Liposomes are prepared from rat brain microsomal lipid and loaded with either TbJ+ or dipicolinic acid (DPA) to test fusion with the Tb·DPA assay. They are also loaded with octadecyl Rhodamine B chloride (R18) to test fusion with the R18 assay. The addition of either Ca2+ or Mg2+ to loaded liposomes develops fluorescence with both assays. The fluorescence elicited by Mg2+ is similar to that elicited by Ca2+ if assessed with R'8, but much higher if determined by Tb-DPA. The Ca2+-dependent fluorescence of the Tb-DPA complex is not suppressed by the addition of EDTA, and therefore it is internai to vesicles. The contrary is true for the Mg2+-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 ofthe 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 R18 assay.
Fusion of Liposomes and Rat Brain Microsomes Examined by Two Assays
CORAZZI, Lanfranco;ARIENTI, Giuseppe
1989
Abstract
Liposomes are prepared from rat brain microsomal lipid and loaded with either TbJ+ or dipicolinic acid (DPA) to test fusion with the Tb·DPA assay. They are also loaded with octadecyl Rhodamine B chloride (R18) to test fusion with the R18 assay. The addition of either Ca2+ or Mg2+ to loaded liposomes develops fluorescence with both assays. The fluorescence elicited by Mg2+ is similar to that elicited by Ca2+ if assessed with R'8, but much higher if determined by Tb-DPA. The Ca2+-dependent fluorescence of the Tb-DPA complex is not suppressed by the addition of EDTA, and therefore it is internai to vesicles. The contrary is true for the Mg2+-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 ofthe 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 R18 assay.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
