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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 28, Issue 6 - Nov 1995
Volume 28, Issue 5 - Sep 1995
Volume 28, Issue 4 - Jul 1995
Volume 28, Issue 3 - May 1995
Volume 28, Issue 2 - Mar 1995
Volume 28, Issue 1 - Jan 1995
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Structural Damage of DNA by 6-Sulfooxymethyl Benzo(a)pyrene
Cho, Young-Sik ; Chung, An-Sik ;
BMB Reports , volume 28, issue 1, 1995, Pages 1~5
The effect of 6-sulfooxymethyl benzo(a)pyrene (SMBP) on conformational changes of calf thymus DNA was investigated. As SMBP is a strong electrophile, the covalent binding of SMBP to DNA should distort three dimensional conformation of DNA at the binding sites. A formaldehyde-unwinding methods were used to determine the rate of DNA denaturation. The increase in absorbance at 251nm was detected by addition of formaldehyde following treatment with SMBP. SMBP changed supercoiled DNA to relaxed and linear DNA as determined by electrophoresis, which was similar to the change in DNA due to in vitro treatment with benzo(a) pyrene diol epoxide. Treatment with SMBP completely denatured DNA under alkaline conditions. However, DNA was nicked or partially denatured under neutral condition. The absorption band of DNA was increased by the treatment with SMBP in V79 cells, which may be explained by the formation of stabilized SMBP-DNA adduct.
Purification and Characterization of a New Galactoside Specific Lectin from Trichosanthes kirilowii Root
Yun, Doo-Hee ; Park, Eun-Ju ; Park, Jong-Ok ; Lee, Young-Han ; Seo, Jeong-Kon ; Ryu, Sung-Ho ; Suh, Pann-Ghill ; Kim, Hee-Sook ;
BMB Reports , volume 28, issue 1, 1995, Pages 6~11
A new lectin, named TRA, was purified from Trichosanthes kirilowii root by acid-treated Sepharose 6B, Mono-Q, and TSK-gel 3000SW column sequential chromatography. The lectin appeared homogeneous by native gel electrophoresis at pH 4.3 and gave two protein bands of Mr=31 and 28 kDa by SDS-PAGE. The N-terminal amino acid sequences of the polypeptides of TRA have not been reported in amino acid sequences of the lectins. TRA lectin formed a precipitate with asialofetuin, neuraminidase-treated fetuin. A sugar inhibition assay indicated that N-acetyl-D-galactosamine, among the monosaccharides tested, was the most potent inhibitor of TRA-induced hemagglutination. Asialofetuin showed a 260-times stronger inhibitory activity than N-acetyl-D-galactosamine. TRA lectin also showed agglutination with normal leukocytes and lymphoma cells, but not with premature hemopoietic cells. These results suggest that TRA is a novel plant lectin.
Determination of Branched-Chain α-Keto Acid Dehydrogenase Activity in Rat Tissues
Kim, Hyun-Sook ; Johnson, Wayne A. ;
BMB Reports , volume 28, issue 1, 1995, Pages 12~16
-keto acid dehydrogenase (BCKAD) complex is a rate limiting enzyme which catalyzes the oxidative decarboxylation of branched-chain
-keto acids. Numerous studies have suggested that BCKAD is subject to covalent modification in vitro via phosphorylation and dephosphorylation, which are catalyzed by a specific kinase and phosphatase, respectively. The biggest difficulty in the assay of BCKAD activity is to arrest the interconversion between the active and inactive forms. BCKAD activity was determined from fresh rat heart and liver tissues using homogenizing and assay buffers containing inhibitors of phosphatase and kinase. The results suggest that a radiochemical assay using
]-isovalerate as a substrate for the enzyme can be applied as a reliable method to determine in vitro enzyme activity with arrested interconversion between the active and inactive forms of the BCKAD complex.
Direct Reduction of DTNB by E. coli Thioredoxin Reductase
Lim, Hye-Won ; Lim, Chang-Jin ;
BMB Reports , volume 28, issue 1, 1995, Pages 17~20
Thioredoxin reductase is a flavoprotein oxidoreductase catalyzing the reduction of a cystine disulfide in thioredoxin. Thioredoxin, in turn, can reduce disulfide bonds in other proteins and serves as a reducing agent in enzymatic reactions such as those of ribonucleotide reductase and methionine sulfoxide reductase. In this work thioredoxin reductase was found to directly reduce DTNB in the absence of thioredoxin. This new reactivity of E. coli thioredoxin reductase was produced by relatively high concentrations of univalent cations such as
, and it appeared with the oxidation of NADPH. These results indicate that E. coli thioredoxin reductase may be slightly modified by univalent cations, and the modified enzyme directly reacts with DTNB. This DTNB-reducing activity offers a new assay method for E. coli thioredoxin reductase.
