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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 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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Chemical Synthesis and Determination of Biological Activity of the Epidermal Growth Factor-Like Domain of Mouse Betacellulin
Shin, Song-Yub ; Kang, Shin-Won ; Ha, Jong-Myung ;
BMB Reports , volume 28, issue 2, 1995, Pages 87~93
To investigate the biological functions of the EGF-like domain of mouse betacellulin (BTC), mouse BTC(33-80), a 48-residue peptide corresponding to the EGF-like domain, was synthesized by stepwise solidphase methods using a 9-fluorenylmethoxycarbonyl (Fmoc) strategy. The homogeneity of synthetic mouse BTC(33-80) was confirmed by analytical reversed phase (RP)-HPLC, amimo acid analysis, and fast atom bombardment mass spectrometer (FAB-MS). Three disulfide bond pairings of synthetic mouse BTC(33-80) were established by amino acid analysis of cysteine-containing fragments derived from thermolytic digestion. These were consistent with the pairings of EGF and transforming growth factor (
). The EGF-Iike domain of mouse BTC showed equipotent activity in both EGF-receptor binding on A-431 epidermoid carcinoma cells, and mitogenesis on NIH-3T3 fibroblast cells, as compared with authentic h-EGF. Results suggest that the EGF-Iike domain of BTC plays a significant role in mitogenic activity with an EGF-receptor mediated system.
Binding Subsites In the Active Site of
Sok, Dai-Eun ; Kim, Mee-Ree ;
BMB Reports , volume 28, issue 2, 1995, Pages 94~99
The properties of binding sites in the active site of
-glycerophosphocholine cholinephosphodiesterase were examined using substrates and inhibitors of the enzyme. Phosphodiesterase hydrolyzed p-nitrophenylphosphocholine, p-aminophenylphosphocholine, and glycerophosphocholine, but did not hydrolyze either acylated glycerophosphocholine or bis (p-nitrophenyl)phosphate, suggesting a size limitation for interaction with a glyceryl moiety-binding subsite. The hydrolysis of p-nitrophenylphosphocholine was competitively inhibited by glycerophosphocholine and p-aminophenylphosphocholine, while glycerophosphoethanolamine was a weak inhibitor. The enzyme was also inhibited by choline, but not by ethanolamine. Thiocholine, a much more potent inhibitor than choline, was more inhibitory than cysteamine, suggesting a strict specificity of an anionic subsite adjacent to a
subsite. Of all oxyanions tested, the tellurite ion was found to strongly inhibit the enzyme by binding to a
subsite. The inhibitory role of tellurite was synergistically enhanced by tetraalkylammonium salts, but not by glycerol. Deactivation of the enzyme by diethylpyrocarbonate was partially protected by choline, but not by glycerophosphate. It is suggested that the active site of phosphodiesterase contains three binding subsites.
Purification and Characterization of S-adenosylmethionine Synthetase from Soybean (Glycine max) Axes
Kim, Dae-Gun ; Park, Tae-Jin ; Kim, Jong-Yeol ; Cho, Young-Dong ;
BMB Reports , volume 28, issue 2, 1995, Pages 100~106
S-adenosylmethionine (SAM) synthetase was purified to homogeneity from soybean (Glycine max) axes. The enzyme was purified 216-fold with a 1.5% yield by ammonium sulfate fractionation, acetone fractionation, ion exchange chromatography with DEAE-sephacel, gel filtration with Sephacryl S-300, and afffinity chromatography with ATP-agarose. The enzyme activity reached a maximum 3 days after germination. SAM synthetase had a subunit molecular weight of 57,000 daltons from a silver stained single band on SDS-PAGE. The molecular weight of the enzyme was 110,000 daltons from Sephacryl S-300 gel filtration. The enzyme was composed of two identical subunits. The
values of the enzyme for L-methionine and ATP were 1.81 and 1.53 mM, respectively. The enzymatic activity was not affected by polyamines, agmatine, or SAM analogues, but was inhibited by SAM. The inhibition pattern was showed non-competitive for L-methionine and uncompetitive for ATP. The activity of SAM synthetase was inhibited by thiol-blocking reagents. The enzyme was induced by treatment with
M putrescine at germination. Experimental data revealed a possible novel regulation mechanism of polyamine biosynthesis through several endogenous intermediates.
