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The Non‐Ionic Detergent Brij 58 Conserves the Structure of the Tonoplast H + ‐ATPase of Mesembryanthemum crystallinum L. During Solubilization and Partial Purification
Author(s) -
Ratajczak R.
Publication year - 1994
Publication title -
botanica acta
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.871
H-Index - 87
eISSN - 1438-8677
pISSN - 0932-8629
DOI - 10.1111/j.1438-8677.1994.tb00787.x
Subject(s) - mesembryanthemum crystallinum , atpase , enzyme , vacuole , biochemistry , tricine , chemistry , fast protein liquid chromatography , proton transport , chromatography , substrate (aquarium) , crassulacean acid metabolism , biology , photosynthesis , membrane , cytoplasm , ecology
The effects of solubilization with Triton X‐100 or Brij 58 on the polypeptide composition and the substrate affinity of the tonoplast H + ‐ATPase of plants of Mesembryanthemum crystallinum performing C 3 photosynthesis or crassulacean acid metabolism (CAM) have been compared. Although all known subunits of the tonoplast H + ‐ATPase were present in the fraction of solubilized proteins after treatment with Brij 58 or Triton X‐100, with Triton X‐100 the apparent K M value for ATP hydrolysis was increased by a factor of 1.8 and 1.5 in preparations from C 3 and CAM plants, respectively, even at low concentrations in contrast to treatment with Brij 58. This is explained by structural changes of the tonoplast H + ‐ATPase due to the Triton X‐100 treatment. After solubilization with Brij 58 the tonoplast H + ‐ATPase was partially purified by Superose‐6 size‐exclusion FPLC. When Brij 58 was present, addition of lipids to the chromatography buffer was not necessary to conserve enzyme activity in contrast to previously described purification methods using Triton X‐100. The substrate affinity of the partial purified H + ‐ATPase was similar to the substrate affinity obtained for ATP‐hydrolysis of native tonoplast vesicles, indicating that the enzyme structure during partial purification was conserved by using Brij 58. The results underline that the lipid environment of the tonoplast H + ‐ATPase is important for enzyme structure and function.