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Biomedical subjects

S M Byun

Publications and source records attributed to S M Byun.

At least 37 records · Page 2Linked to original sources
PubMed1996

Cloning and characterization of one member of the chalcone synthase gene family from Solanum tuberosum L.

We have isolated a chalcone synthase gene 2 (ST-CHS2) from potato by rapid amplification of cDNA ends by PCR. CHS2 cDNA had high homology to tomato LET-CHS2 (98%), petunia PHCHSJ (94%), potato ST-CHS1B (92%), petunia PHCHSA (92%), and LET-CHS1 (90%) at the overall 389-amino acid level. Genomic hybridization analysis indicated that CHS genes of potato comprise a family of at least six individual members.

Acyltransferases↗
PubMed1996

Decreased agglutinability of methoxy-polyethylene glycol attached red blood cells: significance as a blood substitute.

Human Rb(+) AB type red blood cells were modified with methoxy-polyethylene glycol (mPEG) in order to decrease agglutinabilities toward clinically important anti-A, anti-B and anti-D reagents. Attachment of mPEG to membrane proteins of the red blood cell was identified by the shift of well-known blood group active glycoprotein bands such as band 3, 4.5 and PAS-1 on the SDS-polyacrylamide gel electrophoresis. When 4mM concentration of mPEG was added to red blood cells, agglutination was minimum by a blood group typing and microwell agglutination tests. Antibody binding tests showed decreased antibody binding to blood group antigens after mPEG attachment. The decrease of both agglutinability and antibody binding was the result of mPEG attachment to blood group active glycoprotein of the cell membrane. The morphology of red blood cells after mPEG attachment was the usual discocytic cell. Oxygen equilibrium curves of the mPEG-attached red blood cells were similar to unmodified red blood cells. This approach to decrease agglutinability of the red blood cells toward blood group antibodies may be used to develop a universally transfusible blood substitute.

Blood Substitutes↗
PubMed1996

Affinity purification and binding characteristics of Citrobacter freundii AmpR, the transcriptional regulator of the ampC beta-lactamase gene.

The transcriptional regulator of the Citrobacter freundii ampC beta-lactamase gene, AmpR, was purified as a single SDS/PAGE-gel band by using various techniques, including DNA-Sepharose 4B affinity chromatography. The purified AmpR consisted of a 32.5 kDa monomer that interacted with three operator sequences: two binding sequences, at positions -75 to -70 and -67 to -51 with respect to the transcriptional start site, were located in the LysR motif (-72 to -60), and the third sequence was at position -43 to -38. Equilibrium binding studies raise the possibility that the adjacent operator sequence could exert a positive influence on the ability of AmpR to bind to these sites.

Bacterial Proteins↗
PubMed1995

Improvement of thermal stability of subtilisin J by changing the primary autolysis site.

The thermostability of subtilisin J, an extracellular serine protease secreted from Bacillus stearothermophilus, has been improved by changing the primary autolysis site of the Asp-49 mutant protein. Previously we have shown that the Asp-49 mutant protein has proteolytic activity, but so unstable that it was primarily autolyzed in Tyr-58-Gln-59 peptide bond during cultivation (Jang et al. Biochim. Biophys. Acta. 1162, 233-235 1993). In the present study, to mitigate the autolytic degradation and increase the thermostability, we deleted the Tyr-58 residue using the Asp-49 mutant as a template. This mutant (Asp-49/delta Tyr-58 mutant) protein showed an improved resistance to heat treatment without changing the catalytic efficiency of the enzyme. These results show that change of primary autolysis site can stabilize the subtilisin.

Amino Acid Sequence↗
PubMed1995

Use of progesterone-3(O-carboxymethyl oxime)-horseradish peroxidase in a sensitive microtitre-plate EIA and its application to a visual membrane EIA of progesterone.

A simple method of visual membrane enzyme-immunoassay (EIA) for the detection of progesterone is described. When two types of progesterone-horseradish peroxidase (HRP) tracers were challenged for binding, in the presence of progesterone, to the monoclonal anti-progesterone antibody, 15A, coated on the microtitre plate, the HRP conjugated at the C-3 position (A-ring) of progesterone competed more effectively with progesterone to the binding site of the monoclonal antibody (mAb) than HRP conjugated at the C-11 position of the C-ring. By using this combination of mAb, 15A, and progesterone-3(O-carboxymethyloxime)-HRP (P-3CMO-HRP), we developed a visual membrane EIA system in which free progesterone in the sample could be quantified by the degree of color development. In this system, free progesterone competed with P-3CMO-HRP for binding sites of mAb immobilized on the nitrocellulose membrane. The stable grey color was formed on the surface of membrane for progesterone-negative and no color for progesterone-positive sample using 3,3'-diaminobenzidine (DAB) with Co2+ as an insoluble substrate solution. To examine whether tetramethylbenzidine (TMB) can substitute for DAB in membrane EIA, an experiment was conducted where TMB was used as an insoluble substrate.

