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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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Korean Society for Biochemistry and Molecular Biology
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Volume & Issues
Volume 34, Issue 6 - Nov 2001
Volume 34, Issue 5 - Sep 2001
Volume 34, Issue 4 - Jul 2001
Volume 34, Issue 3 - May 2001
Volume 34, Issue 2 - Mar 2001
Volume 34, Issue 1 - Jan 2001
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Compartmental Analysis of the Insulin-induced GLUT4 Recruitment in Adipocytes
Ryu, Ji-Won ; Jung, Chan-Y. ;
BMB Reports , volume 34, issue 4, 2001, Pages 285~292
Insulin stimulates glucose uptake in muscle and adipose tissue and thus maintains normal blood glucose level in our body. Derangement of this process causes many grave health problems. Insulin stimulates glucose transport primarily by recruiting GLUT4 from its intracellular storage sites to the plasma membrane. The process is complex and involves GLUT4 trafficking through multiple subcellular compartments (organelles) and many protein functions, details of which are poorly understood. This review summarizes a recent development to isolate and characterize the individual intracellular GLUT4 compartments and to illustrate how this compartmental analysis will help to identify the insulin-sensitive step or steps in the insulin-induced GLUT4 recruitment in rat adipocytes. The review does not cover the recent exciting development in identification of many proteins implicated in this process.
Expression and Biochemical Characterization of the Bacillus thuringiensis Cry4B
Puntheeranurak, Theeraporn ; Leetacheewa, Somphob ; Katzenmeier, Gerd ; Krittanai, Chartchai ; Panyim, Sakol ; Angsuthanasombat, Chanan ;
BMB Reports , volume 34, issue 4, 2001, Pages 293~298
Tryptic activation of the 130-kDa Bacillus thuringiensis Cry4B
-endotoxin produced protease-resistant products of ca. 47 kDa and ca. 21 kDa. The 21-kDa fragment was identified as the N-terminal five-helix bundle (
,) which is a potential candidate for membrane insertion and pore formation. In this study, we constructed the recombinant clone over-expressing this putative pore-forming (PPF) fragment as inclusion bodies in Escherichia coli. The partially purified inclusions were composed of a 23-kDa protein, which cross-reacted with Cry4B antibodies, and whose N-terminus was identical to that of the 130-kDa protein. Dissimilar to protoxin inclusions, the PPF inclusions were only soluble when the carbonate buffer, pH 9.0, was supplemented with 6 M urea. After renaturation via a stepwise dialysis, the refolded PPF protein appeared to exist as an oligomer and was structurally stable upon trypsin treatment. Unlike the 130kDa protoxin, the refolded protein was able to release entrapped glucose from liposomes, and showed comparable activity to the full-length activated toxin, although it lacks larvicidal activity These results, therefore, support the notion that the PPF fragment that consists of
of the activated Cry4B toxin is involved in membrane pore-formation.
Regulation of 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) Synthase of Bacillus sp. B-6 Producing Phenazine-1-carboxylic acid
Kim, Kyoung-Ja ;
BMB Reports , volume 34, issue 4, 2001, Pages 299~304
The 3-Deoxy-D-arabinoheptulosonate 7-phosphate (DAHP) synthase is the first enzyme of aromatic amino acid-, folic acid-, and phenazine-1-carboxylic acid biosynthetic pathways. DAHP synthase of Bacillus sp. B-6 that produces phenazine-1-carboxylic acid was feedback inhibited by two intermediary metabolites of aromatic amino acid biosynthetic pathways, prephenate and chorismate, but not by other metabolites, such as anthranilic acid, shikimic acid, p-aminobenzoic acid, and 3-hydroxyanthranilic acid. DAHP synthase of Bacillus sp. B-6 was not inhibited by end products, such as aromatic amino acids, folic acid, and phenazine-1-carboxylic acid. The inhibition of DAHP synthase by prephenate and chorismate was non-competitive with respect to erythrose 4-phosphate and phosphoenolpyruvate. Prephenate and chorismate inhibited 50% of the DAHP synthase activity at concentrations of
, respectively The synthesis of DAHP synthase of Bacillus sp. B-6 was not repressed by exogenous aromatic amino acids, folic acid, and phenazine 1-carboxylic acid, single or in combinations.
