In vitro studies of an alkaline phosphatase-cell wall complex from Pseudomonas aeruginosa.
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.
D000469:Alkaline Phosphatase / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism / Q000648:ultrastructure; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D005994:Glycerophosphates / Q000378:metabolism; D006358:Hot Temperature; D007525:Isoelectric Focusing; D008070:Lipopolysaccharides / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D009596:Nitrophenols; D010710:Phosphates / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D011135:Polysaccharides, Bacterial / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D013395:Sucrose / Q000494:pharmacology
D005994:Glycerophosphates; D008070:Lipopolysaccharides; D009596:Nitrophenols; D010710:Phosphates; D010714:Phosphatidylethanolamines; D011135:Polysaccharides, Bacterial; D013395:Sucrose; D000469:Alkaline Phosphatase; D008274:Magnesium