Inositol phospholipid turnover is enhanced during mitogenic stimulation of cells by growth factors and the breakdown of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) may be important in triggering cell proliferation. PtdInsP2 also binds actin-binding proteins to regulate their activity, but it is not yet understood how this control is achieved. The protein alpha-actinin from striated muscle contains large amounts of endogenous PtdInsP2, whereas that from smooth muscle has only a little but will bind exogenously added PtdInsP2. In vitro alpha-actinin binds to F-actin and will crosslink actin filaments, increasing the viscosity of F-actin solutions. We report here that alpha-actinin from striated muscle is an endogenous PtdInsP2-bound protein and that the specific interaction between alpha-actinin and PtdInsP2 regulates the F-actin-gelating activity of alpha-actinin. Although the F-actin-gelating activity of alpha-actinin from smooth muscle is much reduced compared with that from striated muscle, exogenous PtdInsP2 can enhance the activity of smooth muscle alpha-actinin to the level seen in striated muscles. These results show that PtdInsP2 is present in striated muscle alpha-actinin and that it is necessary for alpha-actinin to realize its maximum gelating activity.

Physarum actinin previously isolated [Hatano, S., & Owaribe, K. (1976) in Cell Motility (Goldman, R., Pollard, T., & Rosenbaum, J., Eds.) Vol. 3, Book B, p 499, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY] was found to be a 1:1 complex of actin and fragmin which is a regulatory factor in the formation of actin filaments. Since fragmin did not contain a cysteine residue, it was purified from the complex by the selective cleavage of actin with 2-nitro-5-thiocyanobenzoic acid, followed by column chromatography. Fragmin had nearly the same molecular weight as actin, but had a quite different amino acid composition. When added to G-actin before polymerization, fragmin accelerated the initial viscosity increase of actin solutions induced by salts, but kept the final viscosity much lower than normal F-actin. When added to F-actin after polymerization, fragmin drastically reduced the viscosity of actin solutions. In both cases, the final products of reaction of fragmin with actin were short F-actin filaments. The number average length of the filaments decreased with the increasing molar ratio of fragmin to actin, and the length distribution was always exponential. Fragmin required for its activity a concentration of free Ca2+ higher than 10(-6) M. When the concentration of free Ca2+ was lower than 10(-7) M, fragmin affected neither actin polymerization nor F-actin. The regulation by Ca2+ was reversible.

When Asterias or Thyone sperm come in contact with egg jelly, a long process which in Thyone measures up to 90 µm in length is formed from the acrosomal region . This process can be generated in less than 30 s . Within this process is a bundle of microfilaments . Water extracts prepared from acetone powders of Asterias sperm contain a protein which binds rabbit skeletal muscle myosin forming a complex whose viscosity is reduced by ATP . Within this extract is a protein with the same molecular weight as muscle actin . It can be purified either by collecting the pellet produced after the addition of Mg++ or by reextracting an acetone powder of actomyosin prepared by the addition of highly purified muscle myosin to the extract . The sperm actin can be polymerized and by electron microscopy the polymer is indistinguishable from muscle F-actin . The sperm actin was shown to be localized in the microfilaments in the acrosomal processes by : (a) heavy meromyosin binding in situ, (b) sodium dodecyl sulfate (SDS) gel electrophoresis of the isolated acrosomal processes and a comparison to gels of flagella which contain no band corresponding to the molecular weight of actin, and (c) SDS gel electrophoresis of the extract from isolated acrosomal caps . Since the precursor for the microfilaments in the unreacted sperm appears amorphous, we suspected that the force for the generation of the acrosomal process is brought about by the polymerization of the sperm actin . This supposition was confirmed, for when unreacted sperm were lysed with the detergent Triton X-100 and the state of the actin in the sperm extract was analyzed by centrifugation, we determined that at least 80% of the actin in the unreacted sperm was in the monomeric state .