Kinetic properties of DM-nitrophen binding to calcium and magnesium.
Caged-Ca(2+) compounds such as nitrophenyl-EGTA (NP-EGTA) and DM-nitrophen (DMn) are extremely useful in biological research, but their use in live cells is hampered by cytoplasmic [Mg(2+)]. We determined the properties of Ca(2+) release from NP-EGTA and DMn by using Oregon green BAPTA-5N to measure changes in [Ca(2+)] after ultraviolet flash photolysis in vitro, with or without Mg(2+) present. A large fraction (65%) of NP-EGTA, which has a negligible Mg(2+) affinity, uncages with a time constant of 10.3 ms, resulting in relatively slow increases in [Ca(2+)]. Uncaging of DMn is considerably faster, but DMn has a significant affinity for Mg(2+) to complicate the uncaging process. With experimentally determined values for the Ca(2+) and Mg(2+) binding/unbinding rates of DMn and NP-EGTA, we built a mathematical model to assess the utility of NP-EGTA and DMn in rapid Ca(2+)-uncaging experiments in the presence of Mg(2+). We discuss the advantages and disadvantages of using each compound under different conditions. To determine the kinetics of Ca(2+) binding to biologically relevant Ca(2+) buffers, such as Ca(2+)-binding proteins, the use of DMn is preferable even in the presence of Mg(2+).
Faas
Guido C
GC
Department of Neurology, University of California, Los Angeles, 90095, USA.
Karacs
Kinga
K
Vergara
Julio L
JL
Mody
Istvan
I
D000085:Acetates / Q000378:metabolism*; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D005029:Ethylenediamines / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008954:Models, Biological; D010782:Photolysis
D000085:Acetates; D005029:Ethylenediamines; C057014:DM-nitrophen; C085112:2-nitrophenyl-EGTA; D004533:Egtazic Acid; D008274:Magnesium; D002118:Calcium