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 31, Issue 6 - Nov 1998
Volume 31, Issue 5 - Sep 1998
Volume 31, Issue 4 - Jul 1998
Volume 31, Issue 3 - May 1998
Volume 31, Issue 2 - Mar 1998
Volume 31, Issue 1 - Jan 1998
Selecting the target year
Cytochrome c Peroxidase: A Model Heme Protein
Erman, James E. ; Vitello, Lidia B. ;
BMB Reports , volume 31, issue 4, 1998, Pages 307~327
Cytochrome c peroxidase (CcP) is a yeast mitochondrial enzyme which catalyzes the reduction of hydrogen peroxide to water using two equivalents of ferrocytochrome c. The CcP/cytochrome c system has many features which make it a very useful model for detailed investigation of heme protein structure/function relationships including activation of hydrogen peroxide, protein-protein interactions, and long-range electron transfer. Both CcP and cytochrome c are single heme, single subunit proteins of modest size. High-resolution crystallographic structures of both proteins, of one-to-one complexes of the two proteins, and a number of active-site mutants are available. Site-directed mutagenesis studies indicate that the distal histidine in CcP is primarily responsible for rapid utilization of hydrogen peroxide implying significantly different properties of the distal histidine in the peroxidases compared to the globins. CcP and cytochrome c bind to form a dynamic one-to-one complex. The binding is largely electrostatic in nature with a small, unfavorable enthalpy of binding and a large positive entropy change upon complex formation. The cytochrome c-binding site on CcP has been mapped in solution by measuring the binding affinities between cytochrome c and a number of CcP surface mutations. The binding site for cytochrome c in solution is consistent with the crystallographic structure of the one-to-one complex. Evidence for the involvement of a second, low-affinity cytochrome c-binding site on CcP in long-range electron transfer between the two proteins is reviewed.
Mechanism of Antibiotic Action and Biosynthesis of Centipedin Purified from Scolopendra subspinipes multilans L. Koch (Centipede)
Kim, Ki-Tae ; Hong, Sa-Weon ; Lee, Jong-Ho ; Park, Kyung-Bae ; Cho, Key-Seung ;
BMB Reports , volume 31, issue 4, 1998, Pages 328~332
The 8-hydroxyisocoumarin, named Centipedin, which has a significant antibiotic activity, was separated and solubilized with organic solvents, such as diethyl ether from centipede Scolopendra subspinipes multilans L. Koch. The Centipedin was purified by silicic acid column and high S cation exchange chromatography followed by reverse-phase HPLC. It was confirmed that Centipedin has a potent antibiotic effectiveness against Gram-negative Klebsiella pneumoniae ATCC 8308. The results showed that Centipedin blocks both DNA replication and RNA transcription during the growth of this pathogen in vivo. The biosynthesis of antibiotic 8-hydroxyisocoumarin was studied in vivo by feeding
compound as a precursor to live centipede, in which
was the most efficiently incorporated into the Centipedin within 30 h after injection. Also, in vitro study on the biosynthesis of Centipedin showed that efficient incorporation of
occurred at pH range 5.0-7.0 for 10 h incubation and decreased significantly after then. It is suggested that 8-hydroxyisocoumarin is one of the defense compounds acting on bacterial infection in Scolopendra subspinipes.
Ceramide-Mediated Cell Death Was Accompanied with Changes of c-Myc and Rb Protein
Moon, Soon-Ok ; Lee, Jin-Woo ;
BMB Reports , volume 31, issue 4, 1998, Pages 333~338
The sphingomyelin cycle and ceramide generation have been recognized as potential growth suppression signals in mammalian cells. Ceramide has been shown to induce differentiation, cell growth arrest, senescence, and apoptosis. Although the intracelluar target for the action of ceramide remains unknown, recent studies have demonstrated the role of cytosolic ceramideactivated protein phosphatase(CAPP). In this study, the cytotoxic effect of C2-ceramide, a synthetic cellpermeable ceramide analog, on HEp-2 cells and the mechanism by which ceramide induces cell death were investigated. The addition of exogenous C2-ceramide resulted in a concentration dependent cell death. Okadaic acid, a potent inhibitor of CAPP, enhanced ceramide-mediated cell death, which suggests that CAPP is not involved in this process. To understand the mechanism of action of ceramide, we studied the relationship between ceramide and c-Myc and pRb which are defined components of cell growth regulation. Western blot analyses revealed that C2-ceramide (10
) induced c-Myc down-regulation, but there were no significant changes in pRb. However, treatment of okadaic acid (10 nM) enhanced c-Myc and pRb down-regulation. Reduction of the amount of c-Myc and pRb occurred during HEp-2 cell death. These results suggest that the cytotoxic effect of ceramide in HEp-2 cells may not be mediated through the action of CAPP and that the downstream target for ceramide is c-Myc and pRb.
