title: Exchange of alpha-actinin in isolated rigor myofibrils.
abstract: 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.
authors.affiliation: Anatomy Department, Indiana University Medical School, Indianapolis 46202, USA. dswartz@iupui.edu
authors.forename: D R
authors.initials: DR
authors.lastname: Swartz
mesh_terms: D000185:Actinin / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D008239:Lysine / Q000031:analogs & derivatives; D008856:Microscopy, Fluorescence; D009119:Muscle Contraction / Q000502:physiology*; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011817:Rabbits; D012518:Sarcomeres / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000185:Actinin; C013411:biocytin; D008239:Lysine