THE METABOLISM OF THE ORGANIC ACIDS OF TOBACCO LEAVES

X. EFFECT OF CULTURE OF EXCISE’D LEAVES IN SOLUTIONS OF FUMARATE AND MALEATE

BY HUBERT BRADFORD VICKERY AND JAMES K. PALMER

(From the Biochemical Laboratory of The Connecticut Agricultural Experiment Station, New Haven, Connecticut)

(Received for publication, June 15, 1955)

A preliminary experiment some years ago showed that fumaric acid supplied to tobacco leaves by the leaf culture technique readily enters into the organic acid metabolism (1). Both malic and citric acids increased markedly, and the group of undetermined acids, in which any excess of fumaric acid present would have been included, increased to only a small extent. The effects were in general quite similar to those observed when succinic acid was administered in the same way. The development of chromatographic methods for the determination of organic acids in plant extracm (2) and the improved sampling methods now employed have re- cently made it possible to reinvestigate the behavior of fumaric acid in greater detail under better experimental conditions. In addition, the study has been extended to a consideration of the influence of the isomeric substance maleic acid upon the organic acid metabolism of tobacco leaves.

No previous test of the effect of the administration of maleic acid to leaves has come to our attention although there are a few records of experi- ments with this substance in other connections. For example, Greulach (3) observed that young bean or sunflower plants dipped for a short time into 0.015 M solutions of sodium maleate subsequently exhibited an en- hanced rate of growth. Lundegardh (4) found that the respiration of young wheat roots is stimulated when they are immersed in a 0.002 M solu- tion of potassium maleate at pH 6.8, the stimulation being somewhat diminished in the presence of cyanide. Thimann and Bonner (5) have noted that the inhibition of the growth of Avena coleoptiles by iodoacetate is relieved by a number of organic acids, maleate being one of the moder- ately effective ones, and McRae, Foster, and Bonner (6) have made use of 0.0025 M potassium maleate buffer solutions at pH 4.5 in their studies of the effects of auxins on the growth of coleoptiles. Such observations as these indicate that dilute solutions of salts of maleic acid are not toxic to plant tissues and that the acid may have definite effects upon the general metabolism; in fact, Sacks and Jensen (7) have presented evidence for the

225

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226 ORGANIC ACIDS OF TOBACCO LEAVES. X

conversion of maleic acid into malic acid by an enzyme preparation from maize kernels.

On the other hand, maleic acid is a recognized inhibitor of the activity of certain enzymes, presumably because of its capacity to react with thioi groups under physiological conditions (8). However, enzymes differ widely in their sensitivity to maleic acid, the succinic acid oxidase system of pigeon brain tissue, for example, requiring a far higher concentration for significant inhibition than pyruvic acid oxidase from the same source (9). Furthermore, ample time must be allowed for the interaction with maleic acid to take place if inhibition is to become pronounced.

The present experiments have shown that fumaric acid is taken up by tobacco leaves from 0.2 M solutions at pH 5 or 6 in somewhat smaller amounts than succinic acid under similar conditions. Maleic acid is taken up in much larger amounts. As was found in the earlier test, fumaric acid seems to behave, with respect to its influence upon the me- tabolism of malic and citric acids, in a manner similar to that of succinic acid, for it was extensively metabolized and the production of citric acid was greatly stimulated. Maleic acid, however, although it was also me- tabolized to an appreciable extent, depressed the formation of citric acid. In the presence of either fumaric or maleic acid, the extent to which malic acid was used up in the metabolism was greatly diminished. The evidence indicates, furthermore, that maleic acid made a significant contribution to the respiratory loss of organic solids from the system.

EXPERIMENTAL

Samples of twenty leaves each, ten in number, were collected by the statistical method (10) on September 22, 1953, 55 days after the seedlings had been set out in the greenhouse. The species was Nicotiana tabacum var. Connecticut shade-grown, although, in the preliminary experiment (l), Nicotiana rustica had been used. The coefficient of variation of the fresh weight of the samples was 1.6 per cent, that of the total nitrogen 2.0 per cent. One sample was dried at once for analysis, three were cultured for 24 hours, respectively, in water, in 0.2 M potassium fumarate at pH 5, and in 0.2 M potassium fumarate at pH 6, and three for 48 hours under the same conditions. In addition, three samples were cultured for 48 hours, respectively, in 0.2 M potassium maleate at pH 5, in 0.2 M potassium ma- leate at pH 6, and in 0.2 M potassium succinate at pH 5, the last to serve as a basis of comparison. The dark room was held at 24” and 50 per cent relative humidity during the culture period, and all samples were dried at 80” in a ventilated oven and subsequently equilibrated with air at 24’ and 50 per cent relative humidity in preparation for analysis.

