considered to constitute a model molecule to elucidate the electron transfer process in cytochrome c. Their coordinates in the structure of tuna cytochrome c are extracted, and an extended Htickel molecular orbital calculation has been executed on this molecule. The examination of the shapes and the energy levels of the resulting MOs has suggested that three nearly degenerate HOMOs might play an important role in the electron transfer. These HOMOs are exposed to the protein surface around the heme, Cys-17, and Phe-82. A delocalized electron system developing in these regions is proposed to be the electron transfer pathway of cytochrome c.View full abstractDownload PDF (1428K) -
Jue Wang, Hironao Ueno, Takashi Masuko, Yoshiyuki Hashimoto1994 Volume 115 Issue 5 Pages 898-903
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding of bovine (BSA) and human serum albumin (HSA) to the surface of human and mouse tumor cells in vitro was examined by flow cytometry using monoclonal antibodies against these albumins. Both isologous and heterologous types of albumin bound firmly on several lines of human and mouse tumor cells. The cell-bound albumin was removable by protease (actinase) treatment or by culture in albumin-free medium for more than 2 days, but not by simple washing. The amount of albumin capable of binding to the tumor cell surface differed among the 9 tumor cell lines tested. As determined with radioiodinated BSA, about 2.6×106 BSA molecules/cell could bind to MDA-MB-453 human mammary cancer cells, which exhibit high binding capacity as to BSA. A unique peptide having a molecular weight of 18 kDa was detected on gel electrophoresis in extracts of tumor cells or normal aortic endothelial cells which had been treated with radioiodinated, photocross-linker-labeled BSA or HSA, followed by UV-irradiation. This peptide was also immunoprecipitated with an anti-BSA or anti-HSA monoclonal antibody. With both methods, the yield of the peptide was decreased by previous addition of an excess amount of unlabeled BSA or HSA. These findings indicated that the 18-kDa peptide expressed on both normal aortic endothelial cells and tumor cells is a principal serum albumin-binding protein, and that this protein binds both BSA and HSA.View full abstractDownload PDF (1649K) -
Katsuto Hozumi, Takashi Masuko, Yoshiyuki Hashimoto1994 Volume 115 Issue 5 Pages 904-908
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNonparenchymal cells isolated from Fischer rat liver were separated into subpopulations by passage through a nylon wool column and/or culturing in a plastic plate. Besides typical Kupffer cells, we detected a unique population of cells (called PKu cells) which were plastic adherent but did not spread on a short term culture, were nonadherent on nylon wool, and were nonphagocytic, as opposed to Kupffer cells. Both Kupffer cells and PKu cells expressed CD4 (recognized with W3/25 mAb), CD8 (OX-8), a NK cell antigen (3. 2. 3), and a monocyte antigen (OX-41), as assessed by flow cytofluorometry. However, the proportion of cells bearing high densities of CD8 and 3. 2. 3 antigens was much larger for PKu cells than for Kupffer cells. Reverse transcriptase-PCR analysis of CD8 mRNA revealed that PKu cells expressed the CD8 α chain but not the β chain. When PKu cells were treated with phorbol 12-myristate 13-acetate (PMA), they exhibited spreading and phagocytic activities, and showed a similar morphology to Kupffer cells in the spread form. Moreover, PMA treatment decreased the high density of CD8 antigen on PKu cells. These findings indicated that PKu cells present in normal rat liver are precursors of Kupffer cells.View full abstractDownload PDF (2423K) -
Susumu Nakayama, Hirofumi Tanaka, Eiko Yajima, Tetsuo Maita1994 Volume 115 Issue 5 Pages 909-926
