Self-association of human spectrin. A thermodynamic and kinetic study.

Depending on conditions of extraction from the membrane, spectrin may be recovered either as a dimer or a tetramer. It is demonstrated that these species are linked by a simple equilibrium, and can be readily interconverted. At low temperature, however, either species is kinetically trapped, and no interconversion occurs over periods of up to several days. In the range 25 - 40 "C, equilibrium is achieved on a time scale of hours to minutes. First-order and second-order rate constants and thermodynamic parameters for the equilibrium have been determined. The large activation energy indicates that conformational effects are rate-limiting. In the absence of other proteins, no associated states higher than the tetramer are formed. Neither phosphorylation of the spectrin with endogenous kinase, nor dephosphorylation with phosphatase have any effect on the dimer-tetramer equilibrium, neither do they promote formation of higher associated states. It is therefore improbable that phosphorylation-dependent shape changes in the erythrocyte are related to the association of spectrin per se, and are likely instead to be the direct result of a spectrin-actin interaction. The dimer-tetramer equilibrium is strongly affected by ionic strength, and at low salt concentrations, such as those used to extract spectrin from membranes, the dimer becomes strongly favoured. Thus the recovery of tetramer by extraction at low temperature implies that this is the basic unit present in the membrane. Calcium and magnesium ions cause further association to higher oligomers, though only at high concentrations (in the millimolar range) of the cation.