Mechanism of action of uncouplers on mitochondrial energy coupling

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113

which catalyzes the reaction; 20;+ +2H+ + H,O, +O, [251. Ascorbic acid has also been suggested to be a scavenger of0; [26]. It has been shown that ascorbic acid reacts rapidly with OH ’ [27], which would be formed in the reaction between H,O, and 0; [25]. In its reaction with 0; or OH ’ ascorbic acid loses one electron and becomes a radical. The ascorbic acid radical thus formed is a relatively nonreactive one, and decays mainly by disproportionation, thereby terminating the propagation of free radical reactions [28]. The ascorbic acid radical does not react with molecular oxygen [28]. All these properties of ascorbic acid and its high concentration in both plant and animal tissues indicate that ascorbic acid serves as a defense against oxygen toxicity. If this is true then it is obvious why ascorbic acid is so essential as to be regarded as a vitamin in man. The fact that guinea pigs, monkeys, man and other scurvy-prone animals have survived through evolution with such a serious enzyme defect would suggest that they were generally highly successful in obtaining adequate amounts of ascorbic acid from the environment. Acknowledgement We wish to thank Dr. Paul Sato for his advice and help in writing this review. References 1 Stanbury, J.B., Wyngaarden, J. B. and Frederickson, D.S. (1972) in The Metabolic Basis of Inherited Diseases (Stanbury, J. B., Wyngaarden, J. B. and Frederickson, D.S.. eds), 3rd edn, pp. 3-27, McGraw-Hill, New York Burns, J.J., Peyser, P. and Moltz, A. (1956) Science 124,114fb1149 Burns, J.J. (1957) Nature 180,553 Burns, J. J. (1967) in Metabolic Pathways (Greenberg, D. M., ed.), Vol. I, 3rd edn, pp. 394-411, Academic Press, New York Touster,

6 I 8 9

0. (1969) in Comprehensive

Biochemistry

(Florkin, M. and Stotz, E. H., eds), Vol. 17, pp. 2 19-240, Elsevier, Amsterdam Chatterjee, I.B., Kar, N.C., Ghosh, N.C. and Guha, B.C. (1961) Nature 192, 163-164 Chatterjee, I. B., Ghosh, J. J., Ghosh, N.C. and Guha, B.C. (1958) Biochem. J. 70,50%515 Dutta Gupta, S., Choudbury, P. K. and Chatterjee, I. B. (1973) Int. J. Biochem. 4,309-314 Dutta Gupta, S. and Chatterjee, LB. (1967) Naturwirsenschaften

54,20

10 DuttaGupta, S., Sen Gupta, C., Ray Chaudhuri, C. and Chatterjee, I.B. (1970) Anal. Biochem. 38,46-55

11 Sato, P., Nishikimi, M. and Udenfriend, S. (1976) Biochem. Biophys. Res. Commun. 71,293-299

12 Chatterjee, I.B. (1973) Science 182, 1271-1272 13 Chatterjee, I. B. (1973) Sci. Cult. 93,210-212 14 Ray Chaudhuri, C. and Chatterjee, I.B. (1969) Science 164,435-436

15 Nakagawa, H., Asano, A. and Sato, R. (1975) J. Biochem. Tokyo 77,221-232 16 Bernlohr, R. W. (1973) in Birth Defects: Original Artical Series (Bergsma, D., ed.), Vol. 9, pp. xi-xiii, Williams and Wilkins Co., Baltimore, Md. 17 Nishikimi, M., Tolbert, B. and Udenfriend, S. (1976) Arch. Biochem. Biophys. 175,427-435

