注記 |
In the process of metamorphosis of the housefly, Musca domestica vicina Macquart, phenoloxidase undergoes a sharp fluctuation as shown in Fig.1. The high activity observed at the final stage of larval development disappears completely in the prepupae. However, a homogenate of prepupae, which exhibits no phenoloxidase activity, displays enzyme activity on the addition of either an anionic detergent or an extract of aged pupae as can be seen in Figs.1 and 2. It was inferred from these facts that phenoloxidase survived as a latent enzyme in the prepupae and that its activator was contained in the aged pupae. Although there was a difficulty in the separation of the prepupae from the aged pupae, we were successful in this separation by putting them into water. The prepupae sank to the bottom, while the others floated on the surface of the water. The mode of activation of latent phenoloxidase from prepupae by SDS was studied in detail. As shown in Figs.3 and 4, there was a certain range of ratios between the concentration of SDS and that of latent phenoloxidase which is effective for the activation. It was observed that a narrow pH range between 7 and 8 was effective for the activation as seen in Fig.5. The Fig.6 shows the thermal stability of latent phenoloxidase and temperature dependence of the activation with SDS. Thus, effective activation evidently occurs only with the specific experimental conditions mentioned above. An activator of protein nature was partially purified from the aged pupae. Effects of concentration of activator, pH, and temperature on the activation of latent phenoloxidase were studied. The results obtained are shown in Figs.7-11. From these results it was presumed that the activation occurs catalytically. Inhibition of the activation by various chemical reagents were studied and the results were shown in Table 2. The activation of the latent phenoloxidase was also brought about by dialyzing it against water or buffer solution of low salt concentration below 0.1 M at pH 6 to 7 as seen in Fig. 12. The decrease in salt concentration could affect either ionic strength or osmotic pressure of the solution. The lowering of osmotic pressure would cause the destruction of a granule to which phenoloxidase is bound, hence the liberation of the enzyme. Thus, dialysis of the latent phenoloxidase solution was carried out against 0.01 M phosphate buffer of pH 6.0 containing 0.5 to 1.0 M sugar. Despite of maintaining the osmotic pressure of the solution, activation was brought about in all cases. From this it was inferred that the activation occurred independently on the osmotic pressure and dependently upon the ionic strength of the solution. It was also presumed that the latent form of phenoloxidase is not a bound form but an inactive form of soluble protein. Depending upon the salt concentration of the solution, phenoloxidase fluctuated reversibly from active to inactive form. At a high concentration of 0.2 M phenoloxidase became inactive again. It seems reasonable to conclude that the limited proteolysis which plays an important role in zymogen activation can be excluded from the mechanism of the activation of latent phenoloxidase. The decrease in ionic strength of solution would lead to a conformational change of latent phenoloxidase protein or affect the interaction between protein molecules. As to the effects of various chelating reagents on the activation of latent phenoloxidase by dialysis, EDTA was found to inhibit the activation completely as seen in Table 3. In order to elucidate the common mechanism involved in three different processes mentioned above, attempts were made of the purification of latent phenoloxidase. Scheme 1 shows the partial purification procedure of latent phenoloxidase from prepupae. By 0.4 saturation of the latent phenoloxidase N solution with ammonium sulfate, a rather labile latent phenoloxidase A was precipitated leaving a factor in the supernatant, which can inhibit the activation of latent phenoloxidase by its activator, presumably by stabilizing it. The inhibitory effect of this factor, which was tentatively referred to as factor N, was lost on heating above 60℃ as can be seen in Fig.13. Factor N was non-dialyzable and assumed to be a protein. Although latent phenoloxidase A was fairly unstable, it was in an inactive state in 0.2 M phosphate buffer at pH 6.0 unless any activating agent was present. This means that latent phenoloxidase A solution does not contain free activator. In addition, the latent phenoloxidase A exhibited only a single peak in its gel filtration pattern on Sephadex G-50. However, two peaks were observed after dialysis against water as shown in Fig. 14. Phenoloxidase produced was found in F-2, while F-1 exhibited no apparent phenoloxidase activity but was found to accelerate the rate of activation of latent phenoloxidase A by activator remarkably as can be seen in Fig. 15. In addition, it was found as shown in Fig. 16 that the activation was fulfilled by F-1. Thus, it is believed that an activator exists in the latent phenoloxidase system but in an inactive form. This inactive form of activator was designated as an inert activator. Consequently, it appears that three types of protein; prophenoloxidase, a precursor of phenoloxidese, inert activator, and