| Autor: |
Mwanjewe J; Departments of Medicine, Chemistry, and Psychiatry and Behavioural Neurosciences, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada., Martinez R, Agrawal P, Samson SE, Coughlin MD, Brassard P, Grover AK |
| Abstrakt: |
Non-transferrin-bound iron (NTBI) uptake has been reported to follow two pathways, Ca(2+)-dependent and Ca(2+)-independent (Wright, T. L., Brissot, P., Ma, W. L., and Weisiger, R. A. (1986) J. Biol. Chem. 261, 10909-10914; Sturrock, A., Alexander, J., Lamb, J., Craven, C. M., and Kaplan, J. (1990) J. Biol. Chem. 265, 3139-3145). Studies reporting the two pathways have ignored the weak interactions of Ca(2+) with the chelator nitrilotriacetate (NTA) and the reducing agent ascorbate. These studies used a constant ratio of total Fe(2+) to NTA with and without Ca(2+). We observed Ca(2+) activation of NTBI uptake in PC12 cells with the characteristics reported for other cells upon using 1 mm ascorbate and a constant ratio of total Fe(2+) to NTA with or without Ca(2+). However, Ca(2+) did not affect NTBI uptake in solutions without NTA. We then determined conditional stability constants for NTA binding to Ca(2+) and Fe(2+) by potentiometry under conditions of NTBI uptake experiments (pH, ionic strength, temperature, ascorbate, total Fe(2+), and total Ca(2+) concentrations). In solutions based on these constants and taking Ca(2+) chelation into account, Ca(2+) did not affect NTBI uptake over a range of free Fe(2+) concentrations. Thus, the Ca(2+) activation of NTBI uptake observed using the constant total Fe(2+) to NTA ratio was because of Ca(2+)-NTA chelation rather than an activation of the NTBI transporter itself. It is suggested that the previously reported Ca(2+) dependence of NTBI uptake be re-evaluated. |
