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Home , Aldose reductase, Hexokinase, Sorbitol, Sorbitol dehydrogenase, Strombine dehydrogenase, Triokinase

Proc. Nati. Acad. Sci. USA Vol. 80, pp. 901-905, Februarv 1983

Enzyme relationships in a pathway that bypasses and pentose in (// /reductases/alcoholism) JONATHAN JEFFERY* AND HANS JORNVALLt *Department of Biochemistry, University ofAberdeen, Marischal College, Aberdeen, AB9 lAS Scotland, United Kingdom; and tDepartment of Chemistry I, Karolinska Institutet, S-104 01 Stockholm 60, Sweden Communicated by Sune Bergstr6m, September 17, 1982 ABSTRACT A pathway from glucose via sorbitol bypasses the (8). Increased operation ofthe shut- control points of and in glucose tle could also mediate the effect (9). metabolism. It also may produce glycerol, linking the bypass to The structural relationships, the diverse and unrelated met- lipid synthesis. Utilization of this bypass is favored by a plentiful abolic suggestions, the apparent associations of some interme- supply of glucose-hence, conditions under which glycolysis also diate compounds, and the possible convergent relationships is active. The bypass further involves oxidation of NADPH, so the motivate a search for unifying explanations, detailing the sor- pentose phosphate pathway and the bypass are mutually facilita- bitol pathway and its consequences. tive. Possible consequences in different organs under normal and pathological, especially diabetic, conditions are detailed. with related structures (for example, and MATERIALS AND METHODS , and possibly, reductase and al- Glucose Metabolism via a Five-Step Sorbitol Pathway By- dose reductase, respectively) are linked functionally by this passing the scheme. Some enzymes of the bypass also feature in glycolysis Regulatory Steps of Glycolysis. In glucose metab- (aldolase and alcohol dehydrogenase), and these enzymes, with the olism, flux through the glycolytic and pentose phosphate path- reductases involved, are known to occur in different ways is regulated at phosphofructokinase (10) and glucose-6- classes or multiple forms. Two of the enzymes (aldolase phosphate dehydrogenase (11). Hexokinase is generally also a and alcohol dehydrogenase) both involve classes with and without control point, though the effect in is overcome at high a catalytic metal (). The existence ofparallel pathways and the glucose concentrations by . However, as shown in occurrence of similar enzymic steps in one pathway may help to Fig. 1, a complete pathway similar to glycolysis but fully by- explain the abundance and multiplicity ofenzymes such as reduc- passing the hexokinase and phosphofructokinase steps also is tases, aldolases, and alcohol . possible. It involves oxidation of one or two molecules of NADPH, thus favoring operation of the pentose phosphate Although alcohol dehydrogenase is widespread in nature, is pathway, and is linked to lipid synthesis via glycerol formation common in liver, and has been structurally characterized from (Fig. 1). several sources (1), its exact role in mammalian organs generally The first step of the pathway is conversion of glucose into has remained unclear. In addition to oxidation, func- sorbitol by and NADPH. Oxidation ofsorbitol tions in bile acid formation, degradation, A by sorbitol dehydrogenase and NAD' then yields fructose. metabolism, a hydroxysteroid reaction, and other areas of me- Fructose is not a for glucokinase, but in some circum- tabolism have been considered (2). Different and multiple func- stances hexokinase and ATP could convert it into fructose 6- tions of alcohol dehydrogenase would be consistent with the phosphate, leading into glycolysis at the phosphofructokinase considerable variations and extensive evolutionary reaction. This loop via sorbitol then would serve as a transhy- changes found. drogenase function (reduction of NAD+ and oxidation of Recently, another common liver , sorbitol dehydro- NADPH). Alternatively, and ATP could convert genase, was shown to be structurally, mechanistically, and an- fructose into fructose 1-phosphate. This compound can be cestrally related to alcohol dehydrogenase (3). The substrates of cleaved by to give dihydroxyacetone phosphate and sorbitol dehydrogenase, fructose and sorbitol, are considered . Glyceraldehyde 3-phosphate results from the important in special organs, such as male sexual organs (4), or action oftriosephosphate on dihydroxyacetone phos- special disease states, such as hyperglycemic formation phate or oftriokinase on glyceraldehyde. Entry at this stage into (5) and possibly diabetic neuropathy (6) and glomerulosclerosis glycolysis via glyceraldehyde 3-phosphate bypasses the hexo- (7), but a more general metabolic significance that is compatible and phosphofructokinase reactions. Conversion of glyc- with the structural similarity of these enzymes has not been clear. eraldehyde to glycerol is an alternative to . The The structural link between alcohol and dehydroge- reduction can be effected by alcohol dehydrogenase and NADH nases showed a type ofparallel or convergent evolution ofa sec- or by aldehyde reductase and NADPH. ond, different enzyme in each case with related