CARBOHYDRATE METABOLISM IN HIGHER PLANTS III. BREAKDOWN OF FRUCTOSE DIPHOSPHATE BY PEA EXTRACTS* BY P. K. STUMPF (From the Division of Plant Nutrition, University of California, Berkeley) (Received for publication, June 6,1949) During the past decade evidence has been accumulating concerning the metabolism of sugars in higher plants. Because James et al. (I), Hanes (2), and Hassid (3) have isolated phosphorylated hexoses from plant t.is- sues identical to those found in animal tissues, it can be assumedthat the mode of transformation of sugars in plants is probably similar to that in animals. Little work, however, has been carried out to elucidate the enzyme systems involved in carbohydrate metabolism in higher plants. Recently the enzyme aldolase (4), catalyzing the reaction that reversibly converts fructose diphosphate to triose phosphates, has been isolated from pea seeds,and its properties and kinetics have been studied. Further- more, distribution studies (5) have revealed the presenceof this enzyme in a wide variety of plants, its concentration being especially high in the meristematic tissues. Finally, the enzyme was found to be localized in the cytoplasm of leaf cells rather than in chloroplastic bodies. The present communication will present observations on some of the enzyme systems involved in the further transformation of triose phos- phates to pyruvic acid and acetaldehyde. The results indicate a striking similarity between the enzyme systems found in plants and those found in yeast and animal tissues with respect to the chemist,ry of the reactions and of the enzymes involved. Preparations-Pea seeds (Dwarf Telephone) were employed as the source of the fermentation system. Since the system was stable to ace- tone, a large quantity of pea acetone powder was prepared as follows: Peas were soaked in distilled water at 2” for 12 hours, homogenized in 5 to 10 times their weight of acetone at 0” in a Waring blendor for 10 minutes, filtered through a large Biichner funnel, and washed twice with acetone and twice with dry, peroxide-free ether. The dry, amorphous powder when stored at - 10” proved to be stable for an indefinite period. Three fermentation systems were prepared from pea acetone powder. Preparation A, acetone powder was suspendedin 5 times its weight of dis- tilled water, and adjusted to pH 6.5 with 0.1 M NaHC03. After 10 min- * This paper was presented in part at the meeting of the American Chemical So- ciety at San Francisco, March 28, 1949. 261 by guest on September 18, 2020 http://www.jbc.org/ Downloaded from
Transcript
CARBOHYDRATE METABOLISM IN HIGHER PLANTS
III. BREAKDOWN OF FRUCTOSE DIPHOSPHATE BY PEA EXTRACTS*
BY P. K. STUMPF
(From the Division of Plant Nutrition, University of California, Berkeley)
(Received for publication, June 6,1949)
During the past decade evidence has been accumulating concerning the metabolism of sugars in higher plants. Because James et al. (I), Hanes (2), and Hassid (3) have isolated phosphorylated hexoses from plant t.is- sues identical to those found in animal tissues, it can be assumed that the mode of transformation of sugars in plants is probably similar to that in animals. Little work, however, has been carried out to elucidate the enzyme systems involved in carbohydrate metabolism in higher plants.
Recently the enzyme aldolase (4), catalyzing the reaction that reversibly converts fructose diphosphate to triose phosphates, has been isolated from pea seeds, and its properties and kinetics have been studied. Further- more, distribution studies (5) have revealed the presence of this enzyme in a wide variety of plants, its concentration being especially high in the meristematic tissues. Finally, the enzyme was found to be localized in the cytoplasm of leaf cells rather than in chloroplastic bodies.
The present communication will present observations on some of the enzyme systems involved in the further transformation of triose phos- phates to pyruvic acid and acetaldehyde. The results indicate a striking similarity between the enzyme systems found in plants and those found in yeast and animal tissues with respect to the chemist,ry of the reactions and of the enzymes involved.
