ANIONIC DOMAINS ON THE CYTOPLASMIC SURFACE OF THE PLASMA-MEMBRANE OF ACANTHAMOEBA-CASTELLANII AND THEIR RELATION TO CALCIUM-BINDING MICROREGIONS

In this report we further characterize the plasma membrane microregion s involved in formation of the calcium-dependent, electron-opaque stru ctures. We used ruthenium red, a highly positively charged molecule to reveal clustered anionic sites on the cytoplasmic surface of the plas ma membrane. To examine the presence of microregions of anionic phosph olipids on this membrane polymyxin B was applied. Using conventional e lectron microscopy, X-ray microanalysis and freeze-etch techniques we demonstrated that in permeabilized cells, ruthenium red enters the cel l interior and forms prominent globular structures located on the cyto plasmic surface of the plasma membrane. The size, location and X-ray e lemental microanalysis indicated that ruthenium red-evoked structures are very similar to the submembrane calcium-structures. Polymyxin B si gnificantly interferes with the formation of calcium-induced electron- opaque structures. Our findings indicate that negatively charged plasm a membrane constituents may be organized as microdomains in response t o calcium ions as well as to polycations (ruthenium red, some proteins ) and finally electron-opaque structures on the cytoplasmic surface of the plasma membrane are formed.