Transcriptional Regulation of Escherichia coli serC-aroA Operon : Further Support for cAMP-Dependent Expression
Sa, Jae-Hoon ; Park, Soo-Sun ; Lim, Chang-Jin ;
BMB Reports , volume 28, issue 1, 1995, Pages 21~26
The Escherichia coli mixed-function serC-aroA operon encodes biosynthethic enzymes for unrelated pathways leading to the syntheses of serine and aromatic amino acids. It has been proposed that the operon is expressed in a cAMP-dependent manner. In this work experiments were performed to investigate the cAMP-dependent expression of the operon. Exogenous cAMP increased
-galactosidase synthesis in the
and cya strains harboring the serC-aroA-lac fusion plasmid. This enhancement was more dramatic in the
strain grown in a minimal medium. In a dot blot assay the serC-aroA mRNA content increased in a concentration-dependent pattern after the addition of exogenous cAMP. The activity of phosphoserine aminotransferase, encoded by the serC gene, apparently increased in E. coli cells after the addition of cAMP. All results obtained confirmed that the expression of the E. coli serC-aroA operon is positively regulated by cAMP at the level of transcription.
Production of Egg Yolk Antibody (IgY) Against Human Placental DNA-Dependent RNA Polymerase II
Lee, Yoon-Ik ; Surzycki, Stefan S. ; Lee, Young-Ik ;
BMB Reports , volume 28, issue 1, 1995, Pages 27~32
Polyclonal antibodies against human DNA-dependent RNA polymerase II (HPP II) were generated from chicken egg yolk after immunization with RNA polymerase II as an antigen. The antibodies from egg yolk (IgY) were purified and characterized. IgY showed a specificity against DNA-dependent RNA polymerase II, and was a polyclonal antibody against 12 subunits of polymerase II. An amount of 0.35 mg of IgY was obtained freman HPP II-Sepharose affinity column using 10 eggs from a chicken immunized against RNA polymerase II as an antigen. These antibodies can be used for isolating the genes for RNA polymerase II components, and for in vitro transcription assays using HP-RNA polymerase II.
Purification of Progelatinase A (Matrix Metalloproteinase 2) and a Tissue Inhibitor of Metalloproteinase-2(TIMP-2) from T98G Human Glioblastoma Cells
Lee, Ho-Jae ; Chung, Myung-Chul ; Lee, Choong-Hwan ; Chun, Hyo-Kon ; Kho, Yung-Hee ;
BMB Reports , volume 28, issue 1, 1995, Pages 33~39
The Gelatinases (typeIV collagenases) are metalloproteinases that may play an important role in tumor invasion and metastasis. Progelatinase A was purified from a conditioned medium of T98G human glioblastoma cells. TIMP-2 complexed progelatinase A and free progelatinase A were separated by heparin affinity HPLC. The final product was homogeneous on SDS-PAGE, with a molecular weight of 64,000 daltons without reduction. TIMP-2 and free progelatinase A were separated from TIMP-2 complexed progelatinase A by reverse-phase HPLC in the presence of trifluoroacetic acid. TIMP-2 complexed progelatinase A was resistant to activation by p-aminophenyl mercuric acetate (APMA), and showed less than 20% of the activity of the TIMP-2 free active enzyme. TIMP-2 free progelatinase A was easily activated to the mature form with a molecular weight of 57,000 daltons by APMA and showed high activity compared to the TIMP-2 complexed enzyme.
Chemical Modification of Serratia marcescens Acetolactate Synthase with Cys, Trp, and Arg Modifying Reagents
Choi, Ho-Il ; Kim, Soung-Soo ;
BMB Reports , volume 28, issue 1, 1995, Pages 40~45
Acetolactate synthase purified from Serratia marcescens ATCC 25419 was rapidly inactivated by the thiol specific reagent p-chloromercuribenzoate (PCMB), the tryptophan specific reagent N-bromosuccinimide (NBS), and the arginine modifying reagent phenylglyoxal (PGO). Inactivation by PCMB was prevented by both
-ketobutyrate and pyruvate, and the second order rate constant for the inactivation was
. The reaction order with respect to PCMB was 0.94. The inactivation of the enzyme by NBS was also substantially reduced by both
-ketobutyrate and pyruvate. The second order rate constant for inactivation by NBS was
, and the reaction order was 2.0. On the other hand, inactivation by PGO was partially prevented by
-ketobutyrate, but not by pyruvate. The second order rate constant for the inactivation was
and the order of reaction with respect to PGO was 0.75. These results suggest that essential cysteine, tryptophan and arginine are located at or near the substrate binding site.
