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Estimation of carrier density and turnover rate of the Na+/H+ exchanger in human platelets using 5-(N-methyl-N-[3H]isobutyl)- amiloride

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Rosskopf,  Dieter
Department of Physiology, Max Planck Institute of Biophysics, Max Planck Society;

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Barth,  Cornelia
Department of Physiology, Max Planck Institute of Biophysics, Max Planck Society;

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Siffert,  Winfried
Department of Physiology, Max Planck Institute of Biophysics, Max Planck Society;

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Citation

Rosskopf, D., Barth, C., & Siffert, W. (1991). Estimation of carrier density and turnover rate of the Na+/H+ exchanger in human platelets using 5-(N-methyl-N-[3H]isobutyl)- amiloride. Biochemical and Biophysical Research Communications, 176(2), 639-644. doi:10.1016/s0006-291x(05)80232-3.


Cite as: https://hdl.handle.net/21.11116/0000-0008-67C6-5
Abstract
We used the radiolabelled inhibitor of Na+/H+ exchange 5-(N-methyl-N-[3H]isobutyl)amiloride ([3H]-MIA) for assessment of the amount of N+/H+ exchanger in intact human blood platelets. The inhibition constant, KI, of unlabelled MIA toward the antiport was determined at 100 nM. Washed platelets were incubated for 5 s with different concentrations of [3H]-MIA in the presence or absence of an excess concentration of unlabelled amiloride (400μM). The platelets were rapidly centrifuged and the radioactivity in the pellet was determined. Scatchard analysis revealed one single class of specific binding sites (KD =63 nM) and a maximum binding capacity of 500 sites/cell. The turnover rate of the Na+/H+-exchanger in unstimulated platelets was estimated at 800/sat 25°C.