Actin in the Brush-Border of Epithelial Cells of the Chicken Intestine

Abstract

The major soluble protein of the isolated brush-border of the intestinal epithelium has a molecular weight and net charge indistinguishable from those of skeletal-muscle actin, as determined by polyacrylamide gel electrophoresis. Furthermore, this protein, isolated from acetone powders of the purified brush-border, undergoes a G to F transformation in the presence of Mg++. The filaments have a substructure indistinguishable from muscle actin, as seen by the negative-staining technique, and bind heavy meromyosin with the arrowhead configuration characteristic of actin. The filaments in the microvilli of the intact brush-border also bind heavy meromyosin. Thus, actin seems to be present in intestinal epithelial cells.Intracellular filaments, about 5 nm in diameter, are now recognized to be common constituents of eucaryotic cells. Their localization in cell types other than muscle has led to the speculation that they may function in certain types of contractile processes such as cleavage (1-4), neural-tube formation (5), resorption of ascidian tadpole tails (6, 7), certain types of cytoplasmic streaming (8), shortening of secondary mesenchymal processes during gastrulation (9), and pulsations of intestinal epithelial cell microvilli (10). Recent evidence in support of these speculations has come from experiments with cytochalasin B (11,12). In man-y, but not all, of the above svstems, this compound affects the integrity of the filaments and the contractile function is lost.5-nm Filaments also occur in primitive cell types, such as the acellular slime mold, Physarum, and amoeba. The filaments in both of these organisms have close biochemical homology with skeletal-muscle actin (13)(14)(15)(16). Furthermore, the isolated filament protein of both systems has been shown to bind heavy meromyosin (HMM), with the arrowhead configuration characteristic of muscle actin (16)(17)(18) The chickens were starved for 1 day before they were killed. Before and after filtration with glass wool, the pellet was washed several extra times to remove the yellowish unconsolidated layer on top of the pellet. Purity was assessed by light microscopy (phase-contrast and Nomarski interference) and byr electron microscopy.Polyacrylamide Gel Electrophoresis. The molecular weights of proteins from the whole isolated brush-border and from proteins extracted from acetone powders of the brush-border were determined electrophoretically (24, 25) on 5% polyacrylamide gels that contained 0.1% sodium dodecyl sulfate (SDS) at pH 7.1. The purified brush-border, isolated brushborder actin, chick actin prepared from acetone powders (26), and protein standards were heated for 2 min at 1000C in 1.0% SDS-1.0% 2-mercaptoethanol-10 mM phosphate buffer (pH 7.1) before electrophoresis. The gels were calibrated with bovine serum albumin (molecular weight 68,000 and 136,000), catalase (molecular weight 60,000), chick actin (molecular weight 46,000), and pepsin (molecular weight 36,000). Bromphenol blue was used as a tracking dye.For the determination of net p...

Properties of guanylate cyclase and levels of cyclic GMP in rat small intestinal villous and crypt cells

“…Should the endogenous substrates be transport proteins, the slight stimulation of gunaylate cyclase activity in the brushborder by aminoacids (table 1) might be of physiological significance. The striking resemblance between the tubular structure of microvilli and flagellae [33], the high activity of guanylate cyclase in flagellar membranes from sea urchin sperm [27,34] and in platelets [35], and the possibility that the microfilaments within the core of the intestinal microvilli are actin-like [33,36], lend some support to the idea that cyclic GMP has a role in the assembly or regulation of contractile systems.…”

The contractile basis of ameboid movement. II. Structure and contractility of motile extracts and plasmalemma-ectoplasm ghosts.

“…Cytokinesis (30) involves the transient formation of filaments under the plasmalemma, while the brush border of intestine contains filaments in close proximity to the plasmalemma (24,40). Furthermore, Difflugia (45,9), a testate ameba, extends a pseudopod by cytoplasmic streaming, but then rapidly forms a birefringent filamentous array in the ectoplasm just under the plasmalemma that contracts.…”

Localization of [alpha]-actinin and tropomyosin in muscle and nonmuscle systems

“…All the Pq-SO crude a-actinin fractions also contained sizable quantities of a 42,000-dalton polypeptide that migrated at the same rate as actin (Figure 67). Previous studies have shown that fibro blasts (Bray and Thomas, 1975;Perdue, 1973;Yang and Perdue, 1972) and brush borders (Tilney and Mooseker, 1971) contain actin. The Pq-SQ crude a-actinin fractions from brush borders and fibroblasts also contain and small amounts of a 30,000-dalton polypeptide that may be tropomyosin (Figure 67).…”