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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 29, Issue 6 - Nov 1996
Volume 29, Issue 5 - Sep 1996
Volume 29, Issue 4 - Jul 1996
Volume 29, Issue 3 - May 1996
Volume 29, Issue 2 - Mar 1996
Volume 29, Issue 1 - Jan 1996
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Specific Recognition of Unusual DNA Structures by Small Molecules: An Equilibrium Binding Study
Suh, Dong-Chul ;
BMB Reports , volume 29, issue 1, 1996, Pages 1~10
The binding interaction of ethidium to a series of synthetic deoxyoligonucleotides containing a B-Z junction between left-handed Z-DNA and right-handed B-DNA, was studied. The series of deoxyoligonucleotides was designed so as to vary a dinucleotide step immediately adjacent to a B-Z junction region. Ethidium binds to the right-handed DNA forms and hybrid B-Z forms which contain a B-Z junction, in a highly cooperative manner. In a series of deoxyoligonucleotides, the binding affinity of ethidium with DNA forms which were initially hybrid B-Z forms shows over an order of magnitude higher than that with any other DNA forms, which were entirely in B-form DNA The cooperativity of binding isotherms were described by an allosteric binding model and by a neighbor exclusion model. The binding data were statistically compared for two models. The conformation of allosterically converted DNA forms under binding with ethidium is found to be different from that of the initial B-form DNA as examined by CD spectra. The ratio of the binding constant was interestingly correlated to the free energy of base unstacking and the conformational conversion of the dinucleotide. The more the base stacking of the dinucleotide is unstable, or the harder the conversion of B to A conformation, the higher the ratio of the binding constant of ethidium with the allosterically converted DNA forms and with the initial B-Z hybrid forms. DNA sequence around a B-Z junction region affects the binding affinity of ethidium. The results in this study demonstrate that ethidium could preferentially interact with unusual DNA structures.
Identification of Phosphatidylcholine-Phospholipase D and Activation Mechanisms in Rabbit Kidney Proximal Tubule Cells
Chung, Jin-Ho ; Chae, Joo-Byung ; Chung, Sung-Hyun ;
BMB Reports , volume 29, issue 1, 1996, Pages 11~16
The present study showed that receptor-mediated activation of rabbit kidney proximal tubule cells by angiotensin II, the
ionophore A23187, or the protein kinase C activator phorbol myristate acetate (PMA) all stimulated phospholipase D (PLD). This was demonstrated by the increased formation of phosphatidic acid, and in the presence of 0.5% ethanol, phosphatidylethanol (PEt) accumulation. Angiotensin II leads to a rapid increase in phosphatidic acid and diacylglycerol, and phosphatidic acid formation preceeded the formation of diacylglycerol. This result suggests that some phosphatidic acid seems to be formed directly from phosphatidylcholine hydrolyzed by Pill. On the other hand, EGTA substantially attenuated angiotensin II and A23187-induced PEt formation, and when the cells were pretreated with verapamil angiotensin II-induced Pill activation was completely abolished. These results provide the evidence that calcium ion influx is essential for the agonist-induced Pill activation. In addition, staurosporine, an inhibitor of protein kinase C, strongly inhibited PMA-induced PEt formation, but was ineffective on angiotensin II-induced PEt accumulation.
also stimulates PEt formation in digitonin-permeabilized cells, but pretreatment of the cells with pertussis toxin failed to suppress angiotensin II-induced PEt formation. From these results, we conclude that in the rabbit kidney proximal tubule cells the mechanisms of angiotensin II- and PMA-induced Pill activation are different from each other and mediated via a pertussis toxin-insensitive trimeric G protein.
Site-Directed Mutagenesis of Ile91 of Restriction Endonuclease EcoRV: Dramatic Consequences on the Activity and the Properties of the Enzyme
Moon, Byung-Jo ; Vipond, I. Barry ; Halford, Stephen E. ;
BMB Reports , volume 29, issue 1, 1996, Pages 17~21
Ile91 of restriction endonuclease EcoRV, which has not been known to take part directly in catalytic activity, was substituted with Leu by site-directed mutagenesis. The Ile91Leu mutant shows over 1000-fold less activity than the wild type EcoRV under standard reaction condition. The metal ion dependency of the reaction was altered. In contrast to the wild type EcoRV, the mutant prefers
as the cofactor. In
buffer the mutant is as active as the wild type enzyme in
buffer. Like the wild type enzyme, the mutant shows an unspecific binding of DNA in gel shift experiments. In contrast to the wild type enzyme, the mutant did not cleave at noncognate sites of DNA under star condition.
