Electron Microscopy of Negatively Stained Jackbean Urease at Three Levels of Quaternary Structure, and Comparison with Hydrodynamic Studies

Abstract

EM, with sodium phosphotungstate as negative stain, was carried out on purified jackbean urease prepared at 3 levels of quaternary structure: A1 urease, Mr [relative molecular weight] = 240,000, s20,w = 11.5 S .alpha. urease, Mr = 480,000, s20,w = 18.3 S 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 EM: 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.degree. between each triplet of 3-fold axes. These features correspond reasonably well with sedimentation and electrophoretic studies of the solvated enzyme, which indicated a hemispherical A1, a spherical .alpha. and string-of-beads polymers. Sedimentation constants of the urease polymers up through the pentamer level were compatible with the rosette, straight-chain and zig-zag forms seen in EM and with the suggested protomer arrangement in A1 and .alpha. urease.