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Purification and Characterization of Farnesyl Protein Transferase from Bovine Testis

  • Ryo, Kwon-Yul;Baik, Young-Jin;Yang, Chul-Hak
    • BMB Reports
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    • v.28 no.3
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    • pp.197-203
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    • 1995
  • Famesyl protein transferase involved in the first step of post-translational modification of $p21^{ras}$ proteins transfers the famesyl moiety from famesyl pyrophosphate to a cysteine residue in $p21^{ras}$ proteins. The enzyme was first purified 30,000-fold from bovine testis by use of 30~50% ammonium sulfate fractionation, DEAE-Sephacel ion exchange chromatography, Sephacryl S-300 gel filtration chromatography, Sephacryl S-200 gel filtration chromatography, and hexapeptide (Lys-Lys-Cys-Val-Ile-Met) affinity chromatography. The molecular weight of the purified enzyme was estimated to be ~100 kDa by gel filtration and SDS-polyacrylamide gels showed two closely spaced bands of ~50 kDa protein. These indicate that the enzyme consists of two nonidentical subunits, a and 13, which have slightly different molecular weights. The enzyme was inhibited by hexapeptide (Lys-Lys-Cys-Val-Ile-Met), which acted as an alternative substrate that competed for famesylation. Kinetic analysis by measuring initial velocities showed that famesyl protein transferase is a very slow enzyme. EDTA-treated famesyl protein transferase showed little activity with $Mg^{2+}$ or $Zn^{2+}$ alone, but required both $Mg^{2+}$ and $Zn^{2+}$ for the catalytic activity.

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An Antiproliferative Ribonuclease from Fruiting Bodies of the Wild Mushroom Russula delica

  • Zhao, Shuang;Zhao, Yong Chang;Li, Shu Hong;Zhang, Guo Qing;Wang, He Xiang;Ng, Tzi Bun
    • Journal of Microbiology and Biotechnology
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    • v.20 no.4
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    • pp.693-699
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    • 2010
  • An antiproliferative ribonuclease with a new N-terminal sequence was purified from fruiting bodies of the edible wild mushroom Russula delica in this study. This novel ribonuclease was unadsorbed on DEAE-cellulose, but absorbed on SP-Sepharose and Q-Sepharose. It had a molecular mass of 14 kDa, as judged by fast protein liquid chromatography on Superdex 75 and SDS-polyacrylamide gel electrophoresis. Its optimal pH and optimal temperature were pH 5 and $60^{\circ}C$, respectively. The ranking of its activity toward various polyhomoribonucleotides was poly C> poly G>poly A>poly U. It could inhibit proliferation of HepG2 and MCF-7 cancer cells with an $IC_50$ value of $8.6\;{\mu}M$ and $7.2\;{\mu}M$, respectively. It was devoid of antifungal and HIV-1 reverse transcriptase inhibitory activity.

A Study of Polymorphisms of Proline-rich Protein, Double-band Protein and Pa Protien in Ullung-do and Jawall-do (한국 울릉도,자월도 거주민의 이하선 타액내 Proline-rich protein(Pr), Double-band protein(Db)과 Salivary acidic protein(Pa)의 유전적 다형 현상에 관한 연구)

  • Soon-Min Chung;Chong-Youl Kim
    • Journal of Oral Medicine and Pain
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    • v.15 no.1
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    • pp.91-104
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    • 1991
  • After alkaline slab polyacrylamide gel electrophoresis and 3.3'-DMB staining of parotid saliva from 48 peoples in Ullung-do and 35 peoples in Jawall-do, the author have got following conclusions. 1. The gene frequencies of Proline-rich protein in Ullung-do were Pr1 =0.719, Pr2=0.281 2. The gene frequencies of Proline-rich protein in Jawall-do were Pr1=0.671, Pr2=0.329 3. The gene frequencies of Double band protein in Ullung-do were Db+=0.087, Db-=0.913 4. The gene frequencies of Double band protein in Jawall-do were Db+=0.0.106, Db-=0.0.894 5. The gene frequencies of Pa protein in Ullung-do were Pa+=0.179, Pa-=0.821 6. The gene frequencies of Pa protein in Jawall-do were Pa+=0.167, Pa-=0.833 7. The gene frequencies of Pr1 of Ullung-do and jawall-do were lower than those of Pr1 in Seoul and Onyang, but similar to those of Pr1 in kangnung and Cheju. 8. The gene frequencies of Db- of Ullung-do and Jawall-do were much higher than those of Db+ in Japanese, Chinese and other populations in Korean.

