Analysis of factors affecting lactic dehydrogenase subunit composition determinations

J. Theoret. Biol. (1968) 19, 169-182

Analysis of Factors Affecting Lactic Dehydrogenase Subunit Composition Determinations WILLIAM ROUSLIN AND EMORY BRASWELL BioZogy Group, University

of Connecticut, Storrs,

Connecticut 06268, U.S.A.

(Received 20 July 1967, and in revised form 5 December 1967) The ratios of the activities exhibited at low and high pyruvate levels, here designated as R values, for prepared mixtures of parental LDH tetramers will not fall on a straight line in a plot of R versus per cent A or B subunits (6~ activity) unless either the R value for the AaBo tetramer (RA) or the R value for the A0 B4 tetramer (RB) is equal to one. Intratetrameric catalytic independence is not necessary for such linearity. R values for the five electrophoretically resolved LDH tetramers will not fall on a straight line in a plot of R versus per cent A or B subunits (denoting per cent concentration of the subunits) unless the turnover number of the A subunits at the high substrate level (TAH) is equal to the turnover number of the B subunits at the high substrate level (TBH) for R, or the turnover number of the A subunits at the low substrate level (TAL) is equal to the turnover number of the B subunits at the low substrate level (TBL) for 1/R, and there is intratetrameric catalytic independence. Per cent A or B subunits by activity (f) is not equatable with per cent A or B subunits by subunit concentration (F) unless TAH is equal to TBL, and there is intratetrameric catalytic independence, i.e. the two standard curves will not be the same. The study of one set of data on chicken LDH isozymes indicates that conditions are not met under which f and F will be equivalent, or under which plots for their equations will be linear. This observation leads to the interesting conclusion that concomitant with the formation of hybrid tetramers there may be an alteration of the catalytic properties of at least the A-type subunits. The determination of turnover numbers of all the isozymes of a given series at both conditions of the differential assay is, therefore, necessary to the establishment of an assay procedure for determining the mole fraction of a given subunit present in an isozyme mixture. This conclusion infers the approximate nature of previous differential assay procedures, and removes the need for linearity of R values in such methods. Intratetrameric catalytic dependence is interesting both in terms of enzyme subunit interaction and its metabolic consequences. 1. Introdnction Heart muscle lactic dehydrogenase (L-lactate: NAD oxidoreductase, E.C. 1.1.1.27) or LDH-I has been shown to differ in several of its properties from LDH-5, the form which predominates in white skeletal muscle. One dis169

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tinguishing feature involves the substrate saturation and inhibition kinetics exhibited by the two forms. In a variety of species which have been examined, the heart muscle enzyme, here designated as A,B, in accordance with notations proposed by Markert (1962), has been shown to be inhibited by pyruvate concentrations exceeding approximately 5 x 10e4 moles per liter (Cahn, 1962; Cahn, Kaplan, Levine & Zwilling, 1962; Plagemann, Gregory & Wroblewski, 1960). On the other hand, the skeletal muscle enzyme, LDH A,&, remains uninhibited in the presence of pyruvate levels five times this value (Cahn, 1962; Cahn ei al., 1962; Plagemann et al., 1960). Plagemann et al. (1960), making use of the different substrate saturation and inhibition kinetics exhibited by the two types of LDH, i.e. A$, and A,&,, developed a method for the estimation of the per cent composition of A and B LDH subunits within any given mixture of them. Their method involves the assay of mixtures of LDH isozymes under two sets of conditions, in this case, in the presence of two different levels of pyruvate. A linear relationship was observed to exist between the logarithm of the ratio of the activities (activity at high pyruvate to that at low pyruvate) exhibited by mixtures of LDH A,B, and A,B, and the per cent of LDH A,B, in the mixtures. Because such linearity was exhibited, it was assumed that this differential assay procedure constituted a rapid means for the estimation of the relative amounts of A subunits and B subunits in any mixture of the two regardless of whether these mixtures contained only the two parental tetramer types, A,B, and AOB4, or all five tetramer types, i.e. the two parental ones plus the three hybrid forms. Subsequently, other investigators adopted the differential assay procedure for the rapid determination of LDH isozyme or subunit composition in a large variety of biological materials (Cahn, 1962, 1964; Cahn et al., 1962; Fine, Kaplan & Kuftinec, 1963; Kaplan & Cahn, 1962; Stambaugh & Post, 1966; Wilson, Cahn & Kaplan, 1963). Each group developed its own pair of conditions for a differential LDH assay; one utilized analogs of NADH such as nicotinamide hypoxanthine dinucleotide (NHXDH) (Cahn, 1962; Cahn et al., 1962; Kaplan & Cahn, 1962; Fine et al., 1963), others used NADH alone (Cahn, 1964; Plagemann et al., 1960; Stambaugh & Post, 1966; Wilson et al., 1963). One used lactate (Stambaugh & Post, 1966), while all others used pyruvate as a substrate (Cahn, 1962, 1964; Cahn et al., 1962; Fine et al., 1963; Kaplan & Cahn, 1962; Plagemann et al., 1960; Wilson et al., 1963). Finally, one used activity ratios calculated by dividing the activity measured at a high substrate level by the activity exhibited with a lower substrate concentration (Plagemann et al., 1960), while all the others used the reciprocal of this ratio, i.e. that calculated by dividing activity at a low substrate level by that exhibited at a higher substrate level (Cahn, 1962, 1964; Cahn et al.,