Metabolic Routes of Malonate in Pseudomonas fluorescens and Acinetobacter calcoaceticus
Byun, Hye-Sin ; Kim, Yu-Sam ;
BMB Reports , volume 28, issue 2, 1995, Pages 107~111
In malonate grown Pseudomonas fluorescens, malonate decarboxylase and acetyl-CoA synthetase were induced, whereas in Acinetobacter calcoaceticus malonate decarboxylase, acetate kinase, and phosphate acetyltransferase were induced. In both bacteria malonate decarboxylase was the first, key enzyme catalyzing the decarboxylation of malonate to acetate, and it was localized in the periplasmic space. Acetate thus formed was metabolized to acetyl-CoA directly by acetyl-CoA synthetase in Pseudomonas, and to acetyl-CoA via acetyl phosphate by acetate kinase and phosphate acetyltransferase in Acinetobacter.
Inactivation of Brain Succinic Semialdehyde Reductase by o-Phthalaldehyde
Choi, Soo-Young ; Song, Min-Sun ; Lee, Byung-Ryong ; Jang, Sang-Ho ; Lee, Su-Jin ; Park, Jin-Seu ; Choe, Joon-Ho ; Cho, Sung-Woo ;
BMB Reports , volume 28, issue 2, 1995, Pages 112~117
Succinic semialdehyde reductase was inactivated by o-phthalaldehyde. The inactivation followed pseudo-first order kinetics, and the second-order rate constant for the inactivation process was 28
at pH 7.4 and
. The absorption spectrum (
337 nm) and fluorescence excitation (
340 nm) and fluorescence emission spectra (
409 nm) were consistent with the formation of an isoindole derivative in the catalytic site between a cysteine and a lysine residue approximately about 3
apart. The substrate, succinic semialdehyde, did not protect enzymatic activity against inactivation, whereas the coenzyme NADPH protected against o-phthaladehyde induced inactivation of the enzyme. About 1 isoindole group per mol of the enzyme was formed following complete loss of enzymatic activity. These results suggest that the amino acid residues of the enzyme participating in a reaction with o-phthalaldehyde are cysteinyl and lysyl residues at or near the NADPH binding site.
Inhibition of the Biodegradative Threonine Dehydratase from Serratia marcescens by
-Keto Acids and Their Derivatives
Choi, Byung-Bum ; Kim, Soung-Soo ;
BMB Reports , volume 28, issue 2, 1995, Pages 118~123
Biodegradative threonine dehydratase was purified to homogeneity from Serratia marcescens ATCC 25419 by streptomycin sulfate treatment, Sephadex G-200 gel filtration chromatography followed by AMP-Sepharose 4B affinity chromatography. The molecular weight of the purified enzyme was 118,000 by fast protein liquid chromatography using superose 6-HR. The enzyme was determined to be a homotetrameric protein with subunit molecular weights of 30,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was inhibited by
acids and their derivatives such as
, pyruvate, glyoxlyate, and phosphoenol pyruvate, but not by
. The inhibition of the enzyme by pyruvate and glyoxylate was observed in the presence of AMP. The inhibitory effect of glyoxylate was decreased at high enzyme concentration, whereas the inhibition by pyruvate was independent of the enzyme concentration. The kinetics of inhibition of the enzyme by pyruvate and glyoxylate revealed a noncompetitive and mixed-type inhibition by the two inhibitors with respect to L-threonine and AMP, respectively.
