Protein Phosphorylation and Bioelectrogenesis - American Chemical

18 Protein Phosphorylation and Bioelectrogenesis

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E. S C H O F F E N I E L S Department of General and Comparative Biochemistry, University of Liège, 17 place Delcour, B-4020 Liège, Belgium

High-molecular-weight proteins extracted from the walking nerves of Crustacea with SDS undergo a cycle of phosphorylation-dephosphorylation that is influenced by electrical stimulation, the ionic composition of the medium, and compounds such as local anaesthetics, diphenylhydantoin, veratridine, and TTX. It is suggested that the ion-gating mechanism in axonal membranes is controlled by the net state of specific protein phosphorylation.

T7or the past few years I have entertained the idea that phosphorylation of specific proteins in nerve membranes could be associated directly with the production of an action potential. The rationale behind this idea and the first experimental evidence were presented at the Second International Meeting on Torpedo in 1976 ( I ) . Briefly stated, it is obvious that electrical activity in conducting membranes is a dissipative process, therefore irreversible: the analysis of the ionic currents and of the thermal events contemporary to the conductance changes unmistakably reveal that a chemical has to be used up as i n any irreversible process occurring in a biological system (2,3). For more than 20 years Nachmansohn has proposed repeatedly that acetylcholine i n conjunction with adequate enzymes could be the specific operating substance controlling the configuration of a receptor protein (4). More recently, calcium ions have been included in the process (5). However there are still many uncertainties as to the exact nature of the role of acetylcholine and calcium ions. Moreover, since the enthalpy of hydrolysis i n acetylcholine i n the presence of a complex buffering medium is -f~ 7—h 12 Kcal mol" (6), it is incompatible with the heat absorption (i.e. A H < o according to the sign convention of the biochemists) observed by H i l l 1

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Blank; Bioelectrochemistry: Ions, Surfaces, Membranes Advances in Chemistry; American Chemical Society: Washington, DC, 1980.


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and his associates (7) during the descending phase of the action potential, an event that according to Nachmansohn should be controlled by the acetylcholine hydrolysis. These facts lead to the idea that another energy-producing process must be evoked to explain the dissipative nature of the impedance variation cycle ( I V C ) described for the first time by Cole and Curtis in 1939 (8). I already have proposed that I V C should be controlled by enzymes (9). I would like to report on a phosphorylating system located in nerves and the properties that are affected by the electrical stimulation, the ionic composition of the medium, local anaesthetics, T T X , veratridine, and more generally by those compounds known to affect the electrical activity of nerves in vivo. Two types of methodology have been applied. Membranes are prepared from the nerves of the walking legs of various species of crabs (Carcinus moenas, Eriocheir sinensis, and Maja squinado) and the phosphorylation of this membrane fraction is studied in various incubating medium using {y — P} A T P as the substrate i n concentrations as low as 10" M. (This work is done in collaboration with G . Dandrifosse, is reported elsewhere (11).) Since one assumes that an enzymatically controlled dephosphorylation process is taking place, membrane fragments also are used to study the properties of the phosphoprotein phosphatase(s) that exist in our preparation. This work, carried out i n collaboration with P. Wins and G . Dandrifosse, also w i l l be published elsewhere. In a second type of experiment intact nerves isolated from the above species of Crustacea are submitted to electrical stimulation or conditions known to affect electrical activity, and phosphorylation of the proteins separated by gel electrophoresis is examined. 32

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Experimental Methods Animals. The experiments were done on nerves isolated from the walking legs of three species of Crustacea: Eriocheir sinensis, Carcinus moenas, and Maja squinado. However most of the experiments were done with E . sinensis that had adapted to fresh water. The saline used as incubation medium was either sea water for the sea-water-adapted animals or 50% sea water in the case of E . sinensis. Both media were supplemented with I m M phosphate. A l l of the experiments were done using nerves isolated from the same side of the animal and were compared with control nerves isolated from the opposite symmetrical legs. The temperature of the medium was 18° ± 1°C. Electrical Activity Measurement. A single nerve was stimulated through external silver-silver chloride electrodes using a Grass-Stimulator or a Tectronix pulse generator complemented with a stimulus isolation unit.


