The lysine-binding-site-mediated interaction between plasmin and antiplasmin is of great importance for the fast rate of this reaction. It also plays an important part in regulating the fibrinolytic enzyme system. To identify structures important for its noncovalent interaction with plasmin, we constructed seven single-site mutants of antiplasmin by modifying charged amino acids in the C-terminal part of the molecule. All the variants were expressed in the Drosophila S2 cell system, purified, and shown to form stable complexes with plasmin. A kinetic evaluation revealed that two mutants of the C-terminal lysine (K452E or K452T) did not differ significantly from wild-type antiplasmin in their reactions with plasmin, in either the presence or absence of 6-aminohexanoic acid, suggesting that this C-terminal lysine is not important for this reaction. On the other hand, modification of Lys436 to Glu decreased the reaction rate about fivefold compared with wild-type. In addition, in the presence of 6-aminohexanoic acid, only a small decrease in the reaction rate was observed, suggesting that Lys436 is important for the lysine-binding-site-mediated interaction between plasmin and antiplasmin. Results from computerized molecular modelling of the C-terminal 40 amino acids support our experimental data.

No abstract

The structure of human plasma fibronectin in 50 mM Tris-HCl buffer, pH 7.4, containing varying concentrations of NaCl, has been investigated using the small-angle X-ray method. Below 0.3 M NaCl the overall structure of the molecule is disc-shaped; at 0 M NaCl the axial ratio of the disc is about 1:7 and between 0.1 M to 0.3 M it is slightly more asymmetric, with an axial ratio of 1:10. At about 0.3 M NaCl there is a reversible transition to a more open structure, and, from 0.3 M up to 1.1 M NaCl the small-angle X-ray data can be explained by models consisting of ensembles of flexible, non-overlapping, bead-chains generated by a Monte Carlo procedure. Within this concentration range there is a gradual increase in the stiffness of the chains, as well as a decrease in bead radius, which indicates that the molecule becomes more open when the NaCl concentration is increased. The transition to a more open structure is also demonstrated by the average radius of gyration which increases gradually from 8.26 nm at 0 M NaCl to 8.75 nm at physiological or near-physiological conditions, and up to 16.2 nm at 1.1 M NaCl.

X-Ray scattering study of alpha 2-macroglobulin in solvents of variable electron densities (sucrose in water) shows that alpha 2-macroglobulin obeys the invariant volume hypothesis; thus, the structure of the particle is independent of the sucrose concentration of the solution. The particle structure is quantitatively described by a set of parameters such as the gyration radius, R = 8.0 nm, the volume, V = 1200 nm3, and the maximum distance within the particle, Dmax = 25 nm. The contrast dependence of the gyration radius indicates that in alpha 2-macroglobulin the regions of higher electron density are located closer to the center (core) than the regions of lower electron density. The core, which may be the place occupied by the carbohydrate, has a maximum dimension of 16 nm and it can be described as a flat cylinder. X-Ray scattering titrations indicate that alpha 2-macroglobulin forms a 1:2 complex as the main product with both trypsin and chymotrypsin simultaneously as the particle contracts. The formation of a ternary 1:1:1 complex with trypsin and chymotrypsin and the absence of higher complexes indicate that the sites for these proteases are closely related. This is further substantiated by the p(r) functions which are virtually identical for the 1:1:1 and 1:2 complexes.