Abstract. The microvillus ll0-kD protein-calmodulin complex (designated ll0K-CM) shares several properties with all myosins. In addition to its well-defined ATP-dependent binding interaction with F-actin, ll0K-CM is an ATPase with diagnostically myosin-like divalent cation sensitivity. It exhibits maximum enzymatic activity in the presence of K + and EDTA (0.24 ~tmol Pi/mg per min) or in the presence of Ca ÷+ (0.40 ~tmol P~/mg per min) and significantly less activity in physiological ionic conditions of salt and Mg++ (0.04 ~tmol Pi/mg per min). This MgATPase is activated by F-actin in an actin concentration-dependent manner (up to 2.5-3.5-fold). The specific MgATPase activity of ll0K-CM is also enhanced by the addition of 5-10 ~tM Ca +÷, but in the isolated complex, there is often also a decrease in the extent of actin activation in this range of free Ca +÷. Actin activation is maintained, however, in samples with exogenously added calmodulin; under these conditions, there is an approximately sevenfold stimulation of ll0K-CM's enzymatic activity in the presence of 5-10 lxM Ca ++ and actin, ll0K-CM is relatively indiscriminant in its nucleoside triphosphate specificity; in addition to ATP, GTP, CTP, UTP, and ITP are all hydrolyzed by the complex in the presence of either Mg++ or Ca ++. Neither AMP nor the phosphatase substrate p-nitrophenyl phosphate are substrates for the enzymatic activity. The pH optimum for CaATPase activity is 6.0-7.5; maximum actin activation of MgATPase occurs over a broad pH range of 6.5-8.5. Finally, like myosins, purified ll0K-CM crosslinks actin filaments into loosely ordered aggregates in the absence of ATP. Collectively these data support the proposal of Collins and Borysenko (1984, J. Biol. Chem., 259:14128-14135) that the ll0K-CM complex is functionally analogous to the mechanoenzyme myosin.

Abstract. The actin bundle within each microvillus of the intestinal brush border is tethered laterally to the membrane by spirally arranged bridges. These bridges are thought to be composed of a protein complex consisting of a llO-kD subunit and multiple molecules of bound calmodulin (CM). Recent studies indicate that this complex, termed l l0K-CM, is myosin-like with respect to its actin binding and ATPase properties. In this study, possible structural similarity between the l l0-kD subunit and myosin was examined using two sets of mAbs; one was generated against Acanthamoeba myosin II and the other against the ll0-kD subunit of avian ll0K-CM. The myosin II mAbs had been shown previously to be cross-reactive with skeletal muscle myosin, with the epitope(s) localized to the 50-kD tryptic fragment of the subfragment-1 (St) domain. The ll0K mAbs (CX 1-5) reacted with the ll0-kD subunit as well as with the heavy chain of skeletal but not with that of smooth or brush border myosin. All five of these ll0K mAbs reacted with the 25-kD, NH2-terminal tryptic fragment of chicken skeletal St, which contains the ATP-binding site of myosin. Similar tryptic digestion of ll0K-CM revealed that these five mAbs all reacted with a 36-kD fragment of ll0K (as well as larger 90-and 54-kD fragments) which by photoaffinity labeling was shown to contain the ATPbinding site(s) of the ll0K subunit. CM binding to these same tryptic digests of ll0K-CM revealed that only the 90-kD fragment retained both ATP-and CMbinding domains. CM binding was observed to several tryptic fragments of 60, 40, 29, and 18 kD, none of which contain the myosin head epitopes. These results suggest structural similarity between the ll0K and myosin St, including those domains involved in ATP-and actin binding, and provide additional evidence that ll0K-CM is a myosin. These studies also support the results of Coluccio and Bretscher (1988. J. Cell Biol. 106:367-373) that the calmodulin-binding site(s) and the myosin head region of the ll0-kD subunit lie in discrete functional domains of the molecule.

