QUATERNARY STRUCTURE OF THE ISOLATED RESTRICTION ENDONUCLEASE ENDOR.BGL I FROM BACILLUS-GLOBIGII AS REVEALED BY ELECTRON-MICROSCOPY

The restriction endonuclease EndoR · BglI was purified nearly to homogeneity. BglI samples, when negatively stained with 4% uranyl acetate, show two different particle projections in the electron microscope. Projection A has an outer diameter of 22.5 ± 0.8 nm and is composed of six intensity maxima arranged in a ring; the centre of the ring exhibits slightly visible additional substructures. Projection B is also a ring; its outer diameter is 23.8 ± 0.7 nm; it does not show detailed fine structure, aside from a probable 10-fold rotational symmetry. Variations of the negative staining technique (single carbon layer, 2% uranyl acetate; 'sandwich' preparation with 4% uranyl acetate) revealed additional fine structural details for both projections. From the electron microscopic observations, a model of the enzyme particle was developed containing 20 identical, biologically active monomers of molecular weight around 61,000, arranged as a pentagonal dodecahedron. Tilting experiments established this structure decisively by interconversion of the different appearances of given particles in the expected way. By sodium dodecyl sulphate/polyacrylamide gel electrophoresis, polyacrylamide gel electrophoresis in a continuous molecular sieve gradient and evaluation of negatively stained enzyme particles, a molecular weight of the monomer of 61,000 was estimated, resulting in a total enzyme particle molecular weight of 1.2 × 106 also determined by linear sucrose density-gradient centrifugation. © 1979.