Chemical Modification of the Biodegradative Threonine Dehydratase from Serratia marcescens with Arginine and Lysine Modification Reagents
Choi, Byung-Bum ; Kim, Soung-Soo ;
BMB Reports , volume 28, issue 2, 1995, Pages 124~128
Biodegradative threonine dehydratase purified from Serratia marcescens ATCC 25419 was inactivated by the arginine specific modification reagent, phenylglyoxal (PGO) and the lysine modification reagent, pyridoxal 5'-phosphate (PLP). The inactivation by PGO was protected by L-threonine and L-serine. The second order rate constant for the inactivation of the enzyme by PGO was calculated to be 136
. The reaction order with respect to PGO was 0.83. The inactivation of the enzyme by PGO was reversed upon addition of excess hydroxylamine. The inactivation of the enzyme by PLP was protected by L-threonine, L-serine, and a-aminobutyrate. The second order rate constant for the inactivation of the enzyme by PLP was 157
and the order of reaction with respect to PLP was 1.0. The inactivation of the enzyme by PLP was reversed upon addition of excess acetic anhydride. Other chemical modification reagents such as N-ethylmaleimide, 5,5'-dithiobis (2-nitrobenzoate), iodoacetamide, sodium azide, phenylmethyl sulfonylfluoride and diethylpyrocarbonate had no effect on the enzyme activity. These results suggest that essential arginine and lysine residues may be located at or near the active site.
Reconstitution of Sarcoplasmic Reticulum-
Release Channels into Phospholipid Vesicles : Investigation of Conditions for Functional Reconstitution
Yang, In-Sik ; Lee, Hee-Bong ;
BMB Reports , volume 28, issue 2, 1995, Pages 129~137
release channel protein in the sarcoplasmic reticulum membrane of rabbit skeletal muscle plays an important role in muscle exitation-contraction (E-C) coupling. Various types of detergents were tested, including Chaps, cholate, octylglucoside, Zwittergents, Mega-9, Lubrol PX, and Triton X-100 for solubilization of this protein. Among these, Chaps and Triton X-100 were found to optionally solubilize the channel complex. Optimum conditions for this solubilization were pH 7.4 with a salt concentration of 1 M. The addition of phospholipid in the solubilization step helped in stabilizing the protein. The purification of the receptor was performed using sucrose density gradient centrifugation. Various methods [dilution, freeze-thaw, adsorption (Biobeads), and dialysis] were investigated to incorporate the Chaps-solubilized and purified
release channel protein into liposomes made from different types of phospholipids. Of these, a combined method consisting of a dialysis, freeze-thaw and sonication steps yielded the best results. Reconstituted vesicles produced by this method with 95% phosphatidylcholine (from soybean extract) had good function.
An Anticoagulant/Fibrinolytic Protease from Lumbricus rubellus
Jeon, Ok-Hee ; Moon, Woong-Joon ; Kim, Doo-Sik ;
BMB Reports , volume 28, issue 2, 1995, Pages 138~142
An anticoagulant/fibrinolytic protease was purified to homogeneity from the earthworm Lumbricus rubellus. The protein was a single chain glycoprotein of 32 kDa that exhibited strong proteolytic activity on human thrombin and fibrin clots. Proteolytic degradation of these plasma proteins by the purified enzyme occurred at a neutral pH range. Among several human plasma proteins tested as possible substrates for the protease reaction, the 32 kDa enzyme specifically hydrolyzed both thrombin and fibrin polymers without affecting other proteins, such as serum albumin, immunoglobulin, and hemoglobin. Treatment of the purified enzyme at neutral pH with either phenylmethylsulfonylfluoride or soybean trypsin inhibitor resulted in a loss of catalytic activity. The enzyme hydrolyzed the chromogenic substrate H-D-Phe-L-Pipecolyl-L-Arg-p-nitroanilide with a
value of 1.1
at a neutral pH. These results suggest that the anticoagulant/fibrinolytic enzyme from Lumbricus rubellus is a member of the serine protease family having a trypsin-like active site, and one of the potential clevage sites for the enzyme is the carbonyl side of arginine residues in polypeptide chains.
