The size of the pyruvate dehydrogenase complex of Azotobacter vinelandii. Association phenomena.
Sedimentation analysis and light-scattering studies indicate that the aggregation state of the pyruvate dehydrogenase complex of Azotobacter vinelandii in 50 mM potassium phosphate (pH 7.0) can be described in terms of a monomer-dimer equilibrium with a dissociation constant of 6.8 microM. The apparent molecular mass of the monomeric particle is 750 000-850 000 Da. The equilibrium is shifted to the monomeric species when pressure is applied on the system. Pressure-jump experiments yielded a relaxation time of about 70 ms. In the presence of 3% poly(ethylene glycol) 6000 and 10 mM MgCl2, further association takes place to a system that can be described in terms of dimer-tetramer-octamer equilibria. Upon applying a pressure of 80 MPa to this system these equilibria are shifted to the dimeric state but some monomer formation cannot be excluded. Release of pressure shows that the relaxation time of the dimer-tetramer equilibrium is less than 5 s, that of tetramer-octamer equilibrium is of the order of minutes. The isolated E2 component has a molecular mass of 2 000 000 +/- 100 000 Da; and thus consists of about 30 E2 peptide chains. Electron micrographs are similar to those of the E2 component of the Escherichia coli complex, which were interpreted as cubic structures with an octagonal symmetry. Upon addition of E1 to the pure E2 component, changes in the assembly occur and mixtures of large (E. coli-like, 22-45 S) and small (A. vinelandii-like, 11-18 S) subcomplexes are obtained. The two forms of the subcomplexes are in slow equilibrium (relaxation time 10-30 min). It is proposed that the E2 tetramer of the intact pyruvate dehydrogenase complex of A. vinelandii is represented by the corner structures of the isolated E2 component.
D001395:Azotobacter / Q000201:enzymology*; D055598:Chemical Phenomena; D002621:Chemistry; D008027:Light; D008854:Microscopy, Electron; D008970:Molecular Weight; D011487:Protein Conformation; D011768:Pyruvate Dehydrogenase Complex / Q000302:isolation & purification*; D012542:Scattering, Radiation
D011768:Pyruvate Dehydrogenase Complex