The leaves cultured in water, in fumarate, and in succinate maintained

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H. B. VICKERY AND J. K. PALMER 227

their turgidity and increased somewhat in fresh weight (Table I, Line 1) ; all appeared to have been substantially unharmed. The leaves cultured in maleate, however, soon became flaccid, and in 48 hours had lost nearly one-half of their initial fresh weight; they became extremely thin and fragile and only the midribs retained any turgidity. The physical behav- ior was quite similar to that of tobacco leaves cultured in oxalate (11).

The analytical data were obtained by methods that have been described in recent bulletins from this Station (12, 2), and the results for the samples cultured for 48 hours are collected in Table I. The data for the samples cultured for 24 hours are omitted since they merely showed that the various changes in composition, although usually more extensive during the first 24 hour period than in the second, continued throughout.

The increases of the ash (Line 3) in the leaves cultured in fumarate did not differ greatly from those in the leaves cultured in maleate, but, owing to differences in uptake of the acid, the increases in the corrected organic solids (Line 5) were widely different. The uptake of each of the three acids (Line 7) was computed from the respective increase in the alkalinity of the ash (Line 6) with the use of factors calculated from the dissociation constants of each acid. Since approximately 90 per cent of fumaric acid is neutralized at pH 5 and 99 per cent at pH 6, the data for fumaric acid are probably reliable. Maleic acid, however, is only approximately 53 and 69 per cent neutralized at these two acidities and succinic acid is only 54 per cent neutralized at pH 5; the accuracy of the data derived with use of these quantities is accordingly somewhat less certain, but the figures nevertheless provide reasonably close estimates.

Malic acid (Line 10) diminished and citric acid (Line 11) increased in the control leaves cultured in water in the manner to be anticipated from previous studies, although more extensively than is sometimes seen. This particular lot of tobacco leaves was apparently characterized by an un-

usually vigorous organic acid metabolism. When cultured in fumarate, the loss of malic acid from the leaves was only about one-quarter as great

as in the water control, but the increase in citric acid was nearly doubled. Culture in succinate brought about an increase both in malic acid and in citric acid, but the increase in citric acid, as compared with that of the water control, was only a little more than half as great as the increase stim-

ulated by culture in fumarate. It is clear that both succinic and fumaric acids share the capacity to stimulate the formation of citric acid in tobacco leaves. The difference between them appears in part at least to be refer- able to the relat,ive amounts of these acids taken up by the leaves under

the experimental conditions (Line 7) ; at pH 5, only about three-quarters as much fumaric acid was taken up as succinic acid, and, accordingly, less

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228 ORGANIC ACIDS OF TOBACCO LEAVES. X

TABLE I

Effect of Culture in 0.2 M Solutions of Potassium Fumarate, Maleate, and Succinate upon Composition o-f Excised Tobacco Leaves

Data expressed in gm. or milliequivalents per kilo of initial fresh weight of leaves.

Lint No.

-

1

6

7

8

9

10 11 12 13

14

15

16 17

-

Final fresh weight per kilo of initia fresh weight, gm

pH of extract of dry tissue

Inorganic solids,

gm. Organic solids, gm

‘L solids,

corrected for CO2 of ash, gm.

Alkalinity of ash, m.eq.

Uptake of acid, m.eq.

Uptake of acid,

gm. Total organic

acids, m.eq. Malic acid, m.eq. Citric “ “ Oxalic “ “ Fumaric, maleic,

or succinic acid,* m.eq.

Unknown Acid A, m.eq.

Undetermined acids, m.eq.

Protein N, gm Starch, gm.

(

1r 1 .

,.

hltro1 before culture

000

5.2:

20.6

85.0 93.1

365

283

198 22.7 32.8

0

18

13

3.0 2.0

Changes during culture in darkness for 48 hrs.

