Skeletal Alpha-actinin Research Articles

Exchange of alpha-actinin in isolated rigor myofibrils.

In the current study, the process of alpha-actinin binding to the myofibrillar Z-line was investigated to determine its mechanism. Pretreatment of rigor myofibrils with unlabeled alpha-actinin did not prevent or slow the incorporation of fluorescein skeletal alpha-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 alpha-actinin incorporation and measures of total myofibrillar alpha-actinin. These results showed that there was no change in myofibrillar alpha-actinin content when up to 15% of the total alpha-actinin was the labeled protein. Measurement of the time-course of fluorescein alpha-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 alpha-actinin concentration (2.5-160 nM) and had an apparent rate of 0.008-0.016 min(-1). Pretreatment of myofibrils with fluorescein alpha-actinin followed by incubation with unlabeled alpha-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 alpha-actinin. Thus, the dissociation rate for the in situ interaction of alpha-actinin with actin appears to be three orders of magnitude slower than that determined from solution studies.

Primary structure of chicken skeletal muscle and fibroblast alpha-actinins deduced from cDNA sequences.

The complete 897-amino-acid sequence of chicken skeletal muscle alpha-actinin and the 856-amino-acid sequence (97% of the entire sequence) of chicken fibroblast alpha-actinin have been determined by cloning and sequencing the cDNAs. Genomic Southern analysis with the cDNA sequences shows that skeletal and fibroblast alpha-actinins are encoded by separate single-copy genes. RNA blot analyzes show that the skeletal alpha-actinin gene is expressed in the pectoralis muscle and that the fibroblast gene is expressed in the gizzard smooth muscle as well as in the fibroblast. The deduced skeletal alpha-actinin molecule has a calculated Mr of 104 x 10(3), and each alpha-actinin can be divided into three domains: (1) the NH2-terminal highly conserved actin-binding domain, which shows similarity to the product of the Duchenne's muscular dystrophy locus; (2) the middle rod-shaped dimer-forming domain, which contains the spectrin-type repeat units; and (3) the COOH-terminal two EF-hand consensus regions. Comparison of the skeletal alpha-actinin sequence with the fibroblast and smooth muscle alpha-actinin sequences demonstrated that the EF-hand structure was conserved in all of these alpha-actinin sequences, despite the reported variability of the Ca2+ sensitivities of the actin-gelation by various alpha-actinin isoforms.