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe sequence of the NH2-terminal 830 amino acid residues of chicken cardiac ventricular muscle myosin subfragment-1 (S-1) was determined. S-1 was obtained by limited chymotryptic digestion, and cleaved into three characteristic fragments (23, 41, and 22 kDa fragments) by limited tryptic digestion. These fragments were isolated by gel filtration on a Sephadex G-100 column, followed by cation-exchange chromatography on a CM-52 column and reverse-phase HPLC. The isolated fragments were sequenced completely. Peptides overlapping the 23 and 41 kDa fragments and also overlapping the 41 and 22 kDa fragments were obtained by cleaving S-1 with cyanogen bromide, and sequenced completely. We also obtained a minor fragment, the 20 kDa fragment, in addition to the three characteristic fragments. Amino acid compositions of the cyanogen bromide peptides of the 20 kDa fragment indicated that a portion of S-1 heavy chains had lost their COOH-terminal 21 residues during limited tryptic digestion. Methylated amino acid residues were found at four positions: ε-N-monomethyllysine at position 32, ε-N-trimethyllysine residues at 127 and 549, and 3-N-methylhistidine at 754.View full abstractDownload PDF (1473K) -
Jun-ichi Toyota, Kaoru Omichi, Sumihiro Hase1994 Volume 115 Issue 5 Pages 927-929
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman urine collected from healthy individuals was ultrafiltered and the filtrate was gel-filtered. The fraction including disaccharides was pyridylaminated to convert the reducing sugars to fluorescent pyridylamino (PA)-derivatives. A PA-disaccharide consisting of Fuc and Man was purified by gel filtration, reversed-phase HPLC, and size fractionation HPLC. Structural analysis revealed that the disaccharide was Fucaα1-2Man-PA. The disaccharide is considered to be a metabolite of unknown glycoconjugates.View full abstractDownload PDF (325K) -
Hayao Taguchi, Takahisa Ohta1994 Volume 115 Issue 5 Pages 930-936
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSubstitutions of the conserved Arg-235 with Lys and Gln induced drastic decreases in the catalytic efficiency of Lactobacillus plantarum D-lactate dehydrogenase (D-LDH). Both the mutant enzymes showed a marked resistance to 2, 3-butanedione, by which the wild-type enzyme is rapidly inactivated unless NADH and oxamate are present. The pKa of the catalytic His was markedly shifted to the alkaline side by the Arg-to-Gln substitution, while it was not significantly shifted by the Arg to Lys substitution. The Arg-to-Lys replacement, by which the catalytic efficiency was less damaged, also induced decreases in kcat/Km for alternative substrates, such as 2-ketobutyrate, by approximately 10, 000-fold, virtually the same level as in the case of pyruvate. Although both the wild-type and mutant enzymes exhibited lower kcat/Km for the alternative substrates than that for pyruvate, in the case of the mutant enzyme, the decrease in kcat/Km for the alternative substrates was mostly due to a decrease in kcat, while it was caused mainly by an increase in Km in the wild-type enzyme, suggesting that the mutant enzyme tends to form a nonproductive enzyme-substrate complex, in particular with more unfavorable substrates. The pH-dependence of the kinetic constants also indicated that there is a nonproductive binding that does not require the protonated or deprotonated form of the catalytic His residue. These results strongly suggest that Arg-235 plays an essential role in the tight and correct binding of substrate to the binding site of D-LDH, as in the case of Arg-171 in L-LDH.View full abstractDownload PDF (773K) -
Akihiko Tsuji, Ryoichi Oda, Kohji Sakiyama, Hideaki Nagamune, Kohji It ...1994 Volume 115 Issue 5 Pages 937-944
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSImprovement of the delivery of exogenous enzymes is essential to achieve effective enzyme replacement therapy in lysosomal storage diseases. To test whether α2-macroglobulin, an endogenous plasma protein, could serve as a transport vehicle of therapeutic agents to cells, α2-macroglobulin and acid α-glucosidase or α-galactosidase A were coupled using two heterobifunctional cross-linking reagents. The α-glucosidase-α2-macroglobulin conjugate was internalized and transported into lysosomes of acid α-glucosidase-deficient fibroblasts. The enzyme activity was stable after being taken up by the cells. Uptake of the conjugate resulted in the degradation of glycogen accumulated in lysosomes. The α-galacto-sidase A-α2-macroglobulin conjugate was also internalized into the lysosomes of α-galacto-sidase A-deficient fibroblasts. Internalized α-galactosidase A-conjugate degraded globotriaosylceramide accumulated in lysosomes. The endocytosis of both conjugate was inhibited by α2-macroglobulin-trypsin complex, indicating that the conjugates were endocytosed by an α2-macroglobulin receptor system. These results showed the usefulness of α2-macroglobulin as a transport vehicle of lysosomal enzymes for effective enzyme replacement.View full abstractDownload PDF (2723K) -