Caffeine Indirectly Activates Ca
-ATPases in the Vesicles of Cardiac Junctional Sarcoplasmic Reticulum
Kim, Young-Kee ; Cho, Hyoung-Jin ; Kim, Hae-Won ;
BMB Reports , volume 29, issue 1, 1996, Pages 22~26
Agents that activate or inhibit the
release channel in cardiac sarcoplasmic reticulum (SR) were tested for their abilities to affect the activity of the SR
-ATPase. Vesicles of junctional SR (heavy SR, HSR) from terminal cisternae were prepared from porcine cardiac muscle by density gradient centrifugation. The steady-state activity of
-ATPases in intact HSR vesicles was/
SD). When the HSR vesicles were made leaky, the activity was increased to
protein. This increase is probably due to the uncoupling of HSR vesicles. Caffeine (10 mM), an agonist of the SR
release channel, increased
-ATPase activity in the intact HSR vesicle preparation to
protein. However, caffeine had no significant effect in the leaky vesicle preparation and in the purified
-ATPase preparation. The effect of caffeine on SR
-ATPase was investigated at various concentrations of
. Caffeine increased the pump activity over the whole range of
, in the intact HSR vesicles. When the SR
-ATPase was inhibited by thapsigargin, no caffeine effect was observed. These results imply that the caffeine effect requires the intact vesicles and that the increase in
-ATPase activity is not due to a direct interaction of caffeine with the enzyme. We propose that the activity of SR
-ATPase is linked indirectly to the activity of the
release channel (ryanodine receptor) and may depend upon the amount of
released by the channels.
M Phase-Specific Phosphorylation of DNA Topoisomerase IIα in HeLa Cells
Bae, Young-Seuk ; Lee, Sook-Ja ; Kwak, Sang-Soo ;
BMB Reports , volume 29, issue 1, 1996, Pages 27~31
Using topoisomerase II (topo II) isozyme-specific antibodies, we investigated the phosphorylation of topo
in mitotic HeLa cells. Topo
was specifically modified in the mitotic cells, resulting in slow migration on SDS-polyacrylamide gel electrophoresis. To characterize the nature of this modification, we treated the nuclear extracts prepared from the mitotic cells with alkaline phosphatase. After the treatment with alkaline phosphatase, the slowly migrated band disappeared and instead a normal (170 kDa) topo
band appeared. These results indicate that human topo
is modified at a specific site(s) in M phase by phosphorylation, supporting the possibility that M phase-specific phosphorylation of topo II is critical for mitotic chromosome condensation and segregation.
Spectral Studies of Conformational Change at the Active Site of Mutant O-acetylserine Sulfhydrylase-A (C43S)
Park, Joon-Bum ; Kim, Sung-Kun ; Yoon, Moon-Young ;
BMB Reports , volume 29, issue 1, 1996, Pages 32~37
The cysteine 43, potentially important in the activity of O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium, has been changed to serine. This mutant enzyme (C43S) has been studied in order to gain insight into the structural basis for the binding of inhibitor, substrate and product. UV-visible spectra of C43S exhibit the same spectral change in the presence of OAS as that observed with wild type enzyme, indicating C43S will form an
-aminoacrylate Schiff base intermediate. At pH 6.5, however, the deacetylase activity of C43S is much higher than wild type enzyme indicating that cysteine 43 plays a role in stabilizing the
-aminoacrylate intermediate. The fluoroscence spectrum of C43S exhibits a ratio of emission at 340 to 502 nm of 16.9, reflecting the lower fluorescence of PLP and indicating that the orientation of cofactor and tryptophan are different from that of the wild type enzyme. The emission spectrum of C43S in the presence of OAS gives two maxima at 340 and 535 nm. The 535 nm emission is attributed to the fluoroscence of the
-aminoacrylate intermediate. The visible circular dichroic spectrum was similar to wild type enzyme, but the negative effect observed at 530~550 nm and the molar ellipicity values for the mutant are decreased by about 50% compared to wild type enzyme. The circular dichroic and fluoroscence studies suggest binding of the cofactor is less asymmetric in C43S than in the wild type enzyme.