Condensation of DNA by a Histone-like Protein in Escherichia coli
Kim, So-Youn ; Hwang, Deog-Su ;
BMB Reports , volume 28, issue 2, 1995, Pages 143~148
In E. coli, chromosomal DNA associated with proteins is condensed into an organized structure known as nucleoid. Using a nitrocellulose filter binding assay to identify proteins forming nucleoid, a 21 kDa protein was purified from E. coli. The molecular weight of the purified protein was 21 kDa on SDS-polyactylamide gel electrophoresis and 24 kDa on gel permeation chromatography. A molecular weight of 21 kDa on SDS-polyacrylamide gel electrophoresis is unique among known proteins which are believed to be involved in the formation of nucleoid in E. coli. The 21 kDa protein nonspecifically binds to both double-stranded and single-stranded DNA. Sedimentation in a sucrose gradient revealed that the protein induced significant condensation of both supercoiled plasmid DNA and linear bacteriophage
DNA On the basis of quantitative Western-blot analysis, approximately 40,000 molecules of the protein were estimated to exist in an E. coli. The biochemical properties and cellular abundance of the 21 kDa protein suggest that this protein participates in the formation of nucleoid in E. coli.
Purification and Characterization of Protein Methylase II from Porcine Testis
Jung, Ki-Kyung ; Kwon, Myung-Hee ; Lee, Hoi-Young ; Lee, Hyang-Woo ; Hong, Sung-Youl ;
BMB Reports , volume 28, issue 2, 1995, Pages 149~154
Protein methylase II (S-adenosyl-L-methionine : protein O-methyl-transferase, EC 22.214.171.124; PM II) was purified approximately 1250-fold from porcine testis by fractional precipitation and DEAE-cellulose chromatography, followed by gel filtration on a Sephadex G-75 column and HPLC on a Protein Pak 125 column. The molecular weight of the enzyme was estimated to be 33,000 daltons by SDS-PAGE, which agreed with the value determined by gel filtration. Isoelectric focusing of purified PM II showed a single protein species with an isoelectric point of 6.2. The optimum pH for the reaction was 6.0. The
value of the enzyme was
value of 769 pmol/min/mg of enzyme. S-adenosyl-L-homocysteine is a competitive inhibitor of PM II with a
Immobilization and Characterization of a Liposome-Mediated Reconstituted Nicotinic Acetylcholine Receptor
Suh, Jeong-Ihn ; Palk, Bo-Hyun ; Oh, Se-Zu ; Suh, Jung-Hun ; Cho, Key-Seung ; Palk, Young-Ki ;
BMB Reports , volume 28, issue 2, 1995, Pages 155~161
A nicotinic acetylcholine receptor (nAchR) isolated from the electric tissues of Torpedo californica has been reconstituted into a vesicle comprising a bifunctional azo-ligand (Bae 1) compound, and a liposome containing phospholipids and cholesterol (1 : 1, w/w). The liposome-mediated reconstituted receptor showed a concentration-dependent response to cholinergic drugs in a lithium ion flux assay. This liposome-mediated reconstituted nAchR was immobilized onto an electrode using various synthetic polymers which were tested for their response to the cholinergic ligands. The immobilized nAchR not only exhibited a linear response to a wide range of cholinergic ligand concentrations but also retained an operational stability which lasted for longer than 6 days. Thus, this result provides a basis for application of the immobilized nAchR-based biosensor in detecting cholinergic ligands in vitro.
Purification and Reaction Mechanism of Rat Brain Succinic Semialdehyde Dehydrogenase
Kim, Kyu-Tae ; Joo, Chung-No ;
BMB Reports , volume 28, issue 2, 1995, Pages 162~169
Rat brain succinic semialdehyde dehydrogenase (EC 126.96.36.199 SSADH) activity was detected in mitochondrial, cytosolic and microsomal fractions. Brain mitochondrial soluble SSADH was purified by ammonium sulfate precipitation, DEAE Sephacel, and 5'-AMP Sepharose 4B affinity chromatography. The purified enzyme was shown to consist of four identical subunits, and the molecular weight of a subunit was 55 kD. The
for short chain aliphatic aldehydes and aromatic aldehydes were at the
level but that for succinic semialdehyde was 2.2
can be used as a cofactor but the affinity for
was 10 times higher than that for
. The brain cytosolic SSADH was also purified by ammonium sulfate precipitation, DEAE Sephacel, Blue Sepharose CL-6B and 5'-AMP Sepharose 4B affinity chromatography and its Km for short chain aliphatic aldehydes was at the
level but that for succinic semialdehyde was 3.3
can be used as a cofactor for this enzyme. We suppose that both enzyme might participate in the oxidation of succinic semialdehyde, which is produced during GABA metabolism. The activity of both cytosolic and mitochondrial SSADH was markedly inhibited when the concentration of succinic semialdehyde was high. The reciprocal plot pattern of product inhibition and initial velocity indicated a sequential ordered mechanism for mitochondrial matrix SSADH. Chemical modification data suggested that amino acid residues such as cysteine, serine and lysine might participate in the SSADH reaction.
