TBlll JouBN.u. 01' B10LOGICAL CHllllllTBY
`Vol. 244, No. 20, l1&ue of Oatober 25, pp. 6709-6712, 1969
`Prinwl in U.S.A.
`
`Feedback Jubihition of an Allosteric Triphosphopyridine
`Nucleotide-specific lsocitrate Dehydrogenase*
`
`J. JOSEPH MARRt AND MORTON M. WEBER
`Fram the Department of Microbiology, St. Louis Unwersi,ty School of Medicine, St. Louis, Missouri 63104
`
`(Received for publication, April 1, 1969)
`
`SUMMARY
`Evidence is presented for the concerted inhibition of a
`TPN+-specific
`isocitrate dehydrogenase
`from Crithidia
`fasciculata by two biosynthetic intermediates, oxalacetate
`and glyoxylate. This inhibition is competitive with sub(cid:173)
`strate, and the presence of either inhibitory compound greatly
`increases the affinity of the enzyme for the second. This
`inhibition has been shown not to be due to the formation of
`oxalomalate, the nonenzymatic condensation product. Struc(cid:173)
`tural analogues of the substrate are not inhibitory, so that
`steric configurational analogy is apparently not the mecha(cid:173)
`nism of inhibition. In view of this, it is believed that an
`alteration in the protein must be induced by the combination
`of the two inhibitors, as has been demonstrated previously
`with this enzyme for ATP. This work confirms the allosteric
`nature of the ATP inhibition and substantiates the require(cid:173)
`ment for 2 moles of ATP per mole of enzyme.
`Since isocitrate can either enter the glyoxylate cycle or be
`metabolized via the tricarboxylic acid cycle, it is believed that
`the inhibition of this enzyme by oxalacetate and glyoxylate
`has biological significance.
`
`Earlier studies (1) with the TPN+-specific isocitrate dehy(cid:173)
`drogenase from the protozoan Crithidia f asciculata demonstrated
`that it was subject to inhibition by nucleoside triphosphates. A
`more detailed investigation of this inhibition showed that it was
`cumulative in nature and appeared to require 2 moles of ATP
`per mole of enzyme. Kinetic studies indicated that the inhibitor
`was acting at a site other than the substrate site and that chela(cid:173)
`tion was not the mechanism of inhibition.
`Since these studies were, to our knowledge, the first demonstra(cid:173)
`tion that a TPN+-specific isocitrate dehydrogenase was subject
`to allosteric inhibition by nucleotides, we felt it desirable to
`investigate this system further. Shiio and Ozaki (2) recently
`demonstrated that several TPN+-isocitrate dehydrogenases
`were inhibited by oxalacetate and glyoxylate, competitive with
`isocitrate. We therefore examined the effect of these compounds
`on this enzyme from Crithidia and showed that they were in-
`• This investigation was supported by Grant AI-03046 from the
`National Institutes of Health, United States Public Health Serv(cid:173)
`ice.
`t Postdoctoral Fellow of the American Cancer Society (Pf-280).
`
`hibitory and competitive with the substrate (3). In this report
`the two types of inhibitors are compared to show that simulta(cid:173)
`neous inhibition by nucleotides and a-keto acids can occur.
`This substantiates the previous data (1) which indicated that
`the nucleotides were acting at a locus other than that occupied
`by the substrate and confirms the requirement for 2 moles of
`ATP to inhibit the enzyme. The concerted inhibition produced
`by oxalacetate and glyoxylate is not competitive with that pro(cid:173)
`duced by ATP, and the former do not alter the affinity of the
`enzyme for the latter. The possibility that this may be an
`artificial inhibition due to the "creation" of an alternate sub(cid:173)
`strate from two smaller molecules, as described by Inagami (4)
`and Inagami and Mitsuda (5), has been examined and is con(cid:173)
`sidered not to be the case. We believe, therefore, that: this
`cumulative nucleotide inhibition and concerted inhibition by
`biosynthetic intermediates has biological significance. This is
`discussed in relation to the position of the isocitrate dehy(cid:173)
`drogenase in cell metabolism and the possibilities for alteration
`of lipid and carbohydrate metabolism by inhibition of the cyto(cid:173)
`plasmic TPN+-specific isocitrate dehydrogenase.
`
`EXPERIMENTAL PROCEDURE
`Materials--All chemicals except isocitrate were purchased
`from Sigma.
