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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
Selecting the target year
Properties of Acetyl-CoA Synthetase from Pseudomonas fluorescens
Kim, Yu-Sam ; An, Jae-Hyung ; Yang, Bu-Hyun ; Kim, Kyu-Wan ;
BMB Reports , volume 29, issue 4, 1996, Pages 277~285
In Pseudomonas fluorescens grown on malonate as sole carbon source, acetyl-CoA synthetase was induced, suggesting that malonate is metabolized through acetate and then acetyl-CoA. Acetyl-CoA synthetase was purified 18.6-fold in 4 steps to apparent homogeneity. The native molecular mass of the enzyme estimated by a native acrylamide gel electrophoresis was 130 kDa. The enzyme was composed of two identical subunits with a molecular mass of 67 kDa. Optimum pH was 70. The acetyl-CoA synthetase showed typical Michaelis-Menten kinetics for the substrates, acetate, ATP and CoA, whose
values were calculated to be 33.4, 74.8, and 40.7 mM respectively. Propionate. butyrate and pentanoate were also used as substrates by the enzyme, but the rate of the formation of the CoA derivatives was decreased in the order of the increase in carbon number. The enzyme was inhibited by the group-specific reagents diethylpyro-carbonate, 2,3-butanedione, pyridoxal-5'-phosphate and N-bromosuccinimide. In the presence of substrates the inactivation rate of the enzyme, by all of the group-specific reagents mentioned above decreased, indicating the presence of catalytically essential histidine, arginine, lysine and tryptophan residues at or near the active site. Preincubation of the enzyme with ATP,
resulted in the increase of its susceptibility to diethylpyrocarbonate, suggesting that ATP,
may induce a conformational change in the active site exposing the essential histidine residue to diethylpyrocarbonate. The enzyme was acetylated in the presence of acetyl-CoA, indicating that this is one of acyl-enzyme.
Analysis of the Stability of HLA-A2 Molecules Expressed on the Cell Surface
Lim, Jong-Seok ; Lee, Ki-Young ; Lee, Hee-Gu ; Kim, Ik-Hwan ; Lee, Chong-Kil ; Han, Seong-Sun ; Kim, Kil-Hyoun ;
BMB Reports , volume 29, issue 4, 1996, Pages 286~293
Association of antigenic peptide with class I MHC is believed to be crucial for maintaining stable conformation of class I molecules. T2 cells that are defective in TAP gene function mainly express class I molecules with an unstable conformation due to little or no association with antigenic peptides, whereas T1 cells that are normal in TAP gene function mainly express the stable form of class I molecules. In this work, attempts were made to determine the molecular stability of stable and unstable class I molecules. Dissociation of HLA-A2 molecules on T1 and T2 cells was monitored by flow cytometry using anti-HLA-A2 antibody after the cells were treated with brefeldin A to shut down the transport of newly-assembled HLA-A2. Estimated dissociation rate constants for the stable and unstable forms of HLA-A2 were 0.076
, respectively. It appeared that both T1 and T2 cells express stable and unstable class I complex, but with different ratios of the two forms. Furthermore,
treatment of T1 cells appeared to induce the expression of both the stable and unstable class I molecules. These results demonstrate that class I MHC molecules can be divided into two groups in terms of structural stability and that they exist on the cell surface in both forms in a certain ratio.
Purification and Characterization of Glyoxalase I from Chlamydomonas reinhardtii
Hwang, Sun-Jun ; Chai, Young-Gyu ;
BMB Reports , volume 29, issue 4, 1996, Pages 294~299
Glyoxalase I (Ee 184.108.40.206, lactoylglutathione lyase) from Chlamydomonas reinhardtii was purified to homogeneity by ammonium sulfate fractionation, anion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography on S-hexylglutathione agarose. The purified enzyme was judged to be homogeneous on SDS-PAGE, and consisted of a single polypeptide chain with a relative molecular weight of 24,000. The enzyme was most active at
and pH 7.5. It was catalytically most active with methylglyoxal as substrate. A number of properties of the Chlamydomonas glyoxalase I enzyme, such as substrate specificity, molecular mass, kinetic parameters, pi, metal ion effect, have been determined and compared with those reported for preparations from other sources. It had somewhat different characteristics from mammalian enzymes.
