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Respiratory oxidation of pyruvate by plant mitochondria
Respiratory Oxidation of Pyruvate by Plant Mitochondria’ Adele Millerd2 From
the Kerckhoff
Laboratories
of Biology, California Pasadena, California
Institute
of Technology,
Received June 2, 1952
That pyruvate is an intermediate in the respiratory oxidation of hexose by plant tissue has now been firmly established. The most direct demonstration of the participation of pyruvate in plant respiration is that of James and James (1) who isolated the compound as the 2,4-dinitrophenylhydraxone from barley leaves in which the further metabolism of the material had been inhibited by trapping agents. Other lessdirect experiments have also indicated that pyruvate is produced by the plant. The respiration of spinach leaves was found by Bonner and Wildman (2) to be inhibited by fluoride. Fluoride inhibition of respiration is frequently to be attributed to inhibition of enolase which is responsible for the conversion of 2-phosphoglyceric acid to phosphoenolpyruvic acid. That fluoride inhibition of spinach leaf respiration is actually due to inhibition at this level was indicated by the fact that fluoride-treated leaves were able to respire normally if supplied with pyruvate although they were unable to utilize sucrose or other sugars under these conditions. It was also found that the respiration of spinach leaves is inhibited by malonate and that this inhibition is partially reversed by added pyruvate. It is clear then that pyruvate may be metabolized by plant tisuses in vim. Pyruvate is not only produced by plant tissues but available evidence indicates that it is also oxidatively metabolized by the Krebs cycle. From an examination of the effect of malonate on the accumulation of succinate Bonner (3) concluded that succinic dehydrogenase must func1 Report of work supported by the Herman Frasch Foundation for Agricultural Chemistry. 2 Predoctoral Fellow, Frasch Foundation; recipient of Fulbright travel grant, administered by the Institute of International Education. 149
150
ADELE
MILLERD
tion in the metabolism of pyruvate by sections of the Avena coleoptile. Avena coleopt.iles, partially depleted of endogenous substrates, showed an increased rate of respiration on the addition of pyruvate. This response was inhibited by malonate, and such inhibition could be partially offset by the addition of succinate, indicating the competitive nature of the malonate inhibition. In addition, depleted Avena coleoptiles respiring at the expense of endogenous substrate accumulated succinate in the presence of malonate. The addition of cu-ketoglutarate or fumarate under these conditions causeda marked increase in the extent of succinate accumulation. Similar experiments have been done with other plant tissues. Thus, Laties (4) has demonstrated that accumulation of succinate in the presence of malonate occurred both in spinach leaves and in barley roots. This accumulation was enhanced by feeding pyruvate or fumarate, or especially by feeding both. The interaction of these two compounds indicates that they or their derivatives are together essential to the oxidative formation of succinate. The findings of Bonner and of Laties are interpretable in terms of a Krebs cycle and in fact are quite similar to the observations made by Krebs and Johnson (5) and on which the suggestion of the Krebs cycle was originally based. The participation of the succinoxidase system in the metabolism of higher plants has been established for a number of species,and the earlier work of Laties (6) with cauliflower, of Millerd (7) with potato, of Stafford with pea (8), and of Bhagvat and Hill (9) with various species has established that the succinoxidase activity of plant tissues is associated with the particulate matter of the plant cell. It has also been shown by Millerd et al. (10) that these particles cont.ain additional enzymes and that it is in fact possible to isolate from seedlings of mung bean, particles which can carry out the complete oxidation of pyruvate to carbon dioxide and water by way of an apparent Krebs cycle. It has also been shown that these particles must be considered to be identical with the mitochondria since they stain vitally with Janus Green B. The method adopted for the isolation of particles active in the oxidation of pyruvate is the result of a series of experiments on the effects of varied preparational procedures on subsequent particle activity. Evaluation of the varied factors which affect the oxidative activity of the isolated particles forms the subject matter of the present paper.
