Self-association of CPV3, an avian thymic parvalbumin.

The avian parvalbumin called CPV3 readily forms disulfide-linked oligomers. Sedimentation data presented herein reveal that CPV3 also undergoes noncovalent self-association. Interestingly, the noncovalent interaction is promoted by either Ca2+ or Mg2+, whereas covalent complex formation displays an absolute requirement for the Ca(2+)-bound protein. Apo-CPV3 exhibits an apparent sedimentation coefficient of 2.08 S at 20 degrees C, in 0.15 M NaCl, 0.025 M HEPES-NaOH, pH 7.4. This value increases to 2.85 S or 3.16 S with addition of 1.0 mM Ca2+ or 5.0 mM Mg2+, respectively. Least-squares analysis of sedimentation equilibrium data suggests that 100 microM apo-CPV3 is primarily a mixture of monomeric and dimeric forms. With the addition of Ca2+, the equilibrium becomes exclusively monomer-trimer, with negligible amounts of dimer. A comparable distribution is observed in the presence of Mg2+.

Interaction of histidine-proline-rich glycoprotein with plasminogen: effect of ligands, pH, ionic strength, and chemical modification.

The association of plasma histidine-proline-rich glycoprotein (HPRG) with plasminogen (PLG) was examined using a sucrose density gradient assay in order to evaluate the effects of several relevant conditions on complex formation. Addition of PLG shifts the S-value of 125I-labeled HPRG from 4.8S to 6.8S, providing the first direct evidence that HPRG associates with the zymogen form of plasmin in solution. Complex formation is not sensitive to pH in the range of pH 6.5-8.5, but is abolished at high ionic strength (1 M NaCl). No species differences were found, as either rabbit or human HPRG bound readily to rabbit or human PLG. Of the ligands of HPRG tested, mesoheme (20 microM) but not heparin (M(r) 10,000, 10 microM) inhibits the formation of the HPRG-PLG complex. Modification of lysine residues of HPRG or competitive binding by lysine and anti-fibrinolytic agents containing primary amino groups also inhibits association. Conversely, modification of arginine or histidine residues of HPRG has no effect on complex formation. These results indicate that HPRG has independent binding sites for heparin and PLG and confirm that one or more lysine residues of HPRG are involved in its recognition by PLG. The protein-protein interaction was also quantitatively characterized at thermodynamic equilibrium by analytical ultracentrifugation. The stoichiometry and dissociation constant (KD) of the complex were determined from the equilibrium distribution of fluorescein-isothiocyanate-labeled PLG in the presence of HPRG. The experimental data were analyzed by nonlinear least-squares curve fitting and indicated that a heterodimer is formed.(ABSTRACT TRUNCATED AT 250 WORDS)