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Seo, Jae Ho,Lim, Jung Chae,Lee, Duck-Yeon,Kim, Kyung Seok,Piszczek, Grzegorz,Nam, Hyung Wook,Kim, Yu Sam,Ahn, Taeho,Yun, Chul-Ho,Kim, Kanghwa,Chock, P Boon,Chae, Ho Zoon American Society for Biochemistry and Molecular Bi 2009 The Journal of biological chemistry Vol.284 No.20
<P>Peroxiredoxins (Prxs) are a group of peroxidases containing a cysteine thiol at their catalytic site. During peroxidase catalysis, the catalytic cysteine, referred to as the peroxidatic cysteine (C(P)), cycles between thiol (C(P)-SH) and disulfide (-S-S-) states via a sulfenic (C(P)-SOH) intermediate. Hyperoxidation of the C(P) thiol to its sulfinic (C(P)-SO(2)H) derivative has been shown to be reversible, but its sulfonic (C(P)-SO(3)H) derivative is irreversible. Our comparative study of hyperoxidation and regeneration of Prx I and Prx II in HeLa cells revealed that Prx II is more susceptible than Prx I to hyperoxidation and that the majority of the hyperoxidized Prx II formation is reversible. However, the hyperoxidized Prx I showed much less reversibility because of the formation of its irreversible sulfonic derivative, as verified with C(P)-SO(3)H-specific antiserum. In an attempt to identify the multiple hyperoxidized spots of the Prx I on two-dimensional PAGE analysis, an N-acetylated Prx I was identified as part of the total Prx I using anti-acetylated Lys antibody. Using peptidyl-Asp metalloendopeptidase (EC 3.4.24.33) peptide fingerprints, we found that N(alpha)-terminal acetylation (N(alpha)-Ac) occurred exclusively on Prx II after demethionylation. N(alpha)-Ac of Prx II blocks Prx II from irreversible hyperoxidation without altering its affinity for hydrogen peroxide. A comparative study of non-N(alpha)-acetylated and N(alpha)-terminal acetylated Prx II revealed that N(alpha)-Ac of Prx II induces a significant shift in the circular dichroism spectrum and elevation of T(m) from 59.6 to 70.9 degrees C. These findings suggest that the structural maintenance of Prx II by N(alpha)-Ac may be responsible for preventing its hyperoxidation to form C(P)-SO(3)H.</P>
말론산에서 배양한 Pesudomonas fluorescens 에서 유도된 말산생성효소
채호준,김유삼 ( Ho Zoon Chae,Yu Sam Kim ) 생화학분자생물학회 1987 BMB Reports Vol.20 No.3
Malate synthase (EC 4.1.3.2) was induced in Pseudomonas fluorescens grown on malonate as a sole source of carbon. This enzyme was purified by the combined methods of ammonium sulfate fractionation, DEAE-Sephacel ion exchange chromatography, tartronate ω-aminohexyl Sepharose 4B affinity chromatography and hydroxyapatite chromatography in an electrophoretically homogeneous form. Molecular size of the enzyme was estimated by Sephadex G-150 gel filtration to be 79,000 dalton composed of single polypeptide. pH optimum of the enzyme was 8.5 and the pI was 4.6. The rate of acetyl group incorporation on variable concentration of substrate into malate was well fitted to a typical Michaelis-Menten kinetics. From the Lineweaver-Burk plot, Km and V_(max) for acetyl-CoA were calculated to be 17.5 μM and 21 umoles/min/㎎, respectively and those for glyoxylate to be 7.7 μM and 35.7 μmoles/min/㎎, respectively. The activity of this enzyme was inhibited by glycolate with 16.8 μM of Ki, and oxalate with 25 μM of Ki, competitively. Some metabolic intermediates such as citrate, isocitrate and succinate, also inhibited the enzyme activity. Iodoacetamide and p-chloromercuriphenylsulfonic acid strongly inhibited the enzyme acitivity, suggesting that there may be a reactive thiol group(s) at or near the active site. The purified malate synthase was immunogenic in rabbit and Ouchterlony double diffusion analysis revealed a single precipitation line with the enzyme.