OXIDATION MATERIALS
OF PYRUVATE
151
AND METHODS
Etiolated seedlings of Phaseolus aureus were grom7-n at 26°C. and 90% humidity under a low intensity red light (less than 1 ft.-candle). The seeds were first sterilized by immersion for 10 min. in 0.5% NaOCl, soaked in distilled water for 60 min., and then planted in vermiculite (heat-expanded mica) which had been previously saturated with water. The seedlings were harvested 90 hr. later at which stage they were approximately 8 cm. in length. For the isolation of active cytoplasmic particles from mung bean, 30 g. tissue representing the complete aerial portion of the plant were first ground in a mortar with 10 g. sand and 40 ml. of medium of the desired composition. The brei was strained through muslin, elarified by low-speed centrifugation (500 X g for 5 min.), and the residual suspension recentrifuged at 10,000 X g for 15 min. The supernatant was then removed by suction and the residue resuspended in 3.0 ml. of the grinding medium. One-half milliter of the preparation was used as the enzyme in the reaction mixture which in all cases had a total volume of 1.5 ml. All steps in the preparation wefe carried out at 2°C. or lower and all apparatus (mortars, etc.) was chilled &fore use. Oxygen consumption was estimated by standard Warburg technique at. 30°C. and measurements were made over a I-hr. period. Total nitrogen was estimated by micro-Kjeldahl digestion, followed by nesslerization and calorimetric determination against known standards. RESULTS
E$‘ect of Composition of Grinding Medium The activity and characteristics of the mitochondria isolated from mung bean depend greatly upon the medium in which the tissue is initially ground, a factor which apparently determines the integrity of the particles. Such integrity may be assessed by the degree to which their oxidative capacity is independent of added cofactors, in particular cytochrome c. Thus, as is shown in Table I, particles prepared from seedlings ground in 0.1 M phosphate buffer, pH 7.1, are capable of oxidizing cr-ketoglutarate without the addition of cytochrome c. Particles prepared from the same batch of seedlings but with 0.3 M sucrose as the grinding medium are unable to oxidize cu-ketoglutarate but possess the ability to oxidize succinate. This latter ability is markedly increased by the addition of exogenous cytochrome c. By the use of grinding medium containing both sucrose and phosphate, particles may be obtained which show greater activity toward cu-ketoglutarate than do particles obtained by grinding in phosphate alone. The data of Table I illustrate these relationships, and they show that the addition of sucrose to the phosphate-containing grinding medium results
152
ADELE
MILLERD
in particles of greater activity than particles isolated by grinding in either medium alone. The composition of the medium in which the plants are ground exerts a marked influence not only on the ability of the enzyme to oxidize substrate but also on the autonomy of the particles. Thus particles prepared by grinding tissue in 0.1 M phosphate buffer, pH 7.1, containing 0.3 M sucrose not only oxidize cu-ketoglutarate and succinate at a rapid rate, but this rate is very little affected by the addition of cytochrome c (Table I). These facts may be taken to indicate that the use of phosphate TABLE Oxidative
I
Activity of Cytoplasmic Particles of Mung Bean as a Function of Composition of Medium Used to Grind Tissue
I Substrate= None.................
a-Ketoglutarate. a-Ketoglutarate + cytochrome c.. . Succinate.. . Succinate + cytochrome 0.. . . . . . . .