Growth and Telomerase Inhibition of SK-MEL 28 Melanoma Cell Line by a Plant Flavonoid, Apigenin
Kang, Sang-Sun ; Lim, Seung-Eun ;
BMB Reports , volume 31, issue 4, 1998, Pages 339~344
The plant flavonoids, including apigenin which is found in especially high concentrations in edible plants, are reported to protect against chronic diseases including several types of cancer. We observed that apigenin inhibited not only the growth of human melanoma cell line SK-MEL 28 but also the telomerase activity. We also noted the telomerase activity inhibition by genistein, a tyrosine kinase inhibitor found in soybean, which suggests that the telomerase activity of SK-MEL 28 cells may be regulated by the protein phosphorylation. Furthermore, we observed that apigenin induced SK-MEL 28 cell apoptosis with p53 upregulation. Taken together, our results indicate that apigenin plays a role as an antimelanoma component in edible plants.
Purification and Characterization of Aspartase from Hafnia alvei
Yoon, Moon-Young ; Park, Jae-Ho ; Choi, Kyong-Jae ; Kim, Joung-Mok ; Kim, Yeon-Ok ; Park, Jon-Bum ; Kyong, Jin-Burm ;
BMB Reports , volume 31, issue 4, 1998, Pages 345~349
Aspartase (EC 22.214.171.124) from Hafnia alvei was purified to homogeneity by a combination of DEAE-cellulose, Red A-agarose, and Sepharose 6B chromatography. The purified enzyme appeared homogeneous on denatured SDS-polyacrylamide gel electrophoresis. The purified enzyme was a tetrameric protein composed of identical subunits with a molecular weight of 55,000 daltons. The optimum pH for the enzymatic reaction was 8.5 and the optimum temperature for maximum activity was
. The enzyme has an absolute requirement of divalent metal ions (
) at the alkaline pH. The enzyme, however, was inactivated in the presence of other divalent cations such as
. The helical content of the purified enzyme was estimated by CD spectropolarimetry to be 61%.
Phosphorylation of a 66 kDa Protein, a Putative Protein Kinase C Substrate, is Related to Chondrogenesis of Chick Embryo Mesenchymes In Vitro
Lee, Sun-Ryung ; Sonn, Jong-Kyung ; Yoo, Byung-Je ; Lim, Young-Bin ; Kang, Shin-Sung ;
BMB Reports , volume 31, issue 4, 1998, Pages 350~354
To understand the role of protein kinase C (PKC) in the regulation of chondrogenesis, we examined proteins which are phosphorylated by PKC. Stage 23/24 chick embryo wing mesenchymes were micromass-cultured to induce chondrogenesis and cell extracts were phosphorylated in a condition that activates PKC. Several proteins including 63 and 66 kDa proteins were phosphorylated. The 66 kDa protein was phosphorylated only in the presence of phorbol 12-myristate 13-acetate (PMA) and phosphatidylserine CPS), and the phosphorylation was almost completely diminished by bisindolylmaleimide, a PKC inhibitor. In addition, partially purified PKC increased the phosphorylation of the 66 kDa protein. Treatment of cultures with lysophosphatidylcholine (LPC) promoted chondrogenesis and phosphorylation of 66 kDa protein, while PMA and thymeleatoxin inhibited both of the two events. Our results suggest that the 66 kDa protein is a putative substrate of PKC, and phosphorylation of the 66 kDa protein, probably by
is required for chondrogenesis.