18 Nishikimi, M. and Udenfriend, S., (1976) Proc. Nat. Acad. Sci. U.S.A. 13,2066-2068 S.P. and Rinaldini, L. M. (1965) 19 DeFabro, Develop. Biol. 11,468-488 20 DeFabro, S.P. (1968) C. R. Sot. Biol. Paris 162, 284285 21 Ginter, E. (1976) Int. J. Vit. Nutr. Res. 46, 173179 22 Willis, R.J. and Kratzing, C.C. (1974) Biochem. Biophys. Res. Commun. 59, 1250-1253 23 Jamieson, D. and Van den Brenk, H. A. A. (1964) Biochem. Pharmacol. 13, 1599164 24 Willis, R. J. and Kratzing, C. C. (1972) Am. J. Physiol. 222, 1391-1394 25 Fridovich, I. (1974) in Molecular Mechanisms of

Oxygen Activation (Hayaishi, O., ed.). pp. 453477, Academic Press, New York 26 Nishikimi, M. (1975) Biochem. Biophys. Res. Commun. 63,463-468 27 Adams, G,E., Boag, J.W., Currant, J. and Michael, B. D. (1965) in Pulse Radiolysis (Ebert,

M., Keene, J. P., Swallow, A. J. and Baxendale, J. H., eds). pp. 131-143, Academic Press, New York 28 Bielski, B. H. and Richter, H. W. (1975) Ann. N. Y. Acad. Sci. 258,231-237

Mechanism of action of uncouplers on mitochondrial energy coupling D.E. Green The molecular mechanism by which uncouplers dissociate electron jlow from synthesis of ATP and other coupled processes has finally been unraveled, The elucidation of this uncoupling mechanism provides some new dimensions,fbr defining the mechanism of mitochondrial energy coupling.

Some thirty years ago, R. Hotchkiss [l] discovered that one of the gramicidins severed the link between electron flow and synthesis of ATP in bacterial and animal respiratory systems, suppressing synthesis of ATP but not electron flow. Since then a large family of molecules has been uncovered with the widest variety of structures that duplicate in every respect the action of gramicidin [2]. These molecular species are collectively known as uncouplers and their action on coupled ATP synthesis is generally referred to as uncoupling. In recent years it has been recognized that uncouplers suppress all the known coupled processes - active transport of cations, energized transhydrogenation, and reverse electron flow as well as coupled synthesis of ATP [3]. Again in such suppression, electron flow is not interfered with. In fact, uncouplers generally enhance the rate of electron flow and the most efficient uncouplers can increase the rate manyfold. Other characteristic properties of uncouplers are worthy of note. Coupling is suppressed whether the driving reaction is electron flow or hydrolysis of ATP [3]. The site of coupling is also immaterial. Uncouplers are effective at all coupling sites. It has taken some time to sort out the D.E.G. is at the Institute for Enzyme Research, University of Wisconsin, Madison, Wisconsin 53706, U.S.A.

bona tide uncouplers that share all the properties enumerated above from the pseudo uncouplers that lack this generalized capability [4]. Among the bona fide uncouplers are 2 : 4-dinitrophenol, FCCP*, mClCCP*, SF6847*, S13*, dicoumarol, and TTFB* to mention the better known of the large family of uncouplers. Mechanism of uncoupler action Many investigators have addressed themselves to the question of the diagnostic property shared by all uncouplers. This has not been a simple question to answer. The molecular structures of uncouplers cover the widest spectrum of possibilities from long chain fatty acids to substituted phenols and from aromatics to heterocyclics. Since 1960 the impetus of the chemiosmotic model [5] with its emphasis on proton gradients focused attention on the ability of uncouplers to cycle protons, and indeed there was soon no lack of experimental evidence to support the thesis that uncouplers across the board have the capability of acting as protonophores in the physiological range of pH [6,7]. But there were indications that this property alone could not rationalize the *FCCP, carbonylcyanidep-trifluoromethoxyphenylhydrazone; mClCCP, carbonylcyanide mchlorophenylhydrazone; SF6847, 3,5di-t-butyl-4 hydroxybenzylidinemalonitrite; S13, 5chloro-3-t-butyl-2’chloro-4’-nitrosalicylanilide; TTFB, 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole.

