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REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
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Journal DOI :
Korean Society for Biochemistry and Molecular Biology
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Volume & Issues
Volume 33, Issue 6 - Nov 2000
Volume 33, Issue 5 - Sep 2000
Volume 33, Issue 4 - Jul 2000
Volume 33, Issue 3 - May 2000
Volume 33, Issue 2 - Mar 2000
Volume 33, Issue 1 - Jan 2000
Selecting the target year
ATP and GTP Hydrolytic Function of N-terminally Deleted Annexin I
Hyun, Young-Lan ; Park, Young-Min ; Na, Doe-Sun ;
BMB Reports , volume 33, issue 4, 2000, Pages 289~293
Annexin I is a 37 kDa member of the annexin family of calcium-dependent phospholipid binding proteins. Annexin I plays regulatory roles in various cellular processes including cell proliferation and differentiation. Recently we found that annexin I is a heat shock protein (HSP) and displays a chaperone-like function. In this paper we investigated the function of annexin I as an ATPase using 1 to 32 amino acids deleted annexin I (
I hydrolyzed ATP as determined by thin layer chromatography. The ability of ATP hydrolysis was inhibited by ADP, GTP and GDP, but not by the AMP, GMP and cAMP. In view of the ATP hydrolyzing function of HSP, the results support the function of annexin I as a HSP.
The Two-Component Protease NS2B-NS3 of Dengue Virus Type 2: Cloning, Expression in Escherichia coli and Purification of the NS2B, NS3(pro) and NS2B-NS3 Proteins
Champreda, Veerawat ; Khumthong, Rabuesak ; Subsin, Benchamas ; Angsuthanasombat, Chanan ; Panyim, Sakol ; Katzenmeier, Gerd ;
BMB Reports , volume 33, issue 4, 2000, Pages 294~299
Proteolytic processing of the dengue virus serotype 2 polyprotein precursor is catalyzed by a host signal peptidase and a virus encoded two-component protease consisting of the nonstructural proteins, NS2B and NS3. We expressed in Escherichia coli the NS2B, NS3(pro) and NS2B-NS3 proteins from the dengue virus type 2 strain 16681 as N-terminal fusions with a hexahistidine affinity tag under the control of the inducible trc promoter. All fusion proteins were purified to >90% purity by detergent extraction of inclusion bodies and a single step metal chelate chromatography. Proteins were refolded on-column and recovered with yields of 0.5, 6.0 and 1.0 mg/l of E. coli culture that was grown to
for NS2B, NS3(pro) and NS2B-NS3, respectively. Purified proteins gave strong signals in Western blots using
acid as a probe for the presence of the polyHis tag. During the purification process,
was apparently not autoproteolytically cleaved at the NS2B/NS3 site.
Purification and Characterization of Two Isolectins with Arginase Activity from the Lichen Xanthoria parietina
Molina, M. C. ; Vicente, C. ;
BMB Reports , volume 33, issue 4, 2000, Pages 300~307
Two glycoproteins were purified and biochemically characterized from the lichen X. parietina. Both behaved as enzymes with arginase activity and haemaglutinins. Secreted arginase (SA) contained galactose and glucose in the saccharide moiety and an isoelectric point of 4.54. The algal binding-protein (ABP) had N-acetyl-glucosamine and glucose as glycosidic residues and an isoelectric point of 3.53. Both proteins had the same molecular mass (58.6 kDa) and the same qualitative amino acidic composition. The results allowed us to consider these glycoproteins as isolectins, which have significant physiological roles in the relationship between photobiont and mycobiont of symbiotic association.