Modulation of the Cytochrome c Oxidase Activity by ATP: Implications for Mitochondrial Respiratory Control
Park, Nan-Hyang ; Chun, Sun-Bum ; Han, Tae-Young ; Han, Sang-Hwa ;
BMB Reports , volume 29, issue 4, 1996, Pages 300~307
ATP and ADP are potential regulators of mitochondtial respiration and at physiological concentrations they affect the rate of electron transfer between cytochrome c and cytochrome c oxidase. The electron transfer, however, depends on the electrostatic interaction between the two proteins. In order to exclude any nonspecific ionic effects by these polyvalent nucleotides, we used 2'-O-(2,4,6)trinitro(TNP)-derivatives of ATP and ADP which have three orders of magnitude higher affinity for cytochrome c oxidase. A simple titration of the fluorescence intensity of TNP by cytochrome c oxidase showed a binding stoichiometry of 2:1 cytochrome c:cytochrome c oxidase. Higher ionic strength was required for TNP-ATP than for TNP-ADP to be dissociated from cytochrome c oxidase, indicating that the negative charges on the phosphate group are at least partially responsible for the binding. In both spectrophotometric and polarographic assays, addition of ATP (and ADP to a less extent) showed an enhanced cytochrome c oxidase activity. Both electron paramagnetic resonance and fluorescence spectra indicate that there is no Significant change in the cytochrome c-cytochrome c oxidase interaction. Instead, reduction levels of the cytochromes at steadystate suggest that the increased activity of nucleotide-bound cytochrome c oxidase is due to faster electron transfer from cytochrome
, which is known to be the fate limiting step in the oxygen reduction by cytochrome c oxidase.
Increase in Linolenate Contents by Expression of the fad3 Gene in Transgenic Tobacco Plants
Kang, Young-Hwi ; Min, Bok-Kee ; Park, Hee-Sung ; Lim, Kyung-Jun ; Huh, Tae-Lin ; Lee, Se-Yong ;
BMB Reports , volume 29, issue 4, 1996, Pages 308~313
An 1.4 kb of the fad3 cDNA encoding microsomal linoleic acid desaturase catalyzing the conversion of linoleic acid (18:2,
) to linolenic acid (18:2,
) was introduced into tobacco plants by the Agrobacterium-mediated plant transformation, Among the transgenic tobacco plants conferring kanamycin resistance, five transformants showing increment in unsaturated fatty acid contents were selected and further analyzed for the transgenecity, In genomic Southern blot analyses, copy numbers of the integrated fad3 DNA in chromosomal DNA of the five transgenic tobacco plants were varied among the transgenic lines. By Northern blot analyses, the abundancy of the fad3 mRNA transcript directed by Cauliflower Mosaic Virus 35S promoter was consistent with the relative copy number of the fad3 DNA integrated in the chromosome of transgenic tobacco plants. When compared with the wild type, accumulation of linolenic acid in transgenic tobacco roots was elevated 3.7- to 4.7-fold showing a corresponding decrease in the linoleic acid contents; however, slight increments for linolenic acid were noticed in transgenic leaf tissues. These results indicated that the elevated level of fad3 expression is achieved in transgenic tobacco plants.
Effects of Non-Cytotoxic Concentration of Anticancer Drugs on Doxorubicin Cytotoxicity in Human Breast Cancer Cell Lines
Lee, Yoon-Ik ; Lee, Young-Ik ;
BMB Reports , volume 29, issue 4, 1996, Pages 314~320
The effects of non-cytotoxic concentrations of tamoxifen, verapamil, and trifluoperazine on doxorubicin cytotoxicity in five human breast cancer cell lines were studied. A non-cytotoxic concentration of tamoxifen resulted in enhanced doxorubicin cytotoxicity in HTB-123, HTB-26, and MCF-7. In these three cell lines, a combination of tamoxifen with verapamil resulted in even more increased doxorubicin cytotoxicity. Addition of verapamil or trifluoperazine alone did not influence the doxorubicin cytotoxicity significantly. Only in HTB-19 did coincubation with verapamil increase the doxorubicin cytotoxicity. In HTB-123, combination of tamoxifen with trifluoperazine increased the doxorubicin cytotoxicity significantly. In the cell lines where co-incubation with tamoxifen increased doxorubicin sensitivity, high estrogen receptor expression was detected. However, HTB-20, where tamoxifen did not enhance doxorubicin action, was also estrogen receptor positive. None of the cell lines had multidrug resistance related drug efflux and drug retention was not increased by the treatment with tamoxifen and verapamil. Cell cycle traverses were not altered by incubation with tamoxifen, verapamil or combinations thereof. These observatlons suggest mechanism of non-cytotoxic concentrations of tamoxifen and verapamil on doxorubicin cytotoxicity may involve one or more other cellular processes besides those of interference of estrogen binding to its receptor, cell cycle perturbation, or drug efflux blocking.
