Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.7 (plasmin)
 9,023 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gelsolin is a widely distributed actin binding protein that regulates actin filament length. It exists in both an intracellular and an extracellular form that is derived from a single gene by alternative splicing. Both forms contain the six homologous domains that are responsible for function. Little is known regarding differences between the forms. We have used a combination of cysteine-specific modification with 4-vinylpyridine, HPLC peptide mapping methods, and mass spectrometry to analyze the disulfide structures of human plasma and cytoplasmic gelsolin. Of the five Cys residues in the human gelsolin sequence, all were present in the free thiol form in human cytoplasmic gelsolin, while only three of them were free thiols in the human plasma form. Cys residues 188 and 201 in domain 2 of plasma gelsolin were disulfide linked. Recombinant human plasma gelsolin that had been expressed intracellularly in Escherichia coli and as a secreted protein from Cos green monkey cells was also investigated. The E. coli product lacked the disulfide but could be converted to the plasma-like structure with mild oxidation while the mammalian product formed the correct disulfide prior to isolation. Structural differences were also detected by limited proteolysis with plasmin. The differences in proteolytic susceptibility were also due to perturbations in domain 2. These studies demonstrate that the intracellular and extracellular gelsolins are structurally distinct and suggest that at least some of the preparations of recombinant gelsolin that are being used to study structure/function may be improperly folded. The experiments also demonstrate a general method for the location of disulfide bonds in proteins.
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PMID:The plasma and cytoplasmic forms of human gelsolin differ in disulfide structure. 870 41

Gelsolin is a calcium-regulated actin severing and capping protein that binds two calcium ions and has three sites for actin; two recognize monomeric actin and one attaches to the sides of filaments. It contains six repeating sequence segments (G1-6). Here, we have analyzed the effects of calcium ions on (i) limited proteolysis of bacterially expressed human gelsolin by plasmin and (ii) dynamic light scattering and circular dichroism of gelsolin and various of its subdomains. Following cleavage of gelsolin in the absence of calcium between Lys150 and His151 (the junction between G1 and G2), the molecule does not fall apart, nor does it bind actin without added calcium. This same molecule can be reconstituted by mixing an excess of G1 with G2-6 in EGTA. The noncovalently linked form of gelsolin shows three actin binding sites in calcium and requires 3 microM calcium for 50% activation of actin binding. Measurements of light scattering and circular dichroism revealed structural changes in response to calcium for intact gelsolin and a number of its actin-binding subdomains. Many of these changes occurred at calcium concentrations below 100 nM. These results are discussed in relation to the calcium control of gelsolin function and its three-dimensional structure (Burtnick et al.(1997) Cell 90, 661-670). Nanomolar concentrations of calcium initiate the unlatching of structural constraints that maintain the inaccessibility of the actin binding sites, but actin binding occurs only after additional micromolar calcium sites in both the N-terminal and C-terminal halves of the molecule are occupied.
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PMID:Probing the effects of calcium on gelsolin. 939 17