Tominori Kimura, Akio Ohyama1994 Volume 115 Issue 5 Pages 945-952
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExpression of structural proteins of human immunodeficiency virus type 1 requires the direct interaction of the viral Rev trans-activator with its cis-acting RNA target sequence, the Rev response element (RRE). The originally defined 234-residue RRE, however, failed to show the full HIV-1 regulator of virion expression (Rev) response when inserted into a test plasmid that lacked the flanking env sequences. Here, we demonstrate that an alternative 351-residue complete RRE, which carries an extended Stem I structure, is required for the full Rev-responsiveness in vivo. Mutagenesis studies revealed that the association of Rev with RRE was initiated by the recognition of a functional bubble structure in stem-loop II, followed by interaction with the neighbouring duplex RNA, including the elongated Stem I structure. We propose that, as has been previously shown in vitro, the Rev-RRE interaction in vivo is an ordered assembly process and is based on the association of Rev molecules with the double-stranded region of RRE.View full abstractDownload PDF (2268K) -
Keisuke Horiuti, Naoto Yagi, Koichi Kagawa, Katsuzo Wakabayashi, Kazuh ...1994 Volume 115 Issue 5 Pages 953-957
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe examined the equatorial X-ray diffraction of skinned fibers from rat psoas muscle in isometric contraction induced by photorelease of 1.1mM ATP at 24°C in the absence of Call. At 15-20 ms after release of ATP, tension peaked at 21% of the maximum active tension. From the equatorial intensity ratio (I1, 0/I1, 1), we estimated the amount of myosin heads associated with actin filaments (A•M) at the tension peak. It was 32% of that in rigor and about half of that in the maximum contraction. ADP (0.7mM) enhanced the contraction: tension peaked at 47% of the maximum at 25-30 ms. The estimated A•M at this peak was 50% of that in rigor, which was close to that in the maximum contraction. These results indicate that the association of myosin heads with the actin filaments in the Ca2+-free contraction is more than expected from the tension size, both in the absence and presence of ADP.View full abstractDownload PDF (587K) -
Akihito Yamaguchi, Tomomi Kimura, Tetsuo Sawai1994 Volume 115 Issue 5 Pages 958-964
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUnique hot spots for sulfhydryl inactivation of Cys mutants of the metal-tetracycline/H+ antiporter (TetA) were found at the fourth positions of the dual conserved sequence motifs, GKXXDRXGRR and GRXXXKXGEK [Yamaguchi, A., Kimura, T., Someya, Y., & Sawai, T. (1993) J. Biol. Chem. 268, 6496-6504]. The fourth positions of these motifs are occupied by Ser65 and Ala269, respectively. N-Ethylmaleimide (NEM) rapidly bound to and inactivated the S65C and A269C mutants. M64C and I268C showed low reactivity to NEM, probably due to the partial cripticity of these residues. In contrast, NEM rapidly bound to T270C but did not inactivate it. NEM completely inactivated S65C, whereas a small sulfhydryl reagent, methyl methanethiosulfonate (MMTS), caused only 40% inactivation. [14C] NEM binding to S65C was inhibited by tetracycline. These observations indicated that position 65 is located close to the substrate-protein interaction site and that the inactivation by sulfhydryl reagents comprises volume-dependent steric hindrance. The S65M mutant, having a side chain analogous to one of the thiomethyl cysteines of MMTS-modified S65C, showed about 30% of the Vmax value for the wild-type tetracycline transport, while the S65F mutant had completely lost the activity, confirming