Interrelationship between Cell Differentiation and Expression of mRNA for Transferrin in HL-60 Leukemia Cell Line
Lee, Soo-Young ; Chi, Chung-Hee ; Kim, You-Mie ;
BMB Reports , volume 33, issue 4, 2000, Pages 308~311
The interrelationship between the differentiation and expression of mRNA for transferrin in the HL-60 leukemia cell line was studied. Transferrin mRNA was expressed in HL-60 leukemia cells and the amount was 50% of that in the positive control cell line, HepG-2 cells. The expression of
mRNA in HL-60 cells was not regulated by IL-1, IL-6 and
, respectively. The expression of
mRNA in the differentiated cells into a granulocyte lineage by DMSO, or all-trans RA, was up-regulated (160-170% of control cells); whereas, the expression was not regulated in the differentiated cells into a macrophage lineage by PMA. These results suggest that the differentiation to a granulocyte lineage of HL-60 leukemia cells appear to be related with the upregulation of transferrin mRNA expression.
Peroxidase Activity Boosting by Various Nitrogenous Compounds
Lee, Dong-Joo ; Kim, Soung-Soo ; Lee, Mi-Young ;
BMB Reports , volume 33, issue 4, 2000, Pages 312~316
Effects of various nitrogenous compounds on the peroxidative activity of Korean radish (Rophanus sativus L.) isoperoxidase
were examined by using anilino substrates, such as dianisidine and phenylenediamine. We also used phenolic substrates such as guaiacol, chlorogenic acid, caffeic acid, ferulic acid and esculetin. The peroxidation of dianisidine was stimulated by adenine and imidazole as much as 5 fold and 11 fold, respectively at pH 8. Moreover, about 4.8 fold and 8 fold stimulation of phenylenediamine peroxidation occurred by adenine and imidazole, respectively at pH 8. The stimulation by adenine and imidazole did not occur at the acidic pH range. The peroxidations of phenolic substrates, such as guaiacol, chlorogenic acid, caffeic acid, ferulic acid and esculetin, were not boosted greatly by any of the nitrogenous compounds tested. Notably, ammonium salt, which has been known for the excellent booster of horseradish peroxidase, did not affect the peroxidation of the Korean radish isoperoxidase
. The kinetic studies of dianisidine peroxidation with imidazole, as a model of boosting reaction, showed that neither the affinity of imidazole against dianisidine, nor the activation energy of dianisidine peroxidation changed during the activity boosting of isoperoxidase
Brain Succinic Semialdehyde Dehydrogenase; Reaction of Arginine Residues Connected with Catalytic Activities
Bahn, Jae-Hoon ; Lee, Byung-Ryong ; Jeon, Seong-Gyu ; Jang, Joong-Sik ; Kim, Chung-Kwon ; Jin, Li-Hua ; Park, Jin-Seu ; Cho, Yong-Joon ; Cho, Sung-Woo ; Kwon, Oh-Shin ; Choi, Soo-Young ;
BMB Reports , volume 33, issue 4, 2000, Pages 317~320
The succinic semialdehyde dehydrogenase from bovine brain was inactivated by treatment with phenylglyoxal, a reagent that specifically modifies arginine residues. The inhibition at various phenylglyoxal concentrations shows pseudo-first-order kinetics with an apparent secondorder rate constant of 30
for inactivation. Partial protection against inactivation was provided by the coenzyme
, but not by the substrate succinic semialdehyde. Spectrophotometric studies indicated that complete inactivation of the enzyme resulted from the binding of 2 mol phenylglyoxal per mol of enzyme. These results suggest that essential arginine residues, located at or near the coenzyme-binding site, are connected with the catalytic activity of brain succinic semialdehyde dehydrogenase.
Reduction of Azobenzene by Purified Bovine Liver Quinone Reductase
Kim, Kyung-Soon ; Shin, Hae-Yong ;
BMB Reports , volume 33, issue 4, 2000, Pages 321~325
Quinone reductase was purified to homogeneity from bovine liver by using ammonium sulfate fractionation, ionexchange chromatography, and gel filtration chromatography. The enzyme utilized either NADH or NADPH as the electron donor. The enzyme catalyzed the reduction of several quinones and other artificial electron acceptors. Furthermore, the enzyme catalyzed NAD(P)H-dependent reduction of azobenzene. The apparent Km for 1,4-benzoquinone and azobenzene was 1.64 mM and 0.524 mM, respectively. The reduction of azobenzene by quinone reductase was almost entirely inhibited by dicumarol or Cibacron blue 3GA, potent inhibitors of the mammalian quinone reductase. In the presence of 1.0
Cibacron blue 3GA, azoreductase activity was lowered by 45%, and almost complete inhibition was seen above 2.0
Cibacron blue 3GA.