Chemical Modification of Residue of Lysine, Tryptophan, and Cysteine in Spinach Glycolate Oxidase
Lee, Duk-Gun ; Cho, Nam-Jeong ; Choi, Jung-Do ;
BMB Reports , volume 29, issue 4, 1996, Pages 321~326
Spinach glycolate oxidase was subjected to a series of chemical modifications aimed at identifying amino acid residues essential for catalytic activity. The oxidase was reversibly inactivated by treatment with pyridoxal 5'-phosphate (PLP). The inactivation by PLP was accompanied by the appearance of an absorption peak of around 430 nm, which was shifted to 325 nm upon reduction with
. After reduction, the PLP-treated oxidase showed a fluorescence spectrum with a maximum of around 395 nm by exciting at 325 nm. The substrate-competitive inhibitors oxalate and oxaloacetate provided protection against inactivation of the oxidase by PLP. These results suggest that PLP inactivates the enzyme by fonning a Schiff base with lysyl residue(s) at an active site of the oxidase. The enzyme was also inactivated by tryptophan-specific reagent N-bromosuccinimide (NBS). However, competitive inhibitors oxalate and oxaloacetate could not protect the oxidase significantly against inactivation of the enzyme by NBS. The results implicate that the inactivation of the oxidase by NBS is not directly related to modification of the tryptophanyl residue at an active site of the enzyme. Treatments of the oxidase with cysteine-specific reagents iodoacetate, silver nitrate, and 5,5'-dithiobis-2-nitrobenzoic acid did not affect significantly the activity of the enzyme.
Origin of Chlorophyll
Biosynthetic Heterogeneity in Higher Plants
Kim, Jin-Seog ; Rebeiz, Constantin A. ;
BMB Reports , volume 29, issue 4, 1996, Pages 327~334
In this study, the origin of the monovinyl chlorophyll a carboxylic biosynthetic route was investigated in barley (Hordeum vulgare L.) and com (Zea mays L.). Protoporphyrin IX accumulated in vivo or in vitro was found to be all of the divinyl form. Furthermore, the conversion of divinyl protoporphyrin IX to monovinyl protoporphyrin IX in vitro was not observed. In contrast, the biosynthesis and accumulation of monovinyl Mg-protoporphyrin IX and its methyl ester occurred in etiolated leaves and divinyl Mg-protoporphyrin IX was convertible to monovinyl Mg-protoporphyrin IX in vitro. These results suggest that the monovinyl chlorophyll
carboxylic biosynthetic route in plants may originate from the divinyl Mg-protoporphyrin IX pool.
Studies on the Purification and Partial Characterization of Cysteinesulfinic Acid Decarboxylase from Porcine Liver
Lee, Hong-Mie ; Jones, Evan E. ;
BMB Reports , volume 29, issue 4, 1996, Pages 335~342
Porcine liver cysteinesulfinic acid decarboxylase was purified approximately 460-fold by means of ammonium sulfate fractionation and sequential column chromatographic separation with Sephadex G-100, DEAE-cellulose and hydroxylapatite. The enzyme has a flat pH profile with maximum activity occurring between pH 6.0 and 7.6. Pyridoxal 5'-phosphate must be present in all buffers used for purification procedures in order to stabilize the enzyme. Addition of sulfhydryl reagents such as 2-mercaptoethanol are also necessary to maintain maximum enzyme activity throughout purification. The absorption spectrum shows that cysteinesulfinic acid decarboxylase is a pyridoxal 5' -phosphate-containing protein. The major absorption is at 280 nm with two smaller absorption regions, one at 425 nm which is ascribed to a Schiffs base between pyridoxal phosphate and protein, and another at 325 nm which is thought to be due to the interaction of 2-mercaptoethanol with the Schiffs base. A number of divalent cations tested did not affect enzyme activity with the exception of mercury, copper, and zinc which are inhibitory. The partially purified enzyme has an apparent
of 0.94 mM for cysteinesulfinate. Cysteic acid is a competitive inhibitor of the enzyme with a
of 1.32 mM. The molecular weight of the enzyme was estimated to be about 79,600 by using Sephadex G-200 column chromatography.
