THE NONIONIC DETERGENT BRIJ-58 CONSERVES THE STRUCTURE OF THE TONOPLAST H-ATPASE OF MESEMBRYANTHEMUM-CRYSTALLINUM L DURING SOLUBILIZATION AND PARTIAL-PURIFICATION()

The effects of solubilization with Triton X-100 or Brij 58 on the poly peptide composition and the substrate affinity of the tonoplast H+-ATP ase of plants of Mesembryanthemum crystallinum performing Cp photosynt hesis or crassulacean acid metabolism (CAM) have been compared. Althou gh all known subunits of the tonoplast H+-ATPase were present in the f raction of solubilized proteins after treatment with Brij 58 or Triton X-100, with Triton X-100 the apparent K-M value for ATP hydrolysis wa s increased by a factor of 1.8 and 1.5 in preparations from C-3 and CA M plants, respectively, even at low concentrations in contrast to trea tment with Brij 58. This is explained by structural changes of the ton oplast H+-ATPase due to the Triton X-100 treatment. After solubilizati on with Brih 58 the tonoplast H+-ATPase was partially purified by Supe rose-6 size-exclusion FPLC. When Brij 58 was present, addition of lipi ds to the chromatography buffer was not necessary to conserve enzyme a ctivity in contrast to previously described purification methods using Triton X-100. The substrate affinity of the partial purified H+-ATPas e was similar to the substrate affinity obtained for ATP-hydrolysis of native tonoplast vesicles, indicating that the enzyme structure durin g partial purification was conserved by using Brij 58. The results und erline that the lipid environment of the tonoplast H+-ATPase is import ant for enzyme structure and function.