`Isocitrate was prepared either from the lactone
`purchased from Calbiochem (1), or from the trisodium salt
`(Sigma). As used in this report, isocitrate refers to the threo(cid:173)
`n.L.-racemic mixture (6) unless otherwise specified. The actual
`concentration of the active threo-n. isomer in each solution was
`determined enzymatically and found to be 50% of the total
`isocitrate concentration in all instances.
`Enzyme Preparation-Enzyme was isolated from C. fasciculata
`as previously described (1).
`Enzyme Assay-The reduction of TPN+ was measured at 340
`mµ in a Gilford recording spectrophotometer equipped with a
`Beckman DU monochromator with full scale deflections of Oto
`0.1 optical density unit. Reactions were carried out at 25° and
`were started by the addition of enzyme.
`One unit of activity is defined as the reduction of 0.1 µmole of
`TPN+ per mg of protein per min at 25°.
`Protein was determined by the method of Lowry et al. (7) or
`Warburg and Christian (8).
`
`RESULTS
`Concert,ed Inhibuion by Oxalacetate and Glyoxylate-As shown
`in Table I, the presence of both oxalacetate and glyoxylate is
`required for inhibition of the reaction. Oxalacetate alone shows
`
`This is an Open Access article under the CC BY license.
`
`5709
`
`Rigel Exhibit 1037
`Page 1 of 4
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`

`5710
`
`Feedback Inhibition of TPN+ I socitrate Dehydrogenase
`
`Vol. 244, No. 20
`
`TABLE I
`Concerted inhibition by oxalacetate and glyoxylate
`Reaction mixtures contained 0.5 mmole of Tris-HCl, pH 7.7;
`0.2 µmole of MnCh; 0.3 µmole of NADP+; 0.2 µmole of isocitrate;
`and water to a final volume of 3 ml. Reactions were carried out
`with either oxalacetate or glyoxylate in a final concentration of
`1 mM. Reactions were started by the addition of 22.6 µg of extract
`protein.
`
`Addition
`
`ili("• X
`min-1 X IOI
`
`Inhibition
`
`None ................ . .... . ... . ... . .
`. ....... .
`Oxalacetate. . . . . . . . .
`Glyoxylate .................. .
`Oxalacetate and glyoxylate ..
`
`42
`39
`42
`0
`
`%
`
`8
`0
`100
`
`l.O
`
`~ 0.5
`0
`
`10-7
`
`10·•
`
`10-•
`
`10·2
`
`10·•
`10·•
`log [Glyoxylate], log M
`Fm. 1. Effect of oxalacetate on glyoxylate inhibition. Reac(cid:173)
`tion mixtures contained 0.5 mmole of Tris-HCl buffer, pH 7.7,
`0.2 µmole of Mn Cl 2, 0.3 µmole of TPN+, 0.2 µmole of DL-isocitrate;
`oxalacetate and glyoxylate as indicated, and H20 to 3 ml. Reac(cid:173)
`tions were started by the addition of 45 µg of protein. Vo was
`taken as the rate in the presence of oxalacetate alone, and corre(cid:173)
`sponded to an inhibition of 0, 0, and 31%, respectively, for the
`concentrations 1.7 X 10-5 M, 1.7 X 10-4 M, and 1.7 X 10-3 M.
`
`competitive inhibition with respect to isocitrate and has an ap(cid:173)
`parent Ki of 2.7 mM. Glyoxylate alone produces some slight
`inhibition at a concentration of 5 mM. Neither compound will
`inhibit at concentrations which are physiological. When oxalace(cid:173)
`tate and glyoxylate are present together, there is a striking de(cid:173)
`crease in the Ki to 36 µM. This concerted inhibition has been
`described in detail in a previous report (3).
`Effect of Oxalomalate on Reaction-Since oxalacetate and gly(cid:173)
`oxylate will, in the presence of cations and mildly alkaline condi(cid:173)
`tions, form a nonenzymatic condensation product, experiments
`were carried out to determine whether this product might be the
`true inhibitor. The condensation product, oxalomalate, was
`prepared as previously described (2) and tested. Calculations
`made from previously determined rates of formation of the
`product (2) indicated that when oxalacetate and glyoxylate are
`present in the reaction mixture at a concentration of 1 mM each,
`the maximum concentration of oxalomalate which could ac(cid:173)
`cumulate in 30 sec, the time period of the assay, was 3 µM. How(cid:173)
`ever, this amount of oxalomalate is without effect, and a con(cid:173)
`centration of 333 µM condensation product inhibits the enzyme
`by only 9%, The latter concentration of oxalacetate and gly(cid:173)
`oxylate gives 75% inhibition.