LDH

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171

1962; Fine et al., 1963; Kaplan & Cahn, 1962; Stambaugh & Post, 1966; Wilson et al., 1963). Regardless of these variations on Plagemann’s method, the use of all such techniques has been based on either the observation of an approximately linear relationship, or on the assumption that an actually linear relationship exists between the ratio of activities exhibited in the differential assay and the per cent composition of the mixture of LDH isozymes or subunits in question (Cahn, 1964; Fine et al., 1963; Stambaugh & Post, 1966). 2. Discussion The assumption of a linear relationship between activity ratios obtained by the differential assay procedure, here designated as R values, and the per cent subunit composition of a given mixture of LDH isozymes is based on the observation that R values for the electrophoretically separated hybrid LDH tetramers are evenly spread between those of the parental tetramers (Cahn et al., 1962; Fine et al., 1963; Kaplan & Cahn, 1962; Stambaugh & Post, 1966). This regular gradation of R values has also been interpreted as indicating the catalytic independence of the subunits within the different tetrameric combinations (Cahn et al., 1962; Fine et al., 1963; Stambaugh & Post, 1966). Before the inter-relatedness of the three pertinent elements, i.e. (a) linearity of R values, (b) the per cent subunit composition of LDH isozyme mixtures and (c) the catalytic independence of subunits within tetramers can be explicated, it must be remembered that catalytic independence of subunits is a necessary assumption for the use of R values for the calculation of per cent subunit compositions of LDH isozyme mixtures (Fine et al., 1963; Stambaugh & Post, 1966). The circularity of the reasoning used in developing the abovedescribed differential assay procedures may now be apparent. Linearity of R values, element (a), has been taken to indicate both the catalytic independence of subunits within tetramers, element (c), and the direct relationship between R values and per cent subunit composition, element (b). However, the relationship between R values and per cent subunit composition itself depends upon the catalytic independence of LDH subunits within tetramers. Another point of confusion among studies which have employed differential assay methods concerns the concept of per cent composition. One may speak of per cent composition of a mixture of two species either in terms of the relative concentrations or mole fractions of the two species, or in terms of the relative enzymatic activities exhibited by the two species in mixtures of them. The two are, of course, not equivalent, and can be so only when the turnover numbers of the two different subunits are identical. Where concentrations of LDH are defined in terms of units of activity per given volume (Plagemann et al., 1960; Stambaugh & Post, 1966), one cannot assume that equal numbers

