Electron microscopy of negatively stained jackbean urease at three levels of quaternary structure, and comparison...

Electron microscopy, with sodium phosphotungstate as negative stain, has been carried out on purified jackbean urease prepared at three levels of quaternary structure: (a) A1 urease, Mr = 240 000, S20,W = 11.5 S (b) alpha urease, Mr = 480 000, S20,w = 18.3 S (c) polymers of alpha urease above the tetramer stage. The compatibility of the images from level to level leaves no doubt that the enzyme itself is being visualized, and the following geometry is suggested by electron microscopy: A1 molecules are cyclic trimers, which pair up in eclipsed position across a 1-nm cleft to form the hexameric alpha, which displays D3 (or 32) symmetry of a trigonal prism. Polymers consist of alpha molecules aligned with their clefts coplanar and an angle of 120 degrees between each triplet of 3-fold axes. These features correspond reasonably well with sedimentation and electrophoretic studies of the solvated enzyme, which have indicated a hemispherical A1, a spherical alpha, and string-of-beads polymers. Sedimentation constants of the urease polymers up through the pentamer level were found to be compatible with the rosette, straight-chain, and zig-zag forms seen in the electron microscope, and with the suggested protomer arrangement in A1 and alpha urease.