`Increasing concentrations of
`oxalomalate resulted in 50% inhibition of the reaction at a con-
`
`~oc.Ki "'4. 7mM
`K; • 2.8mM
`O<•l.68
`
`140
`
`120
`
`100
`
`10
`
`50
`
`60
`
`70
`
`40
`30
`20
`(mM)1
`[ATP] 1
`Fm. 2. Lack of interaction of ATP and oxalacetate + glyoxyl(cid:173)
`ate. Reaction mixtures contained 0.5 mmole of Tris-HCI buffer,
`pH 7.7, 0.2 µmole of MnCh, 0.26 µmole of TPN+, 0.2 µmole of DL(cid:173)
`isocitrate, ATP as indicated, and H20 to 3 ml. Reactions were
`started by the addition of 45 µg of protein. Concentrations of
`inhibitors are as follows: 0--0: oxalacetate, 1 IDM; glyoxylate,
`0.01 mM; ,A.--,A.: oxalacetate, 0.03 mM; glyoxylate, 0.03 IDM
`---■ = oxalacetate, 0.01 mM; glyoxylate, 1 mM; •--•: no
`oxalacetate or glyoxylate. 1/Vi is the reciprocal of .c,.A 340 X
`min-1•
`
`centration of 3 mM. The Ki for oxalacetate and glyoxylate,
`however, is 36 µM. Further, no oxalomalate could be detected
`at 1 min in a reaction mixture in which oxalacetate and glyoxylate
`were present at a concentration of 1 mM each. This concentra(cid:173)
`tion of oxalacetate and glyoxylate gave 100% inhibition. The
`above results indicate clearly that inhibition of the enzyme is
`not due to the formation of oxalomalate in the reaction mixture.
`This conclusion is also supported by the observations that when
`either oxalacetate or glyoxylate is present in the reaction mix(cid:173)
`ture, the addition of the other compound results in an immediate
`inhibition which remains constant. The lack of time depend(cid:173)
`ence and the constancy of the degree of inhibition indicate that
`the formation of another compound prior to inhibition of the
`enzyme is unlikely.
`Effect of Oxalacetate on Glyoxylate Inhibition-Fig. 1 illustrates
`the augmentation of the inhibitory effect of the one compound
`Increasing the concentration of oxalacetate 10-
`by the other.
`fold decreases the apparent Ki (taken as the concentration re(cid:173)
`quired for 50% inhibition) for glyoxylate by about the same
`amount. At high concentrations of oxalacetate the requirement
`for glyoxylate reaches extremely low levels. Equimolar con(cid:173)
`centrations of the two compounds are not required for inhibition
`of the reaction and tend to make the construction of an artificial
`substrate less likely (vule infra). n values calculated according
`to the method of Taketa and Pogell (9) for all curves were about
`1.5. This is in agreement with previous data which indicated
`that the reaction is first order in substrate (1). The fact that
`the n values are substantially greater than 1 is probably due to
`augmentation of the glyoxylate inhibition by oxalacetate and
`vice versa.
`Lack of Interaction of ATP and Oxalacetate + Glyoxylate(cid:173)
`Since both nucleotides and other compounds act on this enzyme,
`it was of interest to determine whether they interacted with one
`another as well. Fig. 2 shows the effect of ATP inhibition in
`the presence of three different concentrations of oxalacetate and
`glyoxylate. The graphical method for determining the various
`
`Rigel Exhibit 1037
`Page 2 of 4
`
`

`

`Issue of October 25, 1969
`
`J. J. Marr and M. M. Weber
`
`5711
`
`TABLE II
`Concerted inhibition of isocitrate dehydrogenase
`by glyoxylate and oxalacetate
`Reaction mixtures contained 0.5 mmole of Tris-HCl, pH 7.7;
`0.2 µmole of MnCb; 0.3 µmole of NADP+; 0.2 µmole of isocitrate;
`and water to a final volume of 3 ml. Reactions were carried out
`with either oxalacetate or glyoxylate in a final concentration of
`1 mM, and all other inhibitors were added at the same final con(cid:173)
`centration. Reactions were started by the addition of 38 µg
`of extract protein.