-

1

2

-I

7 7

water

167

+o.or

-0.1

-5.4 -5.5

-6.0

-8.1

-62.6 -50.1 +3.9

+0.3

0.1

-0.2‘ -1.8

Potassium fumarate

PH 5 PH 6 PH 5 PH 6

1077 1054 511 531

Potassium ruccinate

pH5

1129

$O.O! +o.lf

+12.4 +12.6

-1.8 -2.7 +1.6 +0.7

+o.lf +0.6:

+11.5 +16.2

+5.0 +4.2 +8.4 t-9.1

$0.17

+9.3

-0.9 +1.8

t153 t-156 t153 t223 t-123

171 158 289 325 226

9.9 9.1 16.8 18.8 13.4

t165 t155 f-258 t266 t139

-17.0 -17.1 -18.8 -13.7 +3s.3 +96.8 +91.s +21.7 +41.1 +74.1

+3.9 +4.3 +1.1 +2.0 +0.2 +86.4 +76.2 i-239 t205 +1g

-7.2 -6.4 $9.5 +2.5

+6.8 +6.1 $2.4

-0.4‘ -1.9

3 -0.4: -2.0

-O.lf -1.5

+23

+8.3

-O.Of -1.7

+5.5

-0.38 -2.0

Potassium maleate

* Neither fumaric nor maleic acid was detected in normal tobacco leaves by the present methods, but approximately 1 m.eq. per kilo of succinic acid is present. No fumaric acid was detected in the leaves cultured either in maleic or in succinic acid.

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H. B. VICKERY AND J. K. PALMER 229

substrate was available for the presumed transformation into citric acid. As a result, some of the malic acid originally present in the leaves was drawn upon when fumaric acid was supplied. This did not occur when succinic acid was made available. As compared with the behavior of the water control, fumaric acid may, therefore, be said to have “spared” the utilization of malic acid, whereas succinic acid apparently made a direct contribution to the quantity of this substance present. This interpreta- tion of the behavior of fumaric acid is rendered the more probable in the light of the early preliminary experiment (1) in which stimulation of the formation of both malic and citric acids occurred to about the same extent in leaves cultured either in fumarate or succinate at pH 5.5. Nevertheless, the interpretation is dependent upon the assumption that fumaric acid is transported out of the lumina of the vascular system of the leaf into the cells as readily as succinic acid appears to be.

Malic acid diminished in the leaves cultured in maleate at pH 5 to about the same extent as in the leaves cultured in fumarate at the same reaction, i.e. malic acid may seem again to have been “spared”; nevertheless, the formation of citric acid in the culture at pH 5 was less than one-half of that in the water control and was also appreciably diminished in the cul- ture at pH 6. The general course of the organic acid metabolism must, therefore, have been fundamentally altered by the influx of this substance.

Oxalic acid was not affected notably; the changes were all small and near the limit of measurement. However, the increase of 3.9 m.eq. in the leaves cultured in water correlates with the manifestly high level of general met- abolic activity in this lot of leaves. The similar increases in the leaves cultured in fumarate point to the same conclusion, and it was noted that the increases in the leaves cultured for 24 hours either in water or in fuma- rate were nearly as large. The increase of oxalic acid in the leaves cul- tured in succinate was, however, negligible. A hint is provided that the availability of a large amount of exogenous substrate in this case dimin- ished the metabolic load upon the unknown substance which is normally converted into oxalic acid during the culture period. The increases of oxalic acid in the leaves cultured in maleic acid were intermediate between t)he effects upon the leaves in fumarate and succinate, and this is true also of the amounts of acquired acid which were metabolized during the culture period.

Line 13 shows the amounts of fumaric, maleic, and succinic acids found in the leaves cultured in these respective acids. Neither fumaric nor maleic acid could be detected in the control sample, and succinic acid was present in only a trace estimated to be 1 m.eq. per kilo. Furthermore, no fumaric acid could be detected in the samples cultured in either succinic

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230 ORGANIC ACIDS OF TOBACCO LEAVES. X

or maleic acid.l Comparison of the data in Line 13 with the data for up- take in Line 7 shows that approximately one-half of the fumaric acid ac- quired by the leaves was metabolized at both pH 5 and 6. Maleic acid was also moderately well utilized at both pH 5 and 6, but, since the uptake was much greater than that of fumaric acid, the proportions that entered into metabolic reactions were smaller. Succinic acid was, as would be antici- pated from previous results, nearly completely metabolized.