specificity (3). Occurrence of the individual enzymes of the pathway is de- Enhancement of alcohol metabolism by fructose gave one as- scribed in relation to the organs mentioned below. The initial sociation between the substrates for sorbitol and alcohol dehy- steps of the pathway, with the glucose-fructose conversion drogenases. This effect may stem from avoidance of the rate- (sometimes also called the "sorbitol pathway"), have been dis- limiting NADH dissociation off alcohol dehydrogenase by an cussed further in relation to diabetes or effects (12), but in situ coenzyme reoxidation with glyceraldehyde formed from usually not the associated, later combination into glycolysis or lipogenesis. The publication costs ofthis article were defrayed in part by page charge In summary, the essential steps providing the bypass are payment. This article must therefore be hereby marked "advertise- those leading via sorbitol and fructose 1-phosphate to glycer- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. aldehyde 3-phosphate and linking, among other enzymes, sor- 901 Downloaded by guest on September 23, 2021 902 Biochemistry: Jeffery and Jbrnvall Proc. Natl. Acad. Sci. USA 80 (1983) bitol dehydrogenase and alcohol dehydrogenase with various ofthe aldose reductase type (that is, NADPH-linked aldehyde reductases and aldolases. reductases) have little (15) activity towards glucose, and much hepatic sorbitol dehydrogenase is present. These aspects sug- RESULTS AND DISCUSSION gest that liver may not be the main functional site ofthe entire Normal Occurrence. The scheme in Fig. 1 and the enzymes bypass for production but rather for permitting metab- shown demonstrate that the bypass or parts ofit may be offunc- olism of important compounds in any ratios. tional significance. In this respect, five tissues or organs appear . Sorbitol is present in pancreas and glucose-induced of special interest in normal conditions. They are, on the one insulin release evidently requires both NADPH and aldose re- hand, the liver, pancreas, and placenta, where some com- ductase activity within the f3 cells (12). Reduction ofglucose to pounds ofthe scheme may have special roles, and, on the other sorbitol possibly primes or activates an element in the insulin hand, the brain and the reproductive system, where the bypass release mechanism (12). Therefore, it appears possible that in appears capable of having a more general metabolic role. the pancreas, the initial steps ofthe scheme in Fig. 1 may have Liver. As the main processorofingestedcompounds, the liver special functions. As in the liver, they may not be ofenergetic might be expected to encounter sorbitol. However, orally in- value in an entire bypass ofglycolysis but serve other functions, gested sorbitol is poorly absorbed in man and animals, and prod- which in the case of pancreas may be metabolic regulation via ucts ofits metabolism by intestinal , rather than insulin release. sorbitol itself, are absorbed (13, 14). Nevertheless, sorbitol en- Placenta. Glucose is converted into fructose by the aldose tering the livercould be oxidized byhepatic sorbitol dehydroge- reductase and sorbitol dehydrogenase of normal placenta (16) nase in the same way that ethanol is oxidized by alcohol dehy- and umbilical cord (17) in species including man. High concen- drogenase. trations offructose in the fetal ofungulates (for example, Most important, liver deals with high postprandial concen- sheep and pig) apparently result from nonutilization offructose trations of glucose. Accumulation of sorbitol formed from glu- by their fetuses (18). Because fructose is not an important fetal cose within the liver then would be disadvantageous, raising the energy source in these cases, the loop from glucose to fructose osmotic pressure of the cells. Significantly, the liver enzymes may have an osmoregulatory role or serve a transhydrogenase Glucose aldose hexokinase (Iglucokinase)

Glucose-6-P

sorbi tol dehydrogenase Fructose I11 Fructose-6-P phospho- fructokinase fructokinase

Fructose-l-P Fructose-1,6-bisP

aldolase NAI[DPH aldolase FIG. 1. Schematic representation of three pathways of glucose metabo- 11l 3 InHAP lism. A pathway via sorbitol (left) by- DHAP -I- passes the hexokinase and phospho- plus plus fructokinase steps (usually considered glyceraldehyde % > glyceraldehyde-3-P to regulate glucose metabolism through the glycolytic pathway) and leads to -> NADH NADPH the final stages of glycolysis via dihy- alcohol aldehyde droxyacetone phosphate (DHAP) and dehydrogenase reductase Glycolysis glyceraldehyde 3-phosphate. Aldolase and alcohol dehydrogenase serve both NAD'+~ NADP (final steps) the bypass and glycolysis. Sorbitol de- glyce %-^ hydrogenase is homologous to alcohol dehydrogenase, and aldehyde reduc- tase possibly is related to aldose re- ductase. Links with the pentose phos- Lipogenesis]< Acetyl-CoA phate pathway and lipogenesis (via glycerol, acetyl-CoA, and the nicotin- v.1. amide coenzyme requirements) reveal further consequences of the bypass. TCA cycle Loops and branches of lesser impor- < tance in relation to the bypass are NADH omitted, as are all stepreversals. TCA, tricarboxylic acid.