01 consumption, cu. m.lhr.fmg. Ground in 0.1 Y phosphate
Ground in 0.3 Y sucrose
N Ground in 0.1 dl phosphate + 0.3 4f sucrose
3 1
3 220
-
0 15
153
-
64
164
10 69
0 Substrate concentration, 3 X 10-* M. Reaction mixture also contains phosphate buffer, pH 7.1, 10-e M; sucrose, 0.1 M; ATP, 3 X lo-4 M; and MgSOd, 10-* M; cytochrome c where added, 10m6M.
and sucrose together in the grinding medium contributes to the maintenance of the integrity of the particles during their isolation. Cytoplasmic particles prepared in the phosphate-sucrose medium are not only able to oxidize cr-ketoglutarate and succinate but are also capable of oxidizing pyruvate, provided only that a catalytic amount of a Krebs cycle acid such as malate is present. The data of Table II show that neither pyruvate alone nor malate alone in the low concentration used is oxidized at any considerable rate. Only when the two are present together does appreciable oxidation take place. The particles prepared by grinding in and centrifugation from phosphate-sucrose medium may be further purified by a second high-speed centrifugation. The data of Table II indicate that washing the particles
OXIDATION
153
OF PYRUVATE
(obtained by high-speed centrifugation) by resuspension in grinding medium and recentrifugation does indeed result in a lowered endogenous oxygen consumption and higher activity toward pyruvate. Optimal Composition of Grinding Medium It has already been shown (Table I) that the addition of sucrose to the grinding medium has a marked effect on the subsequent activity of isolated particles. In a series of experiments, the concentration and proportions of phosphate buffer and sucrose were varied for determination of TABLE Oxidation
of Pyruvic
II
Acid by Cytoplasmic
Reaction mixtw?
Particles of Mung Bean
Steps used in preparation
02 consumption cu. mm.fhr./mg.
Enzyme Enzyme + pyruvateb Enzyme + L-malateb Enzyme + pyruvate + L-malateb Enzyme Enzyme + pyruvateb
Ground, clarified, used
centrifuged,
I\;
19 24 27 115
Ground, clarified, centrifuged, resuspended,” recentrifuged, used
0
168 ____-
0 Also contains phosphate buffer pH, 7.1,O.l M; and sucrose, 0.1 M. b Pyruvate concentration 3 X low2 M. Also contains ATP 3 X 10e4M, MgSO, 10-a M, and L-malate 1.7 X 1OV M. In all cases where malate is used, nL-malate was added and the concentration given is that of the L-isomer only. c Resuspended in 20 ml. of original grinding medium.
the optimal composition of the grinding medium. It was found that grinding in a sucrose concentration of 0.4 M and a phosphate concentration of 0.1 M yielded particles of higher activity toward pyruvate than any other combination tested. The function of the sucrose in the grinding medium is no doubt osmotic since the osmotic value of the suspending medium is important in the maintenance of particle integrity (11). It has also been shown by Laties (6) that other osmotic agents such as mannitol apparently serve this function as well as sucrose and that the optimal phosphate concentration is relatively high because of several factors. Thus the phosphat’e serves
154
ADELE
MILLERD
in part to satisfy the osmolar requirement. In addition the activity of the particles possessesa requirement for the phosphate ion itself (10). It is apparent that the requirement of the mung bean particles for phosphate ions is twofold. Thus a relatively high phosphate concentration in the grinding medium is necessary for the isolation of cytoplasmic particles possessingmaximal oxidat.ive activity. In addition (lo), the presence of phosphate ion is also necessary during the course of the reaction itself if the maximal rate of substrate oxidation is to be attained. Let us consider the first aspect and examine more closely the nature of the requirement for phosphate in the grinding medium. The experiments described below indicate that the requirement for phosphate in the grinding medium is associated with the inhibition of a phosphorolytic breakdown of someessential substance or substances. In this function the phosphate ion may be replaced by other phosphatase inhibitors such as sodium fluoride. Fluoride cannot, however, completely replace phosphate, and some phosphate must be contained in