The Unknown Acid A in Line 14 represents a component which is eluted from the Dowex 1 analytical column together with citric acid. Citric acid is determined by a pentabromoacetone method (13) in the pooled fractions, and the difference between the titratable acidity of these fractions and the citric acid found in them furnishes an estimate of the quantity of the un- known acid present. It is a substance which occurs in tobacco leaves in amounts almost comparable with those of citric acid (cf. Table I, column 1) and appears to have been drawn upon during culture of the leaves in fumaric acid. It increased materially in the leaves cultured in maleic acid, but did not change significantly in those cultured in succinic acid. Even though the accuracy with which this acid can be determined may not be great, since its stability under the conditions of the analytical method is not known, the presence of a substance which may play an active part in the organic acid metabolism is clearly indicated.

The remaining titratable acidity shown as undetermined acids2 in Line

1 The analytical method involves elution of the organic acids from a column of Dowex 1 with a continuously increasing concentration of formic acid starting at zero and approaching 3.5 N. Fumaric acid is eluted immediately after citric acid and, if

present, forms a clearly defined peak on the titration curve. Maleic acid requires

the use of a considerably higher concentration of formic acid for convenient elution (approaching 6 N) and, even so, is first eluted many fractions after citric acid.

2 The data for “undetermined acids” represent the sums of the titrations of the fractions which are eluted by formic acid from the Dowex 1 analytical column in ad- vance of malic acid. The present “undetermined acids” fraction is usually a much

smaller quantity than has been reported under the same designation in earlier papers, and may thus give the misleading impression that the composition of the tobacco leaf with respect to organic acids is nearly completely known. The difference arises from the chromatographic technique now used to determine the so called “total organic acids.” This method yields data of highly satisfactory precision despite it,s empirical nature (2).

In explanation, it may be pointed out that such substances as aromatic phenolic acids, phosphorylated organic acids, and phosphoric and sulfuric acids (as well as oxalic acid) are retained quantitatively by the Dowex column as ordinarily operated. Such possible components of the tissue as pyruvic, oxalacetic, glycolic, glyoxylic, glyceric, and cu-ketoglutaric acids, if still present in the dried leaves, are eluted, but are in part or even entirely destroyed or lost during the evaporation of the formic acid in a stream of air at 48” in preparation for titration. Thus, many of the possible components that were previously wholly or in part included in the titration between

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H. B. VICKERY AND J. K. PALMER 231

15 increased slightly in all of the samples except the water control. This fraction comprises a mixture of about seven components of which only D-glyceric acid and succinic acid have been identified.

The behavior of the protein nitrogen (Line 16) shows the anticipated stimulation of protein metabolism in the leaves cultured in fumarate or succinate as compared with the water control. The unusual observation is that less protein nitrogen disappeared from the leaves cultured in maleate than from the water control. The small quantity of starch present (Line 17) was nearly completely metabolized in all of the samples.

DISCUSSION

Metabolism of Fumaric and Maleic Acids---Data are assembled in Table II from which certain conclusions can be drawn regarding the fate of fu- maric and maleic acids in comparison with that of succinic acid when these substances are acted upon by the enzyme systems of tobacco leaves. Line 1 shows the differences (rounded to two figures) between the quantities of the respective acids taken up (Table I, Line 7) and the quantities found in the tissues after 48 hours (Table I, Line 13). These differences repre- sent the amounts of the three acids which disappeared as such and accord- ingly must have entered into the metabolism. The ratios to the respec- tive uptakes are shown as percent,ages in Line 2, and it is clear that both fumaric acid and maleic acid are proportionately much less extensively metabolized than succinic acid. It is known from previous work that ap- proximately 90 per cent of the succinic acid supplied at pH 6 under similar conditions is also metabolized (14), and it would thus appear that the ex- tent to which both fumaric acid and succinic acid enter into reaction in the tobacco leaf system is not particularly sensitive to pH in the range between pH 5 and 6. This is not true of maleic acid, and, if the factors used in computing the uptake are reliable, it is of importance to note that less maleic acid was taken up at pH 5 than at pH 6, that a smaller quantity of this substance was metabolized at pH 5 than at pH 6, and that a smaller proportion of the amount taken up was metabolized at pH 5 than at pH 6. Exactly the converse of t,hese three statements has been almost invari- ably found to be true for malic acid and citric acid, and, within narrow limits, for both fumaric and succinic acids. On these grounds alone, maleic acid is seen to behave in an exceptional manner.