`
`Addition
`
`Glyoxylate
`
`Oxalacetate
`
`AA,.., X
`AA1"'X
`min-1 X Inhibition min- 1 x Inhibition
`1()1
`1()1
`
`None .. . ... . .............
`Oxalacetate . .. . .. . . . . . . . . .. .
`Glyoxylate ..... ........ ... ..
`a-Ketoglutarate .. ..... . . . . . .
`L,-Malate . ... . . ...... .. . ... .
`D,-Malate ...................
`Succinate .. . ..... . ..... . . .. .
`Fumarate .. .. . ....... . . . .. ..
`Pyruvate ..... . . .. . .... . . . .. .
`Acetate . .. .. . . . . . . . .. . . ... . .
`Acetaldehyde .. . ..... . ... . . . .
`Glycolic acid . ........ . . . ....
`Glycolaldehyde ....... . ......
`Oxalate .... .. . ........ .... . .
`Ethanol .. . . . ... . ..... ... .. ..
`Formate ........ . . . . . . . ... . ..
`Formaldehyde ....... ... . .. ..
`
`62
`2
`62
`53
`52
`62
`54
`57
`62
`62
`60
`57
`58
`62
`62
`62
`62
`
`%
`0
`97
`0
`15
`16
`0
`13
`8
`0
`0
`3
`8
`6
`0
`0
`0
`0
`
`58
`50
`2
`50
`52
`51
`58
`56
`52
`56
`55
`58
`58
`53
`53
`53
`58
`
`%
`0
`14
`97
`14
`11
`12
`0
`3
`11
`3
`5
`0
`0
`9
`9
`9
`0
`
`constants is essentially a variation of that described by Dixon
`(10). The proof for this method has been discussed in theory by
`Webb (11) and by Yonetani and Theorell (12) with respect to
`the liver alcohol dehydrogenase. The slope of the lines varies
`inversely with a, an interaction constant which is a measure of
`the interaction of the inhibitors 11 and 12 in the El 1l 2 complex.
`In a plot of 1/Vi against! 1 with 12 as the fixed changing variable,
`a series of straight lines will result which intersect at -aK.e11 on
`the abscissa. When the inhibition is purely competitive a = oo
`and the slope remains constant since
`
`It may also be shown that for oo > a > 0 and a ¢ 1, positive
`(a < 1) or negative (a > 1) interactions exist between I 1 and 12
`in the Elil2 complex (11). When a = 1, there is no interaction
`between I 1 and h
`In Fig. 2, -aK.e11 = -4.7 mM, KRri for ATP was deter(cid:173)
`mined separately in the absence of oxalacetate and glyoxylate
`and was shown to be 2.8 mM. a is then equal to 1.68, indicat(cid:173)
`ing that there is no competitive interaction between the nucle(cid:173)
`otides and the a-keto acids, as would be expected if they acted
`at the same site.
`This confirms previous data (1), which indicated that ATP
`was a noncompetitive inhibitor of the substrate, and is in agree(cid:173)
`ment with the other studies (3), which showed that oxalacetate
`and glyoxylate were competitive inhibitors of the substrate.
`The fact that 1/V i must be plotted against [ATP] 2 in order to
`
`TABLE III
`Effect of structural analogues of threo-n,-isocitrate
`Reaction mixtures contained 0.5 mmole of Tris-HCI, pH 7.7;
`0.2 µmole of MnCl2; 0.3 µmole of NADP+; 0.2 µmole of isocitrate;
`and water to a final volume of 3 ml. All inhibitors were present
`in a final concentration of 1 mM. Reactions were started by the
`addition of 45.2 µg of protein.
`
`Addition
`
`None ................. . . . ..... . .... .. ... . .... .
`D,-Malate + formate . .. . ..... . . .. .... ... ..... .
`D,-Malate + formaldehyde ... . .... ..... .. . . . . .
`D,-Malate + CO2 ... .... .. . .. . ... . . .. .. . .. .. . . .
`a-Ketoglutarate . ........ ...... .. . .... . . ...... .
`a-Ketoglutarate + formate ..... . ... . ... .. . ... .