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of activity units of A and B subunits indicates equal numbers of molecules of the two. Differential assay procedures involve two steps which will be discussed in sequence in the following sections. In the first step one constructs a standard curve which relates R values for either electrophoretically separated tetramers or for prepared mixtures of the two parental tetramers, AoB4 and A,B,,, to the per cent composition of A or B subunits in either the electrophoretically resolved isozymes, or in the prepared mixtures. The per cent composition of these prepared mixtures has been expressed as per cent subunit composition, but should have been expressed as per cent composition by activity. That is, a prepared mixture of parental tetramers having the same R value as one of the electrophoretically separated hybrid tetramers may not have the same subunit composition as that hybrid tetramer. The second step of the differential assay procedure involves the application of the standard curve constructed in step one, i.e. the measurement of the R value of a natural mixture of isozymes, and the computation of its subunit composition from the standard curve. By making certain assumptions one may analyse the elements which determine R values for mixtures of LDH isozymes. At present the observed data are not consistent with the conclusions of such an analysis. Therefore one must conclude that at least one of the assumptions is incorrect. This leads to a hypothesis which states that the catalytic properties of LDH subunits are dependent upon the subunit composition of the tetramers in which they reside. As mentioned, one may construct two types of standard curves which relate R values of mixtures of tetramers to their per cent A or B subunit composition. These are (a) curves composed of R values for prepared mixtures of the two parental tetramers, and (b) curves composed of R values of the five electrophoretically separated tetramer types. R values for those two types of mixtures will be analysed separately in the following sections. For the analysis of R values for the first type of mixture, i.e. that composed of the two parental tetramers, AoB, and A&,, two assumptions are necessary. First, it must be assumed that mixtures of the parental tetramers do not equilibrate to form hybrid tetramers. Second, that the properties of a solution of one tetramer type must not be changed by the addition of tetramers of a different composition, i.e. there must be intertetrameric catalytic independence. The total specific activity (units/ml) of a mixture of A,,B, and A,B, LDH tetramers at low substrate (pyruvate) concentration (ATL) is the sum of the specific activities of the two types of tetramers present. Therefore: ATL = AAL + ABL,

(1)

LDH

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173

where AAL is the specific activity of A,B, LDH and ABL is the specific activity of A,B, LDH, both at a low substrate concentration, The same relationship may be stated for assays of tetramer mixtures carried out at the high substrate concentration with H, denoting high substrate concentration, replacing the letter L in equation (1). The ratio of activities measured at low substrate concentration to that at high substrate concentration is therefore, R = AAL+ABL AAH+ABH’

(2)

Replacing AAL and ABH in equation (2) with R values for the parental LDH tetramers, RA and RB, one obtains, (RA)(AAH) $ ABL R = (l/RB)(ABL) + AAH’

(3)

If the concentrations of the A and B LDH subunits in a mixture of parental tetramers are defined in terms of the activities contributed by them at substrate concentration H for A and L for B, then the fraction of activity contributed by subunit A will be designatedfA and is defined as follows, (4) The activity fraction contributed by subunit B, fB, may be similarly defined. Upon substituting the expression offA and fB as stated for fA in equation (4) for AAH and ABL in equation (3), realizing that fA+fB = 1, and then simplifying by eliminating either fB or fA, the result is, R = RN1 +fA@A - 01 [l +fA(RB- l)] ’

(5)

Of

R = RB[RA+fB(l-RA)] [RB+fB(l - RB)]

*

(5a)

It can be seen that neither of these equations nor their reciprocals can be linear for either R versus fA (or fB), or for l/R versus fA (or fB) except under certain conditions. These are, (a) if RB = 1, then R versus fA (or fB) will be linear, and (b) if RA = 1, then l/R versus fA (orfB) will be linear. Plagemann et al. (1960), using mixtures of A,B, and A,B, tetramers could not have obtained a linear grouping of R values had they plotted R (or I/R) versus fB since RA and RB for rabbit LDH had values of 0.44 and 2.33 respectively. However, as will be remembered, they plotted the logarithm of l/R versus fB. It should be noted that equations (5) and (5a) are of the type

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which can be fairly linear on a log Y versus X plot when the constants have certain values. One example is when c = d and CX is less than ca. 0.7~. Using Plagemann’s data the equation is, (I-0.57fB) 1’R = (044+ 0.56fB)

