222 ON THE CEPHALOPOD PHOSPHAGEN BY ERNEST BALDWIN, B.A. (From the Biochemical Laboratory, Cambridge, and the Marine Biological Station, Tamaris, Var, France.) (Received 8th November, 1932.) (With Four Text-figures.) INTRODUCTION. THE comparative researches of Eggleton & Eggleton(s) on the distribution of phosphagen made it clear that while creatine phosphate is very widely distributed amongst the vertebrates, it is not present in the invertebrates. Shortly afterwards, a new phosphagenic substance was isolated from crab muscle by Meyerhof & Lohmann(n, 12) and shown to be arginine phosphate, while the later work of Lundsgaard (9) has made it certain that this compound plays in these tissues a part exactly analogous to that played by the creatine compound in vertebrate muscles. Later, Meyerhof (10) examined a number of invertebrates, representative of several phyla, and came to the conclusion that arginine phosphate is present in Holothuria, Pecten and Sipunculus. Cephalopod muscle contained no phosphagen. The case of the cephalopods was further examined by Needham, Needham, Baldwin & Yudkin (13), who not only found that the muscles of Sepia and of Octopus do contain phosphagen, but were also able to investigate its ontogeny in the former (14). There seemed no reason to think that the compound present was any- thing other than the arginine compound (8). Ackermann, Holtz & Kutscherw claim to have isolated the copper nitrate salt of arginine from extracts of the cephalopod Eledone moschata, while Okuda(is) has made a similar claim in the case of Loligo breekert, whereas Iseki (7) has been able to isolate no arginine from extracts of Octopus, finding in its place a compound which he isolated in the form of its picrate, and which he thinks may be a methyl agmatine. Iseki's compound gave a positive Sakaguchi reaction, and is therefore of the type NH, HN = C NHX and would probably be capable of functioning as a phosphagen, when combined with phosphoric acid. During the work of Needham, Needham, Baldwin & Yudkin, one slight peculiarity was noticed in the behaviour of the phosphagen, and the authors' attention was redirected to this on reading the paper by Iseki to which reference has On the Cephalopod Phosphagen 223 already been made, and further investigations were carried out during a visit to the Marine Biological Station of Tamaris, Var, France. The cephalopod chosen for most of the experiments was Eledone moschata, which closely resembles Octopus except that it possesses one row of suckers on the tentacles instead of two. Experiments on Sepia were also projected, but these animals seldom survived for more than 24 hours after being caught, and would therefore not have been in very good condition for experimental work, since it has been shown that their condition has a very marked effect upon the phosphagen content of the muscles (13). Eledone, on the other hand, lived much longer, and specimens used after 4 or 5 days in the aquarium seemed still to be in excellent condition. When required for an experiment an animal was taken and placed in a large bowl filled with ice and left thus in the ice-chest for 20 min. or longer, according to the size of the animal. A suitable piece of muscle was cut away from the mantle, the animal having become perfectly quiet, then freed from skin, and dropped into an ice-cooled beaker, weighed, and extracted with ice-cold 10 per cent, trichloracetic acid by grinding as rapidly and thoroughly as possible with acid-washed quartz sand. The extracts were filtered under pressure through cooled Gooch crucibles of 16 c.c. capacity, and received in a cooled tube, and used at once for the experiments. The method of Fiske & Subbarow (6) was used throughout for the estimations of the phosphate, and a Klett top-reading colorimeter was employed for the comparisons. EXPERIMENTS. Exp. 1. 4-51 gm. of tissue were extracted with 20 c.c. of the trichloracetic acid. The filtered extract was kept at o° C. and the^H adjusted by adding saturated soda till the colour of a little added thymol blue showed some visible change, and then determining it more accurately by means of a capillator. The value found was 1-7. The whole extract was now rapidly heated to 28° C. by immersion in a warm bath, and then transferred to an incubator at the same temperature. The variation observed in the temperature of the incubator, which was electrically regulated, was ± 0-5° C. during the experiment. 