We have investigated the role of myosin in contraction of the terminal web in brush borders isolated from intestinal epithelium. At 37°C under conditions that stimulate terminal web contraction (1 #M Ca ++ and ATP), most (60-70%) of the myosin is released from the brush border. Approximately 80% of the myosin is also released by ATP at 0°C, in the absence of contraction. Preextraction of this 80% of the myosin from brush borders with ATP has no effect on either the time course or extent of subsequently stimulated contraction. However, contraction is inhibited by removal of all of the myosin with 0.6 M KCI and ATP. Contraction is also inhibited by an antibody to brush border myosin, which inhibits both the ATPase activity of brush border myosin and its ability to form stable bipolar polymers. These results indicate that although functional myosin is absolutely required for terminal web contraction only -20% of the brush border myosin is actually necessary. This raises the possibility that there are at least two different subsets of myosin in the terminal web.Myosin is a major component of the cytoskeletal apparatus associated with the brush border of intestinal epithelial cells (for recent reviews of brush border structure see references 2 and 30). In brush borders, myosin is localized in the terminal web (3,9,19,22,33). Although its function in vivo remains unknown, this myosin has been implicated as the force producer (5, 26) in ATP-dependent contraction of the terminal web first observed in isolated brush borders by Rodewald et al. (41). Further support for this possibility has come from the demonstration that contraction of the terminal web correlates directly with Ca++-calmodulin-dependent phosphorylation of the regulatory light chain of brush border myosin (26). A similar type of contraction has also been stimulated in glycerinated cells from intestinal epithelia (5, 2 l) and retinal epithelia (35).Based on the morphologic changes that occur during contraction, it appears that at least some of the force for this contraction is produced within a ring of filaments that circumscribes the apical ends of both intestinal (24) and retinal epithelial cells (35) at the level of their zonula adherens junctions. This circumferential ring has been isolated from retina and stimulated to contract in vitro (36). In intestinal brush borders, this ring of filaments contains closely apposed, antiparallel actin filaments (21, 22) and myosin (21,22). Presumably, the terminal web contraction that is observed could result from an interaction between actin and myosin within this ring that constricts it in a "purse-string" fashion. However, in our original studies of contraction in isolated brush borders (26) and in glycerinated intestinal epithelial cells (21), we observed that most of the myosin was dissociated from the cytoskeleton during contraction. This observation made us question the role of myosin as a producer of force in terminal web contraction. In this and a companion study (31), we have investigated the nat...

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Abstract. The actin bundle within each microvillus of the intestinal brush border (BB) is tethered laterally to the membrane by bridges composed of BB myosin I. Avian BB myosin I, formerly termed l l0K-calmodulin, consists of a heavy chain with an apparent M, of 110 kD and three to four molecules of calmodulin "light chains." Recent studies have shown that this complex shares many properties with myosin including mechanochemical activity. In this report, the isolation and characterization of a membrane fraction enriched in bound BB myosin I is described. This membrane fraction, termed microvillar membrane disks, was purified from ATP extracts of nonionic detergenttreated microvilli prepared from avian intestinal BBs. Ultrastructural analysis revealed that these membranes are flat, disk-shaped sheets with protrusions which are identical in morphology to purified BB myosin I. The disks exhibit actin-activated Mg-ATPase activity and bind and cross-link actin filaments in an ATPdependent fashion. The mechanochemical activity of the membrane disks was assessed using the Nitella bead movement assay (Sheetz, M. P., and J. A.Spudich. 1983. Nature [Lond.]. 303:31-35). These preparations were shown to be free of significant contamination by conventional BB myosin. Latex beads coated with microvillar membrane disks move in a myosin-like fashion along Nitella actin cables at rates of 12-60 nm/s (average rate of 33 nm/s); unlike purified BB myosin I, the movement of membrane disk-coated beads was most reproducibly observed in buffers containing low Ca 2+.T HE actin bundle within each microvillus of the intestinal brush border (BB) ~ is tethered laterally to the plasma membrane by spirally arranged bridges (Mooseker, 1985). These bridges are composed of 110K-calmodulin (CM), a complex consisting of an ,~ll0-kD protein and multiple molecules of CM (see Coluccio and Bretscher, 1989 for review; this recent study demonstrates the reconstitution of bridges by addition of 110K-CM to actin bundles in vitro). Studies from several laboratories have shown that ll0K-CM purified from chicken BBs shares many properties with myosin (Collins and Borysenko, 1984;Howe and Mooseker, 1983;Conzelman and Mooseker, 1987; Bretscher, 1987, 1988;Krizek et al., 1987;Swanljung-Collins et al., 1987;Carboni et al., 1988) including mechanochemical activity (Mooseker and Coleman, 1989). Because 110K-CM is structurally analogous to the tailless, single-headed myosin I's first characterized in amoeboid cells (for review see Korn and Hammer, 1988), this complex has been named BB myosin I. Recent studies on the domain structure of the ll0-kD subunit indicate that it consists of two discrete domains:I. Abbreviations used in this paper: BB, intestinal brush border; CM, calmodulin.an •80-90-kD myosin head domain; and a 20-30-kD CMbinding domain at the presumed COOH-terminal end of the molecule (Coluccio and Bretscher, 1988;Carboni et al., 1988). Additional support for the conclusion that the I10K-CM isolated from avian BBs is a member of a family of myosin I me...