The data for respiration loss (Table II, Line 3) represent the quantities obtained by subtracting the small increases in the organic solids of the tissues, after correction for the carbon dioxide of the ash (Table ‘I, Line

arbitrarily selected pH limits of the material extracted by ether from acidified tissue are no longer included in the present so called “total organic acid” fraction. It is hoped that an adequate definition of this quantity may ultimately be obtained.

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232 ORGANIC ACIDS OF TOBACCO LEAVES. X

5), from the amounts of acid taken up by each sample (Table I, Line 8). Comparison with the respiration loss from the control sample cultured in water, which is merely the corrected loss of organic solids, shows that the general respiration was stimulated about 60 per cent in the leaves cultured

TABLE II

Metabolism of Fumaric, Maleic, and Succinic Acids by Tobacco Leaf System

Data expressed in terms of 1 kilo of initial fresh weight of leaves. For explana- tions of the derivation of these quantities, see the text.

Line NO.

1

2

7

8 9

10 11

12

13

14 15 16

-

Acquired acid metabolized, m.eq.

Acquired acid metabolized, as y. of uptake

Respiration loss, gm. Loss of organic acids, m.eq.

“ ‘I “ “ gm.

Acquired acid converted into different acid, m.eq.

Acquired acid converted into different acid, mmoles

Loss of malic acid, mmoles Sum, Lines 7 and 8, mmoles Increase of citric acid, mmoles Ratio of sum to increase of

citric acid Sum, A malic + A citric acids,

m.eq. Acquired acid converted into

acid other than malic or citric acid, m.eq.

Uptake of 6.2 M solution, ml. Change in fresh weight, gm. Transpiration, ml.

Water

Potassium fumarate

pH5 PH6 pH5 PH6 PH 5

84 81 209

49 52

51 120

18 37 92

5.5 8.3 8.4 8.4 9.7 11.6 8.1 5.9 2.5 32 59 87 0.5 0.3 0.1: 1.9 3.4 5.1

78 79 19 61 122

39 39.5 61

+31.3 +s.5 47.5

16.7 32.3 1.9 1.5

-12.5+79.s

+8.6 48.1 30.6

1.6

9.5 30.5

+9.4 +6.E 18.9 37.3 7.2 13.7 2.6 2.7

-19.2 41.8 24.7

1.7

-1.6

-112

+10

426 -167 +78

348

t74.7

+4.1

394

t54 340

+2.9 +27.4

+17 +33

724 812 -489 -469 1210 1280

489 -129 360

s1

Potas- sium

lccinate

in fumaric or maleic acid and about 100 per cent in those in succinic acid. No distinction between the effects of fumaric and maleic acids can be

drawn. Line 4 of Table II shows the apparent losses of organic acids from the

several systems. The data are the differences between the amounts of acid taken up (Table I, Line 7) and the corresponding increases in total organic acid acidity (Table I, Line 9), and are expressed in gm. in Line 5.

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H. B. VICKERY AND J. K. PALMER 233

It is assumed that no significant fraction of the metabolized organic acid was converted to substances which are unstable or volatile under the con- ditions of the analytical method. The apparent losses from the leaves cultured in fumaric acid were negligibly small, and, if the sampling and analytical errors are taken into consideration, suggest that, whatever the metabolic fate of the fumaric acid acquired by the leaves may have been, decarboxylation or other reactions which involve loss of base-binding capacity played little if any part in what occurred.

The situation is quite different with both maleic and succinic acids. Substantial losses of titratable acidity took place and, if the highly probable assumption is made that the observed losses actually fell upon these two substances, about 60 per cent of the maleic acid which was metabolized at pH 5 and 50 per cent of that metabolized at pH 6 disappeared completely, as well as 40 per cent of the succinic acid which was metabolized at pH 5. Both of these acids accordingly entered extensively into reactions which involved decarboxylation. There is no evidence regarding the nature of t,he other products of such reactions although they may well have con- tributed to the respiratory loss.