Purification and Characterization of Acidic Chitinases from Gizzards of Broiler (Gallus gallus L.)
Han, Beom-Ku ; Moon, Jong-Kook ; Ryu, Yeon-Woo ; Park, Yun-Hee ; Jo, Do-Hyun ;
BMB Reports , volume 33, issue 4, 2000, Pages 326~331
Acidic chitinases from the gizzards of a broiler were purified to homogeneity, using precipitation with
, ion exchanger chromatography, gel filtration, chromatofocusing and hydrophobic interaction chromatography. The enzymes, GAC1 and GAC2, were purified 180- and 194- folds with a recovery of 4.9% and 2.7%, respectively. The molecular mass of GAC1 and GAC2 were 48.2 kDa and 57.8 kDa, respectively. Chromatofocusing resulted in a pI of 3.1 for both enzymes. The purified enzymes were endochitinases that were devoid of
and lysozyme activity. Kinetic studies using
indicate that GAC1 has a
of 1.97 mg/ml and 185 mg/mg protein/h, respectively. The GAC2 has a
of 0.42 mg/ml and 92.3 mg/mg protein/h, respectively at optimal pH and temperature (pH 5.0 and
). When the pentamer and hexamer of N-acetylglucosamine (GlcNAc) were used as a substrate, the major product by GAC1 was the dimer of GlcNAc with a differential accumulation of the monomer and trimer, depending upon the substrate. However, the GAC2 produced the dimer and trimer in an equal quantity, regardless of the substrate used. The first 9
amino acid residues of the purified gizzard chitinase GAC1 and GAC2 shared a 100% homology. The first 25
amino acid residues of GAC1 also shared 55-60% homology with animal chitinases and some animal proteins, such as whey protein and oviduct-specific proteins. However, little homology was found with either microbial and plant chitinases, or egg white lysozyme.
Phenazine 1-carboxylic acid resistance in phenazine 1-carboxylic acid producing Bacillus sp. B-6
Kim, Kyoung-Ja ;
BMB Reports , volume 33, issue 4, 2000, Pages 332~336
Phenazine 1-carboxylic acid (PCA) is an antifungal antibiotic isolated from a culture filtrate of Bacillus sp. B-6 producing an acyl CoA synthetase inhibitor. This antibiotic is reported as an inhibitor of an acyl CoA synthetase from Pseudomonas sp.. Bacillus sp. B-6 was resistant to PCA up to 350
. We investigated the mechanism of the resistance of Bacillus sp. B-6 to PCA. The rate of growth in a medium containing up to 100
was as rapid as the PCA-free medium. At a PCA concentration of 300
, the growth rate was more than half that of the control. In this work, we purified acyl CoA synthetase from Bacillus sp. B-6 and found that this acyl CoA synthetase was much less sensitive to PCA than the acyl CoA synthetase from other source. These findings suggested that the insensitivity of Bacillus sp. B-6 acyl CoA synthetase plays an important role in the PCA resistance of this bacterium.
