Go to the main menu
Skip to content
Go to bottom
REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
> Journal Vol & Issue
Journal Basic Information
Journal DOI :
Korean Society for Biochemistry and Molecular Biology
Editor in Chief :
Volume & Issues
Volume 32, Issue 6 - Nov 1999
Volume 32, Issue 5 - Sep 1999
Volume 32, Issue 4 - Jul 1999
Volume 32, Issue 3 - May 1999
Volume 32, Issue 2 - Mar 1999
Volume 32, Issue 1 - Jan 1999
Selecting the target year
In vitro Folding of Recombinant Hepatitis B Virus X-Protein Produced in Escherichia coli: Formation of Folding Intermediates
Kim, Sun-Ok ; Sohn, Mi-Jin ; Jeong, Soon-Seog ; Shin, Jeh-Hoon ; Lee, Young-Ik ;
BMB Reports , volume 32, issue 6, 1999, Pages 521~528
The folding of recombinant hepatitis B virus X-protein (rHBx) solubilized from Escherichia coli inclusion bodies was investigated. By sequential dialysis of urea, rHBx was folded into its native structure, which was demonstrated by the efficacy of its transcriptional activation of the adenovirus major late promoter (MLP), fluorescence spectroscopy, and circular dichroism (CD) analysis. The decrease in CD values at 220 nm and a corresponding blue shift of the intrinsic fluorescence emission confirmed the ability of rHBx to refold in lower concentrations of urea, yielding the active protein. Equilibrium and kinetic studies of the refolding of rHBx were carried out by tryptophan fluorescence measurements. From the biphasic nature of the fluorescence curves, the existence of stable intermediate states in the renaturation process was inferred. Reverse phase-high performance liquid chromatography (RP-HPLC) analysis further demonstrated the existence of these intermediates and their apparent compactness.
Asymmetric Polymerase Chain Reaction-Single-Strand Conformation Polymorphism (Asymmetric PCR-SSCP) as a Simple Method for Allele Typing of HLA-DRB
Kang, Joo-Hyun ; Kim, Kyeong-Hee ; Maeng, Cheol-Young ; Kim, Kil-Lyong ;
BMB Reports , volume 32, issue 6, 1999, Pages 529~534
Asymmetric PCR and single-strand conformation polymorphism (SSCP) methods were combined to analyze human leukocyte antigen (HLA)-DRB allele polymorphism. Asymmetric PCR amplification was applied to generate single-stranded DNA (ssDNA) using the nonradioactive oligonucleotide primers desinged for the polymorphic exon 2 region. The conformational differences of ssDNAs, depending on the allele type, were analyzed by nondenaturing polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. The ssDNAs were clearly separated from double-stranded DNA without interference and obviously migrated depending on their allele type. This method was applied to the genomic DNA either from homozygous or from heterozygous cell lines containing the DR4 allele as template DNA using DR4-specific primers, and satisfying results were obtained. Compared to the standard PCR-SSCP method, this asymmetric PCR-SSCP method has advantages of increased speed, reproducibility, and convenience. Along with PCR-SSP or sequence-based typing, this method will be useful in routine typing of HLA-DRB allele.
A Second Thioltransferase of Schizosaccharomyces pombe Contains Glutathione S-transferase Activity
Kim, Hong-Gyum ; Park, Eun-Hee ; Lim, Chang-Jin ;
BMB Reports , volume 32, issue 6, 1999, Pages 535~540
Two types of the thioltransferase (also called glutaredoxin) have been previously detected in the cytosolic extract of Schizosaccharomyces pombe, a fission yeast. Previously, the one with a smaller molecular mass (14kDa) was purified and characterized. In the present study, the second thioltransferase was purified. The purification procedure included ammonium sulfate fractionation (40-80%), Sephadex G-200 gel filtration, DEAE-cellulose ion-exchange chromatography, Sephadex G-50 gel filtration, and glutathione-agarose affinity chromatography. The purified enzyme showed a single band on SDS-PAGE, and its molecular mass was determined to be 23 kDa. It utilizes various compounds as substrates, including 2-hydroxyethyl disulfide. Interestingly, we found that the purified thioltransferase also contains significant glutathione S-transferase activity.
