Actin-binding and dimerization domains of HeLa cell filamin.
HeLa cell filamin is a linear, bivalent, homodimer of high molecular weight subunits (Mr 250,000 that may cross-link actin filaments in vivo into supramolecular structures such as networks and bundles. We used millimolar Ca protease from chicken breast muscle to cleave the subunit into smaller fragments that we mapped with respect to the overall structure of the dimer. The enzyme cleaves HeLa filamin into a larger (Mr 192,000) and a smaller (Mr 104,000) fragment; the smaller fragment is the precursor of a still smaller (Mr 92,000) fragment. Only the larger fragment bound to actin in a cosedimentation test, suggesting that it contains the actin-binding region of the subunit. Digestion of HeLa filamin that had been cross-linked with dimethyl adipimidate produced a good yield of the Mr 192,000 fragment but a poor yield of the Mr 104,000/92,000 fragments. Since native filamins are head-to-head dimers, it was expected that cross-linking would proceed most readily at the dimerization site and, therefore, it appears that the Mr 192,000 fragment is cleaved from cross-linked filamin because it is distal to the dimerization region, while the Mr 104,000/92,000 fragments are absent because they lie at the dimerization region and were cross-linked to a form that was not identifiable by sodium dodecyl sulfate electrophoresis.
Weihing
R R
RR
Department of Biology, Clark University, Worcester, Massachusetts 01610.
D000199:Actins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D003285:Contractile Proteins / Q000378:metabolism*; D004119:Dimethyl Adipimidate; D064448:Filamins; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D046911:Macromolecular Substances; D008840:Microfilament Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding
D000199:Actins; D002352:Carrier Proteins; D003285:Contractile Proteins; D064448:Filamins; D046911:Macromolecular Substances; D008840:Microfilament Proteins; D010446:Peptide Fragments; D004119:Dimethyl Adipimidate