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An Endocrinological Study on Metamorphosis of the Midgut in Silkworm, Bombyx mori (가잠의 중장변태에 관한 내분비학적 연구)

  • 성주일
    • Journal of Sericultural and Entomological Science
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    • v.26 no.1
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    • pp.30-34
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    • 1984
  • In order to investigate an endocrinological effect on metamorposis of the midgut in silkworm, Bombyx mori, 20-hydroxyecdysone and rude extract of brain (PTTH) were injected into pupal isolated abdomens, devoid of prothoracic glands and also in the brainless diapausing pupae with the prothoracic glands (so-called dauer pupae). When ecdysone was assayed abdomens, drastic morphological changes occurred in the midgut. The wet weights of midguts were found decrease as like the normal pupae at metamorphosis. Proteineous changes in epithelial midgut were also observed; pupal specific proteins (PSP) vanished while the adult proteins were newly synthesized. Similar results were also found in the changes of protein in the midgut of dauer pupae by injecting ecdysone and PTTH.

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Purification and Characterization of Aspartase from Hafnia alvei

  • Yoon, Moon-Young;Park, Jae-Ho;Choi, Kyong-Jae;Kim, Joung-Mok;Kim, Yeon-Ok;Park, Jon-Bum;Kyong, Jin-Burm
    • BMB Reports
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    • v.31 no.4
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    • pp.345-349
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    • 1998
  • Aspartase (EC 4.3.1.1) from Hafnia alvei was purified to homogeneity by a combination of DEAE-cellulose, Red A-agarose, and Sepharose 6B chromatography. The purified enzyme appeared homogeneous on denatured SDS-polyacrylamide gel electrophoresis. The purified enzyme was a tetrameric protein composed of identical subunits with a molecular weight of 55,000 daltons. The optimum pH for the enzymatic reaction was 8.5 and the optimum temperature for maximum activity was $45^{\circ}C$. The enzyme has an absolute requirement of divalent metal ions ($Mg^{2+}$, $Mn^{2+}$) at the alkaline pH. The enzyme, however, was inactivated in the presence of other divalent cations such as $Zn^{2+}$, $Ca^{2+}$. The helical content of the purified enzyme was estimated by CD spectropolarimetry to be 61%.

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Cloning and Expression of Mycobacterium bovis Secreted Protein MPB83 in Escherichia coli

  • Xiu-Yun, Jiang;Wang, Chun-Feng;Wang, Chun-Fang;Zhang, Peng-Ju;He, Zhao-Yang
    • BMB Reports
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    • v.39 no.1
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    • pp.22-25
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    • 2006
  • The gene encoding MPB83 from Mycobacterium bovis Vallee111 chromosomal DNA was amplified by using polymerase chain reaction (PCR) technique, and the PCR product was approximately 600bp DNA segment. Using T-A cloning technique, the PCR product was cloned into pGEM-T vector and the cloning plasmid pGEM-T-83 was constructed successfully. pGEM-T-83 and pET28a(+) were digested by BamHI and EcoRI double enzymes. The purified MPB83 gene was subcloned into the expression vector pET28a(+), and the prokaryotic expression vector pET28a-83 was constructed. Plasmid containing pET28a-83 was transformed into competence Escherichia coli BL21 (DE3). The bacterium was induced by isopropyl-$\beta$-D-thiogalactopyranoside (IPTG) and its lysates were loaded directly onto sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), approximately 26 kDa exogenous protein was observed on the SDS-PAGE. The protein was analyzed using Western-blotting. The results indicated that the protein was of antigenic activity of M. bovis. The results were expected to lay foundation for further studies on the subunit vaccine and DNA vaccine of MPB83 gene in their prevention against bovine tuberculosis.

Helical Periodicity of $(dT)_n{\cdot}(dA)_n{\cdot}(dT)_n$ Triple - Stranded DNA

  • Kim, Ki-Hyun;Koo, Hyeon-Sook
    • BMB Reports
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    • v.30 no.6
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    • pp.426-430
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    • 1997
  • The helical periodicity of the triple-stranded $(dT)_n{\cdot}(dA)_n{\cdot}(dT)_n$ sequence was determined by measuring gel-mobilities of bent DNA fragments containing the sequence. In the bent DNA fragments, a $GA_{22}G$ $CT_{22}C$ sequence was located between two bent DNA loci composed of six $A_{6}{\cdot}T_{6}$ repeats. and the DNA length between the bent DNA loci was varied by 1 base pair over a full helical turn. The gel mobility of each bent DNA fragment reflected the overall extent of DNA bending and varied with the DNA length between the two bent loci. Mobilities of the bent DNA fragments in 5% polyacrylamide gel were measured after preincubating the DNA fragments both in the presence and absence of $CT_{22}C$ oligonucleotide. By comparing the bent DNA fragments containing an intermolecular triplex structure with those of a genuine duplex structure in the gel mobilities, the helical periodicity of the $T_n{\cdot}A_n{\cdot}T_n$ triplex DNA was determined to be $11.5({\pm}0.3)bp/turn$.

