Fusion of liposomes and rat brain microsomes examined by two assays (original) (raw)

Liposomes are prepared from rat brain microsomal lipid and loaded with either Tb 3+ or dipicolinic acid (DPA) to test fusion with the Tb-DPA assay. They are also loaded with octadecyl Rhodamine B chloride (Rj8) to test fusion with the RiB assay. The addition of either Ca 2+ or Mg 2+ to loaded liposomes develops fluorescence with both assays. The fluorescence elicited by Mg-' § is similar to that elicited by Ca 2+ if assessed with R~s, but much higher if determined by Tb-DPA. The CaZ+-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 MgZ+-dependent fluorescence. Rat brain microsomes can be disrupted by adding octylgucoside and reconstituted by removing it by dialysis. We use this procedure to toad 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 Rt8 assay.