Evolution of Duplicated Lactate Dehydrogenase Isozymes in Salmon ABORTIVE TERNARY

complex formation and levels the isozymes, of coenzymes. of the isozymes, to be somewhat intermediate between H& and the Ma,-M& series in its potential for abortive ternary complex formation with pyruvate and NAD+, and in the susceptibility of the complex toward NADH-induced dissociation. These results support previous studies on isozyme tissue distribution, low enzyme level catalytic properties, and structural properties in suggesting that the Ha4 isozyme has converged toward M4 in salmon to moderate de- mand for M-type catalytic activity in uiuo. we show here that abortive ternary complexes, including those of higher vertebrates, are susceptible to

Salmon are known to have experienced extensive gene duplication and possess duplicate genes for H-type and for M-type lactate dehydrogenase (Bailey, G. S., Tsuyuki, H., and Wilson, A. C. (1976) J. Fish. Res. Board Can. 33, X0-767). The susceptibilities to abortive ternary complex formation and breakdown have now been examined at high levels of the purified isozymes, using varying nonequilibrium levels of substrates and coenzymes. One of the isozymes, Hcu,, is shown to be somewhat intermediate between H& and the Ma,-M& series in its potential for abortive ternary complex formation with pyruvate and NAD+, and in the susceptibility of the complex toward NADH-induced dissociation. These results support previous studies on isozyme tissue distribution, low enzyme level catalytic properties, and structural properties in suggesting that the Ha4 isozyme has converged toward M4 in salmon sufficiently to satisfy moderate demand for M-type catalytic activity in uiuo.
However, we show here that abortive ternary complexes, including those of higher vertebrates, are highly susceptible to dissociation by NADH. Levels of NADH (1 to 3 PM) which are less than stoichiometric with respect to abortive ternary complex active sites (11.6 PM) induce immediate partial complex dissociation in vitro and limit possible complex formation.
We interpret these results to suggest that abortive ternary complexes are unlikely to be important in uiuo in regulating lactate dehydrogenase activity. We suggest instead that the roles of lactate dehydrogenase isozymes in uiuo can be explained solely on the basis of their K,, and Ki parameters and their responses to substrate fluctuation, without the necessity to invoke abortive ternary complex formation or any other form of lactate dehydrogenase regulation. zymes in salmonid fish tissues does not follow the above wide spread pattern (7,8). The M,-type isozyme in salmon' and trout is found in significant levels only in striated muscle, where it is the only type of lactate dehydrogenase present (7,8). The metabolic requirements of all other cell types examined in salmon and trout are satisfied by tetrameric combinations of two other polypeptides, Ha and HP (7-lo), rather than by various combinations of H and M. The Ha and HP polypeptides have arisen as a result of gene duplication in salmonids and are closely related structurally (7)(8)(9). They may, however, have diverged significantly in function. It is very striking that such tissues as spleen, kidney, lism relative to other fishes, or that one of the special duplicated lactate dehydrogenase components in their tissues has evolved sufficient M-type catalytic behavior to support at least moderate demand for this function. Support for the latter proposal has been provided through recent studies on the catalytic properties at low enzyme levels of highly purified lactate dehydrogenases from salmon (Oncorhynchus tschawytscha) (7,8). The HP4 isozyme was shown to have in uitro, low enzyme level catalytic properties typical of higher vertebrate H, enzymes, whereas the M,-type enzyme from skeletal muscle exhibits typical M, isozyme kinetics. However, the Hal enzyme appeared to be somewhat intermediate between HP4 and the Ma4-MP4 series in most catalytic parameters measured, such as apparent Michaelis constants and resistance to product and substrate inhibition.
These data, together with the unusual tissue distribution of isozymes, were taken to suggest that the Ha polypeptide of salmonid fish may have rapidly converged toward M in functional properties sufficiently to satisfy moderate demand for M-type lactate dehydrogenase activity in many cell types in these fish (7,8).
Recent studies by other workers on the regulation and metabolic significance of lactate dehydrogenase isozymes suggest that regulation of lactate dehydrogenase activity in vivo may be mediated via abortive ternary complexz formation and breakdown, and this would be a major catalytic parameter of functional physiological importance which differentiates H, from M,-type lactate dehydrogenase (for reviews, see Refs. 1 and 11). On this basis we extended our previous studies on the function of salmon lactate dehydrogenase isozymes to examine abortive ternary complex formation of the various highly purified isozymes at high enzyme levels under various equilibrium and nonequilibrium levels of coenzymes and substrates. The results presented here show that the HaI isozyme is intermediate to HP4 and the Ma,-M/3, series in response to parameters which influence formation and breakdown of abortive ternary complexes in vitro and provide further evidence that the HaI isozyme in salmonids has undergone unique and rapid functional evolutionary change. The possible in viva significance of abortive ternary complexes, however, appears from our results to be highly questionable.