Downloaded by guest on September 23, 2021 ~~~~~~~~~~~i Biochemistry: Jeffery and J6rnvaH Proc. Nati Acad. Sci. USA 80 (1983) 903 function. Such a special role may be compatible with the fact morphic and exhibits multiple isozymes. Three loci, with poly- that human and rat fetuses do not have high blood fructose con- morphism at the third locus, correspond to the chains centrations and that the placentae of these species contain an that explain the presence ofthe more cathodic isozymes (cf. ref. estradiol-sensitive soluble transhydrogenase (19, 20). 1); variable alleles at the second locus can explain the "atypical" Brain and Male Accessory Sexual Organs. Male accessory enzyme (cf. ref. 1), the "Indianapolis" enzyme (31), and the sexual organs of most contain both aldose reductase "oriental" enzyme (32); further loci are required to explain al- and sorbitol dehydrogenase and provide the fructose and sor- cohol dehydrogenases ir and y (33, 34) and sorbitol dehydroge- bitol of seminal plasma (4). Sperm contain sorbitol dehydro- nase. The possible importance of this isozyme complexity has genase and utilize sorbitol via fructose, which hexokinase con- been discussed in relation to alcoholism, but it is difficult to verts into fructose 6-phosphate, allowing metabolism by the discern which substrate specificity may be ofspecial relevance classical glycolytic pathway (4). In this case, the special circum- to alcoholism or its secondary disorders. stance is that absence ofglucose allows fructose access to hexoki- In this regard, it is noticed that alcohol dehydrogenase also nase. Provision ofsorbitol and fructose helps sperm to compete works on glyceraldehyde to form glycerol, which is a precursor successfully with , , and other cells that prefer in lipogenesis (Fig. 1). Increased utilization of this part of the glucose (4). Therefore, the initial steps of the scheme in Fig. scheme would be a very effective and direct response to handle 1, as well as possibly the whole bypass, function here to provide increased ethanol metabolism. It also would fit with fatty infil- a dependable and competitive energy source. tration in the liver as a consequence of increased alcohol me- Possibly the same is true of the brain. It contains aldose re- tabolism. Therefore, if alcohol dehydrogenase isozymes are im- ductase (21) and sorbitol dehydrogenase (22) and is a classical portant in alcoholism, the specificities of particular interest, as source ofaldehyde reductases (23). The presence ofall of these evidenced from the present scheme, should include glyceral- enzymes may reflect the constant energy demand ofthe brain, dehyde reduction. This specificity often is not looked for and securing all metabolic routes available from glucose. it is important to check for that activity in future characteriza- Pathological Conditions. In the tissues mentioned so far, the tions. scheme in Fig. 1 or part ofit is evident under normal conditions. Enzyme Relationships. Apart from the metabolic and func- In three other tissues of great importance-lens, nerve, and tional links shown in Fig. 1 and discussed above, the partici- -inappropriate operation of the pathway accompanies pating enzymes also suggest further, structural relationships. . In some cases, clear ancestral connections have been estab- Lens. During continuously high glucose levels, the aldose lished; in others, isozyme relationships can be well docu- reductase of animal lens (24) can produce high enough sorbitol mented. concentrations to cause cataract formation (5). Human lens has Reductases. The NADPH-linked reduction ofglucose to sor- less aldose reductase and more sorbitol dehydrogenase, so the bitol is catalyzed by certain members of a group of aldehyde situation is less unfavorable (25). The decisive factor allowing reductases. Differentactivities havebeen