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. 1975 Nov;152(2):255–265. doi: 10.1042/bj1520255

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

Anthony K Campbell 1, Robert L Dormer 1
PMCID: PMC1172467  PMID: 4062

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+.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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