Chemicals
and Enzymes -Sodium pyruvate, L(+)-lactic acid, NAD+, and NADH were purchased from Sigma. All other chemicals used were of reagent grade quality. Salmon H& Ha,, and M,-type lactate dehydrogenases were purified to homogeneity as previously described (7,8). Crystalline beef heart lactate dehydrogenase for standardization of L(+)-lactic acid was obtained from Sigma. Beef and chicken H, and M, isozymes were also partially purified to isozymic homogeneity by standard methods (7,8 were one-half of those in the syringes, since equal volumes of the two solutions were mixed. All reagents were prepared freshly for each experiment except L(+)-lactate which was prepared from a stock solution.
The L(+)-lactate was carefully standardized each time by the method of Hohorst (14). Pyruvate (15) and NAD+ (16) were also standardized by previously published procedures. NADH concentration was calculated from its known extinction coefficient of 6.22 (cm*I~mol) (16,171. Lactate dehydrogenase concentrations were calculated, by means of standard assay conditions, from the molar catalytic activities (turnover numbers) and known molecular weights of the beef (18) and salmon (7,8) enzymes.

Formation of Abortive Ternary Complex between Lactate Dehydrogenase,
Oxidized Coenzyme, and Pyruvate-Salmon lactate dehydrogenase isozymes form abortive ternary complexes when incubated with NAD+ and pyruvate with absorbance maxima at 322 nm and 388 nm, similar to previous reports for abortive complex formation from mammalian enzymes (1,11,(19)(20)(21)(22)(23). Fig. 1 shows the absorption spectrum of survival of salmon in oxygen-saturated water, and permits a valid comparison of the salmon enzymes with those from higher vertebrates, which have been extensively studied at 25". The final AA,,, value at fixed NAD+ (1.0 mM) and pyruvate (0.25 mM1 is proportional to enzyme concentration; a plot of AA,,, versus enzyme concentration (not shown) is linear. Similar absorption spectra were also obtained for the M, and HCQ isozymes.
The time course of ternary complex formation for the HP, enzyme, as measured by increased absorbance at 322 nm, is shown in Fig. 2. The absorbance change reaches a plateau in 20 to 25 min. At the concentrations shown here (0.25 mM pyruvate, 1 mM NAD+) ternary complex formation is complete within 25 min for all three isozymes. In the experiments described below, changes in A,,, were monitored for up to 2 h to assure that complex formation was complete under the test conditions.
The various isozymes differ in their susceptibility to complex formation over a wide range of pyruvate and NAD+ concentrations. Fig. 3 shows the effect of increasing pyruvate concentration on the final extent of abortive ternary complex formed (NAD+ is maintained at 1.0 mM). From these data the molar extinction coefficients at saturation and the apparent dissociation constants were calculated for each of the abortive ternary complexes (Table I). These results demonstrate that, although all three isozymes form abortive ternary complexes with essentially identical extinction coefficients at full saturation, the ease with which they form differs significantly. The dependence of complex formation on NAD+ concentration is shown in Fig. 4. For all enzymes formation of the abortive complex is essentially complete at 1 mM NAD+, which approximates the concentration of NAD+ found in aerobic cells such as liver and heart (25). The difference in apparent maximum absorbance of the three isozymes at saturating NAD+ levels simply reflects the use of nonsaturating levels (0.25 mM) of pyruvate chosen for this experiment, and is accurately predictable from the results shown in Fig. 3  -Previous proposals (23) have suggested that abortive ternary complex formation may proceed via dissociation of lactate dehydrogenase tetramers followed by formation of complexes between subunits, coenzyme, and substrate, and then reassociation of these subunit complexes into inhibited tetramers. This proposal was tested by incubating a mixture of isozymically pure chicken H, and chicken M, lactate dehydrogenases (2.0 PM each) with 1 mM pyruvate and 1 mM NAD+ under the usual conditions for ternary complex formation.
Control samples of H, and M, were treated separately under the same conditions. To the degree that complex formation might involve CL sociation and reassociation, one would expect the formation of H3M, H,M,, and HM,, heterotetramers in the mixed incubation during the reassociation process. However, starch gel electrophoresis of even vastly overloaded samples failed to reveal the presence of any heterotetramers following complex formation either in the mixed incubation, in single isozyme controls, or in control mixtures which were not subjected to abortive ternary complex formation (starch gels not shown). We conclude from this experiment that abortive. Enzyme Znhibition by Abortive Ternary Complex Formation -It is difficult to directly measure the degree of inhibition of catalytic activity due to abortive ternary complex formation, as any attempt at direct measurement of enzymatic activity involves some form of perturbation of the incubation mixture.