Preparations-Pea seeds (Dwarf Telephone) were employed as the source of the fermentation system. Since the system was stable to ace- tone, a large quantity of pea acetone powder was prepared as follows: Peas were soaked in distilled water at 2” for 12 hours, homogenized in 5 to 10 times their weight of acetone at 0” in a Waring blendor for 10 minutes, filtered through a large Biichner funnel, and washed twice with acetone and twice with dry, peroxide-free ether. The dry, amorphous powder when stored at - 10” proved to be stable for an indefinite period.
Three fermentation systems were prepared from pea acetone powder. Preparation A, acetone powder was suspended in 5 times its weight of dis- tilled water, and adjusted to pH 6.5 with 0.1 M NaHC03. After 10 min-
* This paper was presented in part at the meeting of the American Chemical So- ciety at San Francisco, March 28, 1949.
utes, the suspension was centrifuged for 10 minutes at 2500 R.P.M. and the residue discarded. The opaque supernatant solution was used directly. Preparation B was the same as Preparation A, except that the extract was dialyzed for 2 hours against lo+ M thioglycolate at 4”. A magnetic stirrer was employed to agitate the dialyzing fluid vigorously. Prepa.ra- tion C was the same as Preparation B, except that dialysis was carried out for 12 hours under the same conditions. No attempts were made to separate or isolate the various enzyme systems.
Methods-Conventional Warburg manometric techniques were employed in measuring oxygen uptake and carbon dioxide formation. Fructose diphosphate (FDP) was estimated by first converting it completely to triose phosphates by a pea aldolase-sulfite mixture and then measuring the triose phosphates by their sensitivity to mild alkaline hydrolysis (4). Phosphoglyceric acid was estimated from the difference between total phosphate and phosphate released after 3 hours hydrolysis in 1 N KC1 at 103”, phosphopyruvic acid by phosphate released after iodine-NaOH oxi- dation (6), and pyruvic acid by the Friedemann-Haugen procedure (7). In estimating mixtures of phosphopyruvic acid and adenosine di- and tri- phosphates, clear cut separations of phosphopyruvic acid from the adeno- sine derivatives were effected by barium fractionation as described by Umbreit et al. (6). Inorganic phosphate was measured by the Fiske and Subbarow method (8) and organic phosphate by the perchloric acid diges- tion procedure (9).
Reagents-Acid barium FDP of 85 per cent purity by aldolase analysis was prepared by the method of Neuberg et al. (lo), from barium FDP ob- tained from the Schwarz Laboratories, Inc. In aqueous solutions, how- ever, the concentration of FDP decreased after a week, presumably by hydrolysis to fructose-&phosphate, notwithstanding storage at 0”. There- fore, for quantitative experiments, only freshly prepared solutions of so- dium FDP were used. Adenvlic acid and adenosine diphosphate were obtained from the Sigma Chc mica1 Company, and adenosine triphosphate (Naa) from the Rohm and Haas Company. Coenzyme I was prepared from bakers’ yeast by the method of LePage (11) and assayed 44 per cent spectrophotometrically. We are indebted to Dr. H. A. Barker for gener- ous samples of acid barium 3-phosphoglycerate and silver barium phospho- pyruvate.
Results
FDP s Phosphoglyceric Acid
As summarized in Fig. 1, when FDP is added to Preparation C, no acid (measured as COZ in a bicarbonate medium) is formed anaerobically unless coenzyme I, arsenate, and a suitable oxidant are added. Both coenzyme
I and arsenate are specifically required. Inorganic phosphate with or without adenylic acid is inert in the system in so far as the rapid formation of acid is concerned. A variety of oxidants may be employed to complete the system. It is important to point out that there is a slow formation of acid in the presence of inorganic phosphate. This effect is related to a phosphatase which slowly hydrolyzes 1,3-diphosphoglyceric acid formed during the fermentation. Finally, fluoride was employed to limit the breakdown of FDP to phosphoglyceric acid.
With these factors in mind, the study was resolved into a series of ex- periments designed to demonstrate the component enzymes involved in
I COMPLETE SYSTEM 2 NO As; PLUS Pi *AMP
0 20 40 60 80 TIME,MINUTES
FIG. 1. Breakdown of FDP as affected by components of the system. The condi- tions of the experiment are described in the legend of Table I.
the fermentation system. The components proved to be (1) aldolase, (2) isomerase, (3) triose phosphate dehydrogenase, (4) a phosphotransferase (?), and (5) the adjuncts coenzyme I, arsenate, and a suitable oxidant.