1 c.c. samples were thereafter removed and the phosphorus content determined. In addition, two samples were put up with one- tenth of their volume of 2-5 per cent, ammonium molybdate, and these were also incubated, and their phosphate contents determined in order to get some idea of the effect of the molybdate ion upon the reaction velocity. The results of this experiment are given in graphical form in Fig. 1, and show that the hydrolysis is complete in about 12 hours under the conditions of the experi- ment. The velocity constant for the hydrolysis (calculated in terms of natural logarithms with the minute as time unit) is 6-2 x io~3 for 50 per cent, hydrolysis, a value which agrees well with those in the literature for arginine phosphate. The lower curve in Fig. 1 connects the points obtained in the presence of the molybdate ion, allowance having been made for the dilution of the samples by the addition of the molybdate, and shows that the only effect of the ion is a slight retardation. In addition, a rough estimate of the total free guanidine base of the extract was obtained by the method of Weber (18) which is based on the Sakaguchi reaction (17). 224 ERNEST BALDWIN The base present in the solution gave a coloration which showed no visible difference in tint from that produced in a standard solution of arginine, and it was supposed that the intensity of the colour produced by equivalent amounts of arginine and of the free base would be the same. On this assumption it was possible to calculate the amount of phosphate to which any given amount of base would be equivalent, and thus, in the later experiments, to compare the amounts of base and phosphate liberated in any given time in the same solution. In the present case, the amount of free base corresponded to 0-294 mS- P Per c-c- °f solution, while the total free phosphate was 0-263 mg. per c.c, indicating the presence of a slight excess of free base. The same is the case in frog muscle according to Duliere (4), and in the electrical organ of Torpedo according to the present author 0). 0-281- 0-17 5 10 15 Time (hours) -> Fig. 1. Hydrolysis of Eledone phosphagen. (28°C, pH = 1-7.) Returning to the question of the velocity constants; it has been mentioned that the molybdate ion produced only a small retardation of the hydrolysis. According to Meyerhof & Lohmann(ia) the hydrolysis of arginine phosphate is retarded some 30 times by this ion, or rather less in crude extracts, which suggests that the phos- phagen is not arginine phosphate, as was previously thought. But on the other hand, the molybdate ion is known to have a marked catalytic effect upon a large number of phosphoric compounds, and it was possible that some such compound was breaking down under the influence of the molybdate and masking the effect of the latter upon what might really have been the arginine compound. This possibility was investigated in the next experiment. Exp. 2. An extract was prepared from 4-82 gm. of muscle with 25 c.c. of trichloracetic acid. 2-5 c.c. of 2-5 per cent, ammonium molybdate were added to the On the Cephalopod Phosphagen 225 extract after filtration, when a slight turbidity resulted which, however, cleared up in the subsequent operations. The pH was brought to 1 -6 and the solution heated to 28° C. as before and transferred to the incubator. The fluid was now perfectly clear. 1 c.c. samples were taken at intervals for the estimation of the free base, and further 1 c.c. samples for the estimation of the phosphate. (It has been found by the author and Dr D. M. Needham that the Sakaguchi reaction is given by free arginine, but not by arginine combined in the form of the phosphate.) The results obtained are given in Fig. 2. The estimations of the base are much less accurate than those of the phosphate, for the method used in the former case is accurate at best to only 3-5 per cent. But none the less, it is evident that the amount of guanidine base liberated is approximately equivalent to the phosphate and that the amount of phosphate contributed by the breakdown of other phosphorus compounds is at any rate small. O-33r 0-31 - 10 20 30 Time in hours -* Fig. 2. Hydrolyais of Eledone phosphagen. (28° C, pH = i-6, o'Z5 per cent. amm. molybdate added.) It may therefore be taken that the velocity constant calculated from the phosphate curve, which was 1-9 x 10~s, refers to the hydrolysis of the phosphagen and not to that of another compound. Exp. 3. The third experiment, the results of which are given in Fig. 3, was a repetition of the first, except that the base liberation as well as that of the phosphate was followed. 4-25 gm. of tissue were taken and extracted with 20 c.c. of the tri- chloracetic acid. The curves show that the base and the phosphate are here again liberated in equivalent amounts, thus disposing of the possibDity that two compounds are present which are differently affected by the molybdate ion.