(7)

which is almost linear for log l/R versus@. This equation is similar to that derived by Plagemann et al. (1960). Good agreement was found between their experimental results for mixtures of the parental tetramers and values calculated using either their equations or equation (7). Stambaugh & Post (1966) obtained good agreement between R values for artificial mixtures of the parental tetramers, &Jo and A,$,, and calculated R values for these mixtures. Their calculations agree well with equation (8),

which was obtained by substituting their measured values for RA and RB into equation (5), and rewriting it in the form of equation (6). It is interesting to note that these workers do not attempt to find a plot which would linearize their results, but rather use a plot of R versus fA which yields a curved line. The work of Kaplan & Cahn (1962) is rather puzzling. They obtained RA and RB values for chicken LDH of O-5 and 3.10 respectively. Substituting them into equation (5a) (expressed in fB) we get,

It is evident from equation (9) that it is impossible to obtain a linear relationship between R (or l/R) and fA (or fB), yet these investigators report a plot of R versus fB in which R values for prepared mixtures of the A,B, and A,B,, tetramers fall on a straight line (Kaplan & Cahn, 1962). A plot of equation (9) is shown in Fig. 1. Up to this point the elements which determine R values for prepared mixtures of the parental tetramers have been analyzed where the amounts of A and B subunits were defined in terms of units of activity manifested. Ultimately, one is interested in expressing the per cent composition of any mixture of A and B subunits in terms of the relative concentrations or mole fractions of the two regardless of whether one is dealing with a mixture of the two parental tetramers, or with mixtures of the five electrophoretically separable tetramer types.

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175

As was mentioned, one may construct two types of standard curves which relate R values of isozyme mixtures to their per cent subunit composition. The first type consists of R values for mixtures of the two parental tetramers. The second type, which will now be discussed, consists of R values for the five electrophoretically separated isozymes. This second type of standard curve differs from the first type in some fundamental ways. For the analysis of the second type of standard curve the two assumptions stated previously must be used. These are (a) that mixtures of the parentaf tetramers do not

I I 00

I

I

0.25

O-50

/--B-LDHb,*)

I 0.75

1’ 00

$9-LDHW

FIG. 1. Observed R values for chicken LDH isozymes (Kaplan & Cahn, 1962), 0 ; values for chicken LDH isozymes calculated from equation (14), 0; R for prepared mixtures of the two parental tetramers calculated from equation (9), (); straight line between R values for parental tetramers, (- - -). R

equilibrate to form hybrid tetramers, and (b) that the properties of a solution of one tetramer type must not be changed by the addition of tetramers of a different composition, i.e. there must be intertetrameric catalytic independence. In addition, two more assumptions must be stated. First, it must be assumed that only tetramers of the subunits are enzymatically active. Second, that the turnover numbers and R value of a subunit must remain constant, regardless of the subunit composition of the tetramer in which it resides, i.e. there must be intratetrameric catalytic independence. The specific activity of a given subunit type will now be expressed in terms of two elements, its molar concentration and its turnover number. One has, therefore, AAL = CA x TAL, (10) where CA is the molar concentration of A, and TAL is the turnover number

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of A in moles of substrate converted per mole of subunit per minute at the low substrate concentration. Similarly, one can express the specific activities of the A subunit at the high substrate concentration, and the specific activities of the B subunit at both low and high substrate levels in a manner analogous to that in equation (10). Equation (2) may now be rewritten, R = (CA x TAL)+(CB x TBL) (CA x TAH) +(CB x TBH)’

(11)

The mole fractions of A and B subunits will be designated FA and FB respectively. If one divides the numerator and denominator of equation (1 I) by (CA + CB), expresses the concentrations of the subunits as mole fractions, and realizes that FA + FB = 1, then, solving for FB one obtains, R = TAL+FB(TBL-TAL) TAH+FB(TBH-

TAH)’

w

This equation can be linear only when TAH = TBH. The reciprocal equation, l/R versus FB (or FA), can be linear only when TAL = TBL. Both of these cases are possible, but are not likely to occur without a careful selection of differential assay conditions. Given that TBL/TAH = 1, i.e. that the turnover numbers of A and B are equal at Hand L substrate concentrations respectively and, if we realize that, (fB x TAH) FB = TBL +fB(TAH - TBL)’

