Figure 6.

In vivo redox states of CcmG and CcmH in actively growing cells. Immunoblot analysis of cell extracts from appropriately complemented R. capsulatus mutants MD11/pCS1566 and MD14/pST6 (Table 1) using anti-CcmG- and anti-CcmH-specific polyclonal antibodies, respectively. Aliquots from cell cultures were TCA-precipitated with or without prior DTT reduction. Cell pellets were solubilized in buffer containing SDS with or without AMS. AMS alkylates free Cys thiolates, adding to the total protein molecular mass of ∼0.5 kDa per AMS-modified thiolate. Reduced and AMS-modified proteins show higher molecular weights in SDS-PAGE. The data show that in actively growing cells, CcmG is reduced (A) and CcmH is oxidized (B). NT refers to untreated cell cultures solubilized in buffer without AMS; + AMS indicates untreated cells solubilized in buffer containing AMS; + DTT indicates DTT-reduced cell cultures solubilized in buffer without AMS, and +DDT/AMS indicates cells first reduced with DTT and then reacted with AMS.