`a-Ketoglutarate + formaldehyde .... . .... . ... .
`a-Ketoglutarate + CO2 . ................. . . ... .
`0xalacetate ...... . . . ...... . . . .. .. . ... .
`Oxalacetate + CO2 ... . . . . . .... . .. . .... . .... . . .
`
`80
`83
`76
`78
`64
`64
`62
`61
`72
`70
`
`obtain straight lines confirms our earlier data (1) in which n
`values close to 2 were obtained for plots of log [v/Vo -
`v]
`against log [J]. Two moles of ATP appear to be required for
`inhibition of the enzyme, whereas all other reactants are first
`order.
`Effect of Structuml Analogues of Oxalacetate and Glyoxylate on
`I socitrate Dehydrogenase-In view of the evidence brought forward
`by Koshland (13) for the "induced fit" theory of enzyme sub(cid:173)
`strate interactions, we believed that the concerted inhibition by
`oxalacetate and glyoxylate might be a result of the construction of
`an artificial substrate which had a steric configuration similar to
`threo-D,-isocitrate (6). The use of analogues to form an artifi(cid:173)
`cial substrate has been demonstrated by Inagami (4) and Ina(cid:173)
`gami and Mitsuda (5) with trypsin. Therefore a number of
`other compounds were tested in an attempt to construct a sub(cid:173)
`strate from two smaller molecules. Table II lists these com(cid:173)
`pounds. They were added to the reaction mixture in the pres(cid:173)
`ence of oxalacetate or glyoxylate, as indicated. Although not
`indicated in the table, addition of these compounds in series did
`not result in inhibition unless both oxalacetate and glyoxylate
`were present.
`Because of the apparent specificity of the latter two com(cid:173)
`pounds, the steric configurations of threo-D,-isocitrate, oxalace(cid:173)
`tate, and glyoxylate were examined by the use of Corey-Pauling
`Koltun models. Although some structural similarities were
`evident, there was not a close correspondence between threo-n,(cid:173)
`isocitrate and the combination of oxalacetate and glyoxylate.
`Models of other combinations, D0-malate and formate, formalde(cid:173)
`hyde, or CO2, resembled threo-D.-isocitrate much more closely,
`but none of these was inhibitory (Table III). Even the combina(cid:173)
`tion of a-ketoglutarate and CO2, the end products of the reaction,
`resulted in only a 20% inhibition at a concentration of 1 mM each.
`This is no greater than that found for a-ketoglutarate alone and
`much less than the complete inhibition produced by oxalacetate
`and glyoxylate (Tables II and III). The presence or absence
`of a spatial relationship as demonstrated by models does not
`necessarily apply to the actual molecules. However, the in(cid:173)
`ability of oxalacetate and glyoxylate to conform to the configura(cid:173)
`tion of threo-D,-isocitrate would appear to lend support to the
`concept that simple structural analogy is not a mechanism of
`inhibition for these two compounds.
`
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`5712
`
`Feedback Inhibition of TPN+ Isocitmte Dehydrogenase
`
`Vol. 244, No. 20
`
`DISCUSSION
`
`Previous studies of this enzyme (1) demonstrated that it was
`subject to inhibition by nucleoside triphosphates. The inhibi(cid:173)
`tion was not at any of the substrate loci, and logarithmic plots
`of the inhibition curves indicated that 2 molecules of inhibitor
`were required. Calculations from thermodynamic data showed
`a substantial free energy and entropy change during the enzyme(cid:173)
`ATP interaction without nucleotide hydrolysis. These findings
`led us to postulate that the ATP was acting as an allosteric
`inhibitor.
`This communication describes the concerted inhibition of this
`enzyme by two biosynthetic intermediates of the tricarboxylic
`acid and glyoxylate cycle. The inhibition appears to be specific
`for these two compounds (Table II) and is not due to the syn(cid:173)
`thesis of a nonenzymatic condensation product or the fortuitous
`construction of a sterically similar artificial substrate. The
`best evidence against the latter possibility is the failu're to
`achieve comparable inhibition by a-ketoglutarate and CO2 at
`concentrations considerably higher than those required for
`complete inhibition by the two inhibitors (Fig. 1) (Table III).
`The failure to inhibit with D.-malate and formate (Table III),
`which together closely mimic threo-D,-isocitrate, also argues
`against steric analogy to the substrate as a mechanism of inhibi(cid:173)
`tion.