the idea of volume-dependent steric hindrance at position 65. On the other hand, the A269C mutant was greatly inactivated by both NEM and MMTS. The degree of the inactivation by MMTS (90%) was higher than that of NEM (80%). The synergetic inactivation observed for the S65C/A269C double mutant suggested different inactivation mechanisms for A269C and S65C. [14C] NEM binding to A269C was not affected by tetracycline, thus, position 269 is far from the tetracycline binding site. The Vmax value for the tetracycline transport by the A269M mutant was about 20% of that for the wild-type, while that of the A269F mutant was about 60% of that for the wild type. These observations indicated that the effect of a substituent at position 269 is not directly related to its volume and thus should be a remote conformational one.View full abstractDownload PDF (2046K) -
Shaikh Abu Hossain, Katsuyuki Tanizawa, Yasuaki Kazuta, Toshio Fukui1994 Volume 115 Issue 5 Pages 965-972
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing oligonucleotide probes synthesized on the basis of partial amino acid sequences, we have cloned and sequenced the gene of Escherichia coli K-12 encoding UDP-glucose pyrophosphorylase. The gene consists of 906 base pairs and encodes a polypeptide of 302 amino acid residues with a calculated molecular weight of 32, 941. Its nucleotide sequence was found to be identical with that recently registered (EMBL, X59940) for a gene coding for an unknown 33-kDa protein, which was later annotated as UDP-glucose pyrophospho-rylase on the basis of genetic studies. The UDP-glucose pyrophosphorylase gene, mapped at 27.3 min in the E. coli chromosome, complemented the galU mutation, which renders the bacterium unable to ferment galactose. The recombinant enzyme overproduced in E. coli cells and purified to homogeneity catalyzed the synthesis and pyrophosphorolysis of UDP-glucose by a sequential mechanism. The enzyme required Mg2+ for maximal activity and was inhibited by free UTP and pyrophosphate. The E. coli enzyme shows significant sequence similarities with the enzymes from Acetobacter xylinum and Salmonella typhimurium. However, little or no similarity was found with the eukaryotic enzymes that are involved in the biosynthesis of storage carbohydrates, or with other enzymes acting on similar sugar nucleotides. Thus, UDP-glucose pyrophosphorylases participating in diverse metabolic pathways can be classified structurally into the prokaryotic and eukaryotic groups, even though they have almost identical catalytic properties.View full abstractDownload PDF (2426K) -
Masako Yoshida, Takashi Kanematsu, Yutaka Watanabe, Toshitaka Koga, Sh ...1994 Volume 115 Issue 5 Pages 973-980
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat brain membrane fractions obtained using Triton X-100 were applied to a D-myo-inositol 1, 4, 5-trisphosphate [D-Ins (1, 4, 5) P3] immobilized column, followed by gel filtration and anion-exchange chromatography. Two proteins with molecular masses of 130 and 85 kDa, as assessed by SDS-polyacrylamide gel electrophoresis, were purified to apparent homogeneity as D-[3H] Ins (1, 4, 5) P3-binding proteins with no D-Ins (1, 4, 5) P3-metabolizing activity. Partial amino acid sequence determinations of these proteins revealed that the 130 kDa protein appears to be a new D-Ins (1, 4, 5) P3-binding protein and the 85 kDa protein is a δ1-isozyme of phospholipase C. We have previously purified 130 and 85 kDa proteins, as D-[3H] Ins (1, 4, 5) P3-binding proteins, from rat brain cytosol fraction. Antibodies against the 130 kDa protein from the cytosol cross-reacted with the membrane 130 kDa protein purified in this study, suggesting that the membrane 130 kDa protein is likely to be the same as the protein from the cytosol fraction. The inhibition of D- [3H] Ins (1, 4, 5) P3 binding by D-isomers of inositol phosphates available clarified that the 130 kDa protein has a similar affinity for D-Ins (1, 4, 5, 6) P4 to that for D-Ins (1, 4, 5) P3, while the 85 kDa protein is specific to D-Ins (1, 4, 5) P3.View full abstractDownload PDF (2796K) -
Susumu Tanaka, Kohei Hosaka1994 Volume 115 Issue 5 Pages 981-984