the grinding medium. The data of Table III show that fluoride cannot completely replace phosphate in the grinding medium, since particles prepared from tissue ground in 0.1 M sodium fluoride containing 0.4 M sucrose are unable to oxidize cu-ketoglutarate or pyruvate, and under the same conditions particles prepared in phosphate-sucrose medium show marked activity toward these substrates. Although fluoride alone is thus unable to replace completely phosphate in the grinding medium, it does seem that fluoride may be substituted for the high concentration of phosphate otherwise necessary. This is also shown by the experiment of Table III in which one preparation was ground in 0.011cI phosphate buffer containing 0.1 M sodium fluoride and 0.4 M sucrose.The particles prepared in the high-fluoride, low-phosphate medium are as active toward a-ketoglutarate as the standard particles but are less active toward pyruvate. The preparation of particles highly active in the oxidation of pyruvate requires that phosphate, in low concentrations at least, be present continuously throughout the successive steps of the procedure. Thus phosphate cannot be omitted during the grinding step even though it is present during the washing and subsequent steps. The results summarized in the experiment of Table III are not to be attributed only to the use of unbuffered grinding media since preparations made by grinding tissue in 0.1 M trishydroxymethylaminomethane (THAM) buffer containing 0.4 M sucrose yielded particles which were
OXIDATION
OF
155
PYRWATE
incapable of oxidizing pyruvate. Tissue was ground in 0.1 M THAM containing 0.4 M sucrose, and the residue, after high-speed centrifugation, was resuspended in this medium. Such a preparation, with phosphate present in, or absent from, the reaction medium, shows no capacity to oxidize pyruvate. The experiments described above have defined certain conditions as optimal or near optimal for isolation of active cytoplasmic particles from TABLE
III
For the Isolation
of Active Cytoplasmic Particles from Mung Bean, Fluoride Partially but Not Completely Replace the Requirement for a High Concentration of Phosphate in the Grinding Mixture Reaction mixture”
Composition of grinding medium
Can
02 consumption w. nim.lhr.lmg.
Phosphate, 0.1 Mb and sucrose, 0.4 M
Enzyme
Enzyme + wketoglutarate Enzyme + pyruvateb Enzyme
157 92 Phosphate, fluoride, 0.4 M
0.01 ;Irl; sodium 0.1 M; sucrose,
Enzyme + cu-ketoglutarate Enzyme + pyruvateb Enzyme
N
0
0
151 36 Sodium fluoride, sucrose, 0.4 M
0.1 111; and
Enzyme + a-ketoglutarate Enzyme + pyruvateb
10 15 5
B Also contains phosphate buffer, pH 7.1, 0.05 Jf; sucrose, 0.3 M; ATP, 5 1OWM; and MgSOd, 10-S M. Substrate concentration, 2 X 1OWM. b Also contains r,-malate, 1.7 X 10-S M.
X
mung bean. On the basis of these experiments, the method previously described (10) was selected as the standard procedure and has been employed in all further experiments. E#ect of Concentration of Sucrose in the Reaction Mixture Since the concentration of sucrose employed in the grinding medium has such a marked effect on the subsequent activity of the particles toward pyruvate, it is of interest to determine whether the concentration of sucrose present in the reaction mixture also influences the rate
156
ADELE
MILLERD
of pyruvate oxidation. The data of Fig. 1 show that the concentration of sucrose during the course of the reaction does indeed affect the rate of pyruvate oxidation. The optimal concentration appears to be 0.3 M, a concentration used in all further experiments. Oxidative Capacity of Isolated Cytoplasmic Particles. Oxidation of the Krebs Cycle
of Acids
It has already been reported (10) that isolated cytoplasmic particles are able to oxidize all of the principal acids of the Krebs cycle. The relation of substrate concentration to rate of oxygen consumption is ex-
01
1 0.1
I
, 0.5
0.3 Concentration
of Sucrose : M
1. Effect of concentration of sucrose in the reaction mixture upon the rate of oxidation of pyruvate by cytoplasmic particles of mung bean. The reaction mixture also contains enzyme; phosphate buffer, pH 7.1, 3 X 10-p M; ATP, 3 X 10-d M; MgSO+ lO+ .V; L-malate, 1.7 X lO+ M; and pyruvate, 10-a M. FIG.