Purification and Characterization of Mouse Liver Rhodanese
Lee, Chul-Young ; Hwang, Jae-Hoon ; Lee, Young-Seek ; Cho, Key-Seung ;
BMB Reports , volume 28, issue 2, 1995, Pages 170~176
Rhodanese from mouse liver was purified to near homogeneity by ammonium sulfate precipitation, CM-Sephadex ion exchange, hydroxyapatite and Sephacryl S-200-HR gel filtration chromatographies with a purification of 776 folds. The molecular weight was determined by Sephadex G-150 gel filtration and found to be 34.8 KDa. SOS-PAGE showed molecular weight 34 KDa and two identical subunits splitting by aging for 3 weeks at
the molecular weight of which was 17 KDa. The optimal pH of enzyme activity was 9.4 and the pI value of the enzyme was 6.6. Rhodanese showed the optimal reaction temperature of
and near linear increasing pattern until 10 min. incubation.
values of rhodanese for KCN and
as substrates were 12.5 mM and 8.3 mM, respectively. Rhodanese activity was inhibited by more than 70% at a concentration of 100
. Other metal ions, such as
showed no effect on rhodanese activity.
Purification of Mitochondrial Matrix Aldehyde Dehydrogenase from Pig Brain
Kim, Kyu-Tae ; Lee, Young-Don ;
BMB Reports , volume 28, issue 2, 1995, Pages 177~183
The activity of aldehyde dehydrogenase (ALDH) in the cerebrum, cerebellum, striatum, and medulla oblongata was examined and mitochondrial matrix ALDH was purified prior to immunohistochemical study on the localization of ALDH isozymes in pig brain. Relatively high enzyme activity was found in the striatum and medulla oblongata when using indole-3-acetaldehyde as substrate, and in the striatum when using 3,4-dihydroxyphenylacetaldehyde (DOPAL). The main part of mitochondrial ALDH activities with both acetaldehyde and DOPAL existed in the matrix fraction. The ratio of activity of the matrix to the membrane fraction in the cerebrum was higher than in the cerebellum, suggesting that the distribution pattern of ALDH isozymes was different according to the brain regions. The 276-fold purified mitochondrial matrix ALDH from pig brain was identified to be homologous tetramers with 53 KD subunits. The enzyme showed maximal activity at pH 9.0 and was stable in the temperature range from
. The mitochondrial matrix ALDH activity was considerably inhibited by acetaldehyde in vitro. The
values of the enzyme for acetaldehyde and propionaldehyde were 5.8 mM and 4.9 mM, respectively, whereas
values for indole-3-acetaldehyde and DOPAL were 44
, respectively. The
ratio was the highest with DOPAL as compared with other substrates. These results suggested that mitochondrial matrix ALDH in the present work might be a low Km isozyme involved in biogenic aldehyde oxidation in pig brain.
Sucrose-permeability Induced by Reconstituted Connexin32 in Liposomes.
Rhee, Senng-Keun ; Hong, Eun-Jnng ;
BMB Reports , volume 28, issue 2, 1995, Pages 184~190
Functional study of the gap junction channel has been hindered by its inaccessibility in situ. Identification of forms of this channel in artificial membrane has been elusive because of the lack of identifying channel physiology. Connexin32 forms gap junction channels between neighboring cells in rat liver. Connexin32 was affinity-purified using a monoclonal antibody and reconstituted into artificial phospholipid vesicles. The reconstituted connexin32 formed channels through the vesicle membrane that were permeable to sucrose (Stokes radius:
). The permeability to sucrose was reversibly reduced by acidic pH. In addition, the pH effect on the permeability to sucrose fit well with by the Hill's equation (where, n=2.7 and pK=6.7).