Studies on the Extracellular Alkaline Phosphatase of Micrococcus sodonensis

The secretion of alkaline phosphatase by Micrococcus sodonensis has been studied by observing the production and accumulation of extracellular enzyme by log phase cells which have been resuspended in fresh growth medium. The accumulation of extracellular alkaline phosphatase activity is the result of a selective permeation process and is totally dependent upon the presence of divalent cation. Although calcium is an actual component of the enzyme and is required for both catalytic activity and enzyme stability, magnesium appears to be the divalent cation required for the actual synthesis or release (or both) of alkaline phosphatase from the cell. Studies with various inhibitors of protein synthesis suggest that the bacterial cell may possess at least two distinct sites of protein synthesis, one site functioning to provide intracellular and cell-associated proteins and another for the synthesis of extracellular proteins. The over-all process of enzyme secretion is markedly temperature-dependent and is characterized by an activation energy of 55 kcal per mole. This observation suggests that the rate-limiting step in alkaline phosphatase secretion is probably not an enzyme-catalyzed event. A relatively small but significant amount of alkaline phosphatase activity exists in a cell-bound form and can be quantitatively released from cells by treatment with lysozyme. Although lysozyme-sensitive cell wall components are involved in the binding of alkaline phosphatase to the cell, the cell wall is not an obligatory component of the secretion process since lysozyme-prepared, sucrose-stabilized protoplasts are capable of extracellular enzyme production.