Identification of Amino Acid Residues in the Carboxyl Terminus Required for Malonate-Responsive Transcriptional Regulation of MatR in Rhizobium leguminosarum bv. trifolii
Lee, Hwan-Young ; Kim, Yu-Sam ;
BMB Reports , volume 34, issue 4, 2001, Pages 305~309
MatR in Rhizobium trifolii is a malonate-responsive transcription factor that regulates the expression of genes, matABC, enabling decarboxylation of malonyl-CoA into acetyl-CoA, synthesis of malonyl-CoA from malonate and CoA, and malonate transport. According to an analysis of the amino acid sequence homology, MatR belongs to the GntR family The proteins of this family have two-domain folds, the N-terminal helix-turn-helix DNA-binding domain and the C-terminal ligand-binding domain. In order to End the malonate binding site and amino acid residues that interact with RNA polymerase, a site-directed mutagenesis was performed. Analysis of the mutant MatR suggests that Arg-160 might be involved in malonate binding, whereas Arg-102 and Arg-174 are critical for the repression activity by interacting with RNA polymerase.
Distribution of Chitinases in Rice (Oryza sativa L)Seed and Characterization of a Hull-Specific Chitinase
Baek, Je-Hyun ; Han, Beom-Ku ; Jo, Do-Hyun ;
BMB Reports , volume 34, issue 4, 2001, Pages 310~315
The uneven distribution of acidic and basic chitinases in different parts of rice seed, and also the characterization of hull-specific chitinases, are reported here. After extraction of chitinases from polished rice, bran, and rice hulls, the chitinases were separated into acidic and basic fractions, according to their behavior on an anion exchanger column. Both fractions from different parts of rice seed showed characteristic activity bands on SDS-PAGE that contained 0.01% glycol chitin. The basic chitinases from rice hulls were further purified using chitin affinity chromatography. The chitinase, specific to rice hulls (RHBC), was 88-fold purified with a 1.3% yield. RHBC has an apparent molecular weight of 22.2 kDa on SDS-PAGE. The optimal pH and temperature were 4.0 and
, respectively. With [
]chitin as a substrate, RHBC has
of 13.51 mg/mg protein/hr and
of 1.36 mg/ml. This enzyme was an endochitinase devoid of
-1,3-glucanase, lysozyme, and chitosanase activities.
Biochemical Characterization of Transgenic Tobacco Plants Expressing a Human Dehydroascorbate Reductase Gene
Kwon, Suk-Yoon ; Ahn, Young-Ock ; Lee, Haeng-Soon ; Kwak, Sang-Soo ;
BMB Reports , volume 34, issue 4, 2001, Pages 316~321
Dehydroascorbate (DHA) reductase (DHAR, EC 22.214.171.124) catalyzes the reduction of DHA to reduced ascorbate (AsA) using glutathione (GSH) as the electron donor in order to maintain an appropriate level of ascorbate in plant cells. To analyze the physiological role of DHAR in environmental stress adaptation, we developed transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants that express a human DHAR gene isolated from the human fetal liver cDNA library in the chloroplasts. We also investigated the DHAR activity, levels of ascorbate, and GSH. Two transgenic plants were successfully developed by Agrobacterium-mediated transformation and were confirmed by PCR and Southern blot analysis. DHAR activity and AsA content in mature leaves of transgenic plants were approximately 1.41 and 1.95 times higher than in the non-transgenic (NT) plants, respectively In addition, the content of oxidized glutathione (GSSG) in transgenic plants was approximately 2.95 times higher than in the NT plants. The ratios of AsA to DHA and GSSG to GSH were changed by overexpression of DHAR, as expected, even though the total content of ascorbate and glutathione was not significantly changed. When tobacco leaf discs were subjected to methyl viologen at
transgenic plants showed about a 50% reduction in membrane damage compared to the NT plants.
In Vivo Excision and Amplification of Large Human Genomic Segments Using Cre/loxP-and EBNA-1/oriP-mediated Machinery
Yoon, Young-Geol ; Choi, Ja-Young ; Kim, Jung-Min ; Lee, Jun-Hyoung ; Kim, Sun-Chang ;
BMB Reports , volume 34, issue 4, 2001, Pages 322~328
Excision and amplification of pre-determined, large genomic segments (taken directly from the genome of a natural host, which provides an alternative to conventional cloning in foreign vectors and hosts) was explored in human cells. In this approach, we devised a procedure for excising a large segment of human genomic DNA, the iNOS gene, by using the Cre/loxP system of bacteriophage P1 and amplifying the excised circles with the EBNA-1/oriP system of the Epstein-Barr virus. Two loxP sequences, each of which serves as a recognition site for recombinase Cre, were integrated unidirectionally into the 5'-UTR and 3'-UTR regions of the iNOS gene, together with an oriP sequence for conditional replication. The traps-acting genes cre and EBNA-1, which were under the control of a tetracycline responsive
promoter, were also inserted into the 5'-UTR and 3'-UTR regions of the iNOS gene, respectively, by homologous recombination. The strain carrying the inserted elements was stably maintained until the excision and amplification functions were triggered by the induction of cre and EBNA-1. Upon induction by doxycycline, Cre excised the iNOS gene that was flanked by two ZoxP sites and circularized it. The circularized iNOS gene was then amplified by the EBNA-1/oriP-system. With this procedure, approximately a 45.8-kb iNOS genomic fragment of human chromosome 17 was excised and successfully amplified in human cells. Our procedure can be used effectively for the sequencing of unclonable genes, the functional analysis of unknown genes, and gene therapy.