3,3'-Diaminobenzidine↗
PubMed1995

Identification of immunodominant epitopes in the core and non-structural region of hepatitis C virus by enzyme immunoassay using synthetic peptides.

Thirty-two synthetic peptides, components of the core and non-structural protein of Hepatitis C virus (HCV), were tested for their reactivities against antibodies in sera of healthy, HCV antibody positive of chronic liver disease patients. Among them, 8 of the core peptides, 4 of the NS4 peptides and 3 of the NS5 peptides reacted with the HCV infected sera. In particular, C22 (core peptide) and NS4-1924 (NS4 peptide) were most reactive with the serum samples giving a positive signal with commercially available enzyme-linked immunosorbent assay (ELISA) kit. Our results indicate that the immunodominant regions of the HCV-derived proteins are located at three regions in the core protein, three regions in the NS4 protein, and one region in the NS5 protein. These results indicate that the selected peptides are useful antigens in detecting antibodies in the sera from individuals infected with HCV.

Amino Acid Sequence↗
PubMed1995

Characterization of a metalloprotease inhibitor protein (SmaPI) of Serratia marcescens.

As suggested by Y. Suh and M.J. Benedik (J. Bacteriol. 174: 2361-2366, 1992), Serratia marcescens ATCC 27117 produced very small amounts (0.8 U ml-1) of an inhibitor protein (SmaPI) that shows an inhibitory activity against extracellular 50-kDa metalloprotease (SMP) of S. marcescens and that is localized in the periplasm of cells at the optimal growth temperature of 25 degrees C. A recombinant S. marcescens harboring plasmid pSP2 encoding SMP and SmaPI genes produced 20 U of SmaPI ml-1 that is also localized in the periplasm of cells at 25 degrees C. However, a large amount of SmaPI (86 Uml-1) was extracellularly produced at the supraoptimal growth temperature 37 degrees C from the recombinant S. marcescens (pSP2). We purified SmaPI from the culture supernatant of S. marcescens (pSP2) grown at 37 degrees C, and some biochemical properties were characterized. SmaPI had a pI value of about 10.0 and was a monomeric protein with a molecular mass of 10,000. SmaPI was produced from a precursor SmaPI by cleavage of a signal peptide (26 amino acid residues). The inhibitor was stable in boiling water for up to 30 min. The thermostability of SmaPI can be attributed to its reversible denaturation. SmaPI inhibited SMP by formation of a noncovalent complex with a molar ratio of 1:1 and showed a high protease specificity, which inhibited only SMP among the various proteases we examined.

Amino Acid Sequence↗
PubMed1995

Purification and Characterization of a Maltotetraose-Forming Alkaline (alpha)-Amylase from an Alkalophilic Bacillus Strain, GM8901.

An alkalophilic bacterium, Bacillus sp. strain GM8901, grown at pH 10.5 and 50(deg)C, produced five alkaline amylases in culture broth. At an early stage of the bacterial growth, amylase I (Amyl I) was produced initially and then, as cultivation progressed, four alkaline amylases, Amyl II, Amyl III, Amyl IV, and Amyl V, were produced from proteolytic degradation of Amyl I. A serine protease present in the culture medium was believed to be involved in Amyl I degradation. We purified Amyl I from the culture supernatant by ammonium sulfate precipitation, heparin-Sepharose CL-6B column chromatography, phenyl-Toyopearl column chromatography, and Mono Q HR5/5 high-performance liquid chromatography. The molecular weight of Amyl I was estimated to be about 97,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amyl I had an extremely high optimal pH of 11.0 to 12.0 and was stable in a broad pH range of 6.0 to 13.0. Amyl I had an optimal temperature of 60(deg)C and was stable up to 50(deg)C. Thermostability was increased in the presence of Ca(sup2+) and soluble starch. The enzyme required metal ions such as Ca(sup2+), Mg(sup2+), Cu(sup2+), Co(sup2+), Ag(sup+), Zn(sup2+), and Fe(sup2+) for its enzyme activity and was inhibited by 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride. According to the mode of action of Amyl I on starch, Amyl I was classified as an (alpha)- and exo-amylase. Amyl I produced maltotetraose predominantly from starch via intermediates such as maltohexaose and maltopentaose.

