Separation of non-muscle isoactins in the free form or as profilactin complexes

Profilactin prepared from calf spleen by the standard procedure (Carlsson, L., Nyström, L.-E., Sundkvist, I., Markey, F., and Lindberg, U. (1977) J. Mol. Biol. 115, 465-483) can be further fractionated by chromatography on hydroxyapatite into two major peaks; one containing profilin in combination with gamma-actin (PA gamma) and the other combined with beta-actin (PA beta). Both complexes were shown to consist of 1 mol each of the two proteins. The two forms of profilin, PI (intact form) and PII (lacking the COOH-terminal glutamine and tyrosine residues) (Malm, B., Larsson, H., and Lindberg, U. (1983) J. Muscle Res. Cell Motil. 4, 569-588), were found unequally distributed between the two major peaks of PA, such that PA gamma contained mainly PI and PA beta both PI and PII. The significance of this finding remains unclear. Isoelectrofocusing showed that PI from both isoforms of PA had an isoelectric point of 9.08. Profilin II gave rise to two major bands on isoelectrofocusing having isoelectric points of 9.12 and 9.18, respectively. Finally it was shown that the two isoforms of actin can be separated from each other in the absence of profilin also by chromatography on hydroxyapatite. In this case, ATP remains bound to the actins eluted from the chromatographic matrix and both actins were shown to polymerize on addition of salts as judged by viscometry.