Another aspect of the metabolism of these acids is shown in Lines 6 to 11 of Table II. If from the quantities of acquired acid which were me- tabolized (Line 1) are deducted the quantities of organic acid acidity which disappeared entirely (Line 4), t’he differences shown in Line 6 represent the quantities of acquired acid which had some other fate. This fate must have been conversion into an equivalent quantity of a different organic acid since there was no change in titration value. It is of interest to pur- sue the consequences of the assumption that this other acid may have been malic acid, and that malic acid in turn contributed to the formation of citric acid. Malic acid diminished in all of the samples except the one cultured in succinic acid; in this sample it increased, presumably as a result of trans- formation of some of the acquired succinic acid. Line 7 shows the molar quantities of acquired acid which are assumed to have been converted tran- siently into malic acid, Line 8 the actual losses of malic acid, and Line 9 the sums of these quantities. Since malic acid increased in the sample cultured in succinic acid, the “loss” of malic acid is treated as a negative quantity. Line 10 shows the corresponding molar increases in citric acid. The molar ratios between the calculated amount of malic acid assumed to have been converted to citric acid and the actual increases in citric acid found are shown in Line 11.

The molar ratio 1.9 obtained from the data of the control leaves cultured in mater lends further support to the hypot.hesis that the over-all reaction which occurs in excised tobacco leaves cult.ured in water under the present conditions involves the utilization of 2 moles of malic acid for the forma-

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234 ORGANIC ACIDS OF TOBACCO LEAVES. X

tion of 1 of citric acid. A ratio of this magnitude has repeatedly been ob- served (15). The ratios 1.5 and 1.6 obtained in the experiments with fumaric acid, and 1.7 in that with succinic acid,3 suggest that, when a large quantity of extraneous acid enters the system, the reactions become so complex that stoichiometric relationships between the initial substrate and the final product are no longer evident. The low ratio indicates, how- ever, that extremely efficient use is made of this substrate.

The ratios of 2.6 and 2.7 obtained from the data for the metabolism of maleic acid indicate that the assumptions that have been made are inade- quate to account fully for the behavior of this substance. The constancy of the ratio, in spite of a 3-fold variation in the amount of maleic acid which was presumably metabolized, suggests that there is a real relationship be- tween the metabolism of maleic acid and that of citric acid, but a ratio greater than 2 indicates that a part of the maleic acid had a fate other than conversion through malic acid to citric acid. Data that bear upon this possibility are shown in Lines 12 and 13 of Table II. Line 12 shows the algebraic sum of the changes of malic and citric acid from Table I, Lines 10 and 11. These quantities represent the number of milliequivalents of acidity which were contributed to the formation of citric acid from some source other than malic acid. In Line 13 they are subtracted from the data in Line 6. The differences in the experiments with fumaric acid are negligible, thereby suggesting that all of the fumaric acid which was me- tabolized to another acid was converted ultimately to citric acid. The difference of 10 m.eq. in the case of succinic acid is also of doubtful signifi- cance in view of the analytical and sampling errors involved;4 but the differences of 17 and 33 m.eq. in the two experiments with maleic acid are a strong indication that a part of the maleic acid which was metabolized underwent conversion to substances other than malic or citric acid. Regard- less of the quantitative significance of the data, the evidence seems une- quivocal that the metabolism of maleic acid in tobacco leaves is more com- plex than is that of fumaric or succinic acid.

Maleic Acid As Inhibitor of Enzyme Reactions in Tobacco Leaves-Morgan and Friedmann (16) have shown that maleic acid at 0.08 M concentration produces a marked inhibition of activated papain if the mixture is incu- bated for a few hours before the protein substrate is added. In order to see whether this result has any bearing on the present observations on tobacco leaves, it first becomes necessary to inquire into the concentration of maleic acid which may have been present in the tissues. At the end of

a The value 1.7 confirms the magnitude of the ratio found in four previous experi- ments with succinic acid (11).

4 A similar calculation of the data from another experiment in which succinic acid was administered to tobacco leaves (11) gave four values ranging from 5 to 9.5 m.eq.

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H. B. VICKERY AND J. K. PALMER 235

48 hours, the initial kilo of leaves cultured at pH 5 in maleate weighed 511 gm. and contained 122 gm. of solids; accordingly there were 389 gm. of water present. The leaves then contained 238 m.eq. of maleic acid, and the molar concentration was therefore 0.31 on the assumption that the maleic acid was uniformly distributed in all of this water. The final con- centration in the sample cultured at pH 6 was 0.25 M when calculated in the same way. If maleic acid accumulated in these leaves at a continuous rate, and this was observed to be approximately true for the rate of accu- mulation of fumaric acid, the concentration in the leaves must have been well in excess of 0.1 M for at least 24 hours. This should have provided ample material as well as sufficient time for interaction with any thiol groups present. If, furthermore, the proteolytic enzymes of the tobacco leaf tissue resemble papain with respect to the property of activation by reagents which generate thiol groups and inhibition by reagents which com- bine with such groups, one would expect to observe some interference with the process of protein hydrolysis which normally takes place in excised tobacco leaves and which is usually easily detectable after culture in water for 24 hours, and invariably so after 48 hours. The data in Line 16 of Table I clearly show that the loss of protein nitrogen was much less than that from the water control when the leaves were cultured in maleate.