TIBS - May IF77

114 uncoupling action of uncouplers. The most telling piece of information was that uncouplers abolish not only protonic gradients but also cationic gradients [8,9]. How would one account for this latter property in terms exclusively of the pronotophore hypothesis? At this point an important discovery was made by Mental et al. [9] in respect to the uncoupling action of the combination of two ionophores, valinomycin and nigericin, in presence of K + . This combination duplicated in every respect the uncoupling action of bona fide uncouplers. The importance of this discovery was that the mechanism of action of the combination of ionophores was known; namely, the potentiation of cyclical transport of K + . Electron flow drove the valinomycindependent inward flow of K+ and then nigericin non-electrogenically carried the K + outward. Valinomycin was the neutral electrogenic ionophore and nigericin was the acidic non-electrogenic ionophore that brought a proton on the inward part of its cycle and a K+ on the outward part of the cycle after K +/H + exchange. Since the action of uncouplers is indistinguishable from that of the valinomycin-nigeritin combination, it was logical to assume that the mechanism of uncoupling by uncouplers was identical with the mechanism of uncoupling by the valinomycin-nigeritin combination. The postulate would require that uncouplers induce cyclical transport of cations. A cyclical cation transport mechanism for uncoupler action presupposes that uncouplers are efficient ionophores for cations, and this ionophoric capability would have to extend to both monovalent and divalent cations since active transport of cations is suppressed by uncouplers whether the cation is monovalent or divalent. Was this presupposition correct? Kessler et al. [4] have recently demonstrated that all bona fide uncouplers without exception are highly efficient ionophores for both monovalent and divalent cations but this capability is demonstrable only when the pH of the aqueous phase is above 8. This could mean that the ionophoric capability of uncouplers, though undeniable, cannot be relevant to uncoupling action in the physiological range of pH. Alternatively, one could emphasize the fact that a predicted property was indeed found and anticipate that something additional had yet to be added to the picture to make this property relevant to the uncoupling action of ionophores in the physiological range of pH. Cation requirement A crucial test of the cyclical cation transport hypothesis would be whether a

minimal concentration of cations is essential for uncoupler action. Mitochondrial respiration can be released by uncouplers even when the concentration of added monovalent cation is less than 1 mM in the aqueous suspending medium [3]. At first glance, this would hardly argue for the essentiality of cations for uncoupler action. But there was more to be uncovered before the realities of the uncoupler effect in a medium of low cation concentration could be appreciated. Mitochondria contain a surprisingly large amount of bound divalent cations when in the configurational state appropriate for coupled ATP synthesis. When this complement of bound cations is depleted by exposure of mitochondria to reagents such as the ionophore A23187 or to the chelating agent, EDTA, then uncoupler action is minimal unless salts of monovalent or divalent cations are added to the medium [3]. Apparently uncouplers can readily mobilize the bound divalent cations in the mitochondrion, and as long as this internal supply is available, addition of cations to the medium beyond the minimal amount required for buffering action is unnecessary for efficient uncoupling action. But when the complement of bound divalent cations is largely released from the mitochondrion, then the need of cations (either monovalent or divalent) for uncoupling action can be unambiguously demonstrated. Kessler et al. recognized an important correlation in their study of the cation requirement for uncoupling by A23187treated mitochondria [3]. As the concentration of monovalent cation added to the mitochondrial suspension was progressively increased from 1 to 100 mM, not only was the released respiration by uncoupler enhanced, but also to a smaller degree the rate of respiration in absence of added uncoupler. This parallel enhancement suggested the possibility of some interaction between the uncoupler and the intrinsic electrogenic ionophore in the mitochondrion implicated in active or cyclical transport of K +. Model studies showed that there was indeed a cation-dependent interaction between uncouplers and neutral electrogenic ionophores such as valinomycin, crown ethers, nactins, beauvericin, Triton X-100, etc. [3]. These ionophores were ineffectual in transporting monovalent or divalent cations at neutral pH through an organic phase but given the combination of uncouplers and electrogenic ionophores, the rate of transport increased by one or two orders of magnitude. The synergistic effect of uncoupler and electrogenic ionophore was an across the board phenomenon for all the uncouplers tested and for a wide