The first enzyme system, aldolase, has already been defined in Paper I of this series (4). It cleaves FDP to dihydroxyacetone phosphate and 3-phosphoglyceraldehyde, the equilibrium constant at 37” being about 5 X 10T3 mole per liter. Therefore, on addition of FDP to Preparation C an equilibrium mixture between FDP and the two triose phosphates is rapidly established. As in animal tissues and yeast, an isomerase is also present in Preparation C, which catalyzes an equilibrium between the two triose phosphates (see the scheme in the “Discussion”). The proof for
this conclusion, while not as direct as the actual isolation of isomerase would be, is clearly demonstrated in the following considerations. First, in a complete fermentation system, it is obvious that only if an isomerase is present can 1 equivalent of FDP yield 2 equivalents of acid. Table I summarizes the data of CO2 measuremenbs in which the ratio of 1 equiva- lent of FDP utilized to 2 equivalents of acid formed is demonstrated. This ratio could not have been realized if isomerase had been absent, since of the two triose phosphates formed from the aldolase cleavage of FDP only 3-phosphoglyceraldehyde would have been oxidized by triose phos- phate dehydrogenase to yield phosphoglyceric acid. The other triose phosphate, dihydroxyacetone phosphate, would have accumulated, since it would not be in equilibrium with phosphoglyceraldehyde and is itself not
TABLE I
Formation of Phosphoglyceric Acid from Fructose Diphosphate
Each manometric cup contained 1 cc. of enzyme (Preparation C), different con- centrations of fructose diphosphate, 0.05 cc. of 0.05 M arsenate, 0.1 cc. of 0.1 per cent coenzyme I, 0.5 cc. of 0.05 M sodium bicarbonate, 0.1 cc. of M acetaldehyde, 0.5 cc. of 0.1 M fluoride, and water to a total volume of 3 cc. Temperature 37’; gas phase 95 per cent Nt-5 per cent COI. .-
#Jf wf I
4.3 8.1 4.4 8.6 1 i’ 4.4* 0
I 2.1
PM
7.6 8.1 0.1
* 0.1 PM of iodoacetamide added.
attacked by triose phosphate dehydrogenase. It also follows that on ad- dition of iodoacetamide, which strongly inhibits triose phosphate dehydro- genase, there should be an accumulation of an equilibrium mixture of 97 per cent dihydroxyacetone phosphate and 3 per cent phosphoglyceralde- hyde, provided isomerase is present. If, however, isomerase is absent in Preparation C, then an equimolar mixture of the two triose phosphates should be demonstrated. Analyses of the iodoacetamide-treated fermen- tation system with FDP as the initial substrate showed the presence of 96 per cent dihydroxyacetone phosphate and 4 per cent phosphoglyceral- dehyde instead of a 50 : 50 mixture, which occurs only if isomerase is absent as is the case in purified aldolase preparations (4). Finally, in the absence of a suitable oxidant, such as acetaldehydc or methylene blue, 2 equiva- lents of coenzyme I must be required to oxidize 2 equivalents of triose phosphates derived from FDP through the agency of aldolase and isomer-
ase. Fig. 2 indicates that this condition is completely fulfilled. The evi- dence therefore would appear to justify t,he conclusion that an isomerase occurs in Preparation C which presents triose phosphate dehydrogenase with 2 equivalents of triose phosphate from 1 equivalent of FDP.
The third enzyme, triose phosphate dehydrogenase, is strikingly similar to its counterpart found in yeast zlnd animal tissues. The components of the system include coenzyme I and arsenate. An oxidant is also required to regenerate reduced coenzyme I. It is reversibly inhibited by copper and irreversibly by iodoacetamide.
