title: Phosphatidylserine liposomes can be tethered by caldesmon to actin filaments.
abstract: Rotary shadowing electron microscopy revealed that attachment of caldesmon to phosphatidylserine (PS) liposomes was mainly through its C-terminal end. To determine the PS-binding sites of caldesmon, we have made use of synthetic peptides covering the two C-terminal calmodulin binding sites and a recombinant fragment corresponding to the N-terminal end of the C-terminal domain that contains an amphipathic helix. Interactions of these peptides with the PS liposomes were studied by nondenaturing gel electrophoresis and fluorescence spectroscopy. The results showed that both calmodulin-binding sites of caldesmon were able to interact with PS. The affinity (Kd) of PS for these sites was in the range of 1.8-14.3 x 10(-5) M, compared to 0.69 x 10(-5) M for the whole caldesmon molecule. Fragments located outside of calmodulin-binding sites bound PS weakly (3.85 x 10(-4) M) and thus may contain a second class of lipid-binding sites. Binding of PS induced conformational changes in regions other than the C-terminal PS-binding sites, as evidenced by the changes in the susceptibility to proteolytic cleavages. Most significantly, the presence of caldesmon greatly increased binding of PS to F-actin, suggesting that caldesmon may tether PS liposomes to actin filaments. These results raise the possibility that caldesmon-lipid interactions could play a functionally important role in the assembly of contractile filaments near the membranes.
authors.affiliation: Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
authors.forename: R
authors.initials: R
authors.lastname: Makuch
authors.forename: A
authors.initials: A
authors.lastname: Zasada
authors.forename: K
authors.initials: K
authors.lastname: Mabuchi
authors.forename: K
authors.initials: K
authors.lastname: Krauze
authors.forename: C L
authors.initials: CL
authors.lastname: Wang
authors.forename: R
authors.initials: R
authors.lastname: Dabrowska
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D002645:Chickens; D002918:Chymotrypsin; D005895:Gizzard, Avian; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D008854:Microscopy, Electron; D009130:Muscle, Smooth / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010449:Peptide Mapping; D010718:Phosphatidylserines / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
chemical_list: D000199:Actins; D002148:Calmodulin-Binding Proteins; D008081:Liposomes; D010446:Peptide Fragments; D010718:Phosphatidylserines; D011994:Recombinant Proteins; D014364:Tryptophan; D002918:Chymotrypsin