Specificity of subcellular distribution of alkaline phosphatase in Bacillus licheniformis 749/C.

The objective of this investigation was to examine the in vivo characteristics of binding sites for alkaline phosphatase in Bacillus licheniformis cell surface. An attempt was made to correlate the results from several experimental approaches, namely (i) cell fractionation; (ii) ultrastructural cytochemistry; (iii) MgCl2 extraction and sodium dodecyl sulphate--polyacrylamide electrophoresis of the extracted material; (iv) labelling with 125I-labelled diazonium salt to determine the subcellular origin of MgCl2-extracted material. Results show that 40% of the alkaline phosphatase was bound to the plasma membrane, 35% to the cell wall, and 15% was free in the cytosol. The enzyme was present as aggregates in a few discrete sites in the membrane, wall, and cytoplasm. The membrane enzyme was associated with the inside surface. A few aggregates were enclosed in single-layered vesicles which appeared to protrude through the cell wall. The material extracted with magnesium salt consisted of 8-10 proteins including alkaline phosphatase. The majority of the proteins extracted by MgCl2 originated from the outside half of the plasma membrane, whereas, only a few, including alkaline phosphatase, came from the inside half of the plasma membrane. All of these proteins may have formed a complex which was removed by MgCl2 extraction. Patch formation in the membrane indicated specific aggregation of intramembrane proteins after MgCl2 treatment.