Inactive but Dimeric Form of Lipoprotein Lipase in Human Plasma
Park, Byung-Hyun ;
BMB Reports , volume 34, issue 4, 2001, Pages 329~333
Active lipoprotein lipase (LPL) is known as a noncovalent homodimer of identical subunits, and dissociation of the dimer to a monomeric form renders the lipase inactive. In this study, the oligomerization status of LPL in human and rat plasma was investigated. The LPL activity was barely detectable in the control rat and human plasma. After the injection of heparin, the total lipolytic activity of plasma was rapidly increased, and reached its maximum in 30 min. Changes of the LPL protein correlated well with those of lipolytic activity. The LPL protein that is released by heparin into both human and rat plasma was active and dimeric in the sucrose density gradient ultracentrifugation. In control rat plasma, LPL was inactive, and a great fraction was present as an aggregate. However, the inactive LPL protein in the control human plasma retained the dimeric state, indicating that dimerization can be an entity independent of the catalytic activity of LPL. The released LPL is transported as a complex with lipoproteins in plasma. Lipoprotein profiles, determined by NaBr ultracentrifugation, exhibited typical LDL- and HDL-mammal patterns in humans and rats, respectively, with a smaller amount of the LDL fraction observed in rats. The difference in the lipoprotein profiles might influence the fate of the released LPL in plasma.
Molecular Characterization of a Chinese Cabbage cDNA Encoding Thioredoxin-h that is Predominantly Expressed in Flowers
Lee, Seung-Sik ; Lee, Kyun-Oh ; Jung, Bae-Gyo ; Chi, Yong-Hun ; Yoo, Ji-Young ; Lee, Ji-Yeun ; Lee, Jung-Ro ; Park, Soo-Kwon ; Kang, Soon-Suk ; Jang, Ho-Hee ; Lee, Sang-Yeol ;
BMB Reports , volume 34, issue 4, 2001, Pages 334~341
Even though three isotypes of thioredoxins (-f, -m and -h types) have been identified in a variety of plant cells, there are only a few reports on thioredoxin-h that were recently identified. In this study, a cDNA encoding a h-type of thioredoxin was isolated from a cDNA library of Chinese cabbage, and named here CTrx-h. An open reading frame of the gene contained a polypeptide of 133 amino acids with a conserved active center, WCGPC, which appeared in all of the thioredoxin proteins. A deduced amino acid sequence of the CTrx-h showed the highest sequence identity with those of Arabidopsis thioredoxin-h2 (75.2%) and thioredoxin-h5 (46.6%) proteins, but it shared a low sequence homology to other isotypes of plant thioredoxinm and thioredoxin-f. The CTrx-h protein that is expressed in E. coli represented not only an insulin reduction activity, but also electron transferring activity from NADPH to thioredoxin-dependent peroxidase. A genomic Southern blot analysis using the cDNA insert of CTrx-h revealed that the gene consisted of a small multigene family in Chinese cabbage genome. On the contrary to other thioredoxin-h proteins that were widely distributed in most tissues of the plant, the CTrx-h was predominantly expressed in flowers. The expression was very low in other tissues. The data of the Northern blot analysis suggests that the CTrx-h may have other functions in flower development or differentiation, in addition to its defensive role.
Cloning and Characterization of the Mycobacterium bovis BCG panB Gene Encoding Ketopantoate Hydroxymethyltransferase
Kim, Jin-Koo ; Kim, Kwang-Dong ; Lim, Jong-Seok ; Lee, Hee-Gu ; Kim, Sang-Jae ; Cho, Sang-Hyun ; Jeong, Won-Hwa ; Choe, In-Seong ; Chung, Thi-Wha ; Paik, Sang-Gi ; Choe, Yong-Kyung ;
BMB Reports , volume 34, issue 4, 2001, Pages 342~346
The Mycobacterium bovis BCG panB gene, encoding ketopantoate hydroxymethyltransferase (KPHMT), was cloned from a
genomic library and sequenced. The DNA sequence encodes a protein that contains 281 amino acid residues (M, 29,337) with a high similarity to the KPHMTs. Subcloning of a 846 by open reading frame (ORF), but not a 735 by ORF, into the vector pUC19 led to complementation of the panB mutant of Escherichia coli. The BCG pang gene was overexpressed in E. coli and the KPHMT purified to homogeneity The recombinant protein was further confirmed by an enzymatic assay.
