RESUMO
The oversynthesis of the secreted alkaline phosphatase (PhoA) in E.coli K12802 cells due to transformation with the PhoA+ plasmid pHI-7 leads to a change in its biogenesis--alternative localization and accumulation of the enzyme intermediate forms corresponding to different stages of the its post-translational modification. Instead of the soluble PhoA available in the parent strain mostly as a completely processed mature metazyme III localized in the periplasm, five enzyme forms were discovered in the PhoA overproducer: a cytoplasmic PhoA precursor (prePhoA) as insoluble aggregates; three soluble metazymes of a mature active form localized in the periplasm as in well as in culture medium; and a soluble high-molecular form in the periplasm. PrePhoA was isolated and purified by removal of soluble cell fractions using differential centrifugation, solubilization of membrane proteins with Triton X100, dissolution of the aggregates in the buffer with 8M urea and FPLC on MonoQ. Extracellular PhoA was purified by ultrafiltration, thermal treatment, and gel chromatography on Sepharose CL-4B. It was shown that the isolated prePhoA can be transformed into a mature form in the presence of a leader peptidase in 0.8 urea and is completely cleaved with proteinase K. Three forms of the mature PhoA vary in resistance to proteinase K and trypsin. Metazyme I, the unprocessed mature PhoA, is the most resistant to proteolysis.