selection of known electrogenic ionophores examined., What it meant was that uncoupler and intrinsic electrogenic ionophore were collaborating in the induction of cyclical transport in a fashion similar to that of valinomycin and nigericin. The question raised earlier in the review about the meaning of the ionophoric property of uncouplers expressed only above pH 8 can now be answered satisfactorily. This ionophoric capability is expressed only in combination with an electrogenic ionophore and under such conditions, uncoupler is ionophoretically active at physiological pH. The complex formed by interaction of uncoupler, cation, and electrogenic ionophore was shown to be a 1: 1 :l molar association of the three species in the case of valinomycin with K+ as cation and a 1: 1 :2 association in the case of beauvericin with Ca2+ as cation [3]. In these complexes the cation is coordinated to both the electrogenic ionophore and the uncoupler. This presumably is the complex which plays a key role in the uncouplermediated induction of cyclical transport of cations. Intrinsic mitochondrial ionophores Implicit in the interpretation of Kessler et al. [3] of the synergistic action of antibiotic or synthetic electrogenic ionophores with uncouplers is the postulate that these ionophores are models for and correspond to intrinsic electrogenic ionophores in the mitochondrion. How valid is this postulate? Blondin** has isolated from one of the hydrophobic proteins of beef heart mitochondria a neutral peptidic ionophore active with both monovalent and divalent cations. This isolated ionophore can mediate active transport of K+ in mitochondria. The synergistic action of the peptidic ionophore and uncoupler in mediating cyclical transport of K+ is readily demonstrable. This demonstration provides the first definitive evidence both for the presence of intrinsic electrogenic ionophores in mitochondria and for the synergistic action of uncoupler and intrinsic electrogenic ionophore. On the basis of the evidence summarized above, Kessler et al. [3] have formulated the mechanism for uncoupler-mediated cyclical transport of cations as shown in Fig. 1 for monovalent cations and in Fig. 2 for divalent cations. These formulations are based on the paired moving charge **Summaries of the unpublished studies of R. J. Kessler, G.A. Blondin, and R.A. Haworth, respectively, referred to in the text will be provided in abstracts of the annual meeting of the Biophysical Society (New Orleans, February 1977) and of the American Society of Biological Chemists (Chicago, April 1977).

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Fig. I. Cyclical transport ofK + mediated by the combination of uncoupler (U-) and a neutral electrogenic ionophore such as valinomycin ( VAL). The movement of theelectron (e-) through the electron transfer complex is paired to the movement of valinomycin-K+ (Val-K+) through the membrane. The charge separation of H into e- and H+ in the electron transfer complex is also paired to the charge separation ?f Val-K+U: into Val-K+ and U-. At the terminus of its trajectory the electron is transferred to the final acceptor in the complex (A), and this transfer is simultaneous with the uptake of a proton. Charge elimination in the electron transfer chain is paired to charge elimination of Val-K+ by combination with Ii-. The protonated ,fbrm qfcl- is represented as UH. The diagram shows how the rate of oxidation of substrate by the electron transfer complex can be maximized by drawing off the protons as fast as these are released on one side of the membrane and feeding in the protons required on the other side ?f the membrane. Cyclical transport ?f’K+ mediated by the combination of electrogenic ionophore anduncoupler can accomplish this maximization of the rate of oxidation of substrates in the electron transfer complex.

