Exchange of α-actinin in isolated rigor myofibrils

Abstract

In the current study, the process of α-actinin binding to the myofibrillar Z-line was investigated to determine its mechanism. Pretreatment of rigor myofibrils with unlabeled α-actinin did not prevent or slow the incorporation of fluorescein skeletal α-actinin into myofibrils suggesting that incorporation was not the filling of empty binding sites but rather an exchange reaction. Further support for this was obtained using quantitative measures of labeled α-actinin incorporation and measures of total myofibrillar α-actinin. These results showed that there was no change in myofibrillar α-actinin content when up to 15% of the total α-actinin was the labeled protein. Measurement of the time-course of fluorescein α-actinin incorporation by quantitative fluorescence microscopy showed that the increase in Z-line fluorescence was well described by a rapid (unresolved) incorporation of fluorescence followed by a much slower phase. The slower phase was independent of fluorescein α-actinin concentration (2.5–160 nM) and had an apparent rate of 0.008–0.016 min⁻¹. Pretreatment of myofibrils with fluorescein α-actinin followed by incubation with unlabeled α-actinin resulted in a decrease in Z-line fluorescence with an apparent rate of 0.021 min⁻¹. The slow phase was interpreted as representing the dissociation rate of intrinsic Z-line α-actinin. Thus, the dissociation rate for the in situ interaction of α-actinin with actin appears to be three orders of magnitude slower than that determined from solution studies.