Use of Molecular Replacement to Determine the Phases of Crystal Structure of Taq DNA Polymerase
Kim, Young-Soo ; Suh, Se-Won ;
BMB Reports , volume 29, issue 1, 1996, Pages 38~44
Taq DNA polymerase from Thermus aquaticus has been shown to be very useful in the polymerase chain reaction method, which is being used for amplifying DNA. Not only does Taq DNA polymerase have high commercial value commercial value for the polymerase chain reaction application, but it is also important in studying DNA replication, because it is apparently an homologue to E. coli DNA polymerase I, which has long been used for DNA replication study (Lawyer et ai., 1993). The crystal structure determination of Taq DNA polymerase was initiated. An X-ray diffraction pattern breaks down a crystal structure into discrete sine waves in a Fourier series. The original shape of a crystal object in terms of electron density may be represented as the sum of those sine waves with varying amplitudes and phases in three dimensions. The molecular replacement method was initially employed to provide phase information for the structure of Taq DNA polymerase. The rotation search using the program MERLOT resulted in a solution peak with 5.4 r.m.s. PC-refinement of the X-PLOR program verified the result and also optimized the orientation angles. Next, the translation search using the X-PLOR program resulted in a unique solution peak with 7.35 r.m.s. In addition, the translation search indicated
to be the true space group out of two possible ones. The phase information from the molecular replacement was useful in the MIR phasing experiment.
DNA Polymorphism Analysis of the HLA-DRB1 Gene Using Polymerase Chain Reaction-Sequence Specific Primer (PCR-SSP) among Korean Subjects
Lee, Kyung-Ok ; Park, Taek-Kyu ; Park, Young-Suk ; Oh, Moon-Ju ; Kim, Yoon-Jung ;
BMB Reports , volume 29, issue 1, 1996, Pages 45~51
Most expressed HLA loci exhibit a remarkable degree of allelic polymorphism, which derives from sequence differences predominantly localized to discrete hypervariable regions of the amino-terminal domain of the molecule. In this study, the HLA-DRB1 genotypes were determined in eighteen control cell lines and 112 unrelated Koreans using the PCR-SSP (Polymerase Chain Reaction-Sequence Specific Primer) technique. 29 specific primer pairs in assigning the DRB1 gene were used. The results of control cells correlated well with the data which was previously reported. The heterozygosity and homozygosity of the DRB1 gene were 0.786 and 0.214, respectively. In a total of 41 different DRB1 alleles and 83 genotypes, the most frequent allele and genotype were DRB1*04 and DRB1*0901/1501, respectively. This study shows that the PCR-SSP technique is relatively simple, fast and a practical tool for the determination of the HLA-DRBI genotypes. Moreover, these results-allele and genotype frequency and heterozygosity of the HLA DRB1 gene-could be useful for database study before being applied to individual identification and transplantation immunity.
Isolation, Restriction Mapping, and Promoter Sequence Analysis of an Isoperoxidase Gene from Korean-Radish, Raphanus sativus L.
Park, Jong-Hoon ; Kim, Soung-Soo ;
BMB Reports , volume 29, issue 1, 1996, Pages 52~57
A specific DNA fragment from Korean radish (Raphanus sativus L.) was amplified by performing PCR with oligonucleotide primers which correspond to the highly conserved regions of plant peroxidases. The size of the PCR product was ca. 400 bp, as expected from the known plant peroxidase genes. Comparison of the nucleotide and deduced amino acid sequences of the PCR product to those of other plant peroxidase-encoding genes revealed that the amplified fragment corresponded to the highly conserved region I and III of plant peroxidases. By screening a genomic library of Korean radish using the amplified fragment as a probe, two positive clones, named prxK1 and prxK2, were isolated. Restriction mapping studies indicated that the 5.2 kb Sail fragment of the prxK1 clone and the 4.0 kb EcoRI fragment of the prxK2 clone encode separate isoperoxidase genes. Analyses of the promoter region of the prxK1 clone shows that putative CAAT box, CMT box, and TGA1b binding sequence (5' TGACGT) are present 718 bp upstream from the start codon.