Cytotoxicity of Anti-CD4 Antibody Activated
T-Lymphocytes against Herpesvirus-Infected Target Cells is Dependent on
Protein Tyrosine Kinase Activity
Choi, Sang-Hoon ; Jang, Yong-Suk ; Oh, Chan-Ho ;
BMB Reports , volume 31, issue 4, 1998, Pages 355~363
MHC unrestricted, antigen nonspecific killing by
T-cells against virally-infected target cells was induced following cross-linking of CD4 molecules. The cytotoxicity of antibody-activated
T-cells was abolished by genistein (4',5,7-trihydroxyisoflavone), a protein tyrosine kinase (PTK) inhibitor, but not by H-7, a protein kinase C (PKC) inhibitor. Genisteintreated human or bovine peripheral blood
T-cells lacked PTK activity and failed to kill virally-infected target cells even after cross-linking of CD4 molecules. The cross-linking of CD4 molecules did not induce effector cell proliferation or the transcription of TNF
synthesis was up-regulated by incubating antibody activated effector cells with bovine herpesvirus type 1 (BHV-1) infected D17 target cells. Anti-TNF
antibody partially abrogated direct effector cell-mediated antiviral cytotoxicity. On the other hand, this antibody effectively neutralized antiviral activity of effector and target cell culture supernatants against BHV-1 infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on effector and target cell ratio. These findings have importance to define the mechanisms of how CD4 cytotoxic cells control viral infection.
Purification and Characterization of a 25 kDa Cathepsin L-like Protease from the Hemocyte of Coleopteran Insect, Tenebrio molitor Larvae
Jang, Kyung-Suk ; Cho, Mi-Young ; Choi, Hye-Won ; Lee, Kang-Moon ; Kim, Mi-Hee ; Lee, Young-Un ; Kurata, Shoichiro ; Natori, Shunji ; Lee, Bok-Luel ;
BMB Reports , volume 31, issue 4, 1998, Pages 364~369
Insect plasma protein is abundant in the hemolymph of holometabolous insect larvae and is used as a source of amino acids and energy for construction of adult structures during metamorphosis. In order to understand the mechanism of decomposition of larval plasma proteins by hemocyte protease, we tried to purify a cysteine protease from the hemocyte lysate by using Carbobenzoxy-L-Phenylalanyl-L-Arginine-4-Methyl-Coumaryl-7-Amide (Z-Phe-Arg-MCA) as substrate and to identify plasma proteins that are selectively susceptible to the purified protease. Here, we describe the purification and characterization of a cysteine protease that specifically hydrolyzes the plasma protein of the coleopteran insect, Tenebrio molitor, larvae. The molecular mass of this enzyme was 25 kDa, as determined by SDS-PAGE under reducing conditions. The amino acids sequence of its
was determined to be Leu-Pro-Gly-Gln-Ile-Asp-Trp-Arg-Asp-Lys-Gly. This sequence contained Pro, Asp, and Arg residues, conserved in many papain superfamily enzymes. The specific cysteine protease inhibitors, such as E-64 and leupetin, inhibited its hydrolytic activity. One plasma protein with a molecular mass of 48 kDa was selectively hydrolyzed within 3 h when the purified enzyme and plasma proteins were incubated in vitro. However, the 48 kDa protein was not hydrolyzed by the purified 25 kDa protease in the presence of E-64. Western blotting analysis at various developmental stages showed that the purified enzyme was detected at larvae, pupae, and adult stages, but not the embryo stage.