pressed in Table IV in terms of the K, for each of the Krebs cycle acids. In these experiments, the rates of oxidation of the varied substrates are determined under conditions previously described (10) for maximal pyruvate oxidation. In each case, substrate saturation is reached or essentially reached in the region of 2 X lOA M, and this concentration is used in all later experiments. E$ect of Added Cofactors on the Oxidatioe Capacity of Isolated Cytoplasmic Particles As has already been reported, the only supplement to the reaction mixture required for the oxidation of pyruvate by mung bean particles
OXIDATION
OF
157
PYRUVATE
is a catalytic amount of one of the acids of the Krebs cycle. However, the addition of adenosine triphosphate (ATP), adenosine di- or monophosphate, or of magnesium ions to the reaction mixture markedly inincreases the rate. The effect on the rate of oxidation of other acids of the cycle by the addition of these substances is expressed in Table V. The data show that in each case the addition of magnesium ions causes con-
Substrate-Afinity
TABLE IV Constants (K,) for the Oxidation of Krebs Cycle Acids by Cytoplasmic Particles of Mung Bean
Substrate
K&O-0
Citrate a-Ketoglutarate Succinate Fumarate L-Malate Pyruvate
3.4 4.1 7.9 7.9 3.6 5.4 TABLE
V
Effect of Catalytic Amounts of ATP and Mg Ions on the Rate of Oxidation of Krebs Cycle Acids by Cytoplasmic Particles of Mung Bean (01 consumption in cu. (mm./hr./mg. Substrate (2 X lo-’ M)”
Citrate ................. a-Ketoglutarate ......... Succinate. ............. Fumarate. ............. Malate ....... ........ a Reaction mixture and eucrose, 0.3 M.
Plus ATP 6 x lo-w)
NO addition
67 72 122 2 33
142 113 218 25 74
110 57 79 1150 124
87 89 137 2 28
Per cent increase
30 24 12 0 -15
-
N)) p1us Mg Per cent bns ATPad increase
172 181 262 31 71
157 152 115 1450 115
also contains enzyme; phosphate buffer, pH 7.1, 0.05 M;
siderable increase in the rate of oxygen consumption. The addition of ATP causes small increases in the rate of oxidation of citrate, cY-ketoglutarate, and succinate but not of malate or fumarate. In those cases in which both magnesium ions and ATP individually increase the rate of oxidation, the increase observed in the presence of both together is more than additive.
158
ADELE
MILLERD
E$ect of Concentration of Factors Injluencing
Pyruvate Oxiohtion
In the experiments reported earlier on the oxidation of pyruvate, the concentrations of malate, ATP, and magnesium ions used were those known to be optimal. The relation between the concentration of each of these substances and the rate of oxidation is expressed in Figs. 2 to 5. It is of particular interest that while the concentration of pyruvate needed for maximal rate is approximately the same as for the other Krebs cycle acids, the concentration of L-malate needed to give maximal 120
100
20
0 Pyruvole
10-O Concentrolion
:M
2. Effect of concentration of pyruvate on rate of oxidation by cytoplasmic particles of mung bean. The reaction mixture also contains enzyme; phosphate buffer, pH 7.1, 0.05 M; sucrose, 0.3 M; ATP, 5 X IO-’ M; and MgSOd, lO+ M. Malate concentration, 1.7 X lWa M. FIQ.