The identification of triose phosphate dehydrogenase resolved itself into three considera,tions, (1) the oxidation-reduction of cocnzyme I, (2) the
THEORETICAL HDP - 2 Co1 VALUE 1.35 ---------------------- --_---------- _________
+-CH,CHO
:, I I I I I 0 20 40 60 80 100 I20
TIME, MINUTES
FIG. 2. Reduction of 2 equivslent,s of coenzyme I by 1 equivalent of FDP. Ab- sorption at 340 ml measured with the Beckman spectrophotometer.
rBle of arsenate and phosphate in the oxidation, and (3) the effect of in- hibitors.
Oxidation-Reduction of Coenzyme I-As is indicated in Fig. 3, in the pres- ence of arsenate, triose phosphate (derived from FDP), and triose phosphate dehydrogenase (Preparation C), coenzyme I is reduced to dihy- drocoenzyme I, with its characteristic absorption band at, 340 rnp. Fur- ther, as indicated in Fig. 2, in the presence of alcohol dehydrogenase, which is found in rather large concentrations in Preparation C, the reoxidation of dihydrocoenzyme I can be readily coupled with the reduction of acetalde- hyde to ethyl alcohol. Table II summarizes the data characterizing pea alcohol dehydrogenase.
Pyruvic acid can serve indirectly as a suitable oxidant. Since lactic de-
hydrogenase is absent in Preparation C, reduction of pyruvic acid to lactic acid cannot be coupled with the reoxidation of dihydrocoenzyme I. How- ever, because of the presence of an active carboxylase in Preparation C, pyruvic acid serves as a source of acetaldehyde.
12 4
I .3x10-*M As ,A
*
2 .03x IO-’ M As
E IO 3 .3x IO-’ M Pi 4.3x10-‘M Pi+AMP 5 NO ADDITIONS /
- 0 IO 20 30 40
TIME, MINUTES
FIG. 3. Effect of different concentrations of arsenate (As) and phosphate (Pi) on the fermentation of FDP.
TABLE II Factors Afecting Activity of Pea Alcohol Dehydrogenase
Each manometric cup contained 1 cc. of enzyme (Preparation C), 0.2 cc. of 0.1 M ferricyanide, 0.5 cc. of 0.05 M sodium bicarbonate. As indicated, additions were 0.1 cc. of 0.1 per cent coenzyme I, 0.1 cc. of different absolute alcohols, and 0.1 cc. of 0.001 M iodoacetamide. Water was added to a total volume of 3 cc. Temperature 37’; gas phase 95 per cent Nz-5 per cent CO*.
Methylene blue can replace acetaldehyde provided pea diaphorase is present. Preparation B contains an active diaphorase which catalyzes the oxidation of reduced coenzyme I. Thus in the presence of a bicarbon- ate buffer, the following reactions account for the formation of CO2 as measured in a Warburg manometer.
Furthermore, since methylene blue is autoxidizable, oxygen can be coupled t,o the system
2Mb.H + 2H+ + 202 --) 2Mb+ + 2H20P (4)
2Hz02 plant catalase
+ 2H20 + 02 (5)
Therefore, in theory, for each mole of FDP utilized 1 mole of oxygen should b? consumed, as indicated by Equations 1, 2, 4, and 5, which together yield the over-all equation
FDP + 02 -+ 2-phosphoglyceric acid + 2Hz0 (6)
The data supporting this series of reactions are presented in Table III.
TABLE III Balance Sheet Studies in Breakdown oj Fructose Diphosphafe -+ CPhosphoglyceric Acid
Each manometric cup contained 1 cc. of enzyme, a given concentration of fructose diphosphate, 0.1 cc. of M acetaldehyde, 0.5 cc. of 0.05 M sodium bicarbonate, 0.05 CC. of 0.05 M arsenate, and 0.5 cc. of 0.1 M fluoride. Where indicated 0.1 cc. of 0.05 M methylene blue was employed. Water was added to a final volume of 3 cc.; gas phase 95 per cent Ns-5 per cent CO,; temperature 37”. In oxygen uptake experi- ments, 0.1 cc. of 0.1 per cent methylene blue was employed. and the renter well con- tained 0.2 cc. of 10 per cent sodium hydroxide. Gas phase air: temperature 37’.
