Brush-border calmodulin. A major component of the isolated microvillus core.

Abstract

Calmodulin is present in brush borders isolated from intestinal epithelial cells and is one of the major components of the microvillar filament bundle . Calmodulin was purified from either demembranated brush borders or microvilli by a simple boiling procedure . The boiled supernate derived from the microvillus cores contained one major polypeptide of 20,000 daltons . The supernate from the brushborder preparation contained the 20,000-dalton subunit and a second protein of 30,000 daltons . The 20,000-dalton subunit has been identified as calmodulin by several criteria: (a) heat resistance, (b) comigration with brain calmodulin on alkaline urea gels and SDS gels, both cases in which the 20,000-dalton protein, like calmodulin, exhibits a shift in electrophoretic mobility in the presence of Ca", and (c) 4-5-fold activation of 3',5'-cyclic nucleotide phosphodiesterase in the presence but not the absence of Ca" . With a cosedimentation assay it was determined that brush-border calmodulin does not bind directly to actin . In the presence of Ca" (>5 X 10-' M) there was a partial release of calmodulin from the microvillus core, along with a substantial conversion of microvillus actin into a nonpelletable form. The dissociation of calmodulin was reversed by removal of Ca" . If microvillus cores were pretreated with phalloidin, the Ca"-induced solubilization of actin was prevented, but the partial dissociation of calmodulin still occurred. The molar ratio of calmodulin:actin is 1 :10 in the demembranated brush border and 1 :2-3 in the microvillus core . No calmodulin was detected in the detergent-solubilized brush-border membrane fraction .Movements of microvilli on the apical, brush-border surface of cells lining the intestine may facilitate the absorptive functions of this epithelium . In vivo observations of brush-border motility have been recorded (32, 38), although adequate documentation of these technically difficult observations is still lacking . Studies on the isolated brush border indicate that this organelle is a highly organized, motile apparatus comprised of actin, myosin, and various associated proteins that are organized in a functional configuration analogous to that determined for actin and myosin in striated muscle (5,(25)(26)(27) . In vitro experiments on contractile models of the brush border have helped to 916 reveal possible molecular mechanisms for microvillar movement (25,30) . The addition of MgATP and micromolar levels of free calcium ions to demembranated brush borders isolated from chicken intestine causes rapid retraction of the microvillar cores into and through the terminal web (25). Presumably, this contractile mechanism coupled to a relaxation mechanism, perhaps involving the extensive linkages of the microvillus cores to the plasma membrane (6,24,25,27,28), is responsible for cyclic movements of microvilli in vivo.We hope to determine the molecular basis for the observed calcium sensitivity of this contrac-J . CELL BIOLOGY

Purification of calmodulin from Chlamydomonas: calmodulin occurs in cell bodies and flagella.

“…Interestingly, the fact that calmodulin appeared to be present in axonemes isolated in the presence of EGTA or EDTA indicates that some of the calmodulin remains bound to an axonemal component even in the absence of Ca". An analogous situation exists in the brush border of chicken intestinal epithelial cells, where calmodulin is tightly associated with the microvillus core in the absence of Ca" (23) .…”

Immunocytochemical demonstration for caldesmon and actin in the striated and smooth muscle cells and non-muscular cells of various organs of rats.

“…4a, b x 105, 5a x 350, 5b x 320, 6a x 410, 6b x440 that the smooth muscle-like contraction mechanism exists in the apical portion of these cells. Because calmodulin and myosin have already been shown in the apical portion of some epithelial cells (3)(4)(5)(6)(8)(9)(10), it is thought that the caldesmoncalmodulin system which is considered to regulate the actin-myosin interaction may play an important role for the activities of the apical cytoplasm such as microvillous movement, secretion and endocytosis.…”

Analysis of cytoskeletal proteins and Ca 2+ -dependent regulation of structure in intestinal brush borders from rachitic chicks

“…The role of calcium in the regulation of brush border contractility (22), brush border myosin phosphorylation (23), and the length and bundling of microvillar actin filaments (24)(25)(26)(27)(28)(29) has been established. Also, the microvillus contains considerable amounts of calmodulin (30), for which only one function, activation of the' brush border myosin light chain kinase (23), has as yet been defined. Thus, the significance of calcium in the modulation and regulation of the transmural flow of calcium through the intestinal epithelial cell constituted the basis for the present investigation.…”

“…Brush borders were prepared from chick intestinal epithelium by the procedure of Mooseker et al (34). Microvilli were isolated from brush borders by procedures described by Howe et al (30). To control Abbreviation: kDal, kilodalton(s).…”