Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold

Abstract

Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15to 18-nm colloidal gold particles (gold-immunoglobulin G [IgG] complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles were largely dispersed, and only a few clusters were found. The gold-IgG binding was quantitatively estimated by stereological analysis of labeled, frozen thin sections. This estimation of a variety of control samples showed that the labeling was specific for the alkaline phosphatase. Cluster formation of the gold-IgG particles in association with the plasma membrane suggests that existence of specific alkaline phosphatase binding sites (receptors) in the plasma membrane of B. licheniformis 749/C.

Immunoelectron microscopic double labeling of alkaline phosphatase and penicillinase with colloidal gold in frozen thin sections of Bacillus licheniformis 749/C

“…Alkaline phosphatase was extracted and purified by a previously described method (12). High-titer antibody was produced in rabbits with pure alkaline phosphatase used as an antigen (26). The serum obtained was used for the isolation of IgG by ion-exchange chromatography (8).…”

“…A 100-ml amount of 0.01% HAuC14 * 4H2 was heated, and 320 ,ul of 10% sodium citrate was added as soon as boiling started. Boiling was stopped after 10 min when the color became wine red; after cooling, the pH was adjusted to 7.6 with 0.2% K2CO3 (5,26).…”

“…This solution (1 ml) was added to the diluted HAuCl4, gently mixed for 10 min at Preparation of colloidal gold-IgG label. The procedure for the preparation of the colloidal gold-IgG complex has been described previously (22,26). A purified and dialyzed solution of IgG (5 ,ug/ml) was added to the colloidal gold suspension.…”

“…The pellet containing colloidal gold-IgG conjugate suspended in the above buffer contained about 109 particles per ml and remained stable (i.e., unaggregated) for several months at 4°C. The minimal amount of IgG needed to saturate the colloidal gold particles was 100 IgG molecules per 15to 18-nm colloidal gold particle (26). This ratio was based on the number of IgG molecules needed to saturate the surface area of colloidal gold particles.…”

“…Penicillinase appears to bind to the membrane by hydrophobic interaction, whereas both hydrophobic and ionic interactions may be involved for alkaline phosphatase binding (24). Both enzymes are present in the membrane, but their distribution appears to be significantly different (8,9,11,26). An immunoelectron microscopic double-labeling technique was used to visualize this difference.…”