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An alkaline phosphatase mutant of Pseudomonas aeruginosa exhibiting both regulatory and catalytic changes was isolated. Under repression conditions (i.e. high inorganic phosphate (Pi)) the mutant culture produced an alkaline phosphatase (APase) displaying significant activity against both beta-glycerol phosphate (betaGP) and p-nitrophenyl phosphate (pNPP), while the wild type displayed no activity directed towards these substrates under the same conditions. In vivo, the mutant enzyme's ratio of specific activities was 45:1 in favour of betaGP versus pNPP, whereas this ratio was reversed to 1:9 betaGP versus pNPP for the same enzyme isolated from mutant cells. In addition, the kinetic parameters and stability requirements for the mutant-derived enzyme was altered in comparison with those of the wild type. A study of lipopolysaccharide (LPS) preparations from both the mutant and wild type indicated the mutant to be deficient in the core region of its LPS. The authors propose that the modifications in the catalytic activity of the mutant enzyme, demonstrated in vivo, are due to a change in the enzyme's microenvironment.

Glutaraldehyde, the biological fixative of choice in the cytochemical localization of the phosphatases, was investigated for its effects on Pseudomonas aeruginosa alkaline phosphatase. Comparative studies on the inactivation of alkaline phosphatase by glutaraldehyde showed significant differences when the purified protein was compared with whole, cell-bound enzyme. The effects of the reagent on the kinetics of the purified enzyme were studied and some conclusions drawn as to the mode of inactivation. The reaction of glutaraldehyde with the cell envelope of P. aeruginosa was also investigated, and it was found not to modify the extraction of lipopolysaccharides from the outer membrane. This study emphasizes the care that must be taken to interpret data, cytochemical or otherwise, obtained when glutaraldehyde is used as a fixative or cross-linking reagent.

Alkaline phosphatase (APase) of Pseudomonas aeruginosa exists primarily in the periplasmic region of the cell, i.e., between the cytoplasmic membrane and the outer tripartite layer. The enzyme is also found in the culture filtrate or associated with the outer layer of the cell wall. APase forms a complex with released outer cell wall material, and lipopolysaccharide (LPS) is associated with the complex. Since the enzyme was purified to homogeneity, it became desirable to determine whether complex formation with LPS , or the outer cell wall, affected any properties of the purified phosphatase. The ratio of activities of purified APase with p-nitrophenylphosphate and beta-glycerolphosphate as substrates is about 4:1. The ratio of activities with enzyme complexed with LPS is about 1:1. The energy of activation of sucrose or magnesium released enzyme is 9500 cal/mol whereas the values for purified enzyme plus LPS, purified enzyme, purified enzyme plus phosphatidylethanolamine (PE), and purified enzyme plus LPS plus PE range from 3400 to 8700 cal/mol. These changes occur in the physiological temperature range, 27 to 39C, of this organism. Sucrose-released enzyme in the presence of substrate is inactivated at 47C whereas pure enzyme plus substrate is affected at 41C. The addition of LPS, PE, or a combination of both increases the temperature of inactivation from 45 to 51C. The results suggest that certain properties of the purified enzyme differ from those of the enzyme released from whole cells by either sucrose or magnesium resuspension. The addition of cell wall components such as LPS and PE to purified APase restores these properties. The addition of cell wall components such as LPS and PE to purified APase restores these properties. The evidence suggests that artificial complex formation changes the environment of the enzyme protein such that the environment now resembles that which exists within the whole cell wall.