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA second form (β isoform) of rat phosphatidylinositol transfer protein cDNA was cloned by complementation of the yeast sec14 mutation from a rat brain cDNA expression library. The deduced sequence of the protein comprised 271 amino acids with a calculated molecular mass of 31, 449 Da. The deduced amino acid sequence showed 77% identity to that of the rat phosphatidylinositol transfer protein, recently reported by Dickeson et al. [Dickeson, S. K., Lim, C. N., Schuyler, G. T., Dalton, T. P., Helmkamp, G. M., Jr., & Yarbrough, L. R. (1989) J. Biol. Chem. 264, 16557-16564]. Northern blot analysis revealed that the cDNA probe of the β isoform hybridized to an _??_3-kilobase RNA in various rat tissues. The mRNA was expressed abundantly in brain, kidney, liver, and lung, but in a lesser amount in testis.View full abstractDownload PDF (1583K) -
Noritaka Ariyoshi, Mitsuko Tanaka, Yuji Ishii, Kazuta Oguri1994 Volume 115 Issue 5 Pages 985-990
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn epoxide hydrolase (mEH) in liver microsomes was purified to apparent homogeneity from a dog treated with phenobarbital. The purified enzyme had a minimum molecular weight of 47, 000 as determined by SDS-PAGE. The dog mEH activity was characterized by use of a substrate, 7-glycidoxycoumarin (GOC), and some effectors of this enzyme. In vitro activators, metyrapone, and isoquinoline, stimulated the microsomal activity, but the former had no such effect on the purified enzyme in case of this substrate. All mEH inhibitors, 1, 1, 1-trichloropropene 2, 3-oxide (TCPO), cyclohexene oxide, and 2-bromo-4'-nitroacetophenone (BrNAP), suppressed hydrolase activity. The NH2-terminal amino acid sequence of the purified enzyme was highly homologous (90%) to the sequences deduced from a cDNA clone of rat enzyme. Antiserum to the purified enzyme raised in rabbits cross-reacted with rat and guinea pig epoxide hydrolases. No gender-difference in this enzyme in liver microsomes was observed in dogs.View full abstractDownload PDF (4020K) -
Norihisa Inazu, Yoshio Nagashima, Tetsuo Satoh, Tomoko Fujii1994 Volume 115 Issue 5 Pages 991-999
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSix aldo-keto reductases from rat adrenal gland have been highly purified to apparent homogeneity. These enzymes were identified as carbonyl reductases (CR-K1, CR-K2, CR-A, and CR-B), aldehyde reductase (AR-H), and aldose reductase (AR-L) in terms of substrate specificity, molecular weight (33, 000-39, 000), inhibitor susceptibility, cofactor requirement, and immunochemical properties. Both CR-K1 and CR-K2 were characterized as possessing high affinity towards 13, 14-dihydro-15-ketoprostaglandin F2α and were localized immunohistochemically in the zona glomerulosa and the zona reticularis of adrenal cortex, and in the ganglion cell of adrenal medulla. Immunoreactive proteins to anti-CR-K2 antibody were observed in male and female reproductive tissues of rats. Positive immuno-reactive protein to anti-CR-A antibody was found in mouse, hamster, and rabbit adrenal gland, whereas that to anti-CR-K2 antibody was present only in rat adrenal gland. AR-H and AR-L mainly reduced aromatic and aliphatic aldehydes. All the aldo-keto reductases from rat adrenal gland were completely inhibited by p-chloromercuribenzoate. Barbiturate and 3, 3'-tetramethylene glutarate potently inhibited AR-H, and quercitrin significantly decreased the activity of CR-K1, CR-K2, and AR-L. We propose that these aldo-keto reductases may play important roles in the rat adrenal functions.View full abstractDownload PDF (4302K) -
Jianwei Zhu, Hans Hansen, Lihe Su, Hui-Ling Shieh, Heimo Riedel1994 Volume 115 Issue 5 Pages 1000-1009