Fructose diphosphate Enzyme preparation
w 2 2.5 3 4 5 2
A B “
C Green peas Yeast extract*
-i-
-
co2
CHaCHO -
TheWy ~~~t~;
~- c.?nm. cmm.
90 87 112 101 134 115 182 186
90 80
i Methylene blue
Theory “m”,“,
-- cmm. cmm.
180 180
180 150
-7
i * Prepared according to the method of Neuberg and Lustig (15).
_- c.mm. cmm.
45 41
112 103
____--
Ferricyanide can also be employed, though it appears to be somewhat toxic to the system. Being a strong oxidant, it probably oxidizes the SH groups of triose phosphate dehydrogenase to the disulfide or inactive form, cu-Ketogllltaric acid and diacetyl do not replace acetaldehyde.
R61e of Arsenate and Phosphate-Manometric studies revealed that the addition of phosphate to either Preparation A, B, or C with or without adenylic acid does not lead to the formation of acid. However, an arse- nate effect was consistently observed with the addition of suitable concen- trations of the anion (Fig. 3). Spectrophotomet,ric data revealed that co- enzyme I was reduced in the presence of either phosphate or arsenate; in the absence of these anions there was no reduction (Fig. 4). The results indicated that either arsenate or phosphate is involved in the oxidation of 3-phosphoglyceraldehyde. However, whereas 1-arseno-3-phosphoglyceral- dehyde breaks down spontaneously (12), the corresponding 1,3-phospho complex cannot be coupled with available phosphate acceptors, adenosine monophosphate (AMP) and adenosine diphosphate (ADP), but is slowly
I .3x10-* M As 2 .3x10-‘M Pi 3 NO ADDITIONS
TIME, MINUTES
FIG. 4. Effect of arsenate (As) and phosphate (Pi) on the reduction of coenzyme I by FDP + fermentation system.
hydrolyzed. With phosphate, therefore, either a highly specific phosphate acceptor is missing or a specific transphosphorylase has been inactivated. These results can be duplicated with any of the three preparations and with a preparation made from fresh green peas (adenosine triphosphatase is present in all preparations). Table IV summarizes the data.
Inhibitors-The effects of several reagents were investigated and are listed in Table V. Cyanide was excluded from the list since it combined with triose phosphates to form cyanohydrins. Pea t,riose phosphate dehy- drogenase resembles closely its counterpart in yeast and animal tissues in that traces of copper inactivate the system reversibly. This inhibition is reversed by the addition of cysteine, thioglycolate, or glutathione. Iodo- acetamide inactivates the system irreversibly, since sulfhydryl reagents do not reverse the inhibition. It is therefore obvious from a comparative point of view that the integrity of the SH groups of the protein portion of
When phosphoglyceric acid is added to Preparation B, little if any CO* formation is observed manometrically. However, on addition of magne- sium (or manganese), cocarboxylase, and adenylic acid, activity is re-
stored. The addition of fluoride causes virtually complete inhibition of COZ formation. These results are summarized in Fig. 5. The data can be interpreted to mean that (1) an enolase is present which is sensitive to fluoride and requires magnesium for activation, (2) a phosphotransferase is present in Preparation B which transfers phosphate from phosphopyru- vie acid to adenylic acid, and (3) a pyruvic acid carboxylase is activated by cocarboxylase to decarboxylate pyruvic acid to acetaldehyde and CO2 which is measured manometrically. No additional observations were car- ried out on the properties of pyruvic carboxylase since the enzyme has been clearly defined (14).
I’ COMPLETE SYSTEM 2 NOCOCARBOXYLASE
6 4 NO ADPorAMP
n 5 COMPLETE SYSTEM PLUS F- w I
g4
N
8
J x2
0 IO 30 50 70 90 TIME. MINUTES
FIG. 5. Breakdown of phosphoglyceric acid as affected by components of the sys- tem. The conditions of the experiment are described in the legend of Table VI.
