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We have investigated the effects of Ca2+ on AMP deaminase from human red cells. At variance with the other known modulators, Ca2+ increased the apparent affinity for AMP without modifying the characteristic positive cooperativity of the enzyme towards the substrate. Ca2+ sensitivity was not modified by dialysis, but dilution of the haemolysate produced an activation of the enzyme similar to that induced by Ca2+. Simultaneously, the Ca2+ dependence was lost. The sensitivity to other modulators, such as ATP, diphosphoglycerate or phosphate, was not modified by dilution. Partial purification of the enzyme produced the same effects as haemolysate dilution. These results may be interpreted to mean that Ca2+ acts by antagonizing an endogenous inhibitor present in red cell lysates.

Alamethicin causes a concentration-dependent increase of [Ca2+]i in suspensions of bovine adrenal chromaffin cells loaded with fura-2. The basal levels of Cai2+ (234 +/- 37 nM; n = 4) increased to a maximum of 2347 +/- 791 nM (n = 3) with 100 micrograms/ml alamethicin. In the presence of 1 mM Cae2+ the increase reached a plateau within about 2-5 s. This increase was due to Ca2+ entry into chromaffin cells, since in the absence of Cae2+ alamethicin did not modify [Ca2+]i. This contrasts with ionomycin (1 microM) which produced a Cai2+ transient even in the absence of Cae2+. Mn2+ ions also entered chromaffin cells in the presence of alamethicin, as measured by the quenching of fura-2 fluorescence following excitation at 360 nm. Resting chromaffin cells had a measurable permeability to Mn2+ which was drastically increased by cell depolarization by K+ (50 mM) addition. This suggests that Mn2+ is able to permeate voltage-dependent Ca2+ channels. Ni2+ uptake into either resting or K(+)-stimulated chromaffin cells was undetectable, but addition of alamethicin induced rapid uptake of this cation. The alamethicin-induced entry of Ni2+ was decreased by 50 mM K+. Overall, the results are compatible with the formation by alamethicin of ion channels in chromaffin cell plasma membranes.