Induction of Malate Synthase in Pseudomonas fluorescens Grown on Malonate
채호준,김유삼,Chae, Ho-Zoon,Kim, Yu-Sam 생화학분자생물학회 1987 한국생화학회지 Vol.20 No.3
Malate synthase (EC 4.1.3.2) was induced in Pseudomonas fluorescens grown on malonate as a sole source of carbon. This enzyme was purified by the combined methods of ammonium sulfate fractionation, DEAE-Sephacel ion exchange chromatography, tartronate $\omega$-aminohexyl Sepharose 4B affinity chromatography and hydroxyapatite chromatography in an electrophoretically homogeneous form. Molecular size of the enzyme was estimated by Sephadex G-150 gel filtration to be 79,000 dalton composed of single polypeptide. pH optimum of the enzyme was 8.5 and the pI was 4.6. The rate of acetyl group incorporation on variable concentration of substrate into malate was well fitted to a typical Michaelis-Menten kinetics. From the Lineweaver-Burk plot, Km and $V_{max}$ for acetyl-CoA were calculated to be $17.5\;{\mu}M$ and 21 umoles/min/mg, respectively and those for glyoxylate to be $7.7\;{\mu}M$ and $35.7\;{\mu}moles/min/mg$, respectively. The activity of this enzyme was inhibited by glycolate with $16.8\;{\mu}M$ of Ki, and oxalate with $25\;{\mu}M$ of Ki, competitively. Some metabolic intermediates such as citrate, isocitrate and succinate, also inhibited the enzyme activity. Iodoacetamide and p-chloromercuriphenylsulfonic acid strongly inhibited the enzyme acitivity, suggesting that there may be a reactive thiol group(s) at or near the active site. The purified malate synthase was immunogenic in rabbit and Ouchterlony double diffusion analysis revealed a single precipitation line with the enzyme. 말론산을 유일한 탄소원으로 이용하여 자란 Pseudomonas fluorescens에서 말산 생성효소가 유도되었다. 이 미생물로부터 황산암모니움 분별침전, DEAE-Sephacel 이온교환, 타트론산 Sepharose 4B, hydroxyapatite 크로마로그래피를 통하여 이 효소를 정제하였다. 정제된 효소는 단일 소단위로 되어있으며 분자량은 79,000 달톤이었다. 최적 pH는 8.5였고, pI는 4.6이었다. 아세칠 CoA에 대한 Km과 $V_{max}$는 각각 $17.5\;{\mu}M$과 $21\;{\mu}moles/min/mg$ 이었고 글리옥실산에 대하여는 각각 $7.7\;{\mu}$과 $35.7\;{\mu}moles/min/mg$이었다. 옥살산과 글리콜산은 경쟁적으로 효소활성을 방해하였고 이에 대한 Ki는 각각 $25\;{\mu}M$과 $16.8\;{\mu}M$이었다. 대사중간물인 시트르산, 이소시트르산과 숙신산에 의해서도 효소활성이 방해되었다. 또 요드아세트아미드, 클로로머큐리페닐설폰산이 강력하게 효소활성을 억제하는 것으로 보아 활성자리나 그 근처에 -SH기가 존재할 것이라고 예상된다. 정제된 효소는 토끼에 항체를 형성하게 하였고 이 항체와 정제된 효소사이에 단일띠를 보여주는 침전반응을 하였다.
Ahn, Taeho,Yun, Chul-Ho,Chae, Ho Zoon,Kim, Hyung-Ryong,Chae, Han-Jung Blackwell Publishing Ltd 2009 FEBS JOURNAL Vol.276 No.8
<P>We investigated the functional activity of recombinant Bax inhibitor-1 reconstituted into liposomes. When proteoliposomes were suspended in acidic solutions, encapsulated Ca<SUP>2+</SUP> was released from the membranes, as previously suggested [Kim HR, Lee GH, Ha KC, Ahn T, Moon JY, Lee BJ, Cho SG, Kim S, Seo YR, Shin YJ <I>et al.</I> (2008) <I>J Biol Chem</I><B>283</B>, 15946–15955]. Concomitantly, proton ions were internalized when assayed using the time-dependent change in the fluorescence of the pH-sensitive dye oxonol V entrapped in the proteoliposomes. The influx of proton ions was confirmed by observing tritium accumulation in the membranes. However, the external acidity of the membranes <I>per se</I> did not induce proton ion influx without internalized Ca<SUP>2+</SUP>. These results suggest that reconstituted Bax inhibitor-1 has a Ca<SUP>2+</SUP>/H<SUP>+</SUP> antiporter-like activity.</P>
Characterization of Haemophilus influenzae Peroxiredoxins
Hwang, Young-Sun,Chae, Ho-Zoon,Kim, Kang-Hwa Korean Society for Biochemistry and Molecular Biol 2000 Journal of biochemistry and molecular biology Vol.33 No.6
Two open reading frames of Haemophilus influenzae, HI0572 and HI0751, showing homology to a yeast thioredoxin peroxidase II (TPx II) and an E. coli thiol peroxidase $P_{20}$, respectively, were cloned and expressed in E. coli, and then the proteins were subsequently purified and characterized. HI0751 protein showed the thioredoxin (Trx)-dependent peroxidase activity, whereas HI0572 protein showed glutathione-dependent peroxidase. The HI0572 is the first peroxiredoxin with glutathione peroxidase activity rather than thioredoxin peroxidase. Purified HI0572 and HI0751 proteins protected specifically the inactivation of glutamine synthetase by metal catalyzed oxidation (MCO) systems composed of $Fe^{3+}$, $O_2$ and mercaptans such as dithiothreitol, ${\beta}-mercaptoethanol$ and glutathione (GSH). Unlike the HI0751 protein, the HI0572 protein was more effective in protecting glutamine synthetase from inactivation by the $GSH/Fe^{3+}/O_2$ system. It seems that these unique properties of the HI0572 protein are due to the structure containing a glutaredoxin domain at it's C-terminal in addition to a peroxiredoxin domain.