Establishment of a Binding Assay System for Screening of the Inhibitors of
Kim, Jyn-Ho ; Hur, Eun-Mi ; Yun, Yung-Dae ;
BMB Reports , volume 31, issue 4, 1998, Pages 370~376
Src-Homology 2 (SH2) domains have a capacity to bind phosphotyrosine-containing sequence context and play essential roles in various cellular signaling pathways. Due to the specific nature of the binding between SH2 domains and their counterpart proteins, inhibitors of SID domain binding have drawn extensive attention as a potential candidate for therapeutic agents. Here, we describe the binding assay system to screen for the ligands or blockers of the SH2 domains with an emphasis on the
SH2 domain. In our assay system, SID domains expressed and purified as fusion proteins to Glutathione-S-transferase (GST) were covalently attached to 96-well microtitre plates through amide bond formation, which were subsequently allowed to bind the biotinylated phosphotyrosine (pY)containing synthetic pep tides. The binding of biotinylated pY peptides was detected by the horseradish peroxidase (HRP)-conjugated streptavidin. Using the various combinations of SH2 domain-pY peptides, we observed that: (1) The binding of pY-peptides to its counterpart SH2 domain is concentration-dependent and saturable; (2) The binding is highly specific for a particular combination of SH2 domain-pY peptide pair; and (3) The binding of Lck SH2-cognate pY-peptides is specifically competed by the nonbiotinylated peptides with expected relative affinity. These results indicate that the established assay system detects the SH2-pY peptide interaction with reproducible sensitivity and specificity and is suitable for screening the specific inhibitors of
Thioltransferase (Glutaredoxin) from Chinese Cabbage: Purification and Properties
Cho, Young-Wook ; Park, Eun-Hee ; Lim, Chang-Jin ;
BMB Reports , volume 31, issue 4, 1998, Pages 377~383
Thioltransferase, also known as glutaredoxin, was purified from Chinese cabbage (Brassica campestris ssp. napus var. pekinensis) by a combination of ion-exchange chromatography and gel filtration. Its purity was confirmed by SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be about 12,000 which is comparable with those of most known thioltransferases. The enzyme utilizes 2-hydroxyethyl disulfide, S-sulfocysteine,
, insulin, and trypsin as substrates in the presence of reduced glutathione. The enzyme has Km values of 0.03-0.97 mM for these substrates. It appeared to contain dehydroascorbate reductase activity. The pH optimum of the enzyme was 8.5, when 2-hydroxyethyl disulfide was used as a substrate. It was greatly activated by reduced glutathione. Its activity was not significantly lost when stored at high temperature, indicating its thermostable character. It may play an important role in thiol-disulfide exchange in plant cells.
Analysis of Double-Stranded DNA Fragments by Capillary Electrophoresis Using Entangle Polymer Solutions in Uncoated Fused Silica Capillary Columns
Lee, Jong-Jin ; Lee, Kong-Joo ;
BMB Reports , volume 31, issue 4, 1998, Pages 384~390
DNA fragments (51-587 bp) were separated by capillary electrophoresis using entangled polymer, hydroxyethylcellulose, in uncoated fused silica capillary columns. The factors affecting the separation of DNA fragments with hydroxyethylcellulose media were evaluated, i.e., the concentration of buffer and entangled polymer, effects of additives (methanol, ethidium bromide, EDTA), temperature, and injection methods. Maximum performance was obtained by adding 5% methanol in 0.5% hydroxyethylcellulose solution at
. Addition of methanol in polymer media increased the resolution of small size DNA fragments (< 100 bp). On the other hand, addition of ethidium bromide and EDTA, which are commonly used in conventional DNA separation, reduced the resolution of DNA fragments in the polymer solution. It turns out that the separation behavior of DNA in entangled polymer is more sensitive to the running condition compared to that in polyacrylamide gel-filled capillary, but the reproducibility of DNA separation in entangled polymer is reliable.
In Vitro Enhancement of Microsomal Cytochrome P450-Dependent Monooxygenases by Organic Solvents in Rat Liver
Lee, Dong-Wook ; Lim, Heung-Bin ; Moon, Ja-Young ; Park, Ki-Hyun ;
BMB Reports , volume 31, issue 4, 1998, Pages 391~398
In vitro effects of acetone, methanol, and dimethylsulfoxide (DMSO) on liver microsomal cytochrome P450 (P450) content, and P450-dependent arylhydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase (ECOD) activities were studied in rats. Acetone at 1% (v/v) enhanced the content ofP450, assayed spectrally in 3-methylcholanethrene (MC)- and
(BNF)-inducible microsomes by 18 and 7%, respectively. Methanol, up to 5% (v/v) applied, also showed enhancement effects on P450 content in liver microsomes from rats treated with phenobarbital (PB), MC, and BNF, as well as uninduced microsomes with similar but low strength. DMSO, however, did not show such enhancing effects at the ranges of the concentrations applied. AHH and ECOD activities in MC-inducible microsomes were also enhanced by acetone at 1%, which was in proportion to the increase in P450 content by the same concentration. However, the P450 content, and AHH and ECOD activities, were decreased by increasing the concentration of acetone. Methanol at the same concentration with acetone also enhanced ECOD activity but not AHH activity in MCinducible microsomes. The enhancing effect of acetone on the enzymes was negligible when the microsomes were pretreated with a specific monoclonal antibody of MC-inducible isozyme. The difference in the effects of these solvents on P450 system might be due to their different properties that cause the P450 active site to be exposed in milieu.