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The electrical activity was recorded with external silver-silver chloride electrodes on a Tectronix oscilloscope. The maximum amplitude of the biphasic potential was generally close to 6 m V . The stimulating and recording electrodes were placed at both ends of the nerve thus leaving a large portion of nerve i n between (up to 6 cm) dipping into a beaker containing the saline. In order to keep that portion of the nerve in the liquid, a small hook made of glass was hooked into the middle of the nerve. Electrical stimulation was performed at 10 sec" during 20-min periods. Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 30, 2018 | https://pubs.acs.org Publication Date: June 1, 1980 | doi: 10.1021/ba-1980-0188.ch018

1

After stimulation the nerve is placed i n 250 /xL of a stop solution brought to p H 7.2 and containing 8% sodium dodecyl sulfate ( S D S ) , 25mM mercaptoethanol, l O m M N a H P 0 , and 6 m M E D T A . U p to 12 nerves are pooled i n both experiment and unstimulated control. The nerves then are ground with a small pestle i n the presence of sea sand which has been washed carefully with hydrochloric acid and distilled water. The suspension is transferred into a small dialyzing bag and dialyzed overnight at room temperature against 40 m L of the above stop solution that is changed four times during the process. SDS-Polyacrylamide Gel Electrophoresis. A known volume of solution is collected from the dialyzing bag. One aliquot is used for protein determination according to Ref. 10. T o the known volume 10 fiL of tracking dye (bromophenol) and 50 fxL of a saturated solution of sucrose is added. 2

4

Two concentrations of polyacrylamide have been used. In the first set of experiments the stacking gel had a concentration of 5 % while that of the resolving gel was 10%. In other experiments reported elsewhere the concentrations were 3.2% and 5 % , respectively. The electrophoresis was run for about 4 hr at a constant current of 4 m A per cylindrical gel with a running buffer of 50mM tris H C l , 2 m M E D T A , 375mM glycine, and 0.1% SDS adjusted to p H 8.3. The gel was stained for protein with 0.06% Coomassie blue (G 250) i n 45% methanol-7% acetic acid for at least 4 hr. It was destained overnight i n 7% acetic acid with several changes. The gels were sliced according to the positions of some bands (see Figure 1) and the radioactivity was measured by liquid scintillation counting (Lumagel) after dissolving the gel i n 0.5 m L H 0 overnight in an oven at 50°C. The results are expressed i n counts per 10 m i n after correcting for the protein concentration. 2

2

In some experiments the sub-bands 1, 2, or 3 were not separated, therefore, when the results are given for a letter without a numeral, they refer to the total activity of the slice identified by the letter. Chemicals. SDS, sea sand, glycine, sucrose, bromophenol blue, E D T A , methanol, N a H P 0 , and ammonium persulfate were bought from Merck. Mercaptoethanol, tris, tetrodotoxin ( T T X ) , tetracaine, and ouabain were supplied by Sigma Chemical C o . Bis-acrylamide, acrylamide, Tcmed, and Coomassie blue (G 250) were bought from Serva. 2

4

P as orthophosphate (sodium salt) was prepared as neutral sterile and injectable solution by I.R.E., Fleurus, Belgium, with a specific activity of 100 m C i / m g phosphorus. 32


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Experimental Procedure. The labeling of endogenous A T P was obtained according to three different experimental procedures. 1. Radioactive phosphate is added to the saline i n which the nerves are submitted either to electrical stimulation or to the action of a compound. The maximum activity used is 10 m C i per 80 m L of saline. The experiments performed in this condition are referred to as incubation in ®P. 2. Nerves are incubated for 30 min i n a radioactive saline. Afterwards these are submitted to electrical stimulation or to the action of a compound in a cold saline. This type of experiment is referred to as preincubation i n P . 3. About 2.5 m C i of radioactive phosphate are injected into the intact animals. The nerves subsequently are isolated at different time intervals. 32

Results

Before investigating in detail the various means of controlling the protein phosphorylation in nerves it was necessary to perform some elementary control experiments. Accordingly after obtaining a constant reproducible pattern of electrophoresis (see Figure 1) we would demonstrate that after incubating nerves i n a saline solution containing P as orthophosphate, the radioactivity was distributed among all of the bands. Since we suspected that part of this radioactivity could be the result of contamination by low-molecular-weight components (including orthophosphate) and occurring not only during the electrophoresis but also during the destaining process, we have decided to submit our material to a thorough dialysis prior to electrophoresis. This has improved considerably the results, and after dialysis the contamination by the destaining solution is abolished completely. However all of the bands still remained labeled but the highest activity is located from Band A to D (see Figure 1). Also, if it may be assumed reasonably that the radioactivity detected along the electrophoretic pattern is the result of some sort of phosphorylation of proteins, it was imperious to demonstrate that we were dealing with proteins. Using pronase completely destroys the electrophoretic pattern, while ribonuclease has no effect on it. F r o m these results we conclude that we are dealing with proteins. However at this stage we have no idea as to the type of bonding phosphate involved, but we may be sure that any low-molecular-weight phosphorylated compound that is adsorbed loosely on the protein has been removed during dialysis and electrophoresis. Another question is whether or not inorganic phosphate, as we used it, is the substrate of reaction. Though no inhibitor of oxidative phosphorylation has been used so far in our experiments, we have, together 3 2



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Figure 1. SDS gel electrophoresis pattern obtained with nerves of E . sinensis (E), C. moenas (C), and M . squinado (M) extracted in SDS solution as indicated in the text. The lines and letters on the LHS indicate the position and numbers of cutting. The dots mean 1, 2, or 3, e.g. O O , O ; A A . The apparent molecular weight (X 10~ ) indicated on the RHS has been obtained with a Pharmacia calibration kit. Bands 0 « , O , and O correspond to the stacking gel (concentration of 5%). Bands A to M: resolution gel at a concentration of 10%. iy

t

g

s

lf

3

2

s

with some results reported below, indirect evidence that A T P is indeed the substrate. A membrane fraction of nerves never is phosphorylated when inorganic phosphate is used as substrate, while w i t h the concentration of {y — P} A T P as low as 10" M a significant phosphorylation of the membrane proteins is observed. Moreover this process is affected by agents known to interfere w i t h bioelectrogenesis such as local anaesthetics, veratridine, T T X , etc. ( I I ) . As indicated by the results of Table I electrical stimulation affects the labeling of the proteins. Since we observe a decrease i n the radioactivity this could well mean that (a) the fluxes of orthophosphate are 32

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Table I. Effect of Electrical Stimulation on Protein Phosphorylation in Nerves of E. sinensis and M . squinado (Counts per 10 min): Incubation Was in P ; Gel Concentration Was 10%. 32


E . sinensis

Ax A Bx B C D 2

2

M . squinado

Control

Stimulated (10 sec' ; 20 min)

432

279

504

176

74 546

59 299

1

Stimulated (10 sec' ; &

Control

1

218 183 251 749 558 1262

70 80 18 231 352 492

Table II. Protein Phosphorylation in Nerves of E. sinensis (Counts per 10 min): Effect of 5 X 10" Ouabain; Gel Concentration Was 10%. 5

Preincubation in P (30 min) 32

Incubation in P 32

Ax A Bx B C D 2

2

a

b

Control

Ouabain

ONS

OS

855 229 333 492 461 2074

290 88 93 348 180 1281

66 65 121 1390

58 75 9 445

910

804

—

—

•In this experiment the nerves are incubated for 30 min in a radioactive saline solution with or without ouabain. No stimulation. ONS = incubation in ouabain without stimulation; OS = incubation in ouabain with stimulation. In this experiment the nerves were preincubated in radioactive saline solution for 30 min and then transferred in a cold saline solution containing ouabain. Frequency of stimulation 10 sec" , duration 20 min. 6

1

Table III. Protein Phosphorylation in Nerves of E. sinensis (Counts per 10 min). Effect of Electrical Stimulation (10 sec" During 20 min) in a Cold Saline after Preincubation for 30 min in Radioactive Saline Solution. Gel Concentration Was 10%. 1

Control A B C D

1135 996 414 1916

Stimulated 760 1138 59 2080



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affected by the electrical activity of the nerves; (b) the conductance of the membrane is controlled by the net phosphorylation state of specific proteins; and (c) the metabolic changes associated with repetitive nerve firing induces changes in A T P concentration or specific radioactivity. However, note that (a) and (c) do not necessarily exclude ( b ) . That a decrease in phosphate influx could explain part of the data is illustrated by the results of Table II obtained in the presence of ouabain (c (control) vs. o (ouabain); unstimulated). Ouabain is an agent known to affect the phosphate influx i n squid giant axons ( I I ) although it has been demonstrated with the same biological material that electrical stimulation has no effect on the phosphate influx (12). However, if the nerves are preincubated in radioactive phosphate for 30 min prior to the application of ouabain thus permitting the biosynthesis of radioactive A T P i n the nerve, the situation is rather different. If one applies ouabain to both control nonstimulated ( O N S ) and stimulated nerves ( O S ) , only Bands Bi and B are affected. Therefore, independently of a possible effect on the phosphate influx, electrical stimulation modifies the phosphorylation pattern. The electrical stimulation performed on nerves that have been preincubated i n a radioactive saline solution also shows a modification of the phosphorylation pattern (see Table III). These results hardly could be explained solely through an increase i n phosphate efflux, assuming that electrical activity affects the movements of labeled phosphate in Crustacean nerves in the same way that it affects garfish olfactory and rabbit vagus nerves (13). Electrical activity certainly alters the energy metabolism of the nerves and one therefore should expect a change in A T P concentrations and i n A T P specific radioactivity. In order to further check out this possibility, crabs have been injected with radioactive phosphate and the nerves have been isolated after 2- and 4-hr periods. Table I V shows that electrical stimulation increases the labeling of Bands A , B, and D , which apparently is i n contradiction with the results of Table I. Though other interpretations are possible, it is reasonable to assume that the phos2

Table IV. Protein Phosphorylation in Nerves of E. sinensis (Counts per 10 min). Crabs Injected with P as Indicated. Frequency of Stimulation 10 sec" for 20 min. Gel Concentration Was 10%. 32

1

2Jfi min

120 min A B C D

Control

Stimulated

Control

Stimulated

413 832 303 1082

925 1079 249 1647

3918 2807 1107 3686

6703 5803 577 8098



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phorylation of proteins proceeds via at least two pools of A T P having different size and/or turnover numbers, one of which is being preferentially used during the electrical stimulation. W h e n stimulation occurs in the radioactive saline solution, the specific activity of the A T P pool being used is lower than that of the pool that is used in resting conditions. Thus this explains the results of Table I. I n this interpretation one assumes that electrical activity increases the rate of the phosphorylationdephosphorylation cycle. O n the other hand, a preincubation of 30 min in a radioactive saline solution (see Table III) or the injection of radioactive phosphate into the animals 2 or 4 hr prior to the experiment (see Table I V ) allows the specific activity of the pool used during electrical stimulation to be higher, thus leading to an increase i n the labeling of some proteins after electrical stimulation. Therefore, depending on the specific activity of the pool supplying A T P , the radioactivity of the protein should be lower or higher than that of the unstimulated control. Results obtained with crabs injected with radioactive phosphate 24 or 48 hr prior to the experiments show that the patterns of labeling of stimulated and control nerves are identical; thus they are in agreement with the idea that a steady state has been reached with respect to the specific activity of the different phosphorylated compounds. The results presented so far do not bring any evidence that (a) the proteins—the phosphorylation of which is affected by electrical stimulation—are located i n the axonal membrane and that ( b ) they are specifically involved i n the ion-gating mechanism. Until we have isolated and characterized some of the proteins making up the electrophoretic pattern (a problem we are now working on), we have to use indirect evidence to support further our hypothesis. Since electrical activity is produced by an extra influx of sodium and an added loss of potassium ions, one may expect a temporary change in intramembraneous ion concentration and therefore a differential effect of sodium and potassium ions on the phosphorylation process. Table V Table V . Protein Phosphorylation in Nerves of E. sinensis as Percent of Control in SDS. Nerves Preincubated 30 min in P . Gel Concentration Was 10%. Explanations in Text. 32

KCl + ATP (10' M)

KCl

NaCl

101 181

444 163

4 12

48

43

43

3

Ax A Bx B C D 2

2

a

a

Not measurable.


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Table VI. Effect of T T X (8 X 10* M) on Protein Phosphorylation in Nerves of C. moenas (Counts per min). Preincubation in T T X = 20 min followed by Incubation in T T X + P. Gel Concentration Was 10%. 8

32

Ai A B B C D 2


x 2

Control

TTX

1014 1822 1439 1625 1123 1215

2176 1857 1092 1242 860 1261

shows that this is indeed the case with the proteins located i n Band A i , i.e. the high-molecular-weight components in our electrophoretic pattern (see Figure 1). The experiment is done as follows. Nerves are incubated 30 m i n in a saline solution containing radioactive inorganic phosphate. They are divided into six groups of 12 symmetrical nerves. After the incubation period one group is placed in 250 fih of stop solution (control group) while the symmetrical group of nerves is incubated in a medium containing 120mM KC1 + A T P 10" M. After 10 min of exposure to this condition, the nerves are place i n 250 /xL of stop solution. The same type of procedure is applied to the other groups of symmetrical nerves except that the experimental group is incubated 10 min in KC1 or i n N a C l . It is obvious that K ions favor the phosphorylation process of the proteins located in Band A while N a ions enhance the dephosphorylation process. When the specific radioactivity of endogenous A T P is lowered by adding a large amount of exogenous cold A T P to the K medium, the radioactivity located on the protein Band A i is lower than w i t h K ions alone. There is now direct electrophysiological evidence that compounds such as T T X , veratridine, and local anaesthetics, etc. modify i n a very specific manner the gating processes responsible for the generation of an action potential. In order to investigate further the possibility that protein phosphorylation could be involved directly i n bioelectrogenesis, these compounds have been tried. W i t h nerves isolated from E. sinensis, diphenylhydantoine at saturation decreases markedly the labeling of proteins in Bands Ai and Bi and has a less pronounced effect on the labeling of the other bands. Veratridine and tetracaine also alter markedly the phosphorylation pattern. As shown by the results of Table V I obtained on nerves isolated from C . moenas, T T X increases the phosphorylation i n Band A i and decreases it in Bands Bi, B , and C . 3

u

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The results presented so far show that electrical stimulation and the compounds known to affect the process of bioelectrogenesis interfere with the phosphorylation of proteins mainly located i n a region of the SDS gel electrophoresis where the molecular weights are higher than 60.000 (see Figure 1). Proteins i n the A band seem to be the most affected. A decrease i n the reticulation of the gel i n order to improve the resolution i n that region was indicated. After a few trials, it appeared that a 5 % concentration was adequate: there was indeed a much better separation of the high-molecular-weight components. Results obtained as a result of this improved resolution are discussed elsewhere (15).


x

Discussion Since the phosphorylation of endogenous proteins was reported first by Johnson et al. (16) there has been a large accumulation of data showing that it is a rather general process controlling most significantly biological events (16,17,18). Among the processing reactions that modify the structure of a protein and therefore its biological activity, covalent modifications such as phosphorylation or glycosylation are certainly of the utmost importance. Phosphorylation of serine or threonine residues are transient processes fluctuating i n response to various stimuli. Hence the net state of protein phosphorylation fluctuates accordingly together with their biological activity. Examples may be found i n two reviews recently published (17,18). Surprisingly enough besides our suggestion made some years ago (1,19,20), protein phosphorylation never has been proposed as a means of controlling the I V C responsible for the action potential in axonal membranes. However it generally is accepted that proteins are the molecular structures that control the impedance of the conducting membranes. One of the most pertinent arguments to support this is the high specificity exhibited by the conducting membrane towards the cations carrying the action current. So far there is little direct evidence as to the molecular structure controlling the ion movement. Are we dealing with an oligomeric structure or with a single large protein? A t any rate it may be useful to coin a name for the protein(s) controlling the cation movements during nerve activity and I suggest calling that class of proteins "conductin," leaving the possibility of specifying sodium conductin, potassium conductin, or calcium conductin if it turns out that different molecular species control the sodium, potassium, and calcium currents. The results presented i n this chapter, if they do not demonstrate that the net state of phosphorylation of some specific proteins determines the conductance of the axonal membrane, point nevertheless very strongly



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to the possibility that such a mechanism indeed exists. They are significant enough when taken together to encourage us to explore further this approach in trying to solve the molecular basis of bioelectrogenesis in nerves. W h e n considering the results of the electrophoresis it is the highestmolecular-weight components that are affected consistently either by the electrical stimulation or by tetracaine, T T X , etc. If one assumes that the major proteins making up the I V C are high-molecular-weight components, the results obtained are i n reasonable agreement with the idea that i n resting conditions the sodium conductin is phosphorylated while at the peak of the action potential it is dephosphorylated thus corresponding to the high conductance state of the membrane to sodium ions. Therefore the electrical stimulation should increase the turnover of phosphate from phosphoproteins. Depending on the specific activity of the pool supplying the A T P the radioactivity of the protein should be lower (see Table I) or higher (see Tables III and I V ) and that of the unstimulated control. Results obtained with crabs injected with radioactive phosphate 24 or 48 hr prior to the experiments show that the patterns of labeling of stimulated and control nerves are identical; thus they are in agreement with the idea that a steady state has been reached with respect to the specific activity of the different phosphorylated compounds. On the other hand, since a local anesthetic blocks the action potential without depolarization, its action on the protein could be explained by the inhibition of the dephosphorylation process leading to a slowing down of the phosphate turnover. As for the effect of T T X , an agent known to block the sodium current by preventing the increase i n conductance, its effect could be explained in terms of an increase in the net state of sodium conductin phosphorylation, a situation typical of rest. This is obviously mere speculation. Nothing is known about the proteins that form the pathways for the ion movements. U n t i l we know if we are dealing with oligomeric structures or a single, large protein, it w i l l be difficult to exclude the possibility that some of the lower-molecularweight components might be protomeric units dissociated by our experiment conditions, such as SDS extraction. Moreover until we have isolated and characterized thoroughly the conductins it is obviously impossible to ascertain that the conducting state of the membrane is indeed controlled by the net state of phosphorylation of these intrinsic proteins. The only strong argument so far i n favor of our hypothesis is that compounds that are supposed to act in a specific manner on the generation of an action potential also act on the phosphorylation process not only on the intact nerve but also on membranes prepared from nerves (11).



296


The alternative to our interpretation is that repetitive nerve firing profoundly affects the energy metabolism and therefore the phosphorylation state of proteins that are not necessarily involved directly i n bioelectrogenesis. The sole merit of our results then would be to demonstrate that most of the compounds used to study the ionic currents such as T T X , veratridine, and the like do not have the specificity one likes to believe they have. Finally it may be argued that some of the results observed may be an expression of a change in phosphate influx. W e do not have information regarding the control of phosphate fluxes in Crustacea nerve. However in squid giant axon the influx is independent of electrical stimulation but is inhibited by ouabain (12,13). This is obviously the case with E. sinensis as shown by the results of Table II; the incubation of nerves i n a radioactive saline solution shows a decrease i n the labeling of all the bands with respect to the control in the presence of ouabain. N o w if the nerves are incubated first in a radioactive saline solution i n order to label at least part of the endogenous A T P and then stimulated in a cold saline solution in the presence of ouabain, only Bands Bi and B show a decrease in radioactivity with respect to the unstimulated control also i n contact with ouabain. If one compares these results with those obtained under the same conditions but without ouabain (see Table III), a reasonable conclusion is that the phosphorylation of the various bands must proceed from different pools of A T P that have not yet obtained the same level of specific radioactivity. Other interpretations are certainly as valid as previous proposals but it is too early to comment further on this situation before knowing more about the number of A T P pools as well as their size and turnover rate. 2

Acknowledgments Many thanks to my colleague G . Dandrifosse for many helpful discussions and comments. Thanks also are extended to G . Pirard for her able technical assistance. This work was aided by Grant N o . 2.4544.76 from the Fonds de la Recherche Fondamentale Collective. Literature Cited 1. Schoffeniels, E. Phosphorylation and Bioelectrogenesis, 2nd International Meeting on Torpedo, Arcachon, France, Sept. 1976. 2. Margineanu, D. G.; Schoffeniels, E. Proc. Natl. Acad. Sci. USA 1977, 74, 3810-3813.



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3. Neumann, E. Adv.Biol.Med. Phys. 1977, 16, 235-239. 4. Nachmansohn, D. Harvey Lect. 1955, 49, 57-99. 5. Neumann, E. In "Biochemistry of Sensory Function"; Jaenicke, L., Ed.; Springer-Verlag: Berlin and New York, 1974; pp. 456-514. 6. Sturtevant, J. M. J. Biol. Chem. 1972, 247, 968-969. 7. Abbott, B. C.; Hill, A. V.; Howarth, J. V. Proc. R.Soc.,Ser. B 1958, 148, 149-187. 8. Cole, K. S.; Curtis, O. J. Gen. Physiol. 1939, 22, 649. 9. Schoffeniels, E. Arch. Int. Physiol. Biochim. 1970, 78, 205-223. 10. Schacterle, G. R.; Pollack, R, L. Anal. Biochem. 1973, 51, 654-655. 11. Schoffeniels, E.; Dandifosse, G. Proc. Natl. Acad. Sci. USA 1980, 77, 812. 12. Caldwell, P. C.; Lowe, A. G. J. Physiol. 1970, 206, 271-280. 13. Ibid., 1966, 186, 24P. 14. Ritchie, J. M . Straub, R. W. J. Physiol. 1978, 274, 539-548. 15. Schoffeniels, E. In "Epithelial Transport in the Lower Vertebrates"; Lahlou, B., Ed.; Cambridge Univ. Press: 1979. 16. Johnson, E. M.; Ueda, T.; Maeno, H.; Greengard, P. J. Biol. Chem. 1972, 247, 5650-5652. 17. Greengard, P. Science 1978, 199, 146-152. 18. Williams, M.; Rodnight, R. Progr. Neurobiol. 1977, 8, 183-250. 19. Schoffeniels, E.; Margineanu, D. G. Bull. Acad. R. Med. Belg. 1977, 132, 127-135. 20. Schoffeniels, E.; Margineanu, D. G. "Power Dissipation in Bioelectrogenesis: A Molecular Approach," Biosc. Comm. 1978, 4, 205-216. ;

RECEIVED October 17, 1978.


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