A 36 kDa monomeric protein and its complex with a 10 kDa protein both isolated from bovine aorta are calpactin-like proteins that differ in their Ca2+-dependent calmodulin-binding and actin-severing properties

Abstract

Interaction of plasma membrane with the cytoskeleton involves a large number of proteins, among them a 36 kDa protein that was found to be involved in the interaction with actin filaments. We have isolated a 36 kDa protein from bovine aorta as a monomer and in a complex with a 10 kDa protein. Partial amino acid sequence determinations show that the 36 kDa and 10 kDa proteins isolated from bovine aorta are analogous to or identical with corresponding proteins purified from bovine intestine already described by Kristensen, Saris, Hunter, Hicks, Noonan, Glenney & Tack [(1986) Biochemistry 25, 4497-4503]. We report here that the association of the 10 kDa protein with the 36 kDa protein confers specific calmodulin-binding and actin-severing properties on the complex that are not possessed by the 36 kDa monomer alone. These findings suggest that the protein complex could be involved in thin-filament-related structures or could modulate some Ca2+-regulated events mediated by calmodulin.

Cholesterol Enhances Phospholipid Binding and Aggregation of Annexins by Their Core Domain

“…On the contrary, acidic phospholipids, the family of molecules present in all these membranes seem indicated for the role of a cholesterol-modulated 'receptor'. However, the cholesterol-PS effect does not preclude the possibility that annexin 2 could interact with other protein(s) in a Ca 2ϩ -independent or -dependent manner as has been proposed for endosomes (30,49) and demonstrated for actin (51,52) and other proteins (10,53).…”

Probing nucleation, cutting and capping of actin filaments

“…Also, the rapid fall in viscosity of polymeric actin solutions brought about by cutting proteins has been interpreted by shortening of filaments (Yin & Stossel, 1979;Norberg et al, 1979;Hasegawa et al, 1980;Craig & Powell, 1980;Mooseker et al, 1980;Hinssen, 1981a;Glenney et al, 1981;Yamamoto et al, 1982;Harris & Weeds, 1983;Maruta & Isenberg, 1983;Nishida et al, 1984;Olumucki et al, 1984;Walsh et al, 1984a;Chaponnier et al, 1985;Janmey et al, 1985;Colucio et al, 1986;Cooper et al, 1986;Hosoya et al, 1986;Porte & Harricane, 1986;Southwick & DiNubile, 1986;Martin et al, 1988;Colombo & Milzani, 1988). Although these two methods demonstrate that short filaments are formed, they do not provide information as to whether short filaments are formed by active cutting or by nucleation of new filaments and subsequent subunit exchange.…”

Further characterization of four lipocortins from human peripheral blood mononuclear cells

“…CPBPs include proteins of an apparent Mr of about 70 kDa that are probably identical and of several proteins in the 3 W O kDa range that are not yet fully distinguishable. This group of [30][31][32][33][34][35][36][37][38][39][40] kDa proteins share common properties and can be differentiated from one another essentially by their amino acid sequence [ 1,2,[11][12][13]24,30] or by their characteristic migration on twodimensional electrophoresis [9]. Four have been clearly identified: lipocortin I (identical to calpactin 11), lipocortin I1 (identical to calpactin I heavy chain), protein I1 (similar to endonexin and chromobindin 4), and endonexin 11, which is identical to the coagulation inhibitor (PAP or IBC) and to lipocortin V [30].…”

“…F-actin binding assays were performed using two different techniques: 1) By chromatography using F-actin glutaraldehyde stabilized and linked to sepharose-4B according to Martin et al [36]; briefly, protein samples (10 pg/assay) equilibrated in buffer A (25 mM Tris-HC1, pH 7.8, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 1 mM CaCl,) were allowed to stay in contact with Immobilized F-actin for 16 hours at 4OC, and then unbound material was washed out by six column volumes of buffer A at a flow rate of 2ml/h, and bound proteins were eluted with buffer B (25 mM Tris-HC1, pH 7.8,lOO mM NaCl, 1 mM DTT, and 2 mM EGTA); 2) F-aortic actin mixed with pure protein in actin polymerizing buffer (10 mM imidazole/HCl, pH 7.5, 1 mM NaCl, 1 mM DTT, 0.3 mM ATP, 100 mM KCl, and 2 mM MgCl,, with either 2 mM CaCl, or 2 mM EGTA) [20]. In these experiments, the molar ratio of actin and protein was 5:l. Some additional experiments have been performed without KCI, because some CPBPs have been reported to bind F-actin only without KCl in the sample buffer [21].…”