(13)

then it can be shown that equation (12) is identical with equation (5).t Equation (12) can be used in an interesting manner. If one knows TAL, TAH, TBL and TBH as they are manifested in the parental tetramers, A,B, and A,B,, and if one knows the subunit compositions of the hybrid tetramers in question, then one may calculate expected R values for the hybrid tetramers. If good agreement were found between experimental and calculated or expected values for R, then the assumption of intratetrameric catalytic independence would be borne out. To our knowledge, turnover numbers for LDH isozymes have not been determined under conditions used in differential assays, i.e. under conditions for which R values have been determined for the five tetramer types. However, enough information is available so that it is possible to derive applicable turnover number values for chicken LDH isozymes. It was necessary to t Equation (15) was derived by taking the definition of fB (equation (4)) and substituting for the three specific activities their definitions in terms of molar concentration and turnover number (e.g. equation (10)). Then, in place of the molar concentration, CA, the term (l-FB)(CA+CB) was substituted, remembering that FA+FB = 1. In place of molar concentration, CB, the term FB(CA+CB) was substituted. Upon solving for FB, one obtains equation (13).

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177

DETERMINATIONS

convert turnover numbers for A& and A,&, LDH obtained at pyruvate concentrations of 4 x 10b4M and 3 x 10Y3 M respectively, in the presence of NADH to values which would be obtained had assays been done for both types of LDH at 3 x 10v4~ pyruvate with NHXDH, and 1 x ~O-‘M pyruvate with NADH. The data and conversions are presented in Tables 1 and 2 respectively. TABLE

1~

Kinetic data for chicken LDH at optimal pyruvate concentrations in the presenceof NADH at pH 7.07 Tetramer

Substrate concentration

AI&

3X10-“M 4X10-4M

A&

Turnover number/mole

tetramer

93,400 45,500

t Pesce ef al. (1964). TABLE

1~

Kinetic data for chicken LDH for various pairs of pyruvate concentrations in the presenceof NADH at pH 7-5t Tetramer

_-.-~-

Substrate concentration -..

A,& Ad&

i

33x10-3 x10-4 3x10-4 1 x10-a

A,&

-

R 0.78

i

0.93 3.10

t Cabn (1962). TABLE

lc

Kinetic data for chicken LDH at low (3 x 10m4M) and high (1 x 10e2 M) pyruvate concentrations in the presenceof NHXDH and NADH, respectively, at pH 75t Tetramer

Observedt

Calculated (from equation (16))

Ad-h Aa&

0.50 0.95 160 2.35 3.10

0.50 0.65 0.91 144 3.10

AaBz AI& Aoh j Kaplan & Calm (1962).

3 x 10-4~+NADH 3 x 10-3~+NADH -+ --f 1 x 10da~+NADH 9.34x lo4 x0.78 = 7.29 x 10’ 7.29 x 10*/0.93 =7*83x 9*34x 10’ Optimum substrate See Table 1~ See Table 1~ concentration pH 7.0 pH 7.5 pH 7.5

4x lo-‘M+NADH --f Substrate conditions 3 x lo-*M+NADH --f 1 x 10-a~+NADH Turnover number per tetramer 4.55 x 104 4.55 x 104 4~55x104/3*10=1~47x104 Optimum substrate Near optimum substrate See Table 1~ Comment concentration, concentration, pH 7.0 pH 7.5 pH 7.5