-Glucosidase from Extreme Thermophile Thermus caldophilus GK24
Nashiru, Oyekanmi ; Koh, Suk-Hoon ; Lee, Se-Yong ; Lee, Dae-Sil ;
BMB Reports , volume 34, issue 4, 2001, Pages 347~354
-Glucosidase of an extreme thermophile, Thermus caldophilus GK24 (TcaAG), was purified 80-fold from cells to a homogeneous state and characterized. The enzyme exhibited optimum activity at pH 6.5 and
, and was stable from pH 6.0 to 85 and up to
. The enzyme had a half-life of 85 minutes at
. An analysis of the substrate specificity showed that the enzyme hydrolyzed the non-reducing terminal unit of
-1,6-glucosidic linkages of isomaltosaccharides and panose,
-1,3-glycosidic bond of nigerose and turanose, and
-1,2-glycosidic bond of sucrose. The gene encoding the TcaAG was cloned, sequenced, and sequenced in E. coli. The nucleotide sequence of the gene encoded a 530 amino acid polypeptide and had a G+C content of 68.4% with a strong bias for G or C in the third position of the codons (93.6%). A sequence analysis revealed that TcaAG belonged to the
-amylase family. We suggest that this monomeric, thermostable, and broad-acting
-glucosidase is a departure from previously exhibited specificities. It is, therefore, a novel
Oligomeric Structures Determine the Biochemical Characteristics of Human Nucleoside Diphosphate Kinases
Kim, Sun-Young ; Song, Eun-Joo ; Chang, Keun-Hye ; Kim, Eun-Hee ; Chae, Suhn-Kee ; Lee, Han-Soo ; Lee, Kong-Joo ;
BMB Reports , volume 34, issue 4, 2001, Pages 355~364
Major human Nucleoside diphosphate kinases (NDPKs) exist as hetero-oligomers, consisting of NDPK-A and NDPK-B, rather than homo-oligomer. To investigate their biological function depending on the oligomeric structure in vivo, we characterized the biochemical properties of cellular NDPK. Cellular NDPKs, which are made up of a unique combination of isoforms, were purified from human erythrocyte and placenta. We found that cellular NDPK and recombinant isoforms NDPKs have their own distinct biochemical properties in autophosphorylation, stability toward heat or urea, and DNA binding. Cellular NDPK was found to have unique characteristics rather than the expected additive properties of recombinant isoforms. The mutations in the dimeric interface of NDPK-B (R34G, N69H or K135L) caused defective DNA binding and simultaneously reduced the enzymatic stability These results suggest that the oligomeric interaction could play a major role in the stability of catalytic domain and might be related to the regulation of various cellular functions of NDPK.
Cooperative Activity of Subunits of Human Ferritin Heteropolymers in Escherichia coli
Lee, Jung ; Seo, Hyang-Yun ; Jeon, Eun-Soon ; Park, Ok-Soon ; Lee, Kang-Min ; Park, Chung-Ung ; Kim, Kyung-Suk ;
BMB Reports , volume 34, issue 4, 2001, Pages 365~370
We constructed a comparative expression system in order to produce recombinant human ferritin homo- and heteropolymers in Escherichia coli. Human ferritin H-(hfH) and L-chain (hfL) genes were expressed without amino acid changes under the control of a tac promoter. Ferritin heteropolymers of varying subunit composition were also produced by combining two different expression systems, a bicistronic expression system and a coplasmid expression system. As a result, recombinant H-chain ferritin and ferritin heteropolymers were catalytically active in forming iron core in vivo. In particular, the ferritin heteropolymer that is composed of 7% H-subunit and 93% L-subunit was capable of forming an iron core of the protein, while the L-chain ferritin homopolymer was inactive in vivo. This result indicates that the two H-subunits (i.e., 7% H-subunit content) are important to keep ferritin active in the cells. In addition, human ferritins were identified as the major iron binding proteins in the transformed cells. Also, the amount of iron bound to the recombinant ferritins was proportional to the H-subunit content in ferritin heteropolymers in vivo.
