Characterization of the 110-kdalton actin-calmodulin-, and membrane-binding protein from microvilli of intestinal epithelial cells.

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Abstract
One of the major proteins of the chicken intestinal microvillus is a calmodulin-binding protein of 105-110 Kdaltons (K) which has been tentatively identified as the bridge linking the microvillar filament bundle laterally to the membrane. Isolated, membrane-intact brush borders were treated with ATP, and solubilization of the 110K protein, calmodulin (CM), myosin and lesser amounts of several other cytoskeletal proteins was obtained. EM of ATP-extracted brush borders showed loss of the linkers between the actin filament bundle and the microvillar membrane, with ballooning of the membrane away from the filament bundle, particularly at the tip end. In brush borders treated with Ca and trifluoperazine to solubilize CM, precise arrangement and morphology of lateral bridges was unperturbed, but ATP treatment would no longer solubilize the 110K protein. Associated CM is apparently necessary for the ATP-induced solubilization of the 110K protein. A 110-K protein-CM complex, with 110 protein:CM ratios of 1:1-2, was partially purified from ATP-extracts of brush borders by a combination of gel filtration and hydroxylapatite chromatography. The 110K protein-CM complex is an irregular, elongated molecule that ranged in size from 5 .times. 8 nm to 8 .times. 14 nm, with a Stokes'' radius of 6.1 nm. This 110K protein-CM complex exhibited no Mg2+-ATPase activity and no detectable myosin light chain kinase activity. In cosedimentation assays, the 110K protein-CM bound to F-actin in the absence but not the presence of ATP. Both the interaction of the complex with actin and the binding of CM to the 110K protein were Ca-independent. Negative stains of F-actin and 110K protein-CM in the absence of ATP showed loosely organized aggregates of actin while the 110K protein-CM complex coating the surface of the filaments. On the basis of these data, and in agreement with previous calculations, it is suggested that the lateral bridge of the microvillus is composed of a dimer of the 110K protein with 4 associated calmodulins.