Substrate conditions Turnover number per tetramer Comment

2

Conversion of turnover numbers

TABLE

lo4

+

-+

pH 7.5

3 x lo-‘ht+NHXDH 1~47x1~x3*10=4~55x10* See Table lc

pH 7-S

3 x lo-‘M+NHXDH 7.83 x lo* x0.5 = 3.92 x 10’ See Table lc

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Using turnover numbers calculated in Table 2 for the A&, and A, B, tetramers, dividing these values by four to get the subunit turnover numbers, and substituting these values into equation (12), one obtains, R = 9.8 + 1.6FB (14) 19*0- 159FB’ When the various values of FB (0,0*25,0*50,0*75 and 1 $0) are substituted into equation (14), one arrives at calculated R values. These are listed in Table lc along with actually observed R values (Kaplan & Cahn, 1962). Differences between observed R values and those calculated by means of equations (9) and (14) are seen clearly in Fig. 1. For the hybrid tetramers the differences are significant, and if the turnover number data (Pesce, McKay, Stolzenbach, Cahn & Kaplan, 1964) and conversion factors (Cahn, 1962) are correct, then the assumption of intratetrameric catalytic independence is suspect. Further, upon comparing the observed R values (Kaplan & Cahn, 1962) with those calculated with equation (14), one notices that, under the differential assay conditions of Kaplan and Cahn, the hybrid chicken LDH tetramers behave as though their A subunits were partially converting to B subunit-like substrate inhibition. It should be mentioned that the turnover numbers for A,B, and A,,B, LDH reported by Pesce et al. (1964) used in these calculations were obtained at pH 7.0. The R values of Kaplan & Cahn (1962), and the conversion factors of Cahn (1962) listed in Table 1 were. however, obtained at pH 7.5. The unavailability of data on the effect of pH on the substrate saturation and inhibition of chicken A4Bo and AoB., LDH in the presence of NHXDH make it impossible to estimate the error introduced into these calculations by this pH difference.? Although it is felt that this one example by no means constitutes definite proof for interaction between LDH subunits, or intratetrameric catalytic dependence, it does show how one might approach the problem. Having analyzed the factors which determine the two types of standard curves, the issue remains whether either type of curve can be usefully applied to LDH subunit analysis. From the above treatment it is clear that the curve which expresses subunit composition by activity can also be utilized for estimating subunit composition as subunit concentration only if there is intratetrameric catalytic independence and if TAH = TBL. Further, it can be linear only if either RA or RB is equal to 1 *O. Because one or more of these conditions will almost surely not be met it would be unwise to rely on the accuracy of this type of standard curve. 1 Fritz (1967) has shown that the turnover number of rabbit LDH-5 with 3 x 10e4 M pyruvate and NADH decreases by about one third as the pH is raised from 7-O to 7.4. This data is not applicable to the calculation of the effect of pH on the turnover number of chicken A,Bo LDH in the presence of NHXDH.

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The second type of standard curve, i.e. that constructed from R values of the five electrophoretically separated isozymes, can be utilized satisfactorily for the estimation of per cent subunit composition as subunit concentration only if there is intratetrameric catalytic independence. This type of standard curve will be linear only if TAH = TBH (for R), or if TAL = TBL (for l/R). It should be stressed that linearity of R values cannot be used as a criterion of intratetrameric catalytic independence, and that linearity is no longer considered essential for the utility of this second type of standard curve. The existence of intratetrameric catalytic independence in the electrophoretically isolated forms can only be established through the determination of the turnover numbers of each of the hybrid tetramers. If intratetrameric catalytic independence is lacking, then this second type of standard curve cannot be relied upon either. Stambaugh & Post (1966) show that R values for five rabbit LDH isozymes fall close to a straight line, while R values for artificial mixtures of the parental tetramers fall on a gentle curve. In terms of our assumptions, this discrepancy is probably due to a disparity between the turnover numbers of the A and B subunits under their assay conditions and/or intratetrameric or subunit interaction. Most probably, both factors contribute to the differences between the two lines, but it is impossible to decide which factor is more important without knowing the turnover numbers of the parental tetramers under the conditions of the differential assay. If the assumption of intratetrameric catalytic independence is invalid, then differential assay procedures for the estimation of per cent subunit composition are open to serious criticism. It would be difficult to say at present just how much error is being introduced into these procedures due to unknown degrees of variation in the turnover numbers of subunits in the hybrid tetramers. If there is a lack of intratetrameric catalytic independence one cannot calculate the turnover numbers or R value of any hybrid tetramer from the turnover numbers of the parental tetramers. Therefore, to properly analyze a mixture of hybrids, one must know the turnover numbers of each tetramer type under the two differential assay conditions being used. If one has this information, and if one assumes that a binomial relationship describes the distribution of the subunits within the different tetramers (Markert, 1962) then, having determined the R value for a given isozymic mixture, one now may calculate the mole fractions of the A and B subunits and, also, of the five tetramer types. It should be mentioned that this method of analysis may be applied to isozymic mixtures other than the LDH series, in which distinct catalytic properties allow the performance of a differential assay.

