Purification of human smooth muscle filamin and characterization of structural domains and functional sites

Biochemistry. 1990 Oct 9;29(40):9441-51. doi: 10.1021/bi00492a019.


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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Birds
  • Blood Platelets / chemistry
  • Contractile Proteins / chemistry
  • Contractile Proteins / isolation & purification*
  • Contractile Proteins / ultrastructure
  • Filamins
  • Humans
  • Microfilament Proteins / chemistry
  • Microfilament Proteins / isolation & purification*
  • Microfilament Proteins / ultrastructure
  • Molecular Sequence Data
  • Molecular Structure
  • Muscle, Smooth / chemistry
  • Peptide Mapping
  • Protein Conformation


  • Contractile Proteins
  • Filamins
  • Microfilament Proteins