[Biochemical studies on intramembranous localization of alkaline phosphatase in Bacillus megaterium KM].
Intramembranous localization of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, alkaline optimum, EC 3, 1, 3, 1; AlPase) was observed biochemically in Bacillus megaterium KM grown in 1% polypeptone medium containing 0.5% NaCl at 37 degrees C under aerobic conditions and harvested at the latter logarithmic phase. AlPases from B. megaterium have been separated into soluble and membrane-bound forms by the centrifugation after cell disruption by sonication. The membrane-bound enzyme was further fractionated to two forms by phase separation using a non-ionic detergent, Triton X-114; one was successfully solubilized into the aqueous phase and the other remained in the Triton phase. Both AlPases of sonication- and Triton-solubilized forms were partially purified by gel filtration and anion-exchange column chromatographies. Their molecular weights were different (52,000 for soluble and 66,000 for Triton-solubilized forms) and the Vmax of the sonication-solubilized enzyme (227 nmol/min/mg protein) was 11-fold higher than that of the Triton-solubilized one although similar Km values (1.7 and 2.3 mM) were observed. Optimum pH of these enzymes tended to shift to a neutral range during the purification steps. These results suggest the multiplicity of AlPase anchoring to the membranes; 1) sonication-solubilized form which may be buried within the membrane lipids by its hydrophobic peptide and solubilized by the cell disruption, 2) detergent-solubilized form which may be bound loosely to the membrane by its hydrophobic domain and solubilized due to the amphiphilicity of enzyme protein, and 3) insolubilized form which may be bound fast to the membrane by its strong hydrophobicity and also have the function of enzymatic ability.
Takahashi
K
K
D000469:Alkaline Phosphatase / Q000032:analysis*; D001410:Bacillus megaterium / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D013010:Sonication
D000469:Alkaline Phosphatase