Permeability to calcium of pigeon erythrocyte 'ghosts' studied by using the calcium-activated luminescent protein, obelin.
1. Obelin, the Ca(2+)-activated luminescent protein from the hydroid Obelia geniculata, was sealed inside pigeon erythrocyte ;ghosts' in order to investigate effects on their permeability of different methods of preparation and of the bivalent cation ionophore A23187. 2. Changes in free Ca(2+) within the ;ghosts' were studied by following the rate of luminescence of obelin. The possibility that the obelin might have been released from the ;ghosts' during an experiment was investigated by studying the release of inulin and pyruvate kinase from the ;ghosts'. Less than 10% of the inulin or pyruvate kinase sealed within the ;ghosts' was released under any of the experimental conditions. 3. Triton X-100 (0.1-10%, v/v) made the ;ghosts' highly permeable to Ca(2+). In the presence of 1mm-Ca(2+) and Triton, 95-100% of the obelin was utilized within 10-20s. 4. A time-course of resealing ;ghosts' at 37 degrees C showed that over a period of 90min, the ;ghosts' became gradually less permeable to Ca(2+). ;Ghosts' which remained at 0 degrees C retained only a small concentration of obelin and ATP, and were highly permeable to Ca(2+). 5. Erythrocyte ;ghosts' resealed for 30min at 20 degrees C rather than 37 degrees C were more permeable to Ca(2+), as shown by the fact that 92% of the obelin in the ;ghosts' was utilized during the first 60s after the addition of 1mm-Ca(2+), as opposed to 44% for ;ghosts' resealed at 37 degrees C. 6. Haemolysis at pH6.0 rather than 7.0 resulted in ;ghosts' which were highly permeable to Ca(2+) after resealing for 60min at 37 degrees C. Of the obelin in the ;ghosts', produced by haemolysis at pH6.0, 90% was utilized in the first 60s after the addition of 1mm-Ca(2+) compared with 23% for ;ghosts' produced at pH7.0. 7. The bivalent cation ionophore A23187 increased the permeability of the ;ghosts' to Ca(2+). Maximum effects of the ionophore (16mug/ml) were obtained by preincubating the ;ghosts' with the ionophore A23187 (16mug/ml) in the presence of a low concentration of Mg(2+) and in the absence of Ca(2+).
D000255:Adenosine Triphosphate / Q000032:analysis; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D010856:Columbidae; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006454:Hemoglobins / Q000032:analysis; D006461:Hemolysis; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007444:Inulin / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D008163:Luminescent Measurements; D008164:Luminescent Proteins; D011092:Polyethylene Glycols / Q000494:pharmacology; D011506:Proteins; D011770:Pyruvate Kinase / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D006454:Hemoglobins; D007476:Ionophores; D008164:Luminescent Proteins; D011506:Proteins; C010497:obelin; D000001:Calcimycin; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D007444:Inulin; D011770:Pyruvate Kinase; D002118:Calcium