Abstract
Upon treatment with denaturing agents, vitronectin has been observed to exhibit conformational alterations which are similar to the structural changes detected when vitronectin binds the thrombin-antithrombin complex or associates with the terminal attack complex of complement. Denaturation and renaturation of vitronectin isolated from human plasma were characterized by changes in intrinsic fluorescence. Unfolding by chemical denaturants was irreversible and accompanied by self-association of the protein to form vitronectin multimers. Self-association was evaluated by equilibrium analytical ultracentrifugation which demonstrated that multimers form only during the refolding process after removal of denaturant, that multimeric vitronectin dissociates to constituent subunits readily upon treatment with chemical denaturant, and that intermolecular disulfide cross-linking occurs primarily at the dimer level among a subset of constituent vitronectin subunits within the multimer. The monomeric form of vitronectin isolated from human plasma partially unfolds at intermediate concentrations of denaturant to an altered conformation with a high propensity to associate into multimers. Folding of vitronectin in vivo appears to be regulated by partitioning of folding intermediates toward either of two conformations, one that exists as a stable monomer and another that associates into a multimeric form.
Highlights
Upon treatment with denaturing agents, vitronectin was first isolated by traditional biochemical approaches as has been observed to exhibit conformational alterations S-protein, a species which associates with the terminal attack which are similar to the structural changes detected when vitronectin binds the thrombin-antithrombin complex or associates with the terminal attack complex of complement
Attention has been paid to evaluating the effects of urea on the structure and function of vitronectin since it was noted that reactivity with a conformation-specific monoclonal antibody (8E6, see Refs. 13 and 14) was increased by urea treatment in a manner reminiscent of conformational changes that are induced in vitronectin by interactions with more physiologically relevant molecules, including the thrombin-antithrombin complex and the C5b-9 complex
A first approach was to determine whether chemical denaturants could be used to unfold and subsequently refold vitronectin in a reversible fashion so that some insight could be gained into the intrinsic stability and energetic differences between different conformational forms
Summary
Upon treatment with denaturing agents, vitronectin was first isolated by traditional biochemical approaches as has been observed to exhibit conformational alterations S-protein, a species which associates with the terminal attack which are similar to the structural changes detected when vitronectin binds the thrombin-antithrombin complex or associates with the terminal attack complex of complement. The monomeric form of vitronectin isolated complex of complement and renders the complex soluble and unable to insert into target cell membranes [6, 7]. This form of the protein has a molecular weight of approximately 72,000 and exists predominantly as a monomer. Immunological evidence suggests that this altered form of vitronectin, only detected in plasma in minor amounts, may be the predominant form of the protein found in extravascular sites [24]
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