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Liposomes are prepared from rat brain microsomal lipid and loaded with either Tb or dipicolinic acid (DPA) to test fusion with the Tb-DPA assay. They are also loaded with octadecyl Rhodamine B chloride (R) to test fusion with the R assay. The addition of either Ca or Mg to loaded liposomes develops fluorescence with both assays. The fluorescence elicited by Mg is similar to that elicited by Ca if assessed with R, but much higher if determined by Tb-DPA. The Ca-dependent fluorescence of the Tb-DPA complex is not suppressed by the addition of EDTA, and therefore it is internal to vesicles. The contrary is true for the Mg-dependent fluorescence. Rat brain microsomes can be disrupted by adding octylgucoside and reconstituted by removing it by dialysis. We use this procedure to load microsomes with DPA. This allows the use of the Tb-DPA assay for testing the fusion of rat brain microsomes. Reconstituted microsomes fuse with liposomes. This fusion has characteristics similar to those of liposome-liposome fusion. However, no microsome-microsome fusion could be detected with either method. The two methods give different results, owing to the chemical properties of the assays. Indeed Tb-DPA implies the retention of vesicle content, whereas this is not required by the R assay. [ABSTRACT FROM AUTHOR]

The compartmentation of the phosphatidylethanolamine newly synthesized in brain microsomes in vitro either by base exchange or net synthesis has been studied, using difluorodinitrobenzene as a chemical probe. The experimental results demonstrate that in rat brain microsomes the phosphatidylethanolamine molecules synthesized by base exchange and the bulk membrane lipid belong to different pools. Ca2+ bound to microsomes seems to be involved in the maintenance of the compartmentation of phosphatidylethanolamine. In the presence of Ca2+ the newly synthesized phosphatidylethanolamine molecules react with difluorodinitrobenzene as though they are organized in clusters. After biosynthesis in vivo or in vitro through the cytidine pathway, the compartmentation of the newly formed phosphatidylethanolamine appears less marked than after the synthesis through base exchange. [ABSTRACT FROM AUTHOR]