Electron microscope histochemical localization of alkaline phosphatase(s) in Bacillus licheniformis

Sites of alkaline phosphatase (APase) activity in a facultative thermophilic strain of Bacillus licheniformis MC14 have been localized by electron microscope histochemistry, using a lead capture method. The effects of 3% glutaraldehyde and 3.0 mM lead on APase activity were investigated, and these compounds were found to significantly inhibit enzyme activity, 68 and 18%, respectively. A number of parameters were varied in studies to localize APase activity, including: growth temperature (55 and 37 degrees C); substrate concentration in the histochemical mixture (0.06, 0.15, 0.30, 1.00 mM); fixatives; protoplast preparations and whole cells; phosphate-repressed and -derepressed cells; and age of vegetative cells (mid-log and late log). These variations affected the number but not the location of lead phosphate deposits, which appeared at discrete sites along the inner side of the cytoplasmic membrane. Control cells incubated in histochemical mixtures lacking substrate, lead, or both exhibited no lead phosphate depositis. The histochemical localization at membrane sites correlated well with biochemical localization data, which indicated that greater than 80% of the APase activity was associated with the membrane fraction in logarithmically growing cells.