The Ca2(+)-dependent actin filament-severing activity of 74-kDa protein (adseverin) resides in its NH2-terminal half.
Calcium sensitive actin severing protein, adseverin, with Mr 74,000, was cleaved into two fragments of Mr 42,000 and Mr 39,000 by V8 protease and trypsin, and both fragments were purified by high performance (pressure) liquid chromatography ion-exchange column chromatography. To understand how adseverin can sever actin filaments, we identified the actin-binding domains. The NH2 termini of native adseverin and the Mr 42,000 fragment were confirmed to be blocked by amino acid sequencing. Twelve amino acids of the Mr 39,000 fragment were sequenced from the NH2 terminus; the sequence of this part had a homology to the hinge region between segments 3 and 4 of gelsolin and villin. Thus, the Mr 42,000 fragment is the NH2-terminal half (N42), and the Mr 39,000 fragment is the COOH-terminal half (C39). Each fragment was examined for actin-severing, -nucleating, -capping, and phospholipid binding activities with and without calcium. N42 contained a calcium-dependent actin-severing activity regulated by phospholipid. C39 bound to G-actin in a calcium-dependent manner, but had no severing activity. The sequence homology and similar functional domain structure suggest a common structural basis for the calcium- and phospholipid-regulated actin-severing properties shared by adseverin, gelsolin, and villin.
Sakurai
T
T
Department of Pharmacology, Faculty of Medicine, University of Tokyo, Japan.
Kurokawa
H
H
Nonomura
Y
Y
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000276:immunology; D002417:Cattle; D018260:Gelsolin; D066298:In Vitro Techniques; D008840:Microfilament Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D013329:Structure-Activity Relationship
D000199:Actins; D002135:Calcium-Binding Proteins; D018260:Gelsolin; D008840:Microfilament Proteins; D010446:Peptide Fragments; C065395:scinderin; D002118:Calcium