described-aldehyde accumulation in human lens (26) may be decrease in hexokinase reductase (EC 1.1.1.2), aldose reductase (EC 1.1.1.21), D-gluc- activity. uronate reductase (EC 1.1.1.19), mevaldate reductase (EC Nerve. In sciatic nerve, reduction ofglucose to sorbitol, par- 1.1.1.33), and reductases that work, for example, on L-hexonate ticularly in the Schwann cells, leads to sorbitol accumulation and daunorubicin (35). However, these activities need not all in diabetes mellitus, causing cellular damage consistent with a correspond to separate enzymes, neither needs each activity be segmental demyelinating process (27). Sorbitol dehydrogenase represented byjust one protein form. Relevant members ofthis activity is associated with the axons. The sorbitol and fructose group are monomeric proteins ofMr 35,000-40,000, which cat- in the cerebrospinal fluid of diabetic subjects (28) probably alyze re attack on the aldehyde carbonyl by the 4-pro-(R) hy- come from spinal nerves composing the cauda equina (27). Neu- drogen of NADPH (35). Thus, the glucose/sorbitol and glycer- ropathy at different locations is a common diabetic complica- aldehyde/glycerol steps (Fig. 1) can be catalyzed by closely tion, and the metabolic explanations have been little studied. similar reductases, not excluding something like isozyme re- It does not appear impossible that the availability ofthe sorbitol lationships. bypass in the neural system secures better glucose utilization Dehydrogenases. The glyceraldehyde/glycerol conversion under normal conditions but causes the neuronal localization (Fig. 1) is effected not only by the reductases but also by NADH- of the complications when pathologically high glucose concen- linked alcohol dehydrogenase (EC 1.1.1.1). This mammalian trations persist. enzyme has a similar polypeptide chain length to the reductases Kidney. An increased sorbitol concentration in the kidneys but is oligomeric (dimeric in liver, though tetrameric in ). of chronic diabetics is the consequence of the properties and Unlike aldehyde reductase, which contains no metal (36), liver location ofaldose reductase and sorbitol dehydrogenase within alcohol dehydrogenase has active-site zinc (1), although non- the kidney (29). Here, these enzymes may relate to the devel- metal dehydrogenases with this activity also occur in nature (3). opment ofdiabetic glomerulosclerosis (7), interstitial nephritis, Liver alcohol dehydrogenase is known to be strictly homologous and papillary atrophy (30). In this context and in regard to the with the second enzyme of the present pathway, sorbitol de- scheme in Fig. 1, it also may be noticed that the kidney is one hydrogenase (3). The structural identities place the character- of the internal organs where considerable alcohol dehydroge- ized sorbitoldehydrogenase (from sheep) exactly inbetween the nase activity is present. two best-studied alcohol dehydrogenases (from horse and Alcoholism. All of the above examples show the importance yeast). Furthermore, the enzymatic mechanisms ofsorbitol de- in normal and pathological conditions of different parts of the hydrogenase and these alcohol dehydrogenases are identical, bypass or of the entire pathway to glycerol or glyceraldehyde as demonstrated by a zinc content and zinc ligands 3-phosphate via sorbitol. Functions appear to range from the (3) in both. special cases discussed to the provision of alternative energy Aldolase. This fourth enzyme of the pathway via sorbitol is sources in some tissues. essentially the same enzyme as the aldolase of glycolysis and Alcoholism with its metabolic changes is another pathological , though isozymes B and C rather than A ofthe condition in which some of the enzymes in the pathway are of class I aldolases serve the bypass (37). special interest. Mammalian alcohol dehydrogenase is