Isolation of a domain of villin retaining calcium-dependent interaction with G-actin, but devoid of F-actin fragmenting activity.
Villin is an F-actin binding protein located in the microfilament bundle of intestinal epithelial cell microvilli. Extensive in vitro proteolysis with Staphylococcus aureus V8 protease results in the production of a stable domain (apparent Mr 44000) which can be isolated due to its Ca2+-dependent interaction with G-actin bound to immobilized DNase-I, the standard procedure for the purification of villin. This 44-kDa fragment retains a single Ca2+ binding site with an apparent Kd = 2 X 10(-6) M, binds to G-actin, and inhibits the rate of actin polymerization. However, the 44-kDa domain does not shown any Ca2+-activated severing activity nor does it compete with villin for F-actin binding. These results suggest that villin contains three domains: headpiece containing an F-actin binding site, 44-kDa fragment containing a G-actin binding site, and an amino-terminal fragment responsible for the Ca2+-dependent severing activity.
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001704:Biopolymers; D002118:Calcium / Q000502:physiology; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002645:Chickens; D010450:Endopeptidases; D007413:Intestinal Mucosa / Q000032:analysis; D008840:Microfilament Proteins / Q000302:isolation & purification* / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D012697:Serine Endopeptidases
D000199:Actins; D001704:Biopolymers; D002352:Carrier Proteins; D008840:Microfilament Proteins; D010446:Peptide Fragments; C026615:villin; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D002118:Calcium