Size Heterogeneity of Murine Tumor Necrosis Factors Induced from Mouse Peritoneal Macrophages
Baik, Na-Gyoung ; Jeong, Jee-Yeong ; Kim, Soung-Soo ;
BMB Reports , volume 28, issue 1, 1995, Pages 46~50
Three kinds of mouse tumor necrosis factor (TNF), which have molecular weights of 35 kDa, 45 kDa, and 18 kDa on SDS-PAGE, were partially purified from serum-free culture supernatants of mouse peritoneal macrophages induced with lipopolysaccharide. Analysis of the native molecular weights by gel filtration indicated that the 18 kDa and 45 kDa TNFs aggregate into 50 kDa and 100 kDa molecules, respectively, while the 35 kDa TNF is contained in high molecular weight aggregates of approximately 200 kDa. The three kinds of cytotoxic factors all elicited tumor reducing responses.
Construction and Characterization of Escherichia coli-Corynebacterium nephridii Hybrid Thioredoxins
Sa, Jae-Hoon ; Kim, Kyung-Hoon ; Lim, Chang-Jin ;
BMB Reports , volume 28, issue 1, 1995, Pages 51~56
Thioredoxin is a small redox protein with an active-site disulfide/dithiol, and is ubiquitous in bacteria, plants, and animals. To investigate the structure-function relationship of thioredoxin, the genes encoding Escherichia coli thioredoxin and Corynebacterium nephridii thioredoxin C3 were fused via a common restriction site in the nucleotide sequence coding for the active site of the proteins to generate two chimeric thioredoxins, designated E-C3(N to C-terminal) and C3-E. The hybrid thioredoxin genes were put under the T7 promoter and their productions were confirmed. The two hybrid thioredoxins complemented phenotypes of a thioredoxin-deficient E. coli strain. A strain containing the C3-E hybrid thioredoxin supported growth of the T7 phage, whereas a strain expressing the E-C3 hybrid thioredoxin did not. However, both hybrids supported growth of M13 phages. The two hybrid thioredoxins were also characterized in other aspects. Differences in activity between the hybrid thioredoxins were attributed to altered interactions of the N- and C-terminal domains of the molecule, which produced changes in the three-dimensional structure of the active site region.
Heterologous Introns Enhanced Expression of Human Lactoferrin cDNA in Mouse Mammary Epithelial Cells
Kim, Sun-Jung ; Yu, Dae-Yeul ; Lee, Ko-Woon ; Cho, Yong-Yeon ; Lee, Chul-Sang ; Han, Yong-Mahn ; Lee, Kyung-Kwang ;
BMB Reports , volume 28, issue 1, 1995, Pages 57~61
The expression of a recombinant human lactoferrin is reported in mouse HC11 mammary epithelial cells. Expression of human lactoferrin (hLF) was achieved by placing its cDNA under the control of the bovine
-casein gene. To improve the hLF expression level in a cell culture system, two artificial introns were also introduced to construct expression vectors. One intron was a hybrid-splice signal consisting of bovine
-casein intron 1 and rabbit
-globin intron II. The other intron was a DNA fragment spanning intron 8 of the bovine
-casein gene. The hybrid intron moderately elevated hLF expression, whereas intron 8 alone did not express any detectable amount of hLF as judged by Northem and Western blot analyses. When the two introns were used together they contributed to a synergistic elevation of hLF expression. These data indicate that artificial introns on both sides of the hLF cDNA were necessary to increase expression of cDNA.
Purification and Characterization of Thioredoxin f from Pea Leaves
Kang, Han-Chul ; Hahn, Tae-Ryong ;
BMB Reports , volume 28, issue 1, 1995, Pages 62~67
Thioredoxin f from pea leaves was purified to homogeneity and characterized. The purification steps involved ammonium sulfate fractionation, heat treatment, Sephadex G-75 and G-50 gel filtration, and hydroxyapatite and DEAE ion exchange chromatography. The monomeric molecular weight of purified pea thioredoxin f determined by SDS polyacrylamide gel electrophoresis was 12,000. The purified protein was active in the presence of reducing agents, such as dithiothreitol, at an alkaline pH (7.8~8.5). It was stable against heat such that more than 40% of its maximum activity remained after treatment at
for 10 min. Pea thioredoxin f was able to reduce insulin and was specific only to pea chloroplast fructose-1,6-bisphosphatase.