Structural Changes of the Spinach Photosystem II Reaction Center After Inactivation by Heat Treatment
Jang, Won-Cheoul ; Tae, Gun-Sik ;
BMB Reports , volume 29, issue 1, 1996, Pages 58~62
The structural changes in the electron donor side of the PSII reaction center have been monitored since heat treatment (
for 5 min) of thylakoids is known to decrease the oxygen evolving activity. In heat-treated spinach chloroplast thylakoids, the inhibitory effect of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on the electron transport activity of the PSII reaction center from diphenyl carbazide to dichlorophenolindophenol became reduced approximately 3.8 times and [
]-labeled DCMU binding on the D1 polypeptide decreased to 25~30% that of intact thylakoid membranes, implying that the conformational changes of the DCMU binding pocket, residing on the D1 polypeptide, occur by heat treatment. The accessibility of trypsin to the
-terminus of the cytochrome b-559
-subunit, assayed with Western blot using an antibody generated against the synthetic peptide (Arg-68 to Arg-80) of the COOH-terminal domain, was also increased, indicating that heat-treatment caused changes in the structural environments near the stromal side of the cytochrome b-559
-subunit, allowing trypsin more easily to cleave the
-terminal domain. Therefore, the structural changes in the electron donor side of the PSII reaction center complexes could be one of the reasons why the oxygen evolving activity of the heat-treated thylakoid membranes decreased.
Molecular Mechanism of R1162 Plasmid Incompatibility Exerted by Direct Repeat in the Replicative Origin
Kim, Yung-Jin ;
BMB Reports , volume 29, issue 1, 1996, Pages 63~67
In order to elucidate the molecular mechanism of plasmid incompatibility of broad host-range plasmid R1162, the plasmid-encoded replication protein RepIB was purified and tested for binding to the 20 bp direct repeat (DR) DNA sequence which is reiterated 3 and 1/2 times within the replicative origin of the plasmid. The RepIB protein specifically binds to the DR DNA. Point mutations in the DR which affect expression of plasmid incompatibility also coordinately affect binding. These results indicate that the incompatibility of broad host-range plasmid R1162 is exerted by the DR DNA by titrating the essential replication protein RepIB.
Increased Expression of c-jun in the Bile Acid-Induced Apoptosis in Mouse F9 Teratocarcinoma Stem Cells
Baek, Jin-Hyen ; Kang, Chang-Mo ; Chung, Hae-Young ; Park, Myung-Hwan ; Kim, Kyu-Won ;
BMB Reports , volume 29, issue 1, 1996, Pages 68~72
Ursodeoxycholic acid (UDCA) and lithocholic acid (LCA), secondary bile acids, have been shown to have a cell differentiation activity in mouse F9 teratocarcinoma stem cells. Treatment with bile acids induced morphological changes, including cytoplasmic and nuclear membrane blebbing, aggregation of organelles, and chromatin condensation, corresponding to apoptosis. Moreover, the bile acids induced intemucleosomal DNA fragmentation, a hallmark of apoptosis. In addition, the expression of c-jun was increased, but that of c-myc and laminin was decreased during apoptosis induced by the bile acids in F9 cells. These results suggest that the bile acids can induce apoptosis in F9 cells. Furthermore, the c-jun expression may be related to the apoptosis induced by UDCA or LCA in F9 cells.
Choline-Lipid Release from Normal and Transformed Cells
Hong, Seong-Tshool ; Jang, Yong-Suk ; Park, Kie-In ;
BMB Reports , volume 29, issue 1, 1996, Pages 73~80
The effect of albumin on phosphatidylcholine (PC) metabolism in Hep-G2, 3T3-H.ras, and 3T3 cells pre-labelled with [Me-
]choline was studied. The [
]choline was more efficiently taken up and incorporated into cellular phospholipids in 3T3-H.ras cells than in Hep-G2 and 3T3 cells. In each of the three cell lines, most of the [
]choline metabolized into the phospholipids was incorporated into PC and only minor was incorporated into lysophosphatidylcholine (LPC). Bovine serum albumin stimulated the release of [
]LPC and [
]PC from each of the three cell lines pre-labelled with [
]PC was also released in the absence of albumin but [
]LPC was not. The efficiency of LPC secretion represented as the proportion of medium [
]LPC to cellular [
]choline lipid during a chase period is approximately 9 to 14 times greater in 3T3 cells compared with the transformed 3T3-H.ras and Hep-G2 cells. A similar comparison of published data for rat hepatocytes with Hep-G2 shows secretion to be 35~75 times greater from the rat hepatocytes than from Hep-G2. Also, PC secretion from 3T3 cells was 1.6 times more effective than from 3T3-H.ras, whereas rat hepatocytes secrete PC 2.8~3.8 times more effectively than does Hep-G2. The measurement of specific radioactivity of cellular PC in pre-labelled 3T3 cells showed it to be similar to that of the secreted PC. However, the specific radioactivity of secreted LPC was markedly lower than that of the cellular PC, which suggests that LPC is being secreted from a PC pool distinct from that used for PC secretion.