Such behavior of the protein is unique in the experience of this labora- tory. Tobacco leaves cultured in water for 48 hours normally undergo a loss of protein nitrogen that ranges from 3 to 8 per cent of the amount initially present, and it is known from earlier work (17) that there is a cor- relative increase in soluble amino nitrogen. An exceptionally vigorous hydrolysis of 13 per cent of the protein has been observed in one instance (18). When cultured in 0.2 M inorganic salts or alkali salts of organic acids, e.g. citrate, malate, succinate, fumarate, oxalate, or tartrate, pro- teolysis is invariably increased and, with a few exceptions, ranges in 48 hours from 10 to nearly 20 per cent. The factor by which the extent of proteolysis is increased over that in the water control may be as great as 5 in a given case and is, with few exceptions, at least 2. Accordingly, a case in which interference is observed with so sensitive an index of general metabolic rate as the extent of hydrolysis of the proteins of the tissues re- quires explanation. In view of the known properties of maleic acid in other connections, the hypothesis of inhibition of the proteolytic enzymes seems the most likely explanation.

In view of this, the data were examined for other possible examples of inhibitory effects. What appears to be a second instance is provided by the data for the formation of citric acid (Table I, Line 11). In the water control sample, 50 m.eq. of citric acid were formed. Nearly twice as much was found in the samples cultured in fumarate and about 50 per cent more

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236 ORGANIC ACIDS OF TOBACCO LEAVES. X

than in the water control was formed in the sample cultured in succinate. Both of these substrates therefore stimulated the synthesis of citric acid, and the amounts of acquired acid which were apparently used in this proc- ess are shown in Table II, Line 6. In the presence of maleate, however, citric acid formation was much diminished at pH 5 and was appreciably diminished at pH 6. Comparison of Table II, Line 6, and Table I, Line 7, shows that only a small fraction of the maleic acid taken up was converted into a different acid whereas about one-half of the fumaric and’succinic acids had this fate. The interpretation of these observations as a further example of inhibition of enzyme action by maleate seems reasonable.

Another possible instance is the extent to which malic acid was used up in the course of the formation of citric acid. Table I, Line 10, shows that there was a heavy demand upon the malic acid in the water control sample. The much smaller demand upon malic acid in the samples cultured in

TABLE III

Apparent Inhibitory Action of Maleic Acid on Metabolic Reactions in Il’obacco Leaves

I Inhibition by potassium maleate

I pH5 I

PH 6

Loss of protein N, gm.. Formation of citric acid, m.eq.. Utilization of malic “ “

per cent per celtt

33 75 57 18 67 62

fumarate was interpreted as a possible example of “sparing” action, since practically all of the fumaric acid available for metabolism (Table II, Lines 1 and 6) was apparently used for the sequence of reactions involved in the formation of malic and, subsequently, citric acids. The equally small demand placed upon the malic acid in the samples cultured in ma- leat,e may also be interpreted in this way, but, in view of the fact that only a small amount of maleic acid was converted to a different acid and some of this presumably finally to citric acid (Table II, Lines 7 and 9), an inter- pretation in terms of inhibition seems at least equally likely. The data for the metabolism of succinic acid further emphasize this possibility.

The data which suggest that maleic acid may act as an inhibitor of cer- tain enzymatic reactions in tobacco leaves are summarized in terms of percentage inhibition in Table III. It is implied that the participation of active thiol groups is essential in each of these transformations. The present experiments were not designed to furnish measurements of inhibi- tory action, and the figures given are accordingly only rough estimates of orders of magnitude. The effects observed did not arise merely from the

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H. B. VICKERY AND 3. K. PALMER 237

severe dehydration which occurred, for a similar set of tobacco leaf samples cultured in oxalate (11) were equally seriously dehydrated during the ex- periment but showed no such behavior. A specific effect of the presence of maleic acid therefore seems the most reasonable explanation of the ob- servations.

