Fast, efficient reconstitution of the cyclooxygenases into proteoliposomes
| Autor: | Zahra MirAfzali, Jeffrey R. Leipprandt, John McCracken, David L. DeWitt |
|---|---|
| Rok vydání: | 2005 |
| Předmět: |
chemistry.chemical_classification
Liposome Vesicle Biophysics Phospholipid Immunogold labelling Chemical Fractionation Spodoptera Biochemistry Cell Line chemistry.chemical_compound Enzyme chemistry Membrane protein Cyclooxygenase 2 Prostaglandin-Endoperoxide Synthases Liposomes Cyclooxygenase 1 Animals lipids (amino acids peptides and proteins) Lipid bilayer Molecular Biology Integral membrane protein |
| Zdroj: | Archives of Biochemistry and Biophysics. 443:60-65 |
| ISSN: | 0003-9861 |
| DOI: | 10.1016/j.abb.2005.08.014 |
| Popis: | To study the physical and catalytic properties of purified membrane proteins, it is often necessary to reconstitute them into lipid bilayers. Here, we describe a fast efficient method for the direct incorporation of cyclooxygenase-1 and -2 (COX-1 and -2) isozymes into liposomes without loss of activity. Purified COX-1 and -2 spontaneously incorporate into large unilamellar vesicles produced from a mixture of DOPC:DOPS (7:3) that has been doped with oleic acid. When incorporation was measured by comparing cyclooxygenase activity to total phospholipid in the proteoliposomes, molar reconstitution ratios of 1000:1 (phospholipid:COX) were obtained. Electron paramagnetic resonance spectroscopic spin counting analysis of proteoliposomes formed with nitroxide spin-labeled COX-2 gave a nearly identical phospholipid:COX ratio, confirming that incorporation had no effect on enzyme activity, and demonstrating that the efficiency of protein incorporation is sufficient for EPR spectroscopic analysis. The spontaneous incorporation of cyclooxygenase into intact liposomes allows only insertion into the outer leaflet for this monotopic enzyme, an orientation confirmed by immunogold staining of the proteoliposomes. This method of reconstitution into liposomes may be generally applicable to the class of monotopic integral membrane proteins typified by the cyclooxygenase isozymes. |
| Databáze: | OpenAIRE |
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