model [lo] and the principle that coupling depends upon the electrostatic interaction of a negatively and positively charged species - the latter being an ionophoric species. It should be noted that according to these formulations under coupling conditions, the electrogenic ionophoric species carries a net charge of + 1 corresponding to the - 1 charge of the electron. The nonelectrogenic species is electrically neutral. The mechanism of uncoupler-induced cyclical cation transport as formulated in Figs 1 and 2 requires at least one molecule of uncoupler per electron transfer complex when a monovalent cation is cycled and at least two molecules of uncoupler per electron transfer complex when a divalent cation is cycled. Is this minimal stoichiometry satisfied? Kessler et al. [3] have indeed demonstrated that the minimal concentration of the most efficient uncouplers such as SF6847 required for complete release of respiratory control is in a 1 : 1.5 molecular relation to the concentration of electron transfer complex. Since the conditions imposed in this titration involved the interaction of uncouplers with both monovalent and divalent cations, the observed stoichiometry is close to theoretical. There have been several reports that uncouplers can release respiration at concentrations substoichiometric with the electron transfer complexes [ll]. Kessler et al. [3] point out that this apparent substoichiometry is referable

to the fact that electron transfer was not proceeding at the maximal potential of the mitochondrion. Unless this condition is fulfilled, the titration measures a small percentage of the available electron transfer complexes, and a titration under such conditions would yield a substoichiometric value for the amount of uncoupler required for maximal uncoupling action. Uncoupling is by no means an unphysiological process mediated by pharmacological agents. Mitochondria in the orthodox configuration show uncoupled respiration; that is, respiration that is unaffected by addition of uncouplers [ 121. R. Haworth** has provided direct evidence that this uncoupled respiration is an expression of cyclical transport of either monovalent or divalent cations mediated by intrinsic uncoupler provisionally identified as long chain fatty acids. There is a body of evidence in the literature that fatty acid-mediated cyclical transport of cations is the physiological mechanism for heat production by mitochondria in mammalian and avian cells [13-l 61. We can now conclude with some contidence that uncouplers mediate in mitochondria a coupled process (cyclical cation transport) ; that cyclical cation transport takes precedence over all other coupled processes; and finally that inherent in uncoupling is complex formation involving uncoupler, electrogenic ionophore, and cation. A few of the important sequellae of these conclusions as to the mechanism of uncoupling are somewhat unexpected. First, the concept of uncoupled particles has to be abandoned. There is no longer any experimental support for the notion that electrons can flow without coupling to the movement of some positively charged species. That means that mitochondrial particles are either active or inactive. If active, they must be coupled. The search for the mechanism of uncoupling has also led to the recognition of the nature of respiratory control. We may define respiratory control as the state of a particle in which electron flow cannot be coupled to the flow of some positively charged ionophoric species. Respiratory control can be imposed by reducing the concentration of cations (free or bound) below a critical level or by not providing an ionophore capable of generating a positively charged cationic species or by selecting experimental conditions in which the cycling of the electrogenic ionophore is prevented (for example, using valinomycin without nigericin and without a protonatable anion). Given that uncouplers uncouple all known coupled processes and assuming the mechanism for uncoupling is the same regardless of the nature of the coupled

Fig. 2. Cyclical transport qf CaZ+ mediated by the combination qf’uncoupler (U-) and electrogenic ionophoie active on divalent metal cations ( 0). The principle for uncoupler-mediated cyclical transport is identical whether the cation is monovalent or divalent. The new.feature is that the uncoupler participates in the electrogenic as well as in the nonelectrogenic steps in the cycle. The charge of the electrogenic ionophore is I + in the electrogenic step and 0 in the nonelectrogenie step.