Biochemical Properties of a Chitin-Binding Class III Chitinase in Pumpkin Leaves
Lee, Kyun-Oh ; Kim, Min-Gab ; Jang, Ho-Hee ; Lee, Ji-Yeun ; Kim, Sun-Chang ; Lee, Sang-Yeol ;
BMB Reports , volume 32, issue 6, 1999, Pages 541~546
When we compared the chitinase activity of various plant sources using colorimetric or active gel-staining assay methods, the specific activity of pumpkin leaves was the highest among the samples we analyzed. The highly active chitinase from pumpkin leaves (designated PL-ChtIII) was purified to homogeneity using affinity chitin gel and HPLC Mono-Q anion-exchange cloumn chromatographies. In contrast to other members of the class III chitinase family, PL-ChtIII showed a strong binding affinity to the regenerated chitin gel column. The apparent molecular weight of PL-ChtIII was estimated to be 29 kDa on SDS-PAGE gel, while its optimum pH and temperature were shown to be pH 6.0 and
, respectively. Analyzing the reaction products of PL-ChtIII with swollen chitin as substrate, the dimer and tetramer of N-acetylglucosamine were produced as major products in the first hour of the enzymatic reaction along with a small amount of monomers and trimers. As the reaction time increased, dimeric N-acetylglucosamine became the predominant form of reaction product.
Methionine Analogue Probes Functionally Important Residues in Active Site of Methionyl-tRNA Synthetase
Jo, Yeong-Joon ; Lee, Sang-Won ; Jo, Myung-Kyun ; Lee, Jee-Woo ; Kang, Mee-Kyoung ; Yoon, Jeong-Hyeok ; Kim, Sung-Hoon ;
BMB Reports , volume 32, issue 6, 1999, Pages 547~553
Aminoacyl-tRNA synthetases are essential enzymes catalyzing the attachment of specific amino acids to cognate tRNAs. In the present work, the substrate analogue L-methionine hydroxamate was used to identify functional residues located in the active site of the E. coli methionyl-tRNA synthetase (MetRS). This compound inhibited bacteria, yeast, and human MetRS activities to a similar degree, suggesting a conserved active site structure and mechanism between MetRSs of different phylogenetic domains. Mutants of the E. coli MetRS resistant to methionine hydroxamate were also isolated. These mutants contained a substitution either at T10, Y15, or Y94. These residues are highly conserved among the different MetRSs and the mutants showed decreased aminoacylation activity, suggesting their functional and structural significances. The putative roles of these residues are discussed on a structural basis.
Gene-Specific Repair of 6-4 Photoproducts in Trichothiodystrophy Cells
Nathan, Sheila ; Van Hoffen, Anneke ; Mullenders, Leon H.F. ; Mayne, Lynne V. ;
BMB Reports , volume 32, issue 6, 1999, Pages 554~560
TTD1BI cells are non-hypersensitive to UV irradiation and perform normal genome repair of pyrimidine dimers but fail to excise 6-4 photoproducts and, concomitantly, are unable to restore RNA synthesis levels following UV irradiation. This pointed to a detect in gene-specific repair and this study was undertaken to examine repair of 6-4 photoproducts at the gene-level. The results indicated a defect in gene-specific repair of 6-4 photoproducts in active genes, although strand-specificity of 6-4 photoproduct removal was essentially similar to that of normal cells. These findings indicate that the near normal UV resistance of TTD1BI cells may be due to the inability of these cells to remove DNA lesions preferentially, as well as to the cells opting out of the cell cycle to repair damage before resuming replication.