The Effects of Carbon Sources on the Biosynthesis of the Phospholipid and the Fatty Acid Composition of Mitochondria in Chlorella ellipsoidea
Yoon, Seung-Hee ; Jang, Jae-Seon ; Lee, Chong-Sam ;
BMB Reports , volume 29, issue 4, 1996, Pages 343~352
The biosynthesis of phospholipid and the composition of fatty acid were analyzed in mitochondria isolated from Chlorella ellipsoidea treated with carbon sources (glucose, sucrose, raffinose) during the culture. The growth of Chlorella and total lipid contents in mitochondria treated with various carbon sources was increased to compare with the control. When Chlorella mitochondria was treated with various carbon sources, four kinds of phospholipid were increased predominantly. The major fatty acids utilized for the biosynthesis of the phospholipid were analyzed linoleic acid (average 25.18%) and stearic acid (average 10.52%) in the control. But, it was shown that the major fatty acids in Chlorella mitochondria treated with glucose were stearic acid (average 30.93%), palmitic acid (average 17.47%) and stearic acid (average 20.31%), linoleic acid (average 16.68%) in sucrose treatment and oleic acid (average 17.17%), palmitic acid (average 15.64%) in raffinose treatment.
Interaction between a Blood Vessel-Inducing Protein Angiogenin and Its Binding Protein Actin
Chang, Soo-Ik ; Paik, Seung-Bum ; So, Seung-Ho ; Ahn, Byung-Cheol ;
BMB Reports , volume 29, issue 4, 1996, Pages 353~358
Bovine angiogenin (bAng) is a potent blood vessel inducing protein purified from cow In ilk. fluorescence spectroscopy has been used to study the interaction of bAng with actin in 50 mM Tris-HCl pH 7.5, and 1 mM
. Actin contains four tryptophans but bAng contains no tryptophans. A 50% decrease in intrinsic fluorescence accompanied formation of the bAng/actin complex. By contrast, the interaction of RNase A, a homologous protein to bAng, with actin results in about 10% quenching of the fluorescence. Fluorescence titration experiments were performed by adding increasing concentrations of bAng (0~1.0
) to a constant concentration of actin (0.1
), and the dissociation constant
for the bAng/actin complex and the stoichiometry n were measured as
respectively. These results suggest that the interaction between bAng with actin is specific and that quenching of actin fluorescence has occurred in the bAng/actin complex. The bAng binding sites of actin are discussed in the results of this study, and we propose that Trp-80 in the small domain of bovine actin is responsible for the bAng/actin binding.
Construction of Yeast Vectors Potentially Useful for Expression of Eukaryotic Genes as
-galactosidase Fusion Proteins
Chung, Kyung-Sook ; Choi, Won-Ja ; Lee, Hee-Won ; Kim, Kyu-Won ; Yoo, Hyang-Sook ;
BMB Reports , volume 29, issue 4, 1996, Pages 359~364
By both in vitro hydroxylamine mutagenesis of the wild type 3-phosphoglycerate kinase gene (PGK) promoter DNA and insertion of the leu2-d gene, we have created yeast expression vectors potentially useful for production of eukaryotic genes in yeast. The guanine (G) to adenine (A) change at the -3 position from the ATG start codon of the PGK promoter-based vector rendered a 6~7 times elevated expression of the adjacent eukaryotic gene, and insertion of the leu2-d gene in the vector containing the mutated PGK promoter further enhanced the expression of the gene. When expression of the AIDS virus HIV1-gagP17 gene in a lacZ fusion form was examined with this new vector, a 15 times higher level of expression than that from the original PGK promoter was observed. Northern and Southern analysis showed that this elevated expression is due to the production of a high copy number of mRNA by leu2-d gene functioning and by efficient translation of the produced mRNA. Thus, the vector that contained the A at the -3 position from the ATG start codon in the promoter region and the leu2-d gene shows increased expression capability and will be potentially useful for production of eukaryotic genes in yeast.