Journal Article↗
PubMed1993

Identification of autoproteolytic cleavage site in the Asp-49 mutant subtilisin J by site-directed mutagenesis.

Ser-49 located at the exposed surface loop of subtilisin J was replaced with Asp and Arg. Proteinase activity of the Asp-49 mutant was similar to that of wild-type, but the Arg mutant was inactivated. At 37 degrees C, mature subtilisin J protein of the Asp-49 mutant rapidly degraded, and specific breakdown products were accumulated. These proteins were analyzed by SDS-PAGE, and the N-terminal sequences were determined for the mature and deleted protein. We identified the autoproteolytic cleavage site in the mature Asp-49 mutant protein from sequencing data.

Amino Acid Sequence↗
PubMed1993

Influence of the conjugation site on the specificity of monoclonal antibodies to progesterone and on the performance of direct enzyme immunoassay.

Monoclonal antibodies against progesterone conjugated to carrier protein through substituent on the A-ring (C3 position ) or the C-ring (C11 position) of progesterone were used in the enzyme immunoassay. Antibody specificities were determined by testing the ability of 11 represensitive steroids to displace labelled progesterone in a competitive enzyme immuno-assay (EIA) and a radioimmunoassay (RIA). Immunization with progesterone conjugated to BSA through substituent on the A-ring (C3) resulted in the formation of monoclonal antibody (mAb) which fairly specific for progesterone. While immunization with progesterone at the 11-position (C11) resulted in the mAbs which were very specific for progesterone. The best EIA system was developed by using mAb against progesterone-11 alpha -hemisuccinyl BSA and tracer of progesterone-3(O-carboxymethyl oxmine)-horseradish peroxidase. Our approach is to confirm the overall orientation of the steroid in the binding site. Immunobiochemical analysis of mAb suggested that the D-ring is substantially more buried in the binding pocket than the A-ring. Our assay was designed to use the reagent inactivating cholesterol binding globulin in serum so that the extraction of the hormone into organic solvent was unnecessary. Therefore, our assay system can detect directly and rapidly the progesterone level in serum within one hour.

Animals↗
PubMed1992

Effect of the weak Ca(2+)-binding site of subtilisin J by site-directed mutagenesis on heat stability.

The functional role of the negatively charged amino acid residue in subtilisin J from Bacillus stearothermophilus has been investigated by site-directed mutagenesis. Glu-195 located at the weak Ca2+-binding site was replaced with Gln to examine the role of Glu-195 in the heat stability of subtilisin J. Mutant enzyme was expressed in Bacillus subtilis and was purified from the culture supernatant. When the mutant enzyme was expressed at 37 degrees C in the presence of 2mM calcium chloride, the pattern of enzyme production was quite different from that of wild-type. The purified Gln-195 mutant enzyme was analyzed with respect to optimal temperature, optimal pH, and heat stability. The mutation was found to decrease the heat stability but not catalytic efficiency (kcat/Km) and optimal pH. These results demonstrate the important role of the negatively charged side chains at the weak Ca(2+)-binding site in the heat stability of subtilisin.

Amino Acid Sequence↗
PubMed1992

Molecular cloning of a subtilisin J gene from Bacillus stearothermophilus and its expression in Bacillus subtilis.

The structural gene for a subtilisin J from Bacillus stearothermophilus NCIMB10278 was cloned in Bacillus subtilis using pZ124 as a vector, and its nucleotide sequence was determined. The nucleotide sequence revealed only one large open reading frame, composed of 1,143 base pairs and 381 amino acid residues. A Shine-Dalgarno sequence was found 8 bp upstream from the translation start site (GTG). The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprised of 275 residues. The productivity of subtilisin in the culture broth of the Bacillus subtilis was about 46-fold higher than that of the Bacillus stearothermophilus. The amino acid sequence of the extracellular alkaline protease subtilisin J is highly homologous to that of subtilisin E and it shows 69% identity with subtilisin Carlsberg, 89% with subtilisin BPN' and 70% with subtilisin DY. Some properties of the subtilisin J that had been purified from the Bacillus subtilis were examined. The subtilisin J has alkaline pH characteristics and a molecular weight of 27,500. It retains about 50% of its activity even after treatment at 60 degrees C for 30 min in the presence of 2 mM calcium chloride.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and nucleotide sequence of Streptomyces griseus trypsin gene.

