The permeability to calcium of pigeon erythrocyte ‘ghosts’ studied by using the calcium-activated luminescent protein, obelin

Abstract

1. Obelin, the Ca²⁺-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 Ca²⁺ 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 Ca²⁺. In the presence of 1mm-Ca²⁺ and Triton, 95–100% of the obelin was utilized within 10–20s. 4. A time-course of resealing ‘ghosts’ at 37°C showed that over a period of 90min, the ‘ghosts’ became gradually less permeable to Ca²⁺. ‘Ghosts’ which remained at 0°C retained only a small concentration of obelin and ATP, and were highly permeable to Ca²⁺. 5. Erythrocyte ‘ghosts’ resealed for 30min at 20°C rather than 37°C were more permeable to Ca²⁺, as shown by the fact that 92% of the obelin in the ‘ghosts’ was utilized during the first 60s after the addition of 1mm-Ca²⁺, as opposed to 44% for ‘ghosts’ resealed at 37°C. 6. Haemolysis at pH6.0 rather than 7.0 resulted in ‘ghosts’ which were highly permeable to Ca²⁺ after resealing for 60min at 37°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-Ca²⁺ compared with 23% for ‘ghosts’ produced at pH7.0. 7. The bivalent cation ionophore A23187 increased the permeability of the ‘ghosts’ to Ca²⁺. Maximum effects of the ionophore (16μg/ml) were obtained by preincubating the ‘ghosts’ with the ionophore A23187 (16μg/ml) in the presence of a low concentration of Mg²⁺ and in the absence of Ca²⁺.