rate of pyruvate oxidation is much less, 1.7 X 1e3 M (2.5 pmoles in the reaction mixture). The concentration of magnesium ions needed for maximal rate of pyruvate oxidation is, as is illustrated in Fig. 4, in the range of 2 x 10-s to 1W3 M. A concentration of Ws M was employed in all experiments recorded. Figure 5 illustrates the effect of varied concentration of ATP on the rate of oxidation of pyruvate. The increase in rate brought about by ATP although real is not striking. A concentration of 5 X 10-a M which is
OXIDATION
159
OF PYRUVATE
100
2
00
i? L
; 2 ;
60
“, 0
40
i E 20
0 L-Malale
Cancentratlon
:M
FIG. 3. Effect of concentration of L-malate on the rate of oxidation of pyruvate by cytoplasmic particles from mung bean. The reaction mixture also contains enzyme; phosphate buffer, pH 7.1, 0.05 M; sucrose, 0.3 M; MgSOa, 1W3 M; ATP, 5 X KY4 .lil; and pyruvate, 2 X 10-* M.
lo-’
10-S
Cor~centroiion
of Mg ions
M
10-Z
FIG. 4. Effect of concentration of magnesium ions on the rate of oxidation of pyruvate by cytoplasmic particles. The reaction mixture also contains enzyme; phosphate buffer, pH 7.1, 0.05 I4; sucrose, 0.3 M; ATP, 5 X W4 111;L-malate, 1.7 X lC8 M; and pyruvate 2 X 10-* M.
160
ADELE
within the range needed for experiments.
MILLERD
maximal
activity
Speczj% Activity of Mituchondrial
was used in
all
Preparations
It is of interest to consider the maximal oxidative capacity obtainable with isolated mung bean mitochondria. The highest rates of succinate oxidation as yet attained have been with mitochondria made not by the standard procedure outlined above but prepared from the hypocotyl tissue alone rather than the whole shoot. Particularly high rates of succinate oxidation have been obtained when such hypocotyl tissue is
100z so z % 605 e 2 400” “E E 20-
”
lo-*
ATP Concentration:
M
10-3
FIG. 5. Effect of concentration
of ATP on rate of oxidation of pyruvate by cytoplasmic particles. The reaction mixture also contains enzyme; phosphate buffer, pH 7.1,0.05 M; sucrose, 0.2 M; MgSO,, 10-a M; malate, 1.7 X lO+ M; and pyruvate, 2 X lo-* M.
ground in 0.5 M sucrose rather than in the standard sucrose-phosphate buffer mixture. The activities of mung bean mitochondria prepared in these different ways are compared in Table VI. It seems probable that the relatively low Qo,(N) of preparations made in the presence of phosphate ion is in some part due to effects on the aggregation and subsequent centrifugal separation of extraneous and inert cytoplasmic proteins. Even though particles prepared in the absence of phosphate ions are highly active in the oxidation of succinate, they have nonetheless been damaged in some way since their ability to oxidize pyruvate has not been correspondingly increased and, in addition, they have in some measure lost their ability to carry out oxidative phosphorylations (12).
OXIDATION
OF
161
PYRWATE
The data of Table VI include a comparison of the specific activity of mung bean mitochondria with that of rat liver at the same stage of purification (11). It is apparent that the specific activities, on a C&,(N) basis, are essentially similar if the comparison is made at a common temperature and that the activity of the plant mitochondria measured at 30” compares favorably with that of the liver particles measured at 38’ if allowance is made for the difference in temperatures. TABLE VI Comparison of Oxidative Activity of Cytoplasmic various
Particles
Isolated
by
PTOCeduTeS
(Succinate as substrate) Material
Grinding medium
02 consumption cu. mm.lhr. Imp. N
Mung beana complete aerial portion hypocotyl hypocotyl Ratb liver 0 Activity b Activity
0.4 M sucrose, 0.1 M phosphate 0.4 M sucrose, 0.1 M phosphate 0.5 M sucrose 0.88 M sucrose
266 270 773 2010
measured at 30°C. measured at 38°C. DISCXJSSION
It has been shown above that from seedlings of the mung bean, particles may be isolated which, in the absence of added cofactors, can oxidize the acids of the Krebs cycle. Among the preparational procedures which must be observed in the isolation of these particles, perhaps the most interesting is a requirement for the presence of phosphate in the medium in which the seedlings are ground. