Calcium-regulated cooperative binding of the microvillar 110K-calmodulin complex to F-actin: formation of decorated filaments.
The 110K-calmodulin complex of intestinal microvilli is believed to be the link between the actin filaments comprising the core bundle and the surrounding cell membrane. Although not the first study describing a purification scheme for the 110K-calmodulin complex, a procedure for the isolation of stable 110K-calmodulin complex both pure and in high yield is presented; moreover, isolation is without loss of the associated calmodulin molecules since a previously determined ratio in isolated microvillar cytoskeletons of calmodulin to 110-kD polypeptide of 3.3:1 is preserved. We have found that removal of calmodulin from the complex by the calmodulin antagonists W7 or W13 results in precipitation of the 110-kD polypeptide with calmodulin remaining in solution. The interaction of 110K-calmodulin with beef skeletal muscle F-actin has been examined. Cosedimentation assays of 110K-calmodulin samples incubated with F-actin show the amount of 110K-calmodulin associating with F-actin to be ATP, calcium, and protein concentration dependent; however, relatively salt independent. In calcium, approximately 30% of the calmodulin remains in the supernatant rather than cosedimenting with the 110-kD polypeptide and actin. Electron microscopy of actin filaments after incubation with 110K-calmodulin in either calcium- or EGTA-containing buffers show polarized filaments often laterally associated. Each individual actin filament is seen to exhibit an arrowhead appearance characteristic of actin filaments after their incubation with myosin fragments, heavy meromyosin and subfragment 1. In some cases projections having a 33-nm periodicity are observed. This formation of periodically spaced projections on actin filaments provides further compelling evidence that the 110K-calmodulin complex is the bridge between actin and the microvillar membrane.
D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D000199:Actins / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000302:isolation & purification / Q000378:metabolism*; D002122:Calcium Chloride / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D002462:Cell Membrane / Q000648:ultrastructure; D007413:Intestinal Mucosa / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism / Q000648:ultrastructure; D009994:Osmolar Concentration; D011188:Potassium / Q000494:pharmacology
D000199:Actins; D002147:Calmodulin; D017301:Ca(2+) Mg(2+)-ATPase; D002122:Calcium Chloride; D011188:Potassium