tailieunhanh - Báo cáo Y học: Properties of the Na+/H+ exchanger protein Detergent-resistant aggregation and membrane microdistribution

The Na + /H + exchanger is a ubiquitous membrane protein of bacteria, plants and mammals. The first isoform discov-ered (NHE1) is present on the mammalian plasma mem-brane and transports oneH + out of cells inexchange for one extracellular Na + . With solubilization in standard SDS/ PAGE buffer, this protein had a high tendency to aggregate when subjected to elevated temperature. The aggregates were stable and did not dissociate in high concentrations of SDSor 2-mercaptoethanol. | Eur. J. Biochem. 269 4887-4895 2002 FEBS 2002 doi Properties of the Na H exchanger protein Detergent-resistant aggregation and membrane microdistribution Bonnie L. Bullis1 Xiuju Li Carmen V. Rieder1 Dyal N. Singh2 Luc G. Berthiaume1 3 and Larry Fliegel1 Departments of 1 Biochemistry CIHR Membrane Protein Group 2Anatomy and 3Cell Biology Faculty of Medicine and Dentistry University of Alberta Edmonton Alberta Canada The Na H exchanger is a ubiquitous membrane protein of bacteria plants and mammals. The first isoform discovered NHE1 is present on the mammalian plasma membrane and transports one H out of cells in exchange for one extracellular Na . With solubilization in standard SDS PAGE buffer this protein had a high tendency to aggregate when subjected to elevated temperature. The aggregates were stable and did not dissociate in high concentrations of SDS or 2-mercaptoethanol. We examined the distribution of the Na H exchanger within membrane subfractions. The Na H exchanger was found both in caveolin-containing fractions and in lesser amounts in higher density membrane fractions where the bulk of proteins were contained. Treatment with cytochalasin D caused only a minor reduction of the amount of Na H exchanger present in caveolin- enriched fractions suggesting an intact cytoskeleton was not important for NHE1 localization to these microdomains. Treatment of cells with methyl b-cyclodextrin had a small stimulatory effect on Na H exchanger activity and reduced the amount of Na H exchanger in low density membrane fractions. Our study demonstrates that SDS cannot maintain the protein in a monomeric state suggesting that strong hydrophobic interactions are responsible for this temperature dependent aggregation behavior. In addition a large proportion of the Na H exchanger protein is found to be enriched in low density caveolin-containing fractions. Keywords caveolin intermolecular hydrophobic interactions lipid rafts Na H exchanger .