Estimation of ABCB1 concentration in plasma membrane
| Autor: | Eliska Ruzickova, Yasuhisa Kimura, Petr Mlejnek, Katerina Cizkova, Petr Dolezel, Petr Kosztyu |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Molar concentration ATP Binding Cassette Transporter Subfamily B Cell Survival Cell Blotting Western Fluorescent Antibody Technique chemical and pharmacologic phenomena ATP-binding cassette transporter Biochemistry 03 medical and health sciences 0302 clinical medicine hemic and lymphatic diseases medicine Humans Molecular Biology P-glycoprotein biology Chemistry Cell Membrane Transporter Cell Biology 030104 developmental biology medicine.anatomical_structure Membrane 030220 oncology & carcinogenesis biology.protein Biophysics RNA Interference K562 Cells Quantitative analysis (chemistry) K562 cells |
| Zdroj: | Journal of cellular biochemistry. 120(10) |
| ISSN: | 1097-4644 |
| Popis: | The interaction between ABCB1 transporter and its substrates takes place in cell membranes but the available data precludes quantitative analysis of the interaction between transporter and substrate molecules. Further, the amount of transporter is usually expressed as a number of ABCB1 molecules per cell. In contrast, the substrate concentration in cell membranes is estimated by determination of substrate-lipid partition coefficient, as examples. In this study, we demonstrate an approach, which enables us to estimate the concentration of ABCB1 molecules within plasma membranes. For this purpose, human leukemia K562 cells with varying expression levels of ABCB1 were used: drug selected K562/Dox and K562/HHT cells with very high transporter expression, and K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with gradually decreased expression of ABCB1 derived from K562/Dox cells using RNA interference technology. First, we determined the absolute amount of ABCB1 in cell lysates using immunoblotting and recombinant ABCB1 as a standard. We then determined the relative portion of transporter residing in the plasma membrane using immunohistochemistry in nonpermeabilized and permeabilized cells. These results enabled us to estimate the concentration of ABCB1 in the plasma membrane in resistant cells. The ABCB1 concentrations in the plasma membrane of drug selected K562/Dox and K562/HHT cells containing the highest amount of transporter reached millimolar levels. Concentrations of ABCB1 in the plasma membrane of resistant K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with lower transporter expression were proportionally decreased. |
| Databáze: | OpenAIRE |
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