Purification of human smooth muscle filamin and characterization of structural domains and functional sites.
A method was developed to purify human smooth muscle filamin in high yield and structural domains were defined by using mild proteolysis to dissect the molecule into intermediate-sized peptides. Unique domains were defined and aligned by using high-resolution peptide mapping of iodinated peptides on cellulose plates. The amino- and carboxyl-terminal orientation of these domains within the molecule was determined by amino acid sequence analysis of several aligned peptides. In addition to the three unique domains which were identified, a number of smaller and larger fragments were also characterized and aligned within the intact molecule. These structural domains and related peptides provide a useful set of defined fragments for further elucidation of structure-function relationships. The two known functionally important binding sites of filamin, the self-association site and the actin-binding site, have been localized. Self-association of two monomers in a tail-to-tail orientation involves a small protease-sensitive region near the carboxyl terminal of the intact polypeptide chain. Sedimentation assays indicate that an actin-binding site is located near the blocked amino terminal of the filamin molecule. Sequences derived from large peptides mapping near the amino terminal show homology to the amino-terminal actin-binding site of alpha-actinin (chicken fibroblast and Dictyostelium), Dictyostelium 120-kDa actin gelation factor, beta-spectrin (human red cell and Drosophila), and human dystrophin. This homology is particularly interesting for two reasons. The functional form of filamin is single stranded, in contrast to alpha-actinin and spectrin which are antiparallel double-stranded actin cross-linkers. Also, no homology to the spectrin-like segments which comprise most of the mass of spectrin, alpha-actinin, and dystrophin was found. Instead, the sequence of a domain located near the center of the filamin molecule (tryptic 100-kDa peptide, T100) shows homology to the published internal repeats of the Dictyostelium 120-kDa actin gelation factor. On the basis of these results, a model of human smooth muscle filamin substructure is presented. Also, comparisons of human smooth muscle filamin, avian smooth muscle filamin, and human platelet filamin are reported.
Hock
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Wistar Institute, Philadelphia, Pennsylvania 19104.
Davis
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Speicher
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001717:Birds; D001792:Blood Platelets / Q000737:chemistry; D003285:Contractile Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000648:ultrastructure; D064448:Filamins; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009130:Muscle, Smooth / Q000737:chemistry; D010449:Peptide Mapping; D011487:Protein Conformation
D003285:Contractile Proteins; D064448:Filamins; D008840:Microfilament Proteins