`However, the above data leave open the question of the mech(cid:173)
`anism of this concerted inhibition. The facts that either com(cid:173)
`pound can greatly increase the affinity of the enzyme for the
`other (Fig. 1) and that both are required for competitive in(cid:173)
`hibition indicate that the presence of the two inhibitors makes
`the substrate locus inaccessible to isocitrate. This could be a
`result of alteration in the protein, a possibility already demon(cid:173)
`strated for ATP (1).
`If this is the case, glyoxylate would be
`kinetically undetectable, binding by itself but not altering the
`rate of the reaction unless oxalacetate is also present (Table I).
`Preliminary binding studies, with Sephadex G-25, have shown
`that glyoxylate alone appears to bind the protein without altera(cid:173)
`tion of the reaction. The addition of small amounts of oxalace(cid:173)
`tate to this presumed enzyme-glyoxylate complex results in a
`significant inhibition of 45%, whereas the equivalent amount of
`oxalacetate added to untreated enzyme gives only about 10 to
`15% inhibition. This is also supported by the finding that
`oxalacetate alone will inhibit slightly but that glyoxylate alone
`is without effect until concentrations in excess of 5 mM are at-
`
`tained. These concentrations have probably no physiological
`significance.
`Competition experiments (Fig. 2) between oxalacetate and
`glyoxylate and ATP have confirmed the nonidentity of the loci
`for the nucleotides and a-keto acids and have corroborated the
`requirement for 2 moles of ATP per mole of enzyme. Since the
`reaction is first order with respect to all other ligands, this would
`appear to substantiate the allosteric nature of the nucleotide
`inhibition.
`The fact that both inhibitory molecules are intermediates of
`the glyoxylate cycle, which uses isocitrate as the initiating com(cid:173)
`pound, raises the intriguing question of whether this represents
`a form of feedback inhibition of a
`tricarboxylic acid cycle
`enzyme. The aconitase equilibrium greatly favors the accumula(cid:173)
`tion of citrate, and so the conversion of isocitrate to a-keto(cid:173)
`glutarate represents the first committed step in oxidative car(cid:173)
`bohydrate metabolism. It is also the first point at which reduced
`pyridine nucleotides are produced, and this may provide a
`rationale for the inhibition by ATP.
`Isocitrate has two alterna(cid:173)
`tives: it may go forward to a-ketoglutarate or be diverted to the
`In this event, the
`glyoxylate cycle via the isocitrate lyase.
`concerted inhibition by oxalacetate and glyoxylate, intermediates
`of the latter pathway, may represent a form of feedback inhibi(cid:173)
`tion of a cytoplasmic isocitrate dehydrogenase.
`
`REFERENCES
`1. MARR, J. J., AND WEBER, M. M., J. Biol. Chem. 244 2503
`(1969).
`'
`'
`2. Smro, I., AND OzAKI, H., J. Biochem. (Tokyo), 64, 45 (1968).
`3. MARR, J. J., AND WEBER, M. M., Biochem. Biophys. Res. Com(cid:173)
`mun., 36, 12 (1969).
`4. lNAGAMI, T., J. Biol. Chem., 239, 787 (1964).
`5. lNAGAMI, T., AND MITSUDA, H., J. Biol. Chem. 239, 1388
`096-0.
`'
`6. VICKERY, H.B., J. Biol. Chem., 237, 1739 (1962).
`7. LowRY,0. H., RosEBROUGH, N. J., FARR, A. C., AND RANDALL
`R. J., J. Biol. Chem., 193,265 (1951).
`'
`8. WARBURG, 0., AND CHRISTIAN, W., Biochem. z., 310,384 (1941).
`9. TAKETA, K., AND PooELL, B. M., J. Biol. Chem. 240 651
`(1965).
`'
`'
`10. DIXON, M., Biochem. J., 66, 170 (1953).
`11. WEBB, J. L., Enzymes and metabolic inhibitors, Vol. I, Aca(cid:173)
`demic Press, New York, 1963, pp. 58 and 300.
`12. YoNETANI, T., AND THEORELL, H., Arch. Biochem. Biophys.
`106, 243 (1964).
`'
`13. KosHLAND, D. E., Cold Spring Harbor Symp. Quant. Biol. 28
`473 (1963).
`'
`'
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