Influence of monovalent cation identity on parvalbumin divalent ion-binding properties

Rat alpha- and beta-parvalbumins have distinct monovalent cation-binding properties [HenzI et al. (2000) Biochemistry 39, 5859-5867]. beta binds two Na+ or one K+, and a binds one Na+ and no K+. Ca2+ abolishes these binding events, suggesting that the monovalent ions occupy the EF-hand motifs. This study compares alpha and beta divalent ion affinities in Na+ and K+ solutions. Solvent cation identity seriously affects alpha. In Hepes-buffered NaCl, at 5 degreesC, the macroscopic Ca2+-binding constants are 2.6 x 10(8) and 6.4 x 10(7) M-1 and the Mg2+ constants, 1.8 x 10(4) and 4.3 x 10(3) M-1. In Hepes-buffered KCl, the Ca2+ values increase to 2.9 x 10(9) and 6.6 x 10(8) M-1 and the Mg2+ values to 2.2 x 10(5) and 3.7 x 10(4) M-1. Monte Carlo simulation of a binding data-employing site-specific constants and explicitly considering Na+ binding-yields a K-Na of 630 M-1 and indicates that divalent ion-binding is positively cooperative. NMR data suggest that the lone Na+ ion occupies the CD loop. Solvent cation identity has a smaller impact on beta. In Na+, the Ca2+ constants for the EF and CD sites are 2.3 x 10(7) and 1.5 x 10(6) M-1, respectively; the Mg2+ constants are 9.2 x 10(3) and 1.7 x 10(2) M-1. In K+, these values shift to 3.1 x 10(7) and 3.8 x 10(6) M-1 and the latter to 1.4 x 10(4) and 2.9 x 10(2) M-1. These data suggest that parvalbumin divalent ion affinity, particularly that of rat alpha, can be significantly attenuated by increased intracellular Na+ levels.