Expression of Human Immunodeficiency Virus Type 1 Tat Proteins in Escherichia coli and Application to Study Tat Functions
Park, Jin-Seu ; Lee, Han-Gyu ; Lee, Yoon ; Kang, Young-Hee ; Rhim, Hyang-Shuk ; Choi, Soo-Young ;
BMB Reports , volume 33, issue 4, 2000, Pages 337~343
The human immunodeficiency virus type 1 (HIV-1), transactivator of transcription (Tat), is one of the viral gene products that is essential for HIV-1 replication. The HIV-l Tat protein regulates transcription from an HIV-1 long terminal repeat (LTR) and affects the gene expression of cellular proteins during infection. In order to develop an expression system to overexpress and simply purify HIV-1 Tat proteins, the HIV-1 Tat coding sequences that contain one or two exons were amplified using PCR and cloned into a pET vector, which contains a consecutive stretch of six histidine residues at the amino-terminus. The reconstituted vectors were overexpressed in the E. coli strain and the soluble recombinant proteins were purified to be homogeneity in a single step by
acid Sepharose chromatography under nondenaturing conditions. Recombinant HIV-1 Tat proteins were shown to transactivate the HIV-1 LTR promoter in a dose-dependent manner when introduced into mammalian cells. In addition, treatment of human endothelial cells with purified Tat proteins resulted in a significant increase in the level of vascular cell adhesion molecule-1 (VCAM-1) expression. These results indicate that the recombinant HIV-1 Tat proteins are active in transactivating viral and cellular promoters. The expression and purification system described in this study will facilitate in characterizing the biological functions of the Tat proteins.
Catalase, Glutathione S-Transferase and Thioltransferase Respond Differently to Oxidative Stress in Schizosaccharomyces pombe
Cho, Young-Wook ; Park, Eun-Hee ; Lim, Chang-Jin ;
BMB Reports , volume 33, issue 4, 2000, Pages 344~348
The logarithmically growing Schizosaccharomyces pombe cells were subjected to high heat (
), hydrogen peroxide, and heavy metals such as mercuric chloride and cadmium chloride. Then, the stress responses of catalase, glutathione S-transferase and thioltransferase were investigated. The high heat and cadmium chloride enhanced the catalase activity. The glutathione S-transferase activity of S. pombe cells was increased after treatments with heavy metals. The thioltransferase activity of S. pombe cells was completely abolished by mercuric chloride. Hydrogen peroxide caused no effect on the activities of glutathione S-transferase and thioltransferase. These results suggest that the response of S. pombe cells against oxidative stress is very complicated.
Tyrosine Phosphorylation of Paxillin during Cell Adhesion
Chang, Jong-Soo ; Lee, Hong-Mie ; Min, Do-Sik ;
BMB Reports , volume 33, issue 4, 2000, Pages 349~352
Proteins that are involved in cellular signal cascade experience phosphorylation and dephosphorylation cycles in their tyrosine residue(s) during cell adhesion. In order to identify the protein(s), which tyrosine desidues are specifically phosphorylated when the cells attached to the substrate, we compared the tyrosine phosphorylation level of proteins between suspension and adhered culture condition in rat fibroblast 3Yl cells. We found that a cluster of 70 kDa protein was specifically phosphorylated when the cells adhered to the substrate, but did not effect the cells held in suspension. The phosphorylated protein is identified as paxillin, a focal adhesion protein in immunoprecipitation and immunobloting analysis. These results suggest that the tyrosine phosphorylation of paxillin may play a role in cell-substrate adhesion.
Bioluminescent Assay of
-Oxidase from Cucumis sativus using Bacterial Luciferase-Coupled Reaction
Cho, Ki-Woong ;
BMB Reports , volume 33, issue 4, 2000, Pages 353~357
A new assay method of
(fatty acid : oxygen dioxygenase, 1-decarboxylating) was developed using a bioluminescence reaction system of marine luminous bacterium, Photobacterium phosphoreum.
-Oxidase was isolated from a cucumber (Cucumis sativus). Pentadecanoic acid was used as a substrate, and the product, tetradecanal, was analyzed with a bacterial luciferase-coupled reaction. Initial light intensity was directly related to the concentration of tetradecanal in the range of 1 nM to 10
. Optimal pH and temperature were 7.5 and
, respectively. Optimal pentadecanoic acid concentration in a standard assay of
-oxidase was 0.1 mM. The Km value of pentedecanoic acid was
. This method is straightforward, rapid, convenient, and easy. Its needs no treatment or extraction of reaction mixture.