The structural and functional properties of the uu (2 x 97 kDa) and cc (2 x 94 kDa) isoforms of platelet a-actinin have been compared. Structural differences between aa and cc are revealed by their peptide maps, obtained from limited proteolysis, and by their immunological cross-reactivity. Both isoforms stimulate the Mg ATPase activity of actomyosin, bind to F-actin (high-speed sedimentation) and cross-link or gel actin filaments (low-speed sedimentation and viscometry), in a calcium-dependent manner. The study of the interaction with Factin indicates that the binding of 1 molecule of UN or cc a-actinin/9 -11 actin monomers is sufficient to produce maximal gelation in the presence of EGTA. CaCI, at 0.1 mM strongly inhibits the binding of U U to F-actin and weakly that of cc, while it inhibits similarly the cross-linking of either uu or cc. The cross-linking efficiency of cc is 9, 7, 1.7 and 1.3 times higher than that of uu at 4, 20, 30 and 3 7 T , respectively. The hh form (2 x 96 kDa), which is a proteolytic product of uu [Y. Gache et al. (1984) Biochem. Biophys. Rcs. Commun. 124, 877-8811, behaves roughly as aa, but the calcium sensitivity of its binding to F-actin is intermediate between that of cia and cc. These results suggest that the effect of Ca2' concentration on the binding of platelet a-actinin to F-actin may be partly dissociated from the effect on the cross-linking. Human platelet a-actinin has been characterized as a nonmuscle a-actinin [12, 131. It has been found to exist as multiple isoforms, which differ in chain length [14], iinmunological cross-reactivity and sensitivity to a Ca2 '-dependent protease [15]. In this report we describe a comparative study of the effect on F-actin of the two homodimeric isoforms of platelet a-actinin, aa and cc, as well as of the bb form, which is a proteolytic product ofaa. The uu and cc isoforms differ in the calcium sensitivity of their binding to F-actin and in their efficiency to cross-link actin filaments. MATERIALS AND METHODSIsolution of a-uctinin a-Actinin was purified from human blood platelets as previously described [13]. However, the susceptibility of the uu isoform to calcium-dependent degradation [14, 151 led us to slightly modify the isolation procedure: shortening of the extraction time by ultrasonic thawing of frozen platelets, presence of 5 mM instead of 1 mM EGTA in the extraction and dialysis buffers and of 1 mM EGTA in the DEAE chromatography elution buffers, addition of leupeptin (1 pg/ ml) to the sample applied to the hydroxyapatite column and to the eluting buffers. With these modifications, the fractions FI, FII, and FIII of the hydroxyapatite elution contained more (I subunit (Fig. 1 A)
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