process, then it necessarily follows that coupling always involves the participation of an electrogenic ionophore. Whether the coupled process involves ATP synthesis or active transport or energized transhydrogenation the vehicle for this coupling must be an intrinsic electrogenic ionophore. The essence of cyclical action transport is the elimination of all gradients both protonic and cationic. That means that the most efficient form of coupling is operative under conditions of zero gradients. The primary postulate of the chemiosmotic model [5] is that it is not the electron but a proton gradient that drives coupled processes. It is difficult to see how this postulate can be reconciled with the indisputable fact that coupling is maximal when gradients have been completely eliminated. Hanstein and Hatefi [ 171have made out a compelling case for the thesis that uncoupler action is localized in a limited number of mitochondrial proteins and to a high degree in a 29000 dalton protein. This localization of uncoupler action dovetails very nicely with the evidence of Kessler** and Blondin** for the localization of intrinsic electrogenic ionophores in various species of hydrophobic proteins of which the 29000 dalton protein is by far the most abundant species. Another puzzle that can now be rationalized is the anomalous behavior of picric acid which can uncouple only in presence of nigericin and K+ [9]. Picric acid, like bona tide uncouplers, can react synergistically with electrogenic ionophores but unlike this uncoupler is incapable of cycling protons at physiological pH [17]. Nigericin can fulfill this proton cycling function by K+/H+ exchange [9]. Thus, while the combination of picric acid and nigericin can act as uncoupler, neither picric acid nor nigericin alone has this capability.

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116 References Hotchkiss, R. D. (1944) Adv. Enzymol. 4, 153-199 Parker, V. H. (1965) Biochem. J. 97,658-662 Kessler, R. J., Vande Zande, H., Tyson, C. A., Blondin, G.A., Fairfield, J., Glasser, P. and Green, D.E. (1977) Proc. Nat. Acad. Sci. U.S.A. (in press) Kessler, R. J., Tyson, C.A. and Green, D.E. (1976) Proc. Nat. Acad. Sci. U.S.A. 73, 3141-3145 Mitchell, P. (1966) Biol. Rev. 41,445 Ting, P., Wilson, D.F. and Chance, B. (1970) Arch. Biochem. Biophys. 141,141-146

Skulachev, V.P., Sharaf, A.A. and Lieberman, E.A. (1967) Nature216,718-719 Komai, H., Hunter, D.R., Southard, J.H., Haworth, R. A. and Green, D. E. (1976) Biochem.

10 Blondin, G.A. and Green, D.E. (1975) Chem. Eng. News, Special Repon, Nov. 10, pp. 26-42 11 Terada, H. and Van Dam, K. (1975) Biochim. Biophys. Acta 387,507-5 18 12 Hunter, D.R., Haworth, R.A. and Southard, J. H. (1976) J. Biol. Chem. 251,5069-5077 13 Hittleman, K. J. and Lindberg, 0. (1970) in Brown Adipose Tissue (Lindberg, O., ed.) Ch. 11, American Elsevier, New York, pp. 245-262 14 Wojtczak, L. (1974) PEBSLert. 44,25-30 15 Smith, R.E. and Horowitz, B.A. (1969) Physiol. Rev. 49,330 16 Pressman, B. C. and Lardy, H. A. (1956) Biochim. Biophys. Acta 21,458 17 Hanstein, W. G. and Hatefi, Y. (1974) Proc. Nat. Acad. Sci. U.S.A. 71,282-292

Biophys. Res. Commun. 69,695-704

Montal, M., Lee, B. and Chance, C.-P. (1970) J. Membr. Biol. 2,201-234

aQ

YEARS AGO

Michael Heidelberger reviews his reviews M. Heidelberger (1927) The chemicalnature qfimmune substances, Physiol. Rev. 7,107-128 M. Heidelberger (1927) Immunologically specific polysaccharides, Chem. Rev. 3,403-423