One method of estimating inhibition is to dilute the abortive complex mixture and assay its remaining enzymatic activity spectrophotometrically.
The complex breaks down slowly upon dilution into buffer, with a resultant increase in enzymatic activity. Treatment of the data as a semilogarithmic decay of inhibition function and extrapolation to zero time after dilution (Fig. 5) gives an estimate of the degree of inhibition before dilution. For the H& enzyme incubated at 2 PM enzyme, 0.5 mM pyruvate, 1.0 mM NAD+, the degree of inhibition as measured by this method is 85 to 90%. A similar experiment with the Moq-M& preparation showed 39% initial inhibition with 0.5 mM pyruvate, and 54% inhibition with 2 mM pyruvate.
However, this value represents a minimum estimate of the degree of inhibition, as the enzyme assay mixture contains 0.14 mM NADH, which itself appears to induce partial rapid ternary complex breakdown. This was confirmed by inclusion of 0.14 mM NADH in the dilution medium prior to assay for the HPI isozyme. In this case the measured "recovery" of enzymatic activity appeared at every time interval to be greater than when dilution was carried out in buffer alone (Fig. 5). Most importantly, the extrapolated "zero time" level of inhibition was only 75%, rather than the above 85 to 90%, although the samples were treated identically for abortive ternary complex formation, prior to dilution and assay. It is thus clear that the high level of NADH used in enzyme assay mixtures in itself promotes further rapid dissociation of approximately 15% of the diluted abortive complex, and that a more accurate assessment of the degree of inhibition by this method would be provided after correction for this NADH effect.
Dissociation constants for the breakdown of abortive complex upon dilution were also derived from these experiments. Plots of log (fraction inhibition) versus time give dissociation constants of 0.237 min-* for Mad-M&,, 0.107 min' for H&, and 0.204 min-' for HPI diluted in buffer plus 0.14 mM NADH. These results reinforce those in Fig. 3 and Table I in showing that the abortive ternary complex formed by Mad-M/l4 is less readily formed and more unstable when extensively diluted than is the HPI complex, and also show that NADH enhances complex breakdown.
The initial state of inhibition of abortive ternary complexes was also assessed by stopped flow spectrophotometry, a procedure which is sensitive and allows the recording of very rapid activation events. The three purified salmon lactate dehydros This mechanism has also been criticized by some workers (28) on the ground that the dissociation of the lactate dehydrogenase tetramer may not be sufficiently rapid at neutral pH and room temperature to support such a mechanism. An alternative mechanism was suggested (29)  Following the addition of NADH there is a significant lag period of at least 100 ms during which the final NADH concentration remains static; this is followed by a rate of NADH oxidation which gradually increases to the control value. The fact that the curve is initially horizontal implies that the enzyme is essentially completely inhibited at the start of the perturbation period.
Without prior incubation to allow abortive ternary complex formation, oxidation of NADH occurs immediately upon its addition, without any observable lag. These results support the dilution recovery experiments in demonstrating that the HP, isozyme is largely or fully inactivated upon incubation with 0.5 mM pyruvate and 1 mM NAD+. They also demonstrate that abortive ternary complexes could not exist in viuo should the steady state level of free NADH approach 50 PM. Fig. 6B shows a similar experiment with the Ma,-M/3, isozyme preparation.
In this case the lag period is virtually eliminated, and reaction ensues almost immediately on addition of NADH. From the dilution recovery experiments we would have expected approximately 40% initial inhibition of enzymatic activity and thus a reduced initial slope relative to the control. There is in fact a small but reproducible lag period, implying some degree of initial inhibition.
However, due to the enhanced susceptibility of the Mq-M& complex to NADH-induced dissociation, the lag is insufficient to permit accurate estimation of the degree of inhibition. Fig. 6C shows the experiment performed for the HaI isozyme. A lag period of approximately 75 ms is seen before the complex breaks down completely, a value intermediate between the behavior of the HP4 and Mad-M& isozymes.
