Calcium pump kinetics determined in single erythrocyte ghosts by microphotolysis and confocal imaging.
The activity of the plasma membrane calcium pump was measured in single cells. Human red blood cell ghosts were loaded with a fluorescent calcium indicator and either caged calcium and ATP (protocol A) or caged ATP and calcium (protocol B). In a suitably modified laser scanning microscope either calcium or ATP were released by a short UV light pulse. The time-dependent fluorescence intensity of the calcium indicator was then followed in single ghosts by repetitive confocal imaging. The fluorescence intensity was converted into calcium concentration, which in turn was used to derive the kinetic parameters of the calcium pump, the Michaelis-Menten constant Km, and the maximal transport rate vmax. Km and vmax values derived in this manner were 24 +/- 14 microM and 1.0 +/- 0.6 microM/(ghost s) for protocol A, and 4 +/- 3 microM and 1.0 +/- 0.6 microM/(ghost s) for protocol B, respectively. The difference between A and B is presumably caused by calmodulin, which is inactive in the experiments with protocol A. The possibilities to extend the new method to living nucleus-containing cells transiently transfected with mutants of the plasma membrane calcium pump are discussed.
Kubitscheck
U
U
Institute of Medical Physics and Biophysics, Westfälische Wilhelms University, Münster, Germany.
Pratsch
L
L
Passow
H
H
Peters
R
R
D000255:Adenosine Triphosphate / Q000097:blood; D002118:Calcium / Q000097:blood; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000528:radiation effects; D004910:Erythrocyte Membrane / Q000201:enzymology*; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal / Q000379:methods; D010782:Photolysis; D013997:Time Factors; D014466:Ultraviolet Rays
D005456:Fluorescent Dyes; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium