T H E COUPLED NATURE OF LACTIC ACID-GLYCOGEN SYNTHESIS I N MUSCLE BYDEANBURK*
The view developed so largely by Meyerhof and Hill that the synthesis of glycogen or carbohydrate from lactic acid in isolated muscle requires energy derived from the oxidation of such compounds has been questioned recently by Bancroft and Bancroft,l who have attempted to demonstrate that the formation of glycogen from lactic acid can be explained equally well upon other grounds. They suggest that a reversible equilibrium exists between the two compounds which may be disturbed by extraction of glycogen from solution by adsorption on muscle protein, thereby causing further formation of glycogen. This implies that the free energy of the synthesis is small, or at times zero. It will be shown here that the Bancroft and Bancroft reversible equilibrium explanation is quantitatively inconsistent with the existing available thermodynamic free energy data.* Although aware that “the equilibrium point of this reaction is well over on the lactic acid side” it would appear that these writers failed to appreciate the quantitative completeness of the spontaneous breakdown, as will be evident immediately upon consideration of the free energy data. From data given el~ewhere,~ in a form somewhat different4 from that employed here, however, the free energy of the following synthesis, as it is normally considered to occur in muscle, lactateion(o.002 M=o.o18%)+H+(2.5
x 1o”,orpH7.6)
= n g l y c o g e n ( ~ % ) (I(
is 393 cal/gm., or 353705 cal/mol of lactic acid (the heat of reaction is 268 cal/gm., or 24120 cal/mol). Correspondingly, a t pII 3.28, where the free * Bureau of Chemistry and Soils, Washington, D. C. J. Phys. Chem., 35, 194 (1931). Bancroft and Bancroft state: “The extent of this coupled reaction is remarkable, for Hill has shown that under suitable conditions the ratio of the amount oxidized to the amount synthesized is one to five or one to six.” The ratio actually corresponds to an efficiency of IOO X (5/(325000/35370)), or 46%, where - 325000 cal. is the approximate free energy of combusion of lactic acid under physiological conditions. As a matter of fact, the writer has recently indicated (J. Phys. Chem., 35, 432-56 (1931))that the coupled autotrophic reduction of carbon dioxide by hydrogen has an efficiency of substantially 100%when (as in the glycogen synthesis just considered) the extraneous maintenance energy of the biochemical machine accomplishing the reaction is neglected. Several other fairly highly efficient reactions were likewise discussed, and also a considerable number of reactions with efficiencies of 20-507~. B u k : h o c . Roy. SOC.,104B, 153-170 (1929). 4 Entropy changes rather than free energy changes received chief expression. 6 In accordance with convention, the accuracy of free energy values is not indicated by the number of significant figures given. 2
COUPLED NATURE O F LACTIC ACID-GLYCOGES SYNTHESIS
269
energy of neutralization is zero at the concentration of lactic acid considered, the free energy of the synthesis lactic acid (0.002 M) = n glycogen (17~) (2) is 336 cal/gm., or 30240 cal/mol of lactic acid (the heat of reaction is - 180 cal,’gm., or 16200 cal/mol). n is the reciprocal of the ratio of the molecular weight of glycogen to that of lactic acid, and its significance will be considered later. Bancroft and Bancroft’s statement “From a purely chemical point of view this reaction (lactic acid-glycogen synthesis) should not require much energy” is obviously untrue; on account of the very large free energy requirement (a positive value of 35370 cal.) it is inconceivable that reaction ( I ) as written could occur spontaneously in muscle, at least in experimental, physiologically significant quantity. The question might remain, however, as to how far Bancroft and Bancroft would agree that Equation (I) represents approximately the essential conditions, particularly of concentration, of the reaction taking place in muscle. They affirm (within * Io-fold) the adopted and generally accepted concentration of o.002 M lactic acid dissolved in the plasma of fresh, isolated muscle.’ It may be pointed out that for every Io-fold dilution or concentration of lactic acid the free energy of Equation (I) becomes changed respectively 1365 cal., Le., changed to 36735 or to 3400s cal., respectively. Correspondingly, for every 1 0 5 - fold dilution or concentration, the free energy becomes changed by approximately z 1365 cal. Hence for equilibrium conditions to prevail (i.e., for A F to equal zero) the activity (or approximate concentration) of lactic acid would have to be maintained a t the impossible figure of o.002 X I O (353’O’1385) M, or ca. 1oZ3M. Even neglecting neutralization, which normally takes place more or less completely under physiological conditions, and considering glycogen formation according to Equation (2), as might occur independently of muscle, the figure remains still as high as 0.002 x ~ o ( ~M,~ ~ ~ ~ ’ ~ or ca. 1 0 ~ 0R.I. Likewise, it can easily be shown that adoption of physiological pH values other than 7.6 would influence the figure of 35370 cal. in Equation (I) but relatively little. At pH 6.9, for instance, it is reduced by only 990 cal. to 34380cal. With respect t o the concentration of glycogen, Bancroft and Bancroft have introduced one new factor hitherto considered but littie. They assert (as may very well be the case) that the 1 7glucogen ~ in muscle does not exist in solution but is almost completely adsorbed on muscle protein, the adsorption being reversible. I n other words, the concentration of glycogen but something like 0.01%~or to be reckoned with in Equation (I) is not 17~, even less, i.e., there obtains an adsorption of 99Yc or more. They state qualitatively, “the reaction can be forced back from lactic acid to glycogen by the adsorption of glycogen out of solution on the protein, thereby reducing the amount of free glycogen in solution and causing the formation of more to re-
*
This value would in their view represent the approximate normal equilibrium concentration of lactic acid in the muscle system a t rest.
DEAN BURK
270
establish the equilibrium.” However, as shown in the manner above with lactic acid, a physiologically inconceivable, great reduction in the concentration of free glycogen would be required, ~ o ( ~ ~ ~ ~ ~ or ’ ~ IoZ6-fold, ~ ~ ~ ) - even fold, granting for the moment that the value of n in Equation ( I ) is unity. Assuming a value of n = I/IOOO,the concentration reduction required would be 36S)-fold,or ca. Io26000-fold, to ~ o - ~ As ~ shown ~ ~ ~before x . (loc. cit.), owing to the very small value of n ( I / I O O to I/IOOO),the free energy of dilution of glycogen is substantially zero for all physiological concentrations, as may be seen from the formula: AFdli
=
n R T ln(I%/y%) = 1.365 log (~%/y%).
(3 )
where y is the physiological concentration to be considered, and n is taken to be I/IOOO. Thus, where 9% is 0.001% (instead of I % ) , nFdll becomes only -4 cal. This rooo-fold dilution of glycogen decreases 35370 by only 100 x (4/35370) or ca. .oI%, leaving reaction (I) substantially as spontaneously unreversible as before. Therefore the reversible equilibrium explanation is in disagreement not only with existing free energy data, but also with a przom‘ mass law considerations of the relative molecular weights of glycogen and lactic acid. This a przorz argument is not necessarily so important, however, since the problem of sugar synthesis from lactic acid involves a large positive free energy in the same way that glycogen synthesis does, and yet in sugar synthesis n is I / P , Le., not far from unity. As a corollary to the above reasoning it follows that the free energy of glycogen adsorption on protein is totally insufficient to account for the synthesis, since the adsorption1 is never complete enough, Le., it is never so complete that about less than ~ o mg. -glycogen ~ per ~ gram ~ of muscle ~ ~plasma is unadsorbed. Moreover, as just pointed out with respect to free energy relationships, sugar synthesis is similar to glycogen synthesis qualitatively and quantitatively, so that for sugar synthesis to take place (in the absence of simultaneous glycogen synthesis) postulation of adsorption on protein and removal from solution would likewise be required, as in the case of glycogen synthesis. Whether such adsorption takes place to a great extent in the ordinary sense is questionable; in the sense required by calculations similar t o those given with respect t o glycogen, it unquestionably does not take place. It should be recalled that refutation of the reversible equilibrium explanation had been accomplished more or less successfully by Meyerhof and others on the basis of heat of reaction, rather than free energy of reaction, data. Obviously, however, a critical and ultimate decision must rest upon free 1 The adsorption is of course itself a reversible process, according to Bancroft and Bancroft, so that the calculations of the previous paragraph with respect to glycogen concentration could be based upon either that amount in free solution or that amount adsorbed with exactly the same results or final conclusions. Since, however, in order to employ the latter method, the free energy of adsorption would have to be known (and added to that of Equation ( I ) ) it is obviously much more convenient, and in the present state of knowledge essential, to proceed as has been done.
COUPLED KATURE O F LACTIC ACID-GLYCOGEN STXTHESIS
271
energy data The calculations required to produce the data given here were carried out with considerable precision; so far as the writer is aware, no assumptions were employed which if arbitrarily but judiciously changed would greatly decrease or enhance the large positive free energy value of lactic acid-glycogen synthesis in muscle given. Bancroft and Bancroft concern themselves more with the details of glycogen-lactic acid breakdown than with lactic acid glycogen synthesis. Owing t o the long-established spontaneous nature of the breakdown, the really deciding test of the freely reversible equilibrium explanation must now concern itself with the synthesis; for this reason the present paper is considering chiefly the synthesis.? It may be pointed out with respect to the breakdown, however, that if lactic acid were in equilibrium with glycogen just before a muscle at rest were stimulated, the free energy of the breakdown subsequently called forth by stimulation would, per mol of lactic acid, be so close to zero as to be totally incapable of accounting for the work performed in any contraction of appreciable duration. So far as is known at present, the anaerobic performance of mechanical work derives its free energy ultzmatdy from glycogenlactic acid breakdown; this statement needs no qualification with respect to the finding within the last year or two that a certain amount of mechanical work may be obtained under conditions where lactic acid formation is prevented by iodo-acetic acid poisoning. Although other reactions may even under normal conditions be more immediately responsible for the performance of mechanical work than glycogen-lactic acid breakdown, presumably the latter is normally required later to reverse the other reactions so that they may then re-perform work. Bancroft and Bancroft state: "If this is in reality a coupled reaction, one should be able to take lactic acid, oxidize it in the presence of protein, and form glucose." This is not necessarily true; one or more of the intermediate processes may require enzymatic catalysis. A coupled reaction need not, by 1 I t is interesting to note, also, that upon the basis of the free energy data given in this paper, the possibility suggested by Kluyver (Archiv. Mikrobiol., 1, 181 (1930))is likewise precluded, that in glycogen synthesis from lactic acid the mechanism proceeds by way of pyruvic acid and acetaldehyde. In such an event, from stoichiometric considerations alone, no more than two molecules of lactic acid could disappear in synthesis, per one molecule burned, whereas (see p. 2 ) theoretically ten are possible, and experimentally five have been observed. The theory of the mechanism involving passa e through a 2-carbon molecule stage (such as acetaldehyde) can not be true, therefore. %scape from this conclusion can involve only the hypothesis that the numerous, and variously performed experimental measurements of the ratio have been incorrect, or wrongly interpreted. It is beyond the scope of this paper to trouble to prove that not only does lactic acidglycogen synthesis in muscle require a large amount of free energy but also that this free energy is supplie'd by oxygen consumption. It need scarcely be mentioned that no anaerobic reaction in muscle is known capable of providing such a large amount of free energy for the large amounts of synthesis which may on occasion take place. Although not inconceivable that very small, substantially unmeasurable amounts of synthesis might take place through energy provided by some anaerobic reaction, possibly incidental to the mechanism, this would be beside the point, since we are interested here in explaining the large synthesis transformations observed experimentally. Bancroft and Bancroft state: ' I . . . the oxidation of lactic acid is not coupled with the synthesis of glycogen, but occurs simultaneously in the presence of oxvgen," but offer no explanation for the fact that synthesis does not occur in the absence of oxvgen. Likewise they do not explain, even on the basis of their theory, how stimulation causes elution of glycogen from protein.
DEAN BUHK
272
its very nature, invariably proceed in the absence of an enzyme. Exception must be taken t o Bancroft and Bancroft’s conception of coupling as excluding enzymatic action, a t least so far as the term coupling has been employed by physiologists with respect to t h t synthesis under discussion. In view of Equations (I) and ( z ) , it is not a t all surprising that Bancroft and Bancroft failed in an experimental attempt to show that glycogen could be formed from lactic acid when “Using 15 cc. of a .z% solution of d-lactic acid, IO cc. of M/z KH2PO4, and I O cc. of enzyme solution, and approximately 30 grams of egg white.. . .” I n any such future attempts, great caution will have to be exercised in evaluating positive results, Le., in excluding all other possible reactions, particularly oxygen consumption, which might be responsible for providing the necessary free energy or equilibrium point shift. Indeed, on account of the great heterogeneity of the system in which the synthesis would have to be carried out, the formation only of considerably more than traces of glycogen could give rise t o the suggestion that the thermodynamic data given here are inadequate or inapplicable. summary
The theory recently suggested by Bancroft and Bancroft that in muscle glycogen may be synthesized from lactic acid according to a freely reversible shift in the equilibrium point caused by adsorption of glycogen out of solution is shown to be quantitatively inconsistent with both (I) the existing thermodynamic free energy data and ( 2 ) a priori mass law considerations. The theory is also shown to be unable to account for the production of mechanical work in muscle upon the basis of free energy derived from glycogenlactic acid breakdown. 2. The failure of Bancroft and Bancroft to accomplish the synthesis experimentally in vitro is in accord with the prediction of thermodynamic data given in this paper, according to which one milligram of glycogen would form spontaneously in not less than some billion trillion liters of a . z % solution of lactic acid; in fact, owing to the high molecular weight of glycogen, a vastly greater volume of such a solution would be required. I.
