• Title/Summary/Keyword: protein purification

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Purification and Physicochemical Characterization of a Recombinant Phospholipid Hydroperoxide Glutathione Peroxidase from Oryza sativa

  • Wang, Zebin;Wang, Feng;Duan, Rui;Liu, Jin-Yuan
    • BMB Reports
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    • v.40 no.3
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    • pp.412-418
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    • 2007

  • Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an unique antioxidant enzyme that directly reduces lipid hydroperoxides in biomembranes. In the present work, the entire encoding region for Oryza sativa PHGPx was expressed in Escherichia coli M15, and the purified fusion protein showed a single band with 21.0 kD and pI = 8.5 on SDS- and IFE-PAGE, respectively. Judging from CD and fluorescence spectroscopy, this protein is considered to have a well-ordered structure with 12.2% $\alpha$-helix, 30.7%$\beta$-sheet, 18.5% $\delta$-turn, and 38.5% random coil. The optimum pH and temperature of the enzyme activity were pH 9.3 and 27$^{\circ}C$. The enzyme exhibited the highest affinity and catalytical efficiency to phospholipid hydroperoxide employing GSH or Trx as electron donor. Moreover, the protein displayed higher GSH-dependent activity towards t-Butyl-OOH and $H_2O_2$. These results show that OsPHGPx is an enzyme with broad specificity for hydroperoxide substrates and yielded significant insight into the physicochemical properties and the dynamics of OsPHGPx.

  • https://doi.org/10.5483/BMBRep.2007.40.3.412 인용 PDF

Characterization and Partial Purification of Storage Protein-i Receptor in the Fat Body of Hyphantria cunea (미국흰불나방 지방체에서 저장단백질-1의 수용체의 특성과 부분정제)

  • 채권석;여성문;김학렬
    • The Korean Journal of Zoology
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    • v.38 no.4
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    • pp.490-497
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    • 1995

  • In vitro tissue culture of fat body of Hyphantria cunea in the medium containing [35S]-methionine reveaied that storage protein-i (SP-1) is taken up into fat body of prepupae and 1-day-old pupae. Using Western blotting and ligand binding method, we were able to identify the protein band of the SP-1 receptor protein. For the partial purification, the membrane proteins of fat body cells were solubilixed with 1% Triton X-1OO and applied to anion exchange chromatography. The results revealed the molecular weight of the receptor protein to be about 80 kl)a in SDSPAGE, and the P1 was estimated to be about 6.1. The mobility of the receptor protein in 8D8-PAGE was highly dependent on both temperature during electrophoresls and the condition of samples whether they were in reducing or nonreducing.

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Purification and Sidedness of Sarcolemma from Canine Ventricle (개 심실 형질막의 분리 및 그 방향성에 관한 연구)

  • 이신웅;구정옥;이정수
    • YAKHAK HOEJI
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    • v.30 no.1
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    • pp.31-41
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    • 1986

  • Sarcolemmal membrane fraction from canine ventricle was isolated from the discarded pellet after the first homogenization in the isolation procedure of sarcoplasmic reticulum (Method 1) and the protein yield, purity, and sidedness of this preparation were compared to those of sarcolemmal fraction prepared by method of Lee et al. (Method 2) and a slight modification of original protocol of Jones et al. (Method 3). Method 1 differed from Method 2 essentially only in that vigorous homogenization was carried out by omnimixer and homogenization medium containing 30mM Tris-maleate was used in the first step. The sarcolemmal fraction was enriched from 45 to 50 and 29-fold in [$^3H$] ouabain, [$^3H$] DHA, [$^3H$] QNB binding and $Na^+$, $K^+$-ATPase activity, respectively, compared to homogenate. Total $Na^+$, $K^+$-ATPase activity of highly sarcolemma enriched fraction was 144.6$\pm$16.4$\mu\textrm{mol}$ Pi/mg protein/hr, which was about 85%, of total ATPase activity, and the yield of the preparation was 15.7 mg protein per 100g of starting ventricular tissue. The sarcolemmal preparation supported $^{45}Ca^{2+}$-uptake in the presence of ATP but this uptake was not dependent on oxalate. Sarcolemmal $Na^+$, $K^+$-ATPase activity and detectable [$^3H$] ouabain binding were increased about 32% and 35%, respectively, by pretreatment of sarcolemmal fraction with optimal concentration of sodium dodecylsulfate (0.3-0.4mg/mg protein), suggesting that this preparation contained about 24% of sealed rightside-out vesicles, 26% of sealed inside-out vesicles, and 5001o of freely permeable (leaky) form. This procedure showed the highest protein yield and leaky population, compared to Method 2 and 3. On the other hand, sarcolemmal fraction prepared by Method 2 and 3 showed low value in protein yield but comtained high population of inside-out (46%) and rightside-out (49%) vesicles, respectively, compared to present procedure (Method 1). The results indicate that vigorous homogenization decreases the population of sealed sarcolemmal vesicles but increases the sarcolemmal protein yield per gram tissue and that this procedure is available for further purification of sarcolemmal fraction and for the receptor binding study of sarcolemma.

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