A fluorescent indicator for measuring cytosolic free magnesium.
The previously developed chelator O-aminophenol-N,N,O-triacetic acid (APTRA) (L. A. Levy, E. Murphy, B. Raju, and R. E. London. Biochemistry 27: 4041-4048, 1988) has been modified to yield a fluorescent analogue which can be utilized as an intracellular probe for ionized Mg2+. The fluorescent analogue, FURAPTRA, with a magnesium dissociation constant of 1.5 mM, is structurally analogous to the calcium chelator fura-2 and exhibits a similar excitation shift on magnesium complexation. Hence, data on the intracellular Mg2+ concentration can be obtained using an analogous ratio method. The acetoxymethyl form of the chelator is readily loaded into cells and has been used to determine a cytosolic free Mg2+ concentration of 0.59 mM for isolated rat hepatocytes. As a consequence of the relatively high levels of cytosolic Mg2+, the problem of ion buffering is much less severe than for the analogous calcium indicators.
Raju
B
B
Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Murphy
E
E
Levy
L A
LA
Hall
R D
RD
London
R E
RE
D000818:Animals; D001572:Benzofurans / Q000138:chemical synthesis*; D002478:Cells, Cultured; D003600:Cytosol / Q000032:analysis; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D016257:Fura-2 / Q000031:analogs & derivatives*; D007202:Indicators and Reagents; D008099:Liver / Q000032:analysis / Q000166:cytology*; D008274:Magnesium / Q000032:analysis*; D008297:Male; D010080:Oxazoles / Q000138:chemical synthesis*; D051381:Rats; D011919:Rats, Inbred Strains; D013050:Spectrometry, Fluorescence / Q000379:methods
D001572:Benzofurans; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D010080:Oxazoles; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D008274:Magnesium; D016257:Fura-2