factor N, participate in the organization of latent (dormant) phenoloxidase system in the prepupae of housefly. As for the activation of latent phenoloxidase by dialysis against water, it is possible to conclude that the inert activator is activated by dialysis, hence the mechanism of activation by dialysis is the same as that by activator. As can be seen in Table 4, the activation of inert activator was inhibited by EDTA, whereas prophenoloxidase was not affected. This suggests the important role of the activater which was produced from its inert form by dialysis. The inhibitory action of EDTA, however, was excluded by precipitation with 30-40% saturation of ammonium sulfate, followed by dialysis against water. The precipitation with 100% saturation, however, was not effective in excluding the inhibitory action of EDTA. In order to clarify whether three types of proteins which participate in the organization of the latent phenoloxidase system exist in a form of complex or not, attempts were made to purify the latent phenoloxidase system in the presence of EDTA by the procedure shown in Scheme 2. A fraction D'-4 obtained was not activated by dialysis and moved as a single band in disc electrophoresis performed in the presence of EDTA as seen in Fig.17. However, the sample moved as three bands in disc electrophoresis either in the presence or absence of EDTA after the treatment with ammonium sulfate, followed by dialysis as mentioned above. Consequently, it was presumed that three protein components exist as a complex in the prepupae. The complex was referred to as latent or dormant phenoloxidase complex. This complex was stable only in a limited pH-range near pH 6 as shown in Fig.18. The mechanism of activation of latent phenoloxidase complex is now still obscure in connection with protein structure. However, there was a possibility that the formation of a certain quartenary structure or subunit structure might be involved in the process of activation. Phenoloxidase was purified from the larvae of housefly by the procedure described in Scheme 3 as an electrophoretically homogeneous protein. Fig.19 is an electron micrograph of the purified phenoloxidase. Evidently, phenoloxidase has a subunit structure and a large molecular weight. In sucrose density gradient sedimentation analyses of crude preparations of latent phenoloxidase and phenoloxidase, the latter was sedimented faster than the former as can be seen in Fig.20. Gel filtrations of partially purified latent phenoloxidase and phenoloxidase preparations through Sephadex G-200 were carried out resulting in an assumption that the molecular weight of the latter was greater than that of the former judging from Fig.21. The activator was purified from the aged pupae by the method described in Scheme 4 and obtained as an electrophoretically and ultracentrifugally homogeneous protein. The specific activity of the preparation was 2,000 times as high as that of the crude extract. The molecular weight was assumed to be about 16,000 from the sedimentation coefficient of 2.06 S. The isoelectric point of this protein was determined to be 4.7. The amino acid composition of the activator is shown in Table 6. Neither sulfhydryl group nor disulfide linkage was contained in this protein. In this connection, the conformation of activator was highly sensitive to the salt concentration in solution and actually it became inactive at concentrations higher than 0.5 M of acetate buffer. The activator lost its activity completely by EDTA. The reactivation was accomplished by either precipitation with 40% saturation of ammonium sulfate and subsequent dialysis against water or the addition of metal ion of two valence cited in Table 6. From these facts it was inferred that the activator is not regarded as a metal protein and that negatively charged carboxyl groups of EDTA interact with positively charged groups inside the activator molecule resulting in the conformational change or the restriction of conformational change. Notwithstanding the examination in various experimental conditions shown in Table 7, neither proteolytic nor esterase activities of the activator was detected. From the evidence mentioned above, a hypothesis on the mode of activation and inactivation of phenoloxidase in the process of metamorphosis from larvae to pupae was presented as shown in Fig.22. In this hypothesis it was postulated that changes in subunit structure cause the fluctuation of the activity of phenoloxidase. It is already known that a metamorphosis hormone, ecdyson, is secreted increasingly at the final stage of larval development, resulting in the acceleration of the synthesis of an activator of prophenoloxidase. From this fact, and those reported here, it is possible to postulate that the activator formed changes in an inactive form at the end of the larval stage and combine with prophenoloxidase forming an enzymatically inactive complex which is stabilized by binding to factor N. Such a latent phenoloxidase complex might be passed along to prepupae and stored as a source of phenoloxidase at the initial step of the metamorphosis of the insect. If this is correct, the possibility exists that such a latent enzyme complex can participate in the regulation of the enzyme activity of living organisms.
|