No further work was carried out with enolase. As is indicated in Table VI, it was found that, while Preparations A and B possess phosphotrans- ferase, Preparation C is devoid of the enzyme. It appeared that during prolonged dialysis (the procedure used to obtain Preparation C) the phos- photransferase became inactivated. Activity could not be restored by adding divalent cations or yeast juice. Therefore, by employing Prepara- tion C. it was possible to demonstrate (1) the accumulation of phosphopy- ruvic acid, (2) the inability of the preparation to synthesize adenosine diphosphate from adenylic acid, and (3) the absence of pyruvic acid. How- ever, in Preparation B, pyruvic acid accumulated in the absence of added cocarboxylase, and adenosine diphosphate was formed from adenylic acid. These conclusions were confirmed when phosphopyruvic acid was added to Preparation B in the presence of adenylic acid. Adenosine diphosphate was synthesized and CO2 was formed (decarboxylation of pyruvic acid):
TABLE VI Balance Sheet for Conversion of S-Phosphoglyceric Acid to Pyruvic Acid
Each test-tube contained 1 cc. of enzyme, a given concentration of phospho- glyceric acid, 0.1 cc. of 0.1 per cent magnesium sulfate, and water to a totalvolume of 3 cc. In the cases indicated, the additions were 10 PM of AMP or 0.3 cc. of 0.1 M fluoride. In experiments of CO* formation, the manometric cups contained re- agents as above and in addit,ion 0.1 cc. of 0.1 per cent cocarboxylase and 0.5 cc.of 0.1 M citrate at pH 6.0. Temperature 37”; gas phase air.
FIG. 6. Scheme for the breakdown of fructose diphosphate by pea seed extract. (I) aldolase; (II) isomerase; (III) triose phosphate dehydrogenase plus arsenate; (IV) phosphoglyceromutase plus enolase and Mg++; (V) phosphopyruvic transphos- phorylase plus AMP; (VI) pyruvic carboxylase plus Mg++ and cocarboxylase; (VII) alcohol dehydrogenase.
in the absence of adenylic acid, no COz formation could be detected. If Preparation C was employed, no changes could be observed either in the presence or absence of adenylic acid.
Fig. G summarizes the observations presented in this communication. A careful analysis of each enzyme system involved in the pea fermenta-
tion system may possibly bring to light dissimilarities, but the over-all series of reactions from FDP to acetaldehyde appears to be analogous in plant and yeast cells.
SUMMARY
In the enzymic breakdown of FDP in plant tissue, the enzymes aldolase, isomerase, triose phosphate dehydrogenase, enolase, phosphotransferase, and carboxylase have been demonstrated to be involved. The results in- dicate t,hat in pea seeds the fermentation of FDP is apparently similar to that in yeast and animal tissues.
BIBLIOGRAPHY
1. James, W. O., Heard, C. R. C., and James, G. M., New Phytologist, 43, 62 (1944). 2. Hanes, C. S., Proc. Roy. Sot. London, Series B, 129, 174 (1940). 3. Hassid, W. %., Plant Physiol., 13, 641 (1938). 4. Stumpf, P. K., J. Biol. Chem., 176,233 (1948). 5. Tewfik, S., and Stumpf, P. K., Am. J. Bot., in press. 6. Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric techniques and
related methods for the study of tissue metabolism, Minneapolis, 169 (1945). 7. Freidemann, T. E., and Haugen, G. E., J. BioZ. Chem., 147,415 (1943). 8. Fiske, C. I-I., and Subbarow, Y., J. BioZ. Chem., 66,375 (1925). 9. King, E. J., Biochem. J., 26, 292 (1932).
10. Neuberg, C., Lustig, H., and Rothenberg, M. A., Arch. Biochem., 3,33 (1943-44). 11. LePage, G. A., J. BioZ. Chem., 168,623 (1947). 12. Warburg, O., and Christian, W., Biochem. Z., 303, 40 (1939). 13. Cori, G. ‘I?., Slein, M. W., and Cori, C. F., J. BioZ. Chem., 173, 605 (1948). 14. Venneshuld, B., and Felsher, It. F., Arch. Biochem., 11, 279 (1946). 15. Neuberg, C., and Lustig, H., Arch. Biochem., 1, 191 (194243).
