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III. Discussion
The effect of the various environmental factors on Colpoda, considered in retrospect, indicates first, that the primary effect of all these factors is differential, suppressing one physiological process and not another; and secondly, that the response of Colpoda is similar to the response of other types of plant and animal. The first phenomenon is known as dissociation (Needham, 1942) and the two systems dissociated by these factors are the “activity” and “resting” systems (McElroy, 1947).
Recent study has confirmed that the “activity” system is generally associated with the aerobic carbohydrate metabolism, and in particular oxidative phosphorylation. Inhibitors, therefore, which are most effective in suppressing the “activity” system are those which interfere with the carbohydrate cycle, and it is these inhibitors, and these inhibitors alone, which prevent cells entering the prophase of division (Hughes, 1950). Naturally oxidative phosphorylation will be inhibited in the absence of oxygen and further the reduced carbohydrates of the Krebs cycle will accumulate as with Tetrahymena (Thomas, 1942). The effect of oxygen lack, then, can be attributed simply to the suppression of phosphorylation which normally takes place under aerobic conditions. Anaerobes must either have an alternative system of phosphorylation or else division must be independent of such organic phosphates.
After an exhaustive review of carbon dioxide toxaemia, Chang and Loomis (1945) attributed its inhibitory effect to the formation of a hydrogen bond linkage perhaps with the one-alpha-amino nitrogen of a protein. The coagulation of the cytoplasm indicates that molecular structure has been affected. In other words, the effect of carbon dioxide is more fundamental than anoxia in that the enzyme itself is reversibly affected. Kavanau (1950), in discussing the effect of pH and temperature on enzyme kinetics, also suggests that the inactivation of the enzyme system is caused by the formation of intramolecular hydrogen bridges Kavanau considers that there is a thermodynamic equilibrium between catalytically active and catalytically inactive forms of the enzyme. The active
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configuration of the enzyme is possessed in only a relatively narrow temperature band, being lost at both high and low temperatures. Increasing the temperature from the point of low temperature inactivation results in the equilibrium between inactive and active configuration of the enzyme changing from the inactive to the active form. This continues until the optimum temperature is reached, after which the equilibrium changes from the active to the inactive form. The rate of biological processes can then be explained in terms of an absolute reaction rate with a changing equilibrium of active and inactive enzyme. This theory accounts satisfactorily for the curves illustrating biological activity such as division or excystment in Colpoda. While this interpretation may be accepted it is important to stress the differential aspect of these factors, for it is only the enzymes of the “activity” system that are affected by temperatures outside the range 8 ° C. to 37 ° C. Other enzyme systems function quite normally, such as those connected with encystment and ciliary movement.
It has been emphasised that an organism will tolerate only a limited osmotic difference between its own cytoplasm and the surrounding medium. In most fresh water animals adaptation to increasing salinity cannot be taken past the point where the medium is hypotonic to the organism. It is probable that euryhaline organisms are exceptions to this rule, and this view is strengthened for protozoa by the fact that the contractile vacuole may cease to function in higher salinities. It is apparent that Colpoda steinii is stenohaline and that increasing salinity seriously affects its physiology. It is difficult to attribute the inhibition of division in this case either to metabolic interference or to inactivation of the protein. A simpler explanation is that as the salinity approaches the distal limit the increasing inhibition of division is due to the decreasing osmotic difference of organism and medium and the increasing difficulty of the organism to absorb water against this gradient. (Hughes, 1952).
It is now necessary to consider the action of the excystment promoting substances on the metabolism of Colpoda. It is apparent that the activity of the potassium ion and alcohol is due to their mobility and is not due to any metabolic significance, and this is confirmed by Garnjobst (1947) who found that the basic growth medium did not stimulate excystment. The activity of the K ion in cells is well known. It is known to stimulate respiration and in particular there is an uptake of K following fertilization of sea urchin eggs accompanying increased acidity and increased oxygen uptake (Oddo and Esposito, 1951). It is considered that the role of K in these exchanges is the replacement of H ions. If this is so the effect of the potassium ion in accelerating excystment in Colpoda, a phenomenon known to be comparable to division, may be interpreted in terms of Kavanau's theory. During the encysted period respiration is depressed, but it rises rapidly in response to the excystment stimulus, and this respiratory increase is cyanide sensitive (van Wagtendonk and Taylor, 1942; Thimann and Commoner, 1940). It is not unreasonable to presume that the increasing metabolic activity is due to increasing activity of the enzymes connected with the “activity” system. That the increased respiration is due to increased enzyme activity and not to a new substrate is shown by the fact that distilled water may stimulate excystment, although it is patently not a metabolic substrate. If, then, there is a change-over between the encysted ciliate before and after stimulation, comparable to the difference between the unfertilized and fertilized sea urchin egg, and this change-over is due, as in the case of the sea urchin egg, to the introduction
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of an alternative respiratory mechanism, then its activity will be governed by the principles outlined by Kavanau. Namely, with increasing temperature, up to in optimum, there will be increased acceleration, as the equilibrium increasingly favours the active form of the enzyme. Past the optimum acceleration will decrease, and this is known to be the case (Thimann and Barker, 1934). Accepting this theory, the activity of the K ion can be attributed to the displacement of the hydrogen bond which characterises the inactive form of the enzyme (Seifriz, 1942). With increased concentrations of the K ions up to an optimum there will be increased acceleration of excystment, and this is also known to be the case (Haagen-Smit and Thimann, 1938; Strickland and Haagen-Smit, 1947). However, this initial activation of the enzyme system must be separated from the ultimate respiration of this system for the two are separable, the second is absolutely dependent upon oxygen and the first is not. The effect of alcohol is less certain (Strickland and Haagen-Smit, 1948). It may be that the hydroxl group is active in accelerating the formation of active enzyme, and that its role is complementary to that of the K ions. As the role is performed by a large number of substances, although with greatest efficiency by alcohol, it is difficult to define precisely what this action is (Thimann and Haagen-Smit, 1937; Haagen-Smit and Thimann, 1938). These metabolic changes are accompanied by a marked uptake of water, and as we have seen such uptake is possible only if there is a sufficient gradient of osmotic tension. This water uptake which is definitely dependent upon excystment metabolism is comparable to the water uptake during division which is also dependent upon a favourable osmotic gradient. The fact that considerably lesser salinities are required to inhibit excystment than those required to inhibit division strengthens the view already propose that this water uptake is not dependent upon absolute, but upon relative tensions, within certain limits.
There remains one phenomenon still to be explained—namely, the difference of high temperature on dry and wet cysts. The former are irreversibly affected at 106 ° C. and the latter at 44 ° C. Taylor and Strickland (1936) assume that the temperature of coagulation of a protein in colloidal solution varies with the amount of free water, but they do not discuss why this should be so. It may be, that, as with erythrocytes, with increasing temperature and increasing rate of diffusion there is increased loss of cations, such as K, which is naturally prevented in the dry cyst. The loss of cations, if indeed it takes place, may cause irreversible denaturation of the proteins before the critical temperature is reached. Considering the importance and mobility of the K ions such an explanation is not improbable. It is interesting to note that the lethal limit of the active ciliates is much the same as the lethal limit of the wet cysts (Bodine, 1923). Encystment. per se, does not therefore raise the resistance of the ciliate to high temperatures.