Probe of the (Ca 2+ + Mg 2+)-ATPase in erythrocyte membranes of cystic fibrosis patients

Abstract

There are several conflicting reports regarding a defective (Ca 2+ + Mg 2+)-ATPase in tissues from cystic fibrosis (CF) patients. The work presented in this paper represents an independent assessment of (Ca 2+ + Mg 2+)-ATPase function in CF and, at the same time, provides a somewhat more detailed analysis of the enzyme from CF patients than has previously existed. We found no significant differences in either the K m value for Ca 2+ or the V m value of the (Ca 2+ + Mg 2+)-ATPase in membrane preparations from CF patients and control subjects when the red cell membranes were prepared by methods which utilize Tris-glycylglycine-Mg 2+ buffers. In contrast, the V m value of the (Ca 2+ + Mg 2+)-ATPase in the preparations from CF patients was found to be lower than that from control subjects when the membranes were prepared by a series of washes with EDTA-containing buffers (i.e., 1–10 mmol/l EDTA). The EDTA treatment, however, did not produce any significant difference in the K m between the two groups. The fluorescent ATP analogue, trinitrocyclohexadienylidine-ATP, appeared to interact with erythrocyte ghosts as evidenced from an enhancement of its fluorescence. This enhancement was greater in control preparations than in samples from CF patients. In addition, the kinetic profiles, with respect to ATP, were quite different between the two enzyme populations. The overall results suggest that the lower rate of ATP hydrolysis observed with membranes from CF patients may reflect an impaired utilization of the substrate by the (Ca 2+ + Mg 2+)-ATPase from these individuals.