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3. Conclusion between observed R values (Kaplan

Discrepancies & Cahn, 1962) and those calcutated with equation (14) (see Fig. 1) indicate that the assumption of intratetrameric catalytic independence may be incorrect. These discrepancies indicate increases in hybrid tetramer substrate inhibition over that which would be expected if the subunits were exhibiting catalytic independence. If one assumes that B-type subunits persist in exhibiting a high degree of substrate inhibition within the hybrid tetramers, as they do in the A,B, form, then it follows that deviations from the theoretical hybrid tetramer R values are due to some kind of alteration which primarily involves the A-type subunits. One might speculate that this A subunit alteration is conformational as well as catalytic and is a consequence of the association of A subunits with B subunits. Rabbit muscle A,& LDH has been reported to behave as a regulatory or allosteric enzyme (Fritz, 1965). In the presence of a variety of metabolites its substrate saturation plot shifts from a sigmoid shape to a hyperbola, and its Kin for its substrate decreases. In a more recent report (Fritz, 1967) these effects have been attributed to a decrease in pH which resulted from the addition of acidic metabolites to the assay mixture. Although debatable, the possibility remains that the effect of decreased pH may not be directly upon the active site of the enzyme, but may be mediated indirectly through an altered subunit conformation and subunit interaction (Fritz, 1967). It is possible that the postulated A-type subunit transformations reported by us are not unrelated in nature to the A,B, LDH kinetic changes reported by Fritz. Under the assumption of intratetrameric catalytic independence, the metabolic potentiality associated with any particular LDH isozyme pattern is related simply to the per cent subunit composition of the isozyme mixture. This may be an oversimplification, for, in addition to the per cent subunit composition of a mixture, the degree to which subunits combine to form hybrid as opposed to parental tetramers becomes metabolically important. Thus, deviations of hybrid tetramer R values from the expected, not only bring into question the validity of previously described methods for LDH subunit analysis, but suggest possibilities for studying enzyme subunit interactions and their consequent metabolic implications. This work was supported in part by a Public Health Service Research Grant (HD 00203) from the National Institute of Child Health and Human Development and in part by a Public Health Service Training Grant (GM-317) from the National Institute of General Medical Sciences. This work is contribution no. 155 of the Institute of Cellular Biology. 7.I?. 13

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REFERENCES CAHN, R. D. (1962). Doctoral dissertation, Brandeis Univ., Waltham, Mass. Cm, R. D. (1964). Devl. Biol. 9, 327. CAHN, R. D., KAPLAN, N. O., LEVINE, L. & ZWILLING, E. (1962). Science, N. Y. 136, 962. FINE, I. H., KAPLAN, N. 0. & K-c, D. (1963). Biochemistry, N. Y. 2, 116. mz, P. J. (1965). Science, N. Y. 150, 364. FRITZ, P. J. (1967). Science, N. Y. 156, 82. KAPLAN, N. 0. & CAHN, R. D. (1962). Proc. natn. Acad. Sci., U.S.A. 48,2123. MARKERT, C. L. (1962). In “Hereditary, Developmental and Immunologic Aspects of Kidney Disease”, p. 54. Evanston: Northwestern Univ. Press. PESCE. A., MCKAY, R. H., STOLZENBACH, F., CANN, R. D. & KAPLAN, N. 0. (1964). J. biol. khem. 23$, 1753. PLAGEMANN, P. G. W., GREGORY,K. F. & WROBLEWSKI, F. (1960). J. biol. Chem. X%,2288. STAMBAUGH, R. & POST, D. (1966). Analyt. Biochem. 15,470. WILSON, A. O., CANN, R. D. & KAPLAN, N. 0. (1963). Nature, Land. 197,331.