Active-Site Mutants of Human Glutathione S-Transferase P1-1: Effects of the Mutations on Substrate Specificity and Inhibition Characteristics
Park, Hee-Joong ; Yoon, Suck-Young ; Kong, Kwang-Hoon ;
BMB Reports , volume 31, issue 4, 1998, Pages 399~404
In order to gain further insight on the relationship between structure and function of glutathione S-transferase (GST), the six active-site mutants, R13T, K44T, Q51A, Q64A, S65A, and D98A, of human GST P1-1 were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The active-site mutants showed marked differences in substrate specificity. The substitution of Gln51 with threonine resulted in a drastic decrease in the specific activities to <10% of the wild-type value. The substitution of Arg13 with threonine resulted in more decreased specific activity toward cumene hydroperoxide and in the
values of S-(2,4-dinitrophenyl) glutathione and benanstatin A. These results suggest that the substitution of Arg13 with threonine changes the conformation of the active site to increase the affinity for the product or electrophilic substrate. Lys44 seems to be in the vicinity of the H-site of hGST P1-1 or may contribute to some extents to the electrophile binding.
Comparison of Endonuclease-Sensitive Sites by T4 Endonuclease V and UvrABC Nuclease Treatments Followed by Formamide or Sodium Hydroxide Denaturation
Chang, Yung-Jin ;
BMB Reports , volume 31, issue 4, 1998, Pages 405~408
Endonuclease-sensitive sites detected by T4 endonuclease V or UvrABC nuclease treatments were compared in the dihydrofolate reductase gene of UV-irradiated Chinese hamster ovary B-11 cells. The number of endonuclease-sensitive sites detected by T4 endonuclease V treatment followed by NaOH denaturation was twice that of formamide denaturation. Repeated treatment of damaged genomic DNA with T4 endonuclease V resulted in no further increase in the number of endonuclease-sensitive sites detected. The numbers of endonuclease-sensitive sites detected by UvrABC nuclease using each denaturation condition were similar. Sequential treatment with the two endonucleases using formamide denaturation resulted in twice the number of endonuclease-sensitive sites detected by treatment of each nuclease alone. Due to a lack of AP endonuclease activity these results suggest the presence of T4 endonuclease V-sensitive sites which could be complemented by alkaline gel separation or by UvrABC nuclease treatment.
Existence of "25 kDa Thiol Peroxidase" in Retina: Evidence for An Antioxidative Role
Cha, Mee-Kyung ; Kim, Il-Han ;
BMB Reports , volume 31, issue 4, 1998, Pages 409~412
We isolated and sequenced a human retina cDNA fragment that encodes 25 kDa thiol peroxidase. A search of a databank showed that the 25 kDa thiol peroxidase from retina is the same type of thiol peroxidase which exists in human brain and red blood cells. This type of tbiol peroxidase was distributed in all of the tested tissues including retina. This result suggests a physiological role for the 25 kDa thiol peroxidase as an important antioxidant.
Expression of a Bovine
-Casein/Human Lysozyme Fusion Gene in the Mammary Gland of Transgenic Mice
Lee, Woon-Kyu ; Kim, Sun-Jung ; Hong, Seung-Beom ; Lee, Tae-Hoon ; Han, Yong-Mahn ; Yoo, Ook-Joon ; Im, Kyung-Soon ; Lee, Kyung-Kwang ;
BMB Reports , volume 31, issue 4, 1998, Pages 413~417
Transgenic mice containing a bovine
lysozyme fusion gene (pBZ) were generated in order to produce human lysozyme in their milk. The expression vector was a quadripartite fusion consisting of a 2 kb upstream DNA of the bovine
gene, human lysozyme gene, intron II of the rabbit
gene, and the polyadenylation/termination signals of SV40 DNA. Fertilized mouse zygotes were microinjected with pBZ, then transferred into the oviduct of foster mothers. Out of 20 mice born, 11 survived until postweaning and three were identified as positivetransgenic by Southern blot analysis (one male and two females). The founder mice were mated to BCFl mice to produce transgenic progeny. It was confirmed by RT-PCR and Northern blot analyses that the transgene was specifically expressed in the mammary gland of the founder mice. Furthermore, the artificial introns within the transgenic RNA was proven to be correctly spliced out as judged by RT-PCR analysis. These results indicated that transgenic mice generated in this study properly expressed the human lysozyme RNA in their mammary gland.