Electron microscopic studies of free and proteinase-bound duck ovostatins (ovomacroglobulins). Model of ovostatin structure and its transformation upon proteolysis.

Conformational changes of duck ovostatin (ovomacroglobulin) upon complexing with thermolysin have been studied by electron microscopy. Both free and thermolysin-bound ovostatin preparations were negatively stained with uranyl acetate, a series of three pictures were taken at 10 degrees specimen tilt intervals (+10 degrees, 0 degrees, and -10 degrees), and images of the inhibitor molecules were observed in three dimensions. Four approximately cylindrical subunits were observed in free ovostatin. Two subunits associated approximately midway from both ends to form a dimer of four arms. Two dimers associated with each other at the midpoint to form a tetramer. The proteinase susceptible "bait" regions were located near the center of the molecule. Eight arms of the tetramer take various configurations. The orthogonal extent of free tetrameric ovostatin in a two-dimensional micrograph averages 26.0 +/- 4.7 x 34.0 +/- 5.0 nm. Upon complexing with thermolysin, all eight arms curl toward the center of the molecule, having four arms upward and the other four downward. Thus, proteinase-bound ovostatin has a uniform structure with a 2-fold axis of symmetry. The overall structure of the complex is more compact with average dimensions of 16.9 +/- 0.6 x 16.9 +/- 0.6 x 19.9 +/- 0.4 nm. From these electron microscopic studies we propose that a proteinase reaches to the center of the free ovostatin molecule and attacks the bait region. Subsequent to proteolysis the subunit arms curl and entrap the enzyme within the ovostatin molecule. The results support the unique mechanism of inhibition of proteinases by alpha 2-macroglobulin and ovostatin postulated from biochemical observations (Barrett, A. J., and Starkey, P. M. (1973) Biochem. J. 133, 709-724; Nagase, H., and Harris, E. D., Jr. (1983) J. Biol. Chem. 258, 7490-7498).