Enzymatic Mechanisms in Tobacco Leaves-As information on the be- havior of the organic acids of the tobacco leaf accumulates, the evidence for the presence in this tissue of a number of enzyme systems which pro: duce well known effects becomes more and more impressive. It must be emphasized, however, that proof in any given case calls for nothing less than isolation of the enzyme in substantially purified form and complete reproduction of the reaction in v&o. In the efforts to interpret the pres- ent results, and in spite of the uncertainties involved, it is assumed that malic acid occupies an intermediate position between citric acid, which seems to be the end-product of certain reactions, and the other components of the several systems. Malic acid is a substance the accumulation or utilization of which is relatively easily demonstrated, since it is a stable substance for which there is an excellent analytical method. That it lies in the chain of products of enzymatic reactions in a position after succinic acid seems probable in view of the accumulation which occurs when succinic acid is furnished to the leaves.

With respect to the relative positions of succinic and fumaric acids, the evidence is less convincing. However, the fact that administration of fumaric acid in the early preliminary experiment led to an accumulation of malic acid suggests that fumaric acid also precedes malic acid in the se- quence.

Nevertheless it is not yet possible to conclude that the sequence of re- actions is precisely that characteristic of the tricarboxylic acid cycle. Such a conclusion involves acceptance of the view that the sequence is succinic -+ fumaric -+ malic -+ oxalacetic + citric acid. The last step presumably involves a condensation of oxalacetic acid with acetyl coenzyme A. If the interpretation of the observed 2: 1 molar relationship between malic and citric acid is valid, the acetyl group of the acetyl coenzyme A must have been provided by a different sequence of reactions into which a substan- tial fraction of one of the components of the chain had been shunted. As an illustration of the difficulty of this view, the data of the experiment with fumaric acid may be cited. This substance is taken up less exten- sively than succinic acid; it is far less completely metabolized but it is an even more effective stimulant of the formation of citric acid and, accord- ingly, must have penetrated rather freely into the cells. The data indicate that 122 m.eq. of succinic acid were converted into another organic acid, 38 m.eq. of malic acid and 74 m.eq. of citric acid being formed. No fumaric

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238 ORGANIC ACIDS OF TOBACCO LEAVES. X

acid was detected. Nevertheless all of this acidity must have passed through the stage of fumaric acid if the above sequence of reactions was followed. In contrast, when 171 m.eq. of fumaric acid were made avail- able, only 84 m.eq. passed through the metabolic process; the remainder accumulated unchanged, but 97 m.eq. of citric acid were formed since some of the malic acid was drawn upon. Fumaric acid supplied from without thus behaved differently from the fumaric acid hypothetically formed during the metabolism of succinic acid. It seems obvious that a general theory of the metabolism of the organic acids of tobacco leaves is still to be sought, although some of the details are becoming clear.

Grateful acknowledgment is made to Marjorie D. Abrahams, Katherine A. Clark, and Laurence S. Nolan for technical assistance, to Dr. Israel Zelitch for helpful discussion, and to the National Science Foundation for a grant which supported a part of the expense of this investigation.

SUMMARY

Fumaric acid and maleic acid were made available in 0.2 M solution at pH 5.0 and 6.0 to tobacco leaves (Nicotiana tabacum var. Connecticut shade-grown) by means of the excised leaf culture technique. Succinic acid was similarly administered as a positive control. Fumaric acid was taken up somewhat less freely than succinic acid and about one-half of the quantity acquired by the leaves was metabolized, citric acid being almost the exclusive product. Maleic acid was taken up considerably more freely than succinic acid and the greater part was found unchanged in the tissues. Although a small contribution to the formation of citric acid appears to have been made, much of that part of the maleic acid which was metabo- lized was converted into substances the identity of which has not yet been established.

Maleic acid present in the tissue apparently behaved as an inhibitor of the activity of the proteolytic enzymes and of the enzyme systems con- cerned with the formation of citric acid. It also appeared to have inter- fered with the utilization of malic acid in the transformations that nor- mally occur. The inference may be drawn that the presence of active thiol groups is essential for these activities.

BIBLIOGRAPHY

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H. B. VICKERY AND J. K. PALMER 239

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Hubert Bradford Vickery and James K. PalmerFUMARATE AND MALEATELEAVES IN SOLUTIONS OF

EFFECT OF CULTURE OF EXCISEDACIDS OF TOBACCO LEAVES: X.

THE METABOLISM OF THE ORGANIC

1956, 218:225-239.J. Biol. Chem. 

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