Effects of the Hinge Region of Cecropin A(1-8)-Melittin 2(1-12), a Synthetic Antimicrobial Peptide on Antibacterial, Antitumor, and Vesicle-Disrupting Activity
Shin, Song-Yub ; Kang, Joo-Hyun ; Jang, So-Yun ; Kim, KiI-Lyong ; Hahm, Kyung-Soo ;
BMB Reports , volume 32, issue 6, 1999, Pages 561~566
CA(1-8)-ME(1-12) [CA-ME], composed of cecropin A(1-8) and melittin(1-12), is a synthetic antimicrobial peptide having potent antibacterial and antitumor activities with minimal hemolytic activity. In order to investigate the effects of the flexible hinge sequence, Gly-Ile-Gly, of CA-ME on antibiotic activity, CA-ME and three analogues, CA-ME1, CA-ME2, and CA-ME3, were synthesized. The Gly-Ile-Gly sequence of Ca-ME was deleted in CA-ME1 and replaced with Pro and Gly-Pro-Gly in CA-ME2 and CA-ME3, respectively. CA-ME1 and CA-ME3 showed a significant decrease in antitumor activity and phospholipid vesicle-disrupting ability. However, CA-ME2 showed similar antitumor and vesicle-disrupting activities, as compared with CA-ME. These results suggest that the flexibility or
-turn induced by Gly-Ile-Gly or Pro in the central part of CA-ME may be important in the electrostatic interaction of the N-terminus cationic
-helical region with the cell membrane surface and the hydrophobic interaction of the C-terminus amphipathic
-helical region with the hydrophobic acyl chains in the cell membrane. CA-ME3 exhibited lower antitumor and vesicle-disrupting activities than CA-ME and CA-ME2. This result suggests that the excessive
-turn structure caused by the Gly-Pro-Gly sequence in CA-ME3 seems to interrupt ion channel/pore formation in the lipid bilayer. We concluded that the appropriate flexibility or bilayer. We concluded that the appropriate flexibility or
-turn structure provided by the central hinge is responsible for the effective antibiotic activity of the antimicrobial peptides with the helix-hinge-helix structure.
A Study of the Anticoagulatory DNA from the Earthworm, Lumbricus rubellus, and its Regulatory DNA-Binding Protein
Kim, Gyoung-Mi ; Yu, Kyoung-Hee ; Woo, Jeong-Im ; Bahk, Yun-Kyoung ; Paik, Seung R. ; Kim, Jung-Gyu ; Chang, Chung-Soon ;
BMB Reports , volume 32, issue 6, 1999, Pages 567~572
We have previously shown that a DNA fragment is responsible for the anticoagulatory effect of an earthworm, Lumbricus rubellus. The anticoagluant increased the activated partial thromboplastin time (APTT) and also inhibited the thrombin activity observed with either N-
-p-tosyl-L-arginine methyl ester (TAME) or H-D-phenyl-alanyl-L-pipecoil-L-arginine-p-nitroanilide (S-2238). Since trypsin digestion of the anticoagulant further increased the APTT, the possible presence of a regulatory protein for the anticoagulatory DNA was investigated by digesting the anticoagulant with trypsin and isolating the DNA fragment with C4-reversed phase HPLC. The DNA fragment lacking a regulatory protein was eluted in the flow-through fraction, and analyzed with thrombin and activated factor X. Activated factor X activity was more strongly inhibited than thrombin activity. For DNA digestion, we treated the anticoagulant with DNase and purified the DNA-binding protein with a FPLC Resource-S cation exchange column. The regulatory protein, with an
of 55.0 kDa, reduced the anticoagulatory effect of the DNA fragment.
