Interactions among serum vitamin D binding protein, monomeric actin, profilin, and profilactin.
Human serum vitamin D binding protein (hDBP), a 58-kDa inter-alpha-globulin, is known to bind, monomeric actin (G-actin) in equimolar quantities. Using monoclonal and polyclonal anti-hDBP antibodies, hDBP, and radioiodinated actin, we developed a reliable saturation assay for actin bound to hDBP. By utilizing this assay, kinetic analysis, and ultracentrifugal sedimentation in sucrose gradients, these proteins' binding affinities (Kd = 10(-9) M) were demonstrated to be 10- to 100-fold greater than earlier estimates. At 4 degrees C, hDBP has an association rate constant of 2.2 x 10(4) M-1 s-1 and a rate of dissociation displaying a t1/2 of 22 h. This high affinity binding was largely unaffected by conditions favoring actin filament formation (1 mM MgCl2 and/or 50 mM KCl), by the range of pH from 6.8 to 8.6 or by temperatures from 4 to 37 degrees C. Compared with ATP-alpha-actin, a 2-fold decrease of binding affinity was observed for the nonmuscle isoactins (beta,gamma), ADP-G-alpha-actin, and N'-ethylmaleimide-modified G-alpha-actin. The 25-hydroxyvitamin D3 and 1 alpha,25-dihydroxyvitamin D3 holo-sterol forms of hDBP bound actin in a manner indistinguishable from the apo-sterol hDBP. The common polymorphisms of hDBP (DBP1 slow, DBP1 fast, and DBP2) were shown to have an equal avidity for G-actin binding. Human platelet profilin competed with hDBP for binding to G-actin, but was 1000-fold less potent (Ki = 1.9 microM). When platelet profilactin was incubated with hDBP, profilin was liberated and hDBP-actin complexes formed. DNase I, which forms a triprotein complex with hDBP-G actin, did not alter the affinity of binding of actin by hDBP. The very high affinity binding observed, which was largely unaffected by the state of G-actin, pH, and ionic conditions, appears to support a constitutive role for plasma DBP in the sequestration of actin monomers, as well as actin from actin-profilin complexes, that are liberated during cell injury.
Mc Leod
J F
JF
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-6069.
Kowalski
M A
MA
Haddad
J G
JG
D000199:Actins / Q000378:metabolism*; D003285:Contractile Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008840:Microfilament Proteins / Q000378:metabolism*; D051304:Profilins; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D014809:Vitamin D-Binding Protein / Q000097:blood* / Q000302:isolation & purification
D000199:Actins; D003285:Contractile Proteins; D008840:Microfilament Proteins; C495738:PFN1 protein, human; D051304:Profilins; D011506:Proteins; D014809:Vitamin D-Binding Protein; C024782:profilactin