Experiments with highly calcium dependence

Fig. 12. (A) m -V relationship for fibres perfused with high Cs in 1/4 Na-SW. . , O, fibres perfused with high Cs and bathed in 1/4 Na-SW made with chloride -t-. X, O. V, fibres perfused with high Cs in 1/4 Na-SW made with iscthionate, with 15 mM CaCl2 (the calcium activity in this solution was identical to that of the 1 /4 Na-SW made with chloride). (B) Voltage dependence of the steady-state distribution of charges. C, , O, , , A, V, fibres internally perfused with high Cs" (the different symbols represent separate experiments) O, , fibres internally perfused with low Cs and all experimental points shifted toward more negative values by 9 mV. All experiments were carried out in an Na- and K-free saline made with iscthionate in place of chloride. Curve was computed by the least-squares fit with the experimental points (sec text). (Adapted from Keynes and Rojas [38].)...

Calcium ions are bound with an identical high affinity of 5.106M by the purified ATPase, by the transport protein in the native membranes as well as by partially deli-pidated, reversibly inactivated membrane preparations"8, ll9 173). The amount of calcium which is bound with that high affinity corresponds to two sites per transport molecule. The observed affinity is in good agreement with the affinity derived from the dependence on ionized calcium of the activation of calcium uptake and ATP splitting as well as of the inhibition of calcium release and ATP synthesis18 u2,, 74 17s Since the latter experiments were performed under conditions which provide a constant internal free calcium concentration by the presence of oxalate or phosphate in the system, the reactions must have been activated or inhibited by the calcium ions... 

It is an everyday experience that calcium soaps have a low aqueous solubility while many surfactants with monovalent counterions have an extremely high solubility. As for other compounds, the solubility is to a great extent given by conditions in the solid phase, but for surfactants the strongly cooperative association produces the peculiar temperature dependence of solubility schematized in Fig. 2.8. The so-... 

Calcium-phosphate transfection has been used transiently to introduce plasmids containing the luciferase gene into animal cells (15) and electroporation has proved useful for plant and Dictyostelium cells, (16,26). For monkey CV-1 cells,10 yg of DNA was optimal for high levels of expression and the activity dropped sharply when DNA below or above this amount was used. In contrast, for electroporation of carrot cells (32) the signal increased almost linearly up to the highest amount of DNA used (50 yg/ml). It would be advisable to determine this optimal amount of DNA for other cell lines or transfection protocols. Extracts for luciferase activity are prepared and assayed as described (15,16). The Instruments used to measure activity in extracts must be calibrated periodically with purified luciferase of known specific activity to obtain an idea of the absolute amount of the enzyme being produced. For experiments in which luciferase and CAT genes are cotransfected into cells, the extracts should be made in the luciferase extraction buffer because CAT is stable in this buffer, whereas luciferase exhibits lower activity in the CAT extraction buffer. Furthermore, in such cotransfection experiments, the relative levels of CAT and luciferase expression have been found to depend upon the amounts of the two plasmids used and there is also the possibility of competition between the promoters on the two plasmids. Hence it is advisable to optimize the absolute amounts as well as the ratios of the two plasmids in such experiments. 

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