S-Thiolation and Oxidation of Glycogen Phosphorylase b and Peroxidation of Liposome Initiated by Free Radical Species
Lee, Kyu-Sun ; Lee, Hyung-Min ; Park, Young-Mee ; Chang, Byeong-Doo ; Chung, Tae-Young ; Choi, Eun-Mi ;
BMB Reports , volume 29, issue 1, 1996, Pages 81~87
The relationship of S-thiolation and oxidation of glycogen phosphorylase b and peroxidation of phosphatidyl choline liposome by xanthine oxidase (XOD), 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH), and 2,2'-azobis(dimethylvaleronitrile) (AMVN)-generated free radicals was investigated, Glycogen phosphorylase b was S-thiolated in the presence of glutathione and oxidized in the absence of it by XOD, AAPH and AMVN. In XOD-initiated reaction, the rates of S-thiolation and oxidation of phosphorylase were very similar and addition of liposome to the reaction mixture showed little inhibition of the modifications. In AAPH-initiated reaction, the rate of oxidation was higher than that of S-thiolation and addition of liposome increased oxidation of the protein but had no effect on S-thiolation. In AMVN-initiated reaction, S-thiolation was higher than oxidation and addition of liposome increased S-thiolation remarkably but showed no effect on oxidation. The effect of liposome on modifications of protein in AAPH and AMVN reaction seemed to be caused by certain reactive degradation products or intermediates of liposome by free radical attack. Peroxidation of liposome was not observed in XOD-initiated reaction. Liposome was gradually peroxidized by AAPH reaction. The peroxidation was inhibited by addition of GSH and phosphorylase. Peroxidation of liposome by AMVN was extreamly fast, and was not affected by GSH and phosphorylase.
Fnr, NarL and NarP Regulation and Time Course Expression of Escherichia coli aeg-46.5 Gene
Ahn, Ju-Hyuk ; Choe, Mu-Hyeon ;
BMB Reports , volume 29, issue 1, 1996, Pages 88~91
The anaerobically expressed gene aeg-46.5, which had been identified by the operon fusion technique with a hybrid bacteriophage of
placMu53, was studied for its expression pattern and growth. The expression of aeg-46.5 was studied in the wild-type cell and mutant cells that have mutation (s) in the control gene of anaerobic respiration (fnr) and nitrate response (narL and narP). The
-galactosidase reporter gene showed maximum expression in narL host after two hours of aerobic to anaerobic switch in M9-Glc-nitrate medium. Both 40 mM and 100 mM concentrations of nitrate ion in the medium had little effect on expression level. We propose that aeg-46.5 is subject to multiple regulations of anaerobic activation by Fnr, nitrate activation by NarP and repression mediated by NarL.
Site-Directed Mutation Effect of the Symmetry Region at the mRNA 5'-end of Escherichia coli aeg-46.5 Gene
Ahn, Ju-Hyuk ; Choe, Mu-Hyeon ;
BMB Reports , volume 29, issue 1, 1996, Pages 92~97
The age-46.5 gene of Escherichia coli is induced by nitrate ion and regulated by Fnr, NarL, and NarP during anaerobic growth. aeg-46.5::lacZ fusion gene shows its maximum expression in narL host after two hours of aerobic to anaerobic switch in M9-Glc-nitrate medium. Fnr and NarP act as positive regulators, and NarL acts as a negative regulator. The control region of the aeg-46.5 was identified and the binding sites of regulator proteins have been predicted (Reznikoff and Choe (1993)). It has two symmetry regions. One is located at -52~-37 bp from the anaerobic mRNA 5'-end, which is the binding site of NarL and NarP. The other is located at +37~+56 bp from the 5'-end of mRNA. In this study, the downstream symmetry region from the mRNA 5'-end was investigated by site-directed mutagenesis. The destruction of the symmetry region increases the expression level of aeg-46.5. We propose that the symmetry region interferes with the expression of aeg-46.5 possibly by forming a stem-and-loop structure.