Interaction of plant profilin with mammalian actin
1994
Giehl, K. | Valenta, R. | Rothkegel, M. | Ronsiek, M. | Mannherz, H.G. | Jockusch, B.M.
The mode of interaction of birch and bovine profilins with actin was compared using a number of techniques. Birch profilin was purified from pollen or as a recombinant protein from Escherichia coli, using poly(L-proline) affinity chromatography and a monoclonal antibody for the identification of the isolated product. On two-dimensional gels, the genuine and recombinant proteins were identical in molecular mass and isoelectric point and revealed that birch profilin, in contrast to the basic profilins found in mammals, is an acidic protein, analogous to maize profilins. Bovine profilin was obtained from calf thymus. In viscometric assays, the birch protein was seen to modulate actin filament formation analogous to animal profilin. Birch profilin increased the critical concentration required for muscle and brain actin polymerization in a concentration-dependent manner, supporting the notion of the formation of a heterologous complex between the plant protein and animal actin. The effect was Mg2+-sensitive, as had been described for homologous complexes. The dissociation constants obtained for the plant/vertebrate and the vertebrate/vertebrate system were both in the micromolar range. The affinity of birch profilin for muscle actin was slightly lower than that for nonmuscle (brain) actin. A binary complex of birch profilin and skeletal muscle actin could be isolated by gel chromatography. Cross-linking experiments with actin, birch profilin, the G-actin binding peptide thymosin beta 4 and gelsolin segment 1, the N-terminal fragment of an actin capping protein, showed that profilin competed with thymosin beta 4, but had no effect on segment 1 binding to actin. These data indicate that the actin-binding domains in plant and animal profilins are functionally highly conserved, although the overall sequence similarity is less than 25%.
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