poly- Aldolases are oftwo types, which are distributed differently. Downloaded by guest on September 23, 2021 904 Biochemistry: Jeffery and Jornvall Proc. Natl. Acad. Sci. USA 80 (1983) Class I aldolases, mentioned above, function without metal compatible with the widespread abundance ofthe correspond- [classically in animals and higherplants, although such aldolases ing proteins. Finally, the use ofidentical or similar enzymes in with other specificities also have been described in prokaryotes subsequent steps or parallel pathways may be the functionally (38)]. Class II aldolases (typically in fungi, yeast, and bacteria) guided evolutionary explanation why isozymes and class split- contain active site zinc (39). tings are common. This applies to alcohol dehydrogenases, Interestingly, the same kind ofrelationship exists among al- where the isozyme substrate specificities at the end ofthe pres- cohol dehydrogenases though the distribution of the two en- ently discussed pathway may influence metabolism in alcohol- zyme types is different. Here, the "classical" enzyme (in mam- ism. mals and yeast) contains active site zinc, whereas the other type (in Drosophila) does not and apparently has evolved a similar This work was supported by grants from the Swedish Medical Re- activity by convergence, although ancient ancestral connections search Council (project 13X-3532) and the European Molecular Biology probably also exist between building units of dehydrogenases Organization. (3). Thus, aldolases and alcohol dehydrogenases demonstrate a 1. Branden, C.-I., Jornvall, H., Eklund, H. & Furugren, B. (1975) in The Enzymes, ed. Boyer, P. D. (Academic, New York), 3rd similar separation, each into two enzyme types. Furthermore, Ed., Vol. 11, pp. 103-190. they form parts of both the present scheme (Fig. 1) and gly- 2. Jeffery, J. (1980) in Dehydrogenases Requiring colysis. Interestingly, they also are enzymes for which limited Coenzymes, ed. Jeffery, J. (Birkhauser, Basel, Switzerland), pp. structural similarities have already been noticed (40). Thus, as- 85-125. suming aone-residue gap, an NH2-terminal part (residues 6-27) 3. Jornvall, H., Persson, M. & Jeffery, J. (1981) Proc. Natl Acad. ofrabbit muscle aldolase (class I) and a similarpart (residues 12- Sci. USA 78, 4226-4230. 4. Mann, T. (1964) in Biochemistry of Semen and of the Male Re- 32) of horse liver steroid-active alcohol dehydrogenases have productive Tract (Methuen, London), pp. 237-307. 11 of 22 residues identical (40). 5. Chylack, L. T. & Kinoshita, J. H. (1969) Invest. Ophthalmol. 8, Possible Control. Aldose reductases have high Km values for 401-412. glucose (20-200 mM) (21, 41). ATP stimulates and ADP inhibits 6. Ward, J. D., Baker, R. W. R. & Davis, B. H. (1972) Diabetes 21, the enzyme by about ±20% over the physiological concentra- 1173-1178. tion range of these nucleotides (41). Sorbitol dehydrogenase 7. Takazakura, E., Nakamoto, Y., Hayakawa, H., Kawai, K., Mur- amoto, S., Yoshida, K., Shimizua, M., Shinoda, A. & Takeuchi, forms an unproductive complex with sorbitol and NADH (42). J. (1975) Diabetes 24, 1-9. Therefore, conversion ofglucose into fructose is favored by high 8. Sund, H. & Theorell, H. (1963) in The Enzymes, eds. Sumner, glucose and high ATP concentrations and by low ADP and low J. B. & Myrback, K. (Academic, New York), 2nd Ed., Vol. 7, pp. NADH concentrations, which implies that it accompanies ef- 25-80. ficient glycolysis and respiration when there is an ample supply 9. Berry, M. N. & Kun, E. (1978) Eur.J. Biochem. 89, 237-241. of glucose. 10. Van Schaftingen, E., Jett, M.-F., Hue, L. & Hers, G.