Cross-linked with Thymosin
1 is Biologically Active
Jeong, Jee-Yeong ; Chung, Hye-Shin ;
BMB Reports , volume 29, issue 4, 1996, Pages 365~371
Partially reduced interferon-a (
) was cross-linked with thymosin
) using sulfo-succinimidyl (4-iodoacetyl) amino benzoate (SIAB), a bifunctional cross-linking reagent. The partially reduced
optimal for the cross-linking reaction was obtained by incubating native
with 0.5 mM DTT at
for 60~100 min.
was activated by incubating with sulfo-SIAB at
for 30 min to produce
cross-linking was achieved by the reaction of the partially reduced
. This cross-linking was between the sulfhydryl group of Cys1 in
and the N-terminal amino group of
through acetyl amino benzoate as a spacer. The immunological activity of the cross-linked molecule showed the same extent as that of
, and most of the antiviral activity was retained compared to that of the partially reduced
Biochemical Characterization of the Interaction between Small Phosphoproteins and Transducin in Frog Photoreceptors
Suh, Kyong-Hoon ;
BMB Reports , volume 29, issue 4, 1996, Pages 372~379
Components I and II (CI&II) are major phosphoproteins in the frog rod outer segments (ROS) of retina, whose phosphorylation is light- and cyclic nucleotide-dependent. Although it was reported that CI & II could be chemically cross-linked to
of transducin (
), it was not clear whether CI&II physically interact with
, under native conditions. CI&II extracted by hypotonic washing fo ROS membranes showed an overlapped migration with
, in sucrose density gradient centrifugation. The elution profile of CI&II in the peripheral membrane fractions from gel filtration chromatography also overlapped that of
. These hydrodynamic parameters indicate that the native molecular state of CI&II in the peripheral membrane fraction appears to be within a complex, most likely with
. CI&II coeluted with
, showed no phosphorylation by endogenous kinase which phosphorylates a serine of CI&II in other fractions. The purified CI&II were not able to inhibit trypsin-activated cGMP-phosphodiesterase, and CI&II were not recognized by a monoclonal antibody against the
of transducin, indicating that CI&II are not y-subunit of PDE or transducin. Thus, it is likely that native CI&II, which undergo a light-dependent phosphorylation/dephosphorylation cycle, can associate with
, in frog photoreceptor membranes, and the complex formation has an inhibitory effect on the endogenous phosphorylation of CI&II.
Intramolecular Hydrogen Bonds in Proteinase Inhibitor Protein, A Molecular Dynamics Simulation Study
Chung, Hye-Shin ;
BMB Reports , volume 29, issue 4, 1996, Pages 380~385
Ovomucoid third domain is a serine proteinase inhibitor protein which consists of 56 amino acid residues. A fifty picosecond molecular dynamics (MD) simulation was carried out for ovomucoid third domain protein with 5
layer of water molecules. A comparison of main chain atoms in the MD averaged structure with the crystal structure showed that most of the backbone structures are maintained during the simulation. Investigation of the intramolecular hydrogen bondings indicated that most of the interactions between main chain atoms were conserved, whereas those between side chains were reorganized for the period of the simulation. Especially, the side chain interactions around the scissile bond of reactive site P1 (Met18) were found to be more extensive for the MD structures. During the simulation, hydrogen bonds were maintained between the side chains of Glu19 and Arg21 as well as those of Thr17 and Glu19. Extensive side chain interactions observed in the MD structures may shed light on the question of why protein proteinase inhibitors are strong inhibitors for proteinases rather than good substrates.
Substrate Specificity of the Yeast Protein Tyrosine Phosphatase, PTP1, Overexpressed from an Escherichia coli Expression System
Kwon, Mi-Yun ; Oh, Min-Su ; Han, Jun-Pil ; Cho, Hyeong-Jin ;
BMB Reports , volume 29, issue 4, 1996, Pages 386~392
A Saccharomyces cerevisiae Protein Tyrosine Phosphatase, PTP1, was expressed from an Escherichia coli expression system and milligram quantities of active PTP1 were purified chromatographically. The substrate specificity of the recombinant PTP1 was probed using synthetic phosphotyrosine-containing peptides corresponding to the regulatory phosphorylation sites of the yeast MAP kinase homologues
. Peptide sequences derived from the MAP kinase homologues were chosen arbitrarily as starting points for sequence variation studies even though they are not likely to be candidates for physiological substrates of PTP1. Phosphotyrosyl-
peptide showed a
value of 877
peptides showed lower
values of 74
each. To study the effect of sequence variations of the peptide, amino acids of the undecapeptide
(DPQMTGpYVSTR) were sequentially substituted by an alanine residue. More extensive variations of each amino acid revealed positional importance of each amino acid residue. Based on these results, we derived a peptide sequence (DADEpYDA) that is recognized by PTP1 with an affinity (
) significantly higher than that of the peptides derived from the phosphorylation sites of Fus3, Kss1, and Hog1.