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Recombinant Expression and Enzyme Activity of Chymotrypsin-like Protease from Black Soldier Fly, Hermetia illucens (Diptera: Stratiomyidae)

  • Park, Kwan Ho;Choi, Young Cheol;Nam, Sung Hee;Kim, Won Tae;Kim, A Young;Kim, Sin Young
    • International Journal of Industrial Entomology and Biomaterials
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    • v.25 no.2
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    • pp.181-185
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    • 2012
  • Chymotrypsin serine protease is one of the main digestive proteases in the midgut of and is involved in various essential processes. In a previous study, a gene encoding a chymotrypsin-like protease, Hi-SP1, was cloned from the larvae of Hermetia illucens and characterized. In this study, we produced the recombinant chymotrypsin-like protease Hi-SP1 in Escherichia coli cells. The molecular weight of the recombinant Hi-SP1 was estimated to be approximately 26 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western-blotting. Chymotrypsin activity was detected when AAPF was used as the substrate. Examination of the effects of temperature and pH revealed that the proteolytic activity of recombinant Hi-SP1 decreased markedly at temperatures above $30^{\circ}C$, and the optimum pH was found to be 10.0.

Application of Cationic PVAm - Anionic PAM Dry Strength Aids System on a Kraft Paper Mill (양이온성 PVAm - 음이온성 PAM 건조지력증강제 시스템의 크라프트지 적용 사례)

  • Cho, Byoung-Uk;Ryu, Jeong-Yong;Son, Dong-Jin;Song, Bong-Keun
    • Journal of Korea Technical Association of The Pulp and Paper Industry
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    • v.42 no.3
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    • pp.50-57
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    • 2010
  • A mill trial was performed in a kraft paper mill in order to evaluate the possibility of utilizing dual polymer dry strength aids system consisting of cationic PVAm and anionic PAM. It was found that the cationic PVAm - anionic PAM dry strength additives can improve paper strength without significantly disturbing the stability of the kraft papermaking process when virgin UKP was used as a furnish. Tensile strength (25.3% in machine direction, 48.4% in cross machine direction), elongation of paper (31.6%, 15.6%) and tensile energy absorption (48%, 54%) were improved. Air permeability of the kraft paper was improved as well (22%). Tear strength was decreased with PVAm dry strength aids system, but it can be compensated with decreasing refining degree. In addition, the mill trial results indicate that highly air permeable kraft sack paper can be produced by the addition of PVAm dry strength agents at the stock with reduced freeness.

Flocculation Behavior and properties of Montmorillonites Mixed with Organic Polymer Solutions (유기폴리머 용액에 혼합한 몬모릴로나이트의 응집 거동 및 특징)

  • 황진영
    • Economic and Environmental Geology
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    • v.32 no.3
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    • pp.307-315
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    • 1999
  • Four organic polymers were mixed with mothmorillonite. Two cationic polymers a hi로 molecular weight polyacrylamide (494C) and a low molecular weight polymer (587C).Two anionic polymers include a high molecular weight polymer (aerotil). Each clay supension series were allowed to stand for 24 hours and were centrifuged, and the clay plugs were washed and dried. The dried samples investigated by XRD, IR and CEC measurement. The suspended clay containing anionic polymers was not flocculated at any concentratuons of polymer. But the suspendions containing two cationic polymers were rapidly flocculated at almost all concentrations. the d(001) spacings of Na-montmorillonite after being with cationic polymer 587C show about 15$\AA$ suggesting the polymers may have entered the interlayer spaces. The polymer 494C-treated sample produced double peaks of about 12 and 15$\AA$ in XRD. It indicates that the high molecular weight polymer. And cationic polymer 494C may be adsorbed mainy on the outside surface of clay, and some polymers may peretrate into olny interlayers in the margin of montmorillonite particles because of its high molecular weught. CEC of polumer 587-treated sample was reduecd mmarkedly suggesting polymer blocks CEC sites. The d(001) spacings of Ca-montmorillonite after being treated with cationic polymers show about 15$\AA$ suggesting that the interlayer spaces have not been expanded. In the experiment using a dilute Ca-bearing solution, the suspended caly containinf anionic polymers was flocculated. The results indicate that the flocculation behavior of montmorillonite-polymer supension depends on not only polymer properties such as concentration, electric charge and molecular weight but also compositions of solvent.

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