-G. (1981) Proc. NatW Acad. Sci. USA 78, 3483-3486. After glyceraldehyde 3-phosphate, the steps are common to 11. Levy, H. R. (1979) Adv. Enzymol 48, 97-192. both pathways (Fig. 1). Interestingly, these include the steps 12. Gabbay, K. H. & Tze, W. J. (1972) Proc. Natl. Acad. Sci. USA 69, that differ between organisms. Pyruvate reduction is linked to 1435-1439. NADH oxidation in mammals and many other organisms by 13. McClain, C. J., Kromhout, J. P., Zieve, L. & Duane, W. C. direct reduction to lactate (), in yeast by (1981) Arch. Intern. Med. 141, 901-903. and reduction of the acetaldehyde (pyruvate 14. Schell-Dompert, E. & Siebert, G. (1980) Hoppe-Seyler's Z. Phys- decarboxylation iol. Chem. 361, 1069-1075. decarboxylase and alcohol dehydrogenase), and in various ma- 15. Tulsiani, D. R. P. & Touster, 0. (1977)J. Biol. Chem. 252, 2545- rine invertebrates by ketoxime formation and reduction (alan- 2550. opine dehydrogenase, strombine dehydrogenase, or octopine 16. Britton, H. G., Huggett, A. St. G. & Nixon, D. A. (1967) dehydrogenase). The complex kinetic behavior of glyceralde- Biochim. Biophys. Acta 136, 426-440. hyde 3-phosphate dehydrogenase from several sources, of a few 17. Brachet, E. A. (1973) Biol. Neonate 23, 314-323. 18. Randall, G. C. B. & L'Ecuyer, C. (1976) Biol. Neonate 28, 74-82. lactate dehydrogenases (43), and of octopine dehydrogenase (44) 19. Kara;;las, H. J., Orr, J. C. & Engel, L. L. (1969) J. Biol. Chem. affords possibilities for regulation. 244, 4413-4421. Interrelationships. Most of the similarities mentioned, in 20. Shirasu, H. (1964) J. Biochem. (Tokyo) 55, 462-463. structure, metabolic pathways, and splitting of enzyme types, 21. Moonsammy, G. I. & Stewart, M. A. (1967) J. Neurochem. 14, might result individually from random changes in unrelated 1187-1193. structures. However, the clear relationship between alcohol 22. Rehg, J. E. & Torack, R. M. (1977)J. Neurochem. 28, 655-660. a 23. Ris, M. M. & von Wartburg, J.-P. (1973) Eur. J. Biochem. 37, and sorbitol dehydrogenases could not be explained in such 69-77. way. Also, the multiple links mentioned more likely seem to 24. Sheaff, C. M. & Doughty, C. C. (1976)J. Biol. Chem. 251, 2696- indicate very old and profound connections. In particular, the 2702. reductases ofthe sorbitol bypass, as noted above, have subunits 25. Jedziniak, J. A., Chylack, L. T., Cheng, H.-M., Gillis, M. K., ofthe same size classes as dehydrogenases and other oligomeric Kalustian, A. A. & Tung, W. H. (1981) Invest. Ophthalmol Visual of further functional or ancestral Sci. 20, 314-326. enzymes glycolysis. Therefore, 26. Varma, S. D., Schocket, S. S. & Richards, R. D. (1979) Invest. links between all three metabolic pathways of glucose metab- Ophthalmol. Visual Sci. 18, 237-241. olism are possible. 27. Gabbay, K. H. & O'Sullivan, J. B. (1968) Diabetes 17, 239-243. The question remains open whether regulated steps via hexo- 28. Servo, C. & Pitkanen, E. (1975) Diabetologia 11, 575-580. kinase and phosphofructokinase in glycolysis evolved as im- 29. Corder, C. N., Braughler, J. M. & Culp, P. A. (1979) Folia His- provements over a parallel primordial pathway via sorbitol or tochem. Cytochem. 17, 137-146. evolution of a route sorbitol 30. Gabbay, K. H. (1975) Annu. Rev. Med. 26, 521-536. whether through improved upon 31. Bosron, W. F., Li, T.-K. & Vallee, B. L. (1980) Proc. NatW Acad. classical glycolysis. Relationships between the proteins extend Sci. USA 77, 5784-5788. across the well-known pathways and indicate possible new ap- 32. Yoshida, A., Impraim, C. C. & Huang, I.-Y. (1981)J. Biol. Chem. proaches to clinically important metabolic problems. In addi- 256, 12430-12436. tion, they place alcohol dehydrogenase and several of the oth- 33. Bosron, W. F., Li, T.-K., Dafeldecker, W. P. & Vallee, B. L. erwise scattered enzymes in a pathway with a general function (1979) Biochemistry 18, 1101-1105. Downloaded by guest on September 23, 2021 Biochemistry; Jeffery and Jomvall Proc. Natl. Acad. Sci. USA 80 (1983) 905

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