Plasma-gelsolin-binding sites on the actin sequence.
Plasma gelsolin was cross-linked to fluorescently labeled actin in order to identify plasma-gelsolin-binding sites on the primary sequence of actin. Plasma gelsolin can be cross-linked to actin with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide (EDC), resulting in the formation of 1:1 and 1:2 plasma gelsolin:actin cross-linked complexes with apparent molecular masses of 130 kDa and 180 kDa respectively. The cross-linked complexes were isolated separately, partially digested with cyanogen bromide and subjected to sodium dodecyl sulfate gel electrophoresis to analyze fluorescent fragments. The electrophoretic pattern showed that fluorescent CNBr fragments obtained from a free actin molecule were all found in those obtained from both the complexes. Since the fluorescent probe was attached to an actin molecule through the penultimate cysteine residue (Cys-374), the agreement in the fluorescent patterns indicated that the NH2-terminal CNBr fragments of both the actin molecules were involved in cross-linking with plasma gelsolin. This was also suggested by hydroxylamine cleavage of the two complexes as the cleavage gave the fluorescent 41-kDa fragment which could not be produced unless plasma gelsolin was cross-linked at the NH2-terminal segment comprising 12 amino acids. Since EDC cross-links an amino group with a carboxyl group only when they are direct contact, the characteristic acidic amino acid residues at the NH2 terminus of actin are suggested to participate in binding plasma gelsolin.
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000097:blood*; D003488:Cyanogen Bromide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D018260:Gelsolin; D006801:Humans; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D008840:Microfilament Proteins / Q000097:blood*; D008970:Molecular Weight; D011817:Rabbits
D000199:Actins; D002135:Calcium-Binding Proteins; D018260:Gelsolin; D006898:Hydroxylamines; D008840:Microfilament Proteins; D019811:Hydroxylamine; D003488:Cyanogen Bromide; D005022:Ethyldimethylaminopropyl Carbodiimide