It is a sobering assignment to review reviews one wrote fifty years ago. How lenient, or how severe, ought one be with youthful follies? I had forgotten entirely about these summaries and my first reaction was to regret that the editors of TIBS had not forgotten them, too. But a rereading after so many years agreeably surprised me by their restraint, maturity, and forward outlook, though I could take no personal credit for these qualities. The writing was done shortly before I left the Hospital of the Rockefeller Institute for Medical Research after nearly five years of enlightening collaboration with Oswald T. Avery, and most of the philosophy and even the language are reflections of the pervasive influence of this remarkable man. Thus there is little I would wish to revise. The first review was an attempt to record progress critically after the writing and publication of H. Gideon Wells’ sti,mulating book in 1925 [I]. For example, although there was much evidence by 1927 that antibodies were actually proteins, rigorous proof was lacking, and methods for separating and isolating pure proteins were limited and imperfect. It was necessary seriously to discuss ‘protein-free antibodies’ because a solution purporting to

contain such had been proposed for the cure of pneumococcal pneumonia. This situation was the result of the far greater sensitivity of immunological tests than of chemical tests for protein, facts neglected by many and pointed out in the review. Proof that antibodies were globulins came only after the introduction of quantitative

May 1977

microanalytical methods for their estimation [2] and the ‘subsequent isolation of 100 % analytically pure antibody globulin 131. Following the discussion of proteins is a short section on lipid antigens that would require expansion if written today. The longest part of the review, as well as more extended coverage in the one in Chemical Reviews, deals with the chemistry and implications of the then newly discovered immunologically specific polysaccharides. Since each of the serological types I, II, and III of Pneumococcus was shown to be characterized by a chemically different polysaccharide which precipitated the homologous antiserum and was unreactive, or nearly so, with antisera of the other two types [4], this was termed a chemical corroboration of the biologically discovered multiple type-specificity of pneumococci [5]. In contrast, the cellular protein of pneumococci reacted with antisera to all three types, and removal of these antibodies yielded more strictly typespecific antisera. This work was rapidly extended to other microorganisms [6] with a resulting transformation of microbiology. Moreover, the nitrogen-free type II and type III pneumococcal polysaccharides subsequently proved to be the antigens of chaise in the early development of microanalytical chemical methods for the estimation of antibodies in weight units [2] which ushered in a new era in immunology. In closing, I cannot help mentioning that the few grams of type III pneumococcal substance available were shown to be a polymer of a new type of disaccharidic acid [7]. Only later were the aldobiouronic acids, as they are now named, rediscovered in enormous tonnage as constituents of plant gums and the hemicelluloses. MICHAEL HEIDELBBRGER

References 1’Wells, H. G. (1925) The Chemical Aspects of ImmuChemical nity. Am. Chem. Sot. Monographs, Catalog Co., New York 2 Heidelberger, M. and Kendall, F. E. (1929) J, Exp. Med. 50, 809-823; (1935) 61, 559-562; 563-591; Heidelberger, M. and Kabat, E.A. (1934) J. Exp. Med. 60,643-653 3 Heidelberger, M. and Kabat, E.A. (1938) J. Exp. Med. 67, 181-199 4 Heidelberger, M., Goebel, W.F. and Avery, 0. T. (1925) J. Exp. Med. 42,727-745; Avery, O.T. and Heidelberger, M. (1925) J. Exp. Med. 42,367-376 5 Dochez, A.R. and Gillespie, L.J. (1913) J. Am. Med. Assn. 61,727-730 6 For example, Heidelberger, M., Goebel, W. F. and Avery, 0. T. (1925). J. Exp. Med., 42, 701-707; Michael Heidelberger, who is Emeritus Professor of Immunology of the Coilege of Physicians and Surgeons, Columbia University and Adjunct Professor in the Department of Pathology. New York University Medical Center, celebrated his 89th birihday on April 29th.

Avery, 0. T., Heidelberger, M. and Goebel, W. F. (1925). J. Exp. Med., 42, 709-725; Laidlaw, P. P. and Dudley, H. W. (1925). Brit. J. Exp. Path., 6, 197-201 7 Heidelberger, M. and Goebel, W. F. (1926) J. Biol.

Chem., 70,6lti24;

(1927) 74,613-629