SDS-PAGE resolved the purified complex into at least five proteins that could be microsequenced and identified. The enzyme is found almost identical in both the plasma membrane and in recycling vesicles where the yield of its purification is much more satisfactory. Highly purified plasma membranes from NB41A3 also display PMO but less tightly associated. The association of PMO into a tight complex was confirmed by its immunoprecipitation from cellular or membrane extracts of NB41A3, using antibodies directed against any component proteins of the complex followed by immunodetection with antibodies directed against the other members.
Antibodies also inhibit the enzyme activity synergistically.
In addition the induction of several components of the complex during oxidative stress could be demonstrated by means of S1-RNAse protection assay in synchronized NB41A3 cells. Also the genes of the five components of the PMO complex have been prepared, coupled to GFP and cloned into an expression vector for transfection into NB41A3. Cells overexpressing individual members of the complex separately or in combination were stressed and observed by fluorescence microscopy. From its composition and properties PMO's may play a role in the cellular response against extracellular oxidative stress during growth and development.