The inclusion of lactate enhances the effect of NADH at inducing abortive ternary complex breakdown (Fig. 6AA); at 1 mM lactate the lag period before NADH oxidation commences is reduced to 40 ms, and at 25 mM lactate (not shown) NADH oxidation begins virtually immediately. However, lactate alone, in the absence of NADH, has no effect on the stability of the complex. Abortive ternary complexes formed either at

Effects
of NADH on Abortive Ternary Complex Stability -Attempts to quantitate the effects of very low steady state levels of NADH (1 to 5 pM) on abortive ternary complex dissociation by direct enzyme assay gave unsatisfactory results. Even slight dissociation, or residual enzymatic activity, would immediately oxidize this small amount of NADH, and the kinetics of this event could not be accurately followed at 340 nm due to the small change in absorbance. However, perturbation of abortive complexes by low levels of NADH can be shown to cause substantial complex breakdown by directly measuring changes in complex Asz2 absorption upon addition of NADH. As shown in Fig. 7, perturbation of abortive ternary complexes by even very low levels of NADH (2.5 pM) results in a very rapid drop in A,,,, followed by a slow re-formation of complex after the small amount of added NADH is oxidized. Controls including perturbation with buffer or with lactate as high as 6 mM show no such drop. Note that in this experiment the concentration of NADH used (2.5 PM) is less than the concentration of tetrameric subunits (11.6 PM). Hence in this experimental system, where initially the enzyme is essentially fully inhibited, addition of 2.5 pM NADH produces a nonequilibrium and physiologically reasonable mixture of substrates where production of lactate is favored. This system comes very close to mimicking the putative (1) nonequilibrium conditions in aerobic tissues where abortive complex is proposed to be required to prevent the reduction of pyruvate to lactate. Yet under these conditions it is in fact seen that abortive complex is rapidly and substantially dissociated, and lactate is produced following the reactivation of lactate dehydrogenase activity. The level of NADH in the resulting equilibrium mixture of substrates is unphysiologically low (lo-!' M), and under these conditions abortive complex re-forms.
Unfortunately the maximum degree of NADH-induced complex dissociation could not be accurately determined in the above experiment since Asz2 monitoring was of necessity discontinuous, although from the shape of the recovery curve, dissociation would clearly exceed 25%. This experiment, therefore, provides only a semiquantitative assessment of the amount of abortive complex which could exist at low, steady state NADH concentrations.
We have, therefore, attempted to quantitate the possible effects of steady state levels of NADH in the 1 to 3 FM range in a different way. Initial perturbation of pre-formed abortive complex with sufficiently high NADH dissociation of abortive ternary complexes. Abortive complexes were formed with 2.9 pM beef heart lactate dehydrogenase (Sigma), or with salmon Ma,MPI with the use of 1.0 mM NAD+, 0.5 mM pyruvate as under "Materials and Methods." The complexes were perturbed by injection of 1 ~1 of lactate or NADH, of varying initial concentrations, or water, and the change in A,,, measured ucrss~~s time. In this experiment, the final concentration of NADH added was 2.5 PM, and for lactate was 1 mM. Addition of lactate up to 6 rnM also failed to produce a significant response.
assures that the equilibrium mixture formed upon dissociation and enzyme activation will still contain micromolar levels of NADH. As shown in Fig. 8, perturbation of abortive complex (2.9 PM enzyme, 3 mM pyruvate, 1 mM NAD+) with 2.6 mM NADH induces breakdown and produces an equilibrium mixture containing 2.5 @M NADH, 0.38 mM pyruvate, 3.6 mM NAD+, and 2.6 mM lactate. (Final values are calculated assuming an apparent equilibrium constant for the lactate dehydrogenase reaction (31) at pH 7.5, 25", of 1.1 x 101.) The levels of pyruvate, and in particular, NAD' in this final equilibrium mixture were perhaps 3-fold above normal physiological (25) concentrations; this was necessary to achieve a mixture which was reasonably physiological and yet still contained 2.5 pM NADH. Note, however, that the effect of elevated pyruvate and NAD+ is to maximize the possible degree of abortive complex formed, yet even under these conditions, re-formation of the dissociated complex was severely limited (final A,,, = 0.011) when compared to the amount of complex formed by the control in the presence of enzyme, pyruvate, and NADH alone (final A,,, = 0.054). Hence in a steady state situation where the levels of substrates constitute an equilibrium mixture of reasonably physiological proportions, lactate dehydrogenase would be approximately 75% active. The inhibitory effect of NADH on the initial formation of abortive complex was also examined directly, without prior though different lots of pyruvate are standardized for total pyruvate content, the ability of these lots to elicite abortive complex may vary due to variable keto:enol ratios. For example the results of Figs. 1 to 4 cannot be directly compared to those in Figs. 5 to 10, since two different lots of pyruvate were used, possibly differing in enol content. For our studies this factor was unimportant since we were interested in comparing the relative behavior of different isozymes. Determination of the rate and of the absolute potential of a given lactate dehydrogenase for abortive complex formation would of course require that the initial enol pyruvate levels be known accurately, and that the rate of approach to tautomeric equilibrium be known in the system.

