Mapping the cysteine residues and actin-binding regions of villin by using antisera to the amino and carboxyl termini of the molecule.
Peptide antisera specific for either the amino- or carboxyl-terminal regions of villin were used to locate the position of cysteine residues in immunoblots of villin cleaved with 2-nitro-5-thiocyanobenzoic acid. Maps constructed from the cleavage pattern suggest that villin contains six cysteine residues, two located in its amino-terminal peptide of Mr 44,000, and four located in the carboxyl-terminal peptide of Mr 51,000. Gel overlays of the partial cleavage fragments with 125I-labeled actin identified a calcium-dependent actin-binding region located within the amino-terminal peptide of Mr 32,000 of villin. The peptide antibody method used, called cleavage mapping, should be a convenient technique for mapping residues and ligand binding sites in proteins.
P
P
Matsudaira
R
R
Jakes
L
L
Cameron
E
E
Atherton
D000199:Actins / Q000378:metabolism*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000276:immunology / Q000378:metabolism*; D002645:Chickens; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004848:Epithelium / Q000378:metabolism; D000939:Epitopes / Q000032:analysis; D007106:Immune Sera; D007413:Intestinal Mucosa / Q000378:metabolism; D008840:Microfilament Proteins / Q000276:immunology / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis
D000199:Actins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D000939:Epitopes; D007106:Immune Sera; D008840:Microfilament Proteins; D010446:Peptide Fragments; C026615:villin; D004228:Dithionitrobenzoic Acid