Cation binding to gizzard and brain alpha-actinins: Effects on structure and function

Abstract

alpha-Actinin was purified from chicken gizzards (muscle) and horse brains (nonmuscle), and their calcium binding properties were characterized. Flow dialysis revealed two binding components when gizzard alpha-actinin was dialyzed and Chelex-100 treated in 20mM Hepes, pH 7.5. The first component had 3.5 ± 1.8 (S.D.) high affinity calcium binding sites per dimer with a kd1 of 6.36 ± 0.34 x 10^-6M. The second component had 87.3 ± 7.3 low affinity sites per dimer and a kd2 of 1.66 ± 0.44 x 10^-4M. Under physiological conditions (120mM KCl, 20mM Hepes, pH 7.5) only one binding component was measured, 82.5 ± 3.3 low affinity calcium binding sites per dimer with a kd in the millimolar range (1.12 ± 0.12 x 10^-3M). Only one binding component was measured when calcium was bound to brain alpha-actinin under physiological conditions (120mM KCl, 20mM Hepes, pH 7.5), 177 ± 17 low affinity calcium binding sites per dimer with a kd of 1.65 ± 0.65 x 10^-4M. Isoelectric focusing revealed at least six different isoforms each of brain and gizzard alpha-actinins. Two-dimensional gels and calcium overlay indicated that none of these isoforms bound calcium in 120mM KCl, 10mM Imidazole-HCl pH 7.5. Increasing concentrations of KCl (0,60 and 120mM) led to a conformational change in both gizzard and brain alpha-actinins as shown by circular dichroism, bisANS fluorescence and proteolysis. Brain alpha-actinin showed a decrease in alpha-helix conformation while gizzard alpha-actinin showed a slight increase in alpha-helix conformation. No beta-sheet conformation was detected in both alpha-actinins. Under physiological KCl concentrations (120mM), increasing concentrations of magnesium or calcium had no significant effect on the secondary structure of both gizzard and brain alpha-actinins. Gizzard alpha-actinin (like other muscle isoforms) binds F-actin in a calcium-insensitive manner while brain alpha-actinin (like other nonmuscle isoforms) binds F-actin in a calcium sensitive manner. Increasing concentrations of calcium led to a decrease in viscosity of F-actin in the presence of brain alpha-actinin and had no effect on viscosity of F-actin in the presence of gizzard alpha-actinin. However this reduction in viscosity is observed only under non-physiological calcium concentrations (> 10^-7M). The differences in calcium sensitivities of nonmuscle alpha-actinins as opposed to their muscle counterparts are not due to high affinity calcium binding to nonmuscle isoforms vs. low affinity calcium binding to muscle isoforms as previously predicted from their structure, because we show that both alpha-actinins bind calcium only with a low-affinity under physiological concentrations of KCl.