Cloning, Sequencing and Baculovirus-based Expression of Fusion-Glycoprotein D Gene of Herpes Simplex Virus Type 1 (F)
Uh, Hong-Sun ; Choi, Jin-Hee ; Byun, Si-Myung ; Kim, Soo-Young ; Lee, Hyung-Hoan ;
BMB Reports , volume 34, issue 4, 2001, Pages 371~378
The Glycoprotein D (gD) gene of the HSV-1 strain F was cloned, sequenced, recombinated into the HcNPV (Hyphantria cunea nuclear polyhedrosis virus) expression vector and expressed in insect cells. The gD gene was located in the 6.43 kb BamHI fragment of the strainF. The open reading frame (ORF) of the gD gene was 1,185 by and codes 394 amino acid residues. Recombinant baculoviruses, GD-HcNPVs, expressing the gD protein were constructed. Spodoptera frugiperda cells, infected with the recombinant virus, synthesized a matured gX-gD fusion protein with an approximate molecular weight of 54 kDa and secreted the gD proteins into the culture media by an immunoprecipitation assay The fusion gD protein was localized on the membrane of the insect cells, seen by using an immunofluorescence assay The deduced amino acid sequence presents additional characteristics compatible with the structure of a viral glycoprotein: signal peptide, putative glycosylation sites and a long C-terminal transmembrane sequence. These results indicate the utility of the HcNPV-insect cell system for producing and characterizing eukaryotic proteins.
Cloning and Regulation of Schizosaccharomyces pombe Gene Encoding Ribosomal Protein L11
Kim, Hong-Gyum ; Lee, Jin-Joo ; Park, Eun-Hee ; Sa, Jae-Hoon ; Ahn, Ki-Sup ; Lim, Chang-Jin ;
BMB Reports , volume 34, issue 4, 2001, Pages 379~384
The cDNA encoding ribosomal protein was identified from a cDNA library of Schizosaccharomyces pombe. The nucleotide sequence of the 548 by cDNA clone reveals an open reading frame, which encodes a putative protein of 166 amino acids with a molecular mass of 18.3 kDa. The amino acid sequence of the S. pombe L11 protein is highly homologous with those of rat and fruit, while it is clearly less similar to those of prokaryotic counterparts. The 1,044 by upstream sequence, and the region encoding N-terminal 7 amino acids of the genomic DNA were fused into the promoterless
-galactosidase gene of the shuttle vector YEp357 in order to generate the fusion plasmid pHY L11. Synthesis of
-galactosidase from the fusion plasmid varied according to the growth curve. It decreased significantly in the growth-arrested yeast cells that were treated with aluminum chloride and mercuric chloride. However, it was enhanced by treatments with cadmium chloride (
), zinc chloride (
), and hydrogen peroxide (0.5 mM). This indicates that the expression of the L,11 gene could be induced by oxidative stress.
The IGFBP-1 mRNA Expression in HepG2 Cells is Affected by Inhibition of Heme Biosynthesis
Park, Jong-Hwan ; Park, Tae-Kyu ; Kim, Hae-Yeong ; Yang, Young-Mok ;
BMB Reports , volume 34, issue 4, 2001, Pages 385~389
Insulin-like growth factor binding protein-1 (IGFBP-1) appears to be an important modular of the insulin growth factor (IGF) bioactivity in metabolic disease and chronic hypoxia. Treatment of desferrioxamine (Dfo), cobalt, or nickel in HepG2 cells stimulated the expression of IGFBP1 mRNA as hypoxia. However, the presence of ferric ammonium citrate (FAC) in the 1%
decreased the upregulation of the IGFBP-1 mRNA expression. In addition, actinomycin D and cycloheximide abolished the increase in the expression of IGFBP-1 mRNA that was induced by Dfo and transition metals (cobalt and nickel). To obtain further information about the putative oxygen sensor, we postulate that putative heme proteins, responsible for the oxygen-sensing process in HepG2 cells, should be sensitive to hypoada. The mechanism of these upregulations of the IGFBP-1 mRNA expression by Dfo and transition metals was investigated by treatment with 2 mM of 4,6-dioxoheptanoic acid (DHA), an inhibitor of heme biosynthesis. The results showed that 1%
-, Dfo-, cobalt-, or nickel induced IGFBP-1 mRNA expressions in HepG2 cells were all markedly inhibited when the heme synthesis was blocked by DHA. We suggest that the IGFBP-1 mRNA expression in the HepG2 cell is regulated by 1%
, Dfo, cobalt, or nickel, implicating the involvement of the putative heme-containing oxygensensing molecule.