Abstract
A mutant of Escherichia coli that has an alteration in the structural gene for the alkaline phosphatase has been found to synthesize an anomalous subunit that is unable to dimerize under normal conditions of cell growth and, thus, cannot form active enzyme. This subunit was detected by the use of antibodies prepared against a chemically modified subunit obtained from the wild type enzyme. The mutationally altered subunit is detected only when the mutant's growth is limited by inorganic phosphate except in a constitutive strain in which this subunit is formed even though inorganic phosphate is present in the growth medium. When fully induced, cells produce 6% of their total protein as the defective subunit which can be released into the medium when spheroplasts are prepared with lysozyme. Inability to make the active dimeric form of this enzyme does not, therefore, affect the regulation of the synthesis of this protein or its localization to the bacterial cell's envelope. The addition of zinc ions and inorganic phosphate to cultures of this mutant or to extracts of sonically disrupted cells leads to the appearance of enzymatically active protein which differs in several respects from wild type alkaline phosphatase. Subunits prepared from a highly purified sample of the active form of the mutationally altered protein are similar to those found in the growing cells.
Highlights
Alkaline Phosphatase Activity in Strains C. 10 F. 27 and S ’ 6Wild type E. coli K. 10 produces alkaline phosphatase only when the cells are deprived of Pi, while constitutive mutants with defects in the regulatory genes for this enzyme synthesize the protein even when Pi is present in the medium [12]
F-27, active enzyme is observed during exponential growth of the cells very shortly after Zn* and Pi are Alkaline Phosphatase Subunit Formation by E. coli Mutant
Chloramphenicol does not inhibit the initial activation but leads to a decrease in the amount of total enzyme formed (Fig. 2). These results suggested that a precursor protein was present in these cells and this hypothesis was confirmed when it was found that extracts prepared from sonically disrupted cells can be treated to yield active enzyme (Table II)
Summary
PurijicaGm of Active Enzyme from C .lO F-27---Procedures identical with those employed for purification of active wild type enzyme [8] were used to obtain a highly purified preparation of the C. 10 F. 27 alkaline phosphatase. The normal medium was supplemented with Pi (6.5 mM) and ZnS04(10 PM), and cells were harvested from a 40-liter batch grown in a Fermacel (New. Brunswick). Lysozyme (100 pg per ml) was added to washed cells that were suspended in 50 mM Tris-Cl (pH 8.0), 2 mu EDTA, and 0.5 M sucrose. The active material from a second fractionation on DEAE-cellulose gave one major protein band on acrylamide gel electrophoresis and on this basis is estimated to be of the order of 90% pure. The electrophoretic mobility in starch gel of the enzymatically active protein from C s10 F.27 is identical with that of wild type enzyme, and it has the same three isoenzyme bands generally found in similar preparations of wild type enzyme [8]
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