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RISS 인기검색어
- 검색키워드
- 저자 : Theeraporn Puntheeranurak (검색결과 3 건)
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Single-Molecule AFM Studies of Substrate Transport by Using the Sodium-Glucose Cotransporter SGLT1
Theeraporn Puntheeranurak,Rolf K. H. Kinne,Hermann J. Gruber,Peter Hinterdorfer 한국물리학회 2008 THE JOURNAL OF THE KOREAN PHYSICAL SOCIETY Vol.52 No.5
In an apical membrane of epithelial cells from the small intestine and the kidney, the high-affinity Na+/D-glucose cotransporter type 1 (SGLT1) plays a crucial role in intestinal glucose absorp- tion and in renal glucose reabsorption. Here, the over-expression of rabbit SGLT1 in rbSGLT1- transfected Chinese hamster ovary (CHO) cells was first characterized using the immuno-staining method on non-permeabilized cells. The cells were then imaged with atomic force microscopy (AFM), revealing live and fixed cells strongly attached to the glass surfaces. A bioconjugate chem- istry approach was employed to functionalize the surfaces of the AFM tips with D-glucose molecules via three dierent heterobifunctional crosslinkers. The D-glucose binding site and the translocation pathway of SGLT1 were investigated by studying the interaction forces between tip-bound D-glucose and SGLT1 in live cells on the single-molecule level. Analysis of these forces suggested that a long crosslinker with a small end group might be suitable for probing the D-glucose transport pathway of SGLT1. We show that single-molecule AFM technology is a powerful method for investigating transmembrane proteins and transporter functions in live cells. In an apical membrane of epithelial cells from the small intestine and the kidney, the high-affinity Na+/D-glucose cotransporter type 1 (SGLT1) plays a crucial role in intestinal glucose absorp- tion and in renal glucose reabsorption. Here, the over-expression of rabbit SGLT1 in rbSGLT1- transfected Chinese hamster ovary (CHO) cells was first characterized using the immuno-staining method on non-permeabilized cells. The cells were then imaged with atomic force microscopy (AFM), revealing live and fixed cells strongly attached to the glass surfaces. A bioconjugate chem- istry approach was employed to functionalize the surfaces of the AFM tips with D-glucose molecules via three dierent heterobifunctional crosslinkers. The D-glucose binding site and the translocation pathway of SGLT1 were investigated by studying the interaction forces between tip-bound D-glucose and SGLT1 in live cells on the single-molecule level. Analysis of these forces suggested that a long crosslinker with a small end group might be suitable for probing the D-glucose transport pathway of SGLT1. We show that single-molecule AFM technology is a powerful method for investigating transmembrane proteins and transporter functions in live cells.
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Puntheeranurak, Theeraporn,Leetacheewa, Somphob,Katzenmeier, Gerd,Krittanai, Chartchai,Panyim, Sakol,Angsuthanasombat, Chanan Korean Society for Biochemistry and Molecular Biol 2001 Journal of biochemistry and molecular biology Vol.34 No.4
Tryptic activation of the 130-kDa Bacillus thuringiensis Cry4B $\delta$-endotoxin produced protease-resistant products of ca. 47 kDa and ca. 21 kDa. The 21-kDa fragment was identified as the N-terminal five-helix bundle (${\alpha}1-{\alpha}5$,) which is a potential candidate for membrane insertion and pore formation. In this study, we constructed the recombinant clone over-expressing this putative pore-forming (PPF) fragment as inclusion bodies in Escherichia coli. The partially purified inclusions were composed of a 23-kDa protein, which cross-reacted with Cry4B antibodies, and whose N-terminus was identical to that of the 130-kDa protein. Dissimilar to protoxin inclusions, the PPF inclusions were only soluble when the carbonate buffer, pH 9.0, was supplemented with 6 M urea. After renaturation via a stepwise dialysis, the refolded PPF protein appeared to exist as an oligomer and was structurally stable upon trypsin treatment. Unlike the 130kDa protoxin, the refolded protein was able to release entrapped glucose from liposomes, and showed comparable activity to the full-length activated toxin, although it lacks larvicidal activity These results, therefore, support the notion that the PPF fragment that consists of ${\alpha}1-{\alpha}5$ of the activated Cry4B toxin is involved in membrane pore-formation.
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(Theeraporn Puntheeranurak),(Somphob Leetacheewa),(Gerd Katzenmeier),(Chartchai Krittanai),(Sakol Panyim),(Chanan Angsuthanasombat) 생화학분자생물학회 2001 BMB Reports Vol.34 No.4
Tryptic activation of the 130-kDa Bacillus thuringiensis Cry4B δ-endotoxin produced protease-resistant products of ca. 47 kDa and ca. 21 kDa. The 21-kDa fragment was identified as the N-terminal five-helix bundle (α1-α5), which is a potential candidate for membrane insertion and pore formation. In this study, we constructed the recombinant clone over-expressing this putative poreforming (PPF) fragment as inclusion bodies in Escherichia coli. The partially purified inclusions were composed of a 23-kDa protein, which cross-reacted with Cry4B antibodies, and whose N-terminus was identical to that of the 130-kDa pmtein. Dissimilar to protoxin inclusions, the PPF inclusions were only soluble when the carbonate buffer, pH 9.0, was supplemented with 6 M urea. After renaturation via a stepwise dialysis, the refolded PPF protein appeared to exist as an oligomer and was structurally stable upon trypsin treatment. Unlike the 130kDa protoxin, the refolded protein was able to release entrapped glucose from liposomes, and showed comparable activity to the full-length activated toxin, although it lacks larvicidal activity These results, therefore, support the notion that the PPF fragment that consists of α1-α5 of the activated Cry4B toxin is involved in membrane pore-formation.
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