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBased on the finding by others that the conserved region C1 of conventional protein kinase C isoforms carries two independent, cysteine-rich phorbol ester binding sites, we have mapped the structural elements of the C1 region for their role in the phorbol ester- and phospholipid regulation of PKC α responses. We have prepared two amino terminal truncation mutants of bovine PKC α, ND91 lacking the first Cys-repeat of C1, and ND153 lacking both Cys-repeats of C1, as well as two internal deletion mutants, D162-245 lacking most of C2, and D109-263 lacking most of C2 and the second Cys-repeat of C1. The mutants were expressed in the yeast Saccharomyces cerevisiae which allows the rapid biochemical and physiological characterization of mammalian PKC isoforms. We found that all mutants displayed an elevated basal level of enzymatic activity in vitro but retained the basic catalytic PKC characteristics: regulation by Ca2+ and (except for ND153) by phospholipid or phorbol ester. In vivo we observed proportional physiological responses, the stimulation of Ca2+ uptake, and an increase in the cell doubling time for all mutants upon phorbol ester stimulation (constitutive for ND153) similar to the response of normal PKC α. Our findings indicate that after partial PKC activation by deletion mutagenesis, the presence of either Cys-repeat in C1 still allows phospholipid- and phorbol ester regulation of protein kinase C α responses.View full abstractDownload PDF (2554K) -
Tominori Kimura, Masao Nishikawa, Akio Ohyama1994 Volume 115 Issue 5 Pages 1010-1020
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe membrane traffic of human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins has been investigated in COS-1 cells transiently expressing the HIV-1 env, vpu, and rev genes. Analysis of oligosaccharide processing revealed that the majority of gp160 remained fully endo-H sensitive throughout a 21-h chase period, and hence cleavage of gp160 to gp120-gp41 took place prior to the creation of hybrid and complex oligosaccha-rides on gp120. Immunofluorescence microscopy demonstrated that in the absence of CD4 both gp160 and Vpu are targeted to the Golgi apparatus, that can be stained with wheat germ agglutinin or antibodies to the human KDEL receptor. In contrast, gp160 complexed with CD4 was retained in the ER and thus failed to reach the cis-Golgi compartment. Although gp160-bound CD4 has its own half life of 4 h 35 min in the endoplasmic reticulum (ER), co-expression of Vpu accelerated the turnover of CD4 by 5.5-fold and thereby enabled gp160 to be translocated out of the ER to the cis-Golgi compartment. We concluded that Vpu prevents the formation of stable CD4-gp160 complexes in the ER and thus indirectly allows gp160 to accumulate in the Golgi apparatus, where it is selectively retained to produce gp120-gp41.View full abstractDownload PDF (6486K) -
Yasuo Sugiyama, Yasuo Mukohata1994 Volume 115 Issue 5 Pages 1021-1026
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSArchae-opsin-1 (aO-1) has been expressed efficiently as a fusion protein with 13 heterologous amino acids at the amino terminus of the mature aO-1 in Escherichia coli under the control of T7 promoter. The E. coli-expressed aO-1, designated as aO-1002, which was located in the membrane fraction, was extracted with 8 M urea and partially purified by gel filtration chromatography in the presence of SDS. When all-trans retinal was added, aO-1002 in dimyristoylphosphatidylcholine and detergent-mixed micelles was converted to a purple pigment with λmax at 558 nm at 20°C via a 435/460 nm intermediate. Conversion of the intermediate to purple pigment was the rate-limiting step and proceeded as a two-state transition, because an isosbestic point was seen at 485 nm. Similar spectral changes were also observed in the regeneration process of hydroxylamine-bleached claret membranes and aO-1 isolated from claret membranes. Thus, the polypeptide of aO-1002 is considered to fold and form a retinal binding pocket in phospholipid and detergent micelles similarly to aO-1 isolated from the halobacterial membranes. Purple pigment showed a light-driven proton-pumping activity when reconstituted into phosphatidylcholine liposomes.View full abstractDownload PDF (1853K) -
1994 Volume 115 Issue 5 Pages 1027c
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (40K) -
1994 Volume 115 Issue 5 Pages 1027b
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (40K) -
1994 Volume 115 Issue 5 Pages 1027a
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (40K)
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