Properties ofDuplicated
Salmon Enzymes -The results presented here support our earlier suggestion (7,s)  We believe this to be an evolutionary trend of functional significance to the organism; the Hal isozyme would appear to be capable of providing M-type catalytic capacity in the many tissues in salmon from which M, is missing. Unfortunately little is known about the dynamics of organ utilization of lactate, pyruvate, glycogen, and glucose in salmonid fish. Although the static tissue levels of these metabolites have been measured in rested and exercised trout using older methods (27), the rates and degrees of lactate utilization by heart, liver, kidney, red muscles, and other tissues in trout are unknown. Such information is clearly important in establishing whether our proposals derived i?om in vitro catalytic studies adequately reflect in uivo isozyme behavior.
In Vivo Significance ofAbortive Ternary Complexes -Abortive ternary complexes of enzyme. NAD+ . pyruvate have been extensively studied in vitro, and we have reported here the conditions under which such complexes form and break down for the various purified salmon isozymes and for bovine H4 lactate dehydrogenase. It is the purported in uiuo formation of abortive complexes which has been suggested to be essential for preventing lactate formation from pyruvate in aerobic cells such as cardiac muscle (1). Under conditions of high lactate availability, the complex is proposed to break down to provide active enzyme for lactate scavenging.
Ideally this proposal would be tested by some form of in uivo measurement of the degree of inhibition of lactate dehydrogenase activity in aerobic cells under steady state conditions at equilibrium and displaced from equilibrium where either lactate or pyruvate formation would be favored. Although such experiments have yet to be devised, we believe on the basis of the in vitro experiments reported here that the proposed functional significance of abortive ternary complex formation in uivo is very questionable.
Using a variety of approaches we show that very low levels of NADH induce rapid complex breakdown in uitro, and may thus limit the possible degree of complex formation in the cell. (These results are contrary to previous suggestions that NADH-induced dissociation is a slow process (11,32).) Total tissue NADH levels in aerobic cells are estimated to range from 0.04 to 0.33 mM (251, and the levels of free unbound NADH to be approximately 1 PM (see below). Any NADH level in this range causes at least partial dissociation of abortive complexes, and at steady state NADH concentrations greater than 0.04 mM, abortive complexes could probably neither form nor exist.
We further question on the basis of thermodynamic and kinetic considerations whether abortive ternary complex formation, or any other form or reversible in viuo inhibition, need even be invoked as an essential means of controlling H4 enzyme behavior in the cell. Recent arguments suggest that the lactate dehydrogenase reaction in aerobic cells always lies at, or very near, equilibrium (33,34) and that, because of the position of the equilibrium constant and of the measured concentrations of lactate and pyruvate, the ratio of free, unbound NAD+:NADH will always be very high, approximately lo3 in rat liver. Similar values have been derived from measurements made with a number of aerobic tissues, including cardiac muscle (351, mammary gland (361, renal cortex (371, and adipose tissue (38), indicating that the oxidation-reduction states of the pyridine nucleotides in these tissues are all similar. Since the level of NAD+ has been found to range from 0.2 to 1.0 mM in such tissues (25) it follows that the concentration of free NADH must be very low, approximately 1 pM under normal metabolic conditions for aerobic cells. This concentration is 30-to 50-fold lower than the NADH optima or apparent Michaelis constants for NADH of typical vertebrate lactate dehydrogenases (39,401, and as long as pyruvate levels are maintained at a low level (by pyruvate dehydrogenase (41-43)) the lactate dehydrogenase isozyme would be operating far below optimum. Hence, unless there were excessively high levels of enzyme in these tissues, lactate formation could proceed at best only very slowly under such conditions. In contrast, the levels of NAD+ and lactate which exist during cardiac lactate utilization are in fact very near or even above the K,,, values for these substrates, and lactate to pyruvate conversion could occur much more efficiently and rapidly than the reverse reaction. One is, therefore, led to question the need for abortive ternary complex formation in order to prevent pyruvate reduction by lactate dehydrogenase under aerobic conditions; the rates of reaction in either direction may reach the appropriate levels simply as determined by K,,, values and substrate/product levels, as defined by Haldane (44,45). Only under anaerobic conditions where pyruvate and NADH levels climb, such as occurs in skeletal muscle during vigorous exercise or in cardiac muscle during myocardial infarction or experimentally induced ischemia, could either lactate dehydrogenase isozyme efficiently catalyze pyruvate reduction. Under such conditions the M, isozyme continues to function efficiently due to its resistance to product inhibition by lactate