Synthesis of Peptides by Bovine Gastricsin
Yoon, Joo-Ok ; Kim, Young-Jun ;
BMB Reports , volume 28, issue 1, 1995, Pages 68~71
Bovine gastricsin catalyzes peptide synthesis over an optimum range of pH 4~5, resulting in satisfactory yields of methyl esters and p-nitroanilides of benzyloxycarbonyl tetra- to hexa-peptides, provided that hydrophobic amino acid residues form new peptide bonds. The effectiveness of the enzyme also depends on the nature of adjacent amino acid residues. An aspartic proteinase with a characteristic gastricsin specificity pattern would be useful for the synthesis of middle-length peptides.
Affinity Labeling of E. coli GTP Cyclohydrolase I by a Dialdehyde Derivative of Guanosine Triphosphate
Ahn, Chi-Young ; Park, Sang-Ick ; Kim, Ju-Myeong ; Yim, Jeong-Bin ;
BMB Reports , volume 28, issue 1, 1995, Pages 72~78
Time-dependent inactivation of E. coli GTP cyclohydrolase I with a 2',3'-dialdehyde derivative of GTP (oGTP) was directed to the active site of the enzyme, and was dependent on the concentration of oGTP. The kinetics of inactivation were biphasic with a rapid reaction occurring immediately upon exposure of the enzyme to oGTP followed by a slow rate of inactivation. The
value of oGTP for the enzyme was 0.25 mM. Inactivation was prevented by preincubation of the enzyme with GTP, the substrate of the enzyme. At 100% inactivation, 2.3 mol of [8.5'-$^3H$]oGTP were bound per each enzyme subunit, which consists of two identical polypeptides. The active site residue which reacted with the affinity label was lysine. oGTP interacted selectively with the
-amino group of lysine in the GTP-binding site to form a morpholine-like structure which was stable without sodium borohydride treatment. However, triphosphate group was eliminated during the hydrolysis step. To identify the active site of the enzyme, [8.5'-
]oGTP-labeled enzyme was cleaved by endoproteinase Lys-C, and the
-labeled peptide was purified by HPLC. The amino acid sequence of the active site peptide was Pro-Ser-Leu-Ser-Lys, which corresponds to the aminoterminal sequence of the enzyme.
An Additional Mechanism for the Cytotoxicity of 2-Chloroethylethyl Sulfide in Spleen Lymphocytes; Lysosomal Labilization
Choi, Dae-Sung ; Shin, Sung-Ho ; Kim, Yun-Bae ; Cha, Seung-Hee ; Sok, Dai-Eun ;
BMB Reports , volume 28, issue 1, 1995, Pages 79~82
Exposure of spleen lymphocytes to 2-chloroethylethyl sulfide (CEES) leads to a reduction of the intracellular ATP level, followed by a decrease in cell viability. Addition of nicotinamide, an inhibitor of poly(ADP-ribose) polymerase (PADPRP), restores both ATP level and viability, indicating that an activation of PADPRP is responsible for the cytotoxicity of CEES. The involvement of a
-mediated process in cytotoxicity is suggested. Verapamil, EGTA, trifluoperazine, and butacaine exhibit a partial protection (20 to 58%) against the cytotoxicity of CEES. Investigation of the causative role of proteolytic degradation in cell death indicate that pepstatin and leupeptin exert a substantial protective effect (60 to 70%), suggesting the involvement of lysosomal destabilization in CEES-induced cytotoxicity. Also, lysosomotropic agents markedly decrease the cytotoxicity. Lysosomal labilization may be a mechanism for the cytotoxicity of CEES.
The Novel Synthetic Substance MR-387C[(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl-L-prolyl-L-leucine] as an Aminopeptidase M Inhibitor
Chung, Myung-Chul ; Chun, Hyo-Kon ; Lee, Ho-Jae ; Kho, Yung-Hee ;
BMB Reports , volume 28, issue 1, 1995, Pages 83~86
In the course of screening for new aminopeptidase M inhibitors which were expected to be analgesic, immunopotentiating, or anti-metastatic agents, the novel synthetic substance MR-387C[(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl-L-prolyl-L-leucine] (M.W. 504 daltons) was obtained. It was competitive with the substrate and had an
) and an inhibition constant (
. This novel MR-387C was compared with various known inhibitors of aminopeptidase M. It inhibited the enzyme more strongly than any other microorganism-originated inhibitor, except probestin.