Streptomyces griseus trypsin (E.C. 3.4.21.4) is one of the major extracellular proteinase, which is secreted by S. griseus. The gene encoding S. griseus trypsin was isolated from a S. griseus genomic library by using a synthetic oligonucleotide probe. Fragments containing the gene for S. griseus trypsin were characterized by hybridization and demonstration of proteolytic activity in S. lividans. Deduced amino acid sequence from the nucleotide sequence suggests that S. griseus trypsin is produced as a precursor, consisting of three portions; an amino-terminal pre sequence (32 amino acid residues), a pro sequence (4 residues), and the mature trypsin. The S. griseus trypsin consists of 223 amino acids with a computed molecular weight of 23,112. The existence of proline at the pro and mature junction suggests that the processing of S. griseus trypsin is non-autocatalytic.

Amino Acid Sequence↗
PubMed1991

Elongation factor-2 in chick embryo is phosphorylated on tyrosine as well as serine and threonine.

An endogenous 95 kDa chick embryo cytosolic protein (p95) was phosphorylated in the presence of [gamma-32P]ATP and the kinase activity for p95 was mostly associated with particulate fraction. Phosphorylation of p95 was prominent in embryos of early developmental stage. Hydrolysis of p95 phosphoprotein yielded phosphotyrosine in addition to phosphothreonine and phosphoserine. Native p95 was also tyrosine-phosphorylated. p95 phosphoprotein was purified by DEAE-Sephacel chromatography and immunoprecipitation with anti-phosphotyrosine antibody and the amino acid sequence was determined. The N-terminal sequence, Val-Asn-Phe-Thr-Val-Asp-Gln-Ile-Arg-Ala-Ile-Met-Asp- Lys-Lys-Ala-Asn-Ile-Arg-Asn-Met-, was found to be identical to those of elongation factor-2 (EF-2) of both rat and hamster. Our results suggest the presence of other EF-2 kinase in chick embryo cell than the previously reported Ca2+/calmodulin-dependent protein kinase III.

Age Factors↗
PubMed1991

The leader sequence of streptokinase is responsible for its post-translational carboxyl-terminal cleavage.

When the expression of streptokinase from two tac promoter-controlled expression vectors, one of these deleted a putative leader sequence of streptokinase and the other not, was compared, both normal and degraded streptokinase were detected in proteins expressed from the leader-contained vector, but only normal streptokinase was detected from the leader-deleted vector. These findings indicate that the characteristic carboxyl-terminal cleavage of streptokinase is correlated with its leader sequence and occurs during the defective secretion. The homogeneous preparation of streptokinase was facilitated by expressing from this leader-deleted vector.

Base Sequence↗
PubMed1991

The sites of cleavage on Escherichia coli glutamine synthetase by dithiothreitol, Fe(III) and O2.

Native conformation of an enzyme molecule is required for the specific non-enzymatic cleavage of Escherichia coli glutamine synthetase by a metal-catalyzing oxidation system comprised of dithiothreitol, Fe(III) and O2. The cleavage reaction is greatly inhibited by the addition of Mg(II). Two major cleavage sites are identified between amino acid residues 264 and 268, and roughly between amino acid residues 31 and 34, which are located on the protein segments forming the active site of the enzyme. These results suggest that the cleavage reaction is a largely site-specific process involving active oxygen species generated at the divalent cation binding sites on glutamine synthetase.

Binding Sites↗
PubMed1990

Affinity enhancement of bispecific antibody against two different epitopes in the same antigen.

For the enhancement of antibody binding affinity, a bispecific antibody against two different epitopes in human chorionic gonadotropin hormone, one is in alpha-subunit and the other is in beta-subunit, was prepared by chemical recombination using 5,5'-dithiobis(2-nitrobenzoic acid). The epitopes recognized by antibodies were investigated by competitive radioimmunoassay, two-site sandwich radioimmunoassay and additivity assay and a proper epitope pair was chosen for preparation of the bispecific antibody. This bispecific antibody has dual specificity and as much as 17.2-fold higher affinity than that of monoclonal antibody with higher affinity by dual antigen binding radioimmunoassay and Scatchard plot analysis.

Animals↗