Biosynthetic utilization of ethanolamine by Erwinia carotovora.

The purified obelin was electrophoresed on 7 % polyacrylamide gels in Tris-glycine buffer, pH 8.3, containing 0.1 mM-EDTA. Gels stained with Coomassie Brilliant Blue showed a single band at the buffer front, which correlated in position with luminescent activity. This activity could be eluted from unstained gel slices by soaking them overnight in 0.1 mM-EDTA-lOmM-Hepes, pH 7.0, at 4°C. Obelin is qualitatively similar to the luminescent protein aequorin in its response to bivalent cations (Campbell, 1974), although there are quantitative differences in the responses to Ca2+ (Moisescu et al., 1975). By assaying obelin in 1OmM-Tes [N-tris(hydroxymethyl)methyl-2-aminoethanesulphonic acid] buffer which had been passed through a column of Chelex 100 to remove Ca2+, it was possible to measure Caz+ at concentrations from 0.1 p~ upwards, without the use of CaZ+-EGTA [ethanedioxybis(ethy1amine)tetra-acetic acid] buffers, which may cause problems in interpreting results (Blinks, 1973). Caz+ was injected from a plastic syringe and the rate of luminescence followed on a chart recorder. By using this sytem, it has been shown that univalent cations affect obelin luminescence in response to Ca2+. Changes in intracellular free Caz+ concentrations have been measured, by using luminescent proteins, only in systems into which the protein can be micro-injected. Substances can be introduced into small cells by first trapping them in liposomes and subsequently fusing the liposomes with the cells (Papahadjopoulos et al., 1974). Obelin can be trapped by positively charged vesicles prepared from a mixture of lipids (Table 1). Addition of CaZ+ to the liposomes stimulated obelin luminescence; this stimulation was greater after treatment of the liposomes with Triton X-100. The percentage of obelin activity which was apparent on addition of CaZ+, before treatment with Triton X-100, was smaller in sonicated liposomes, suggesting that sonication renders the liposomes more impermeable to CaZ+. The loss of obelin activity in the sonicated liposomes was paralleled by loss of total activity in the medium during sonication. Comparison of the initial obelin activity in the medium with that in the liposomes gave an 'obelin space' of 5p1 per pellet. Similarly, the 'inulin space' was 1 .24 per pellet for both sonicated and unsonicated preparations, suggesting that up to 75 % of the obelin may be bound to the positively charged vesicles. The total amount of obelin associated with the liposomes was approx. 10% of the initial activity in the medium. If 1 % of liposome-entrapped obelin could be transferred to other cells, there would be sufficient activity to measure Caz+ concentrations in the physiological range, if obelin with an activity of lolo or 10" counts was added to the initial medium.