Properties of Trypsin-Mediated Activation of Aspartase from Hafnia alvei
Lee, Min-Sub ; Choi, Kyoung-Jae ; Kwom, Si-Joong ; Kang, In-Sug ; Ha, Joo-Hun ; Kim, Sung-Soo ; Han, Myung-Soo ; Yoon, Moon-Young ;
BMB Reports , volume 32, issue 6, 1999, Pages 573~578
Treatment of Hafnia alvei aspartase with limited tryptic digestion resulted in a marked increase in enzymatic activity. The activation required a few minutes to attain maximum level and, thereafter, the activity gradually decreased to complete inactivation. The degree of cleavage associated with the activation was extremely small as judged by SDS-PAGE. Upon activation, the optimum pH and temperature were essentially unchanged. When trypsin-activated enzyme was denatured in 4 M guanidine-HCI followed by removal of the denaturant by dilution, the restoration of activity was similar (40%) to that of the native enzyme, indicating a degree of stability. The
obtained on the acidic side and the
obtained on the basic side of trypsin-activated aspartase were 6.6 and 8.6, respectively, the same as those of the native aspartase, indicating that aspartase may exist in a stable conformation after limited tryptic digestion. These results indicate that the activation of H. alvei may be mediated by a conformational change away from the active site of individual subunits.
Purification and Characterization of a Novel Serine Protease with Fibrinolytic Activity from Tenodera sinensis (Chinese Mantis) Egg Cases
Cho, So-Yean ; Hahn, Bum-Soo ; Kim, Yeong-Shik ;
BMB Reports , volume 32, issue 6, 1999, Pages 579~584
Mantis egg fibrolase (MEF-3) was purified from the egg cases of Tenodera sinensis using ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, DEAE Affi-Gel blue gel affinity chromatogragphy, and MONO-Q anion-exchange chromatography. This protease had a molecular weight of 35,600 Da as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions and its isoelectric point was 6.0. The N-terminal amino acids sequence was Ala-Thr-Gln-Asp-Asp-Ala-Pro-Pro-Gly-Leu-Ala-Arg-Arg. This sequence was 80% homologous to the serine protease from Tritirachium album. MEF-3 readily digested the
-chains of fibrinogen and more slowly the
-chains. It showed strong proteolytic and fibrinolytic activities. Phenylmethanesulfonyl fluoride and chymostatin inhibited its proteolytic activity, while EDTA, EGTA, cysteine,
-mercaptoethanol, elastinal, tosyl-lysine chloromethylketone, and tosyl-amido-2-phenylethyl chloromethyl ketone did not affect its proteolytic activity. Among the chromogenic protease substrates, the most sensitive one to the hydrolysis of MEF-3 was benzoyl-Phe-Val-Arg-p-nitroanilide. Based on these experimental results, we speculated that MEF-3 is a serine protease with a strong fibrin(ogen)olytic activity.
Antiproliferative Effect of Artemisia argyi Extract against J774A.1 Cells and Subcellular Superoxide Dismutase (SOD) Activity Changes
Lee, Tea-Eun ; Park, Sie-Won ; Min, Tae-Jin ;
BMB Reports , volume 32, issue 6, 1999, Pages 585~593
The water and methanol extracts of Artemisia argyi showed significant cytotoxicities against J774A.1 cells but not so much against normal leukocytes. The cytotoxicities were found to be dependent on the extract concentration and the incubation time. The concentration of water and methanol extracts inhibiting 50% of cell proliferation (
) were estimated to be 44.2 mg/ml and 71.6 mg/ml, respectively. In the presence of Artemisia argyi water extract, total superoxide dismutase (CuZnSOD and MnSOD) activities of media, cytoplasmic and mitochondrial fractions of J774A.1 cells increased in accordance with cytotoxicity. MnSOD was found to be the main component of enhanced total SOD activities, particulary in the mitochondrial fraction. In contrast to SOD, catalase and glutathione peroxidase (GPx) were not found in any instance of the current investigation. In addition, substantial amount of
appeared to be generated in the mitochondrial fraction under the influence of Artemisia argyi. All data put together, it is postulated that Artemisia argyi extracts seem to stimulate
generation in mitochondria of J774A.1 cells with concomitant increases of SODs. Since
, the reaction product of SOD on
, is known to be readily converted to very toxic
in the absence of catalase and/or GPx cooperation, toxicity derived from ROS such as
may be the main cause of necrosis and/or apoptosis of J774A.1 cells.
Quantitative Assay for the Binding of Jun-Fos Dimer and Activator Protein-1 Site
Lee, Sang-Kyou ; Park, Se-Yeon ; Jun, Gyo ; Hahm, Eun-Ryeong ; Lee, Dug-Keun ; Yang, Chul-Hak ;
BMB Reports , volume 32, issue 6, 1999, Pages 594~598
The Jun and Fos families of eukaryotic transcription factors form heterodimers capable of binding to their cognate DNA enhancer elements. We are interested in searching for inhibitors or antagonists of the binding of the Jun-Fos heterodimer to the activator protein-1 (AP-1) site. The basic-region leucine zipper (bZIP) domain of c-Fos was expressed as a fusion protein with glutathione S-transferase, and allowed to form a heterodimer with the bZIP domain of c-Jun. The heterodimer was bound to glutathione-agarose, to which were added radiolabeled AP-1 nucleotides. After thorough washing, the gel-bound radioactivity was counted. The assay is faster than the coventional electrophoretic mobility shift assay because the gel electrophoresis step and the autoradiography step are eliminated. Moreover, the assay is very sensitive, allowing the detection of picomolar quantities of nucleotides, and is not affected by up to 50% dimethylsulfoxide, a solvent for hydrophobic inhibitors. Curcumin and dihydroguaiaretic acid, recently known inhibitors of Jun-Fos-DNA complex formation, were applied to this Jun-GST-fused Fos system and revealed to decrease the dimer-DNA binding.
Biochemical Properties of Second Site Mutation of Human Immunodeficiency Virus Integrase
Kim, Do-Jin ; Oh, You-Take ; Lee, Sang-Kwang ; Shin, Cha-Gyun ;
BMB Reports , volume 32, issue 6, 1999, Pages 599~604
A highly conserved amino acid, glutamic acid (Glu), present at position 152 in the catalytic domain of the human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein has been known to be critical for enzymatic function since substitution of Glu 152 with other residues results in a complete loss of enzymatic activities. In order to better understand the role of Glu 152 as a conserved residue in enzymatic action, intragenic second site mutations have been introduced around residue 152 of a mutant IN (E152A), and their biochemical properties were analyzed in terms of enzymatic activities. Disintegration activities were found to be significantly restored in several second site mutant INs, while integration activities were only recovered weakly. However, endonucleolytic activities were not discovered in all the mutant INs. These findings indicate that the second site mutations can partially restore that catalytic structure of the active site disturbed by the E152A mutation and lead to the regaining of integration and disintegration activities. In addition, it is also suggested that endonucleolytic activity requires a more accurate structure of the catalytic site than that for the integration and disintegration activities.
An L-Type Thioltransferase from Arabidopsis thaliana Leaves
Kim, Tae-Soo ; Cho, Young-Wook ; Kim, Joon-Chul ; Jin, Chang-Duck ; Han, Tae-Jin ; Park, Soo-Sun ; Lim, Chang-Jin ;
BMB Reports , volume 32, issue 6, 1999, Pages 605~609
Thioltransferase, also called glutaredoxin, is a general GSH-disulfide reductase of importance for redox regulation. Previously, the protein thioltransferase, now called S-type thioltransferase, was purified and characterized from Arabidopsis thaliana seed. In the present study, a second thioltransferase, called L-type thioltransferase, was purified to homogeneity from Arabidopsis thaliana leaves. The purification procedures included DEAE-cellulose ion-exchange chromatography, Sephadex G-50 gel filtration, and glutathione-agarose affinity chromatography. The purified enzyme was confirmed to show a unique band on SDS-PAGE and its molecular weight was estimated to be 26.6 kDa, which appeared to be atypical compared with those of most other thioltransferase. It could utilize 2-hydroxyethyl disulfide, S-sulfocysteine, and insulin as substrates, and also contained dehydroascorbate reductase activity. Its optimum pH was 8.5 and its activity was greatly activated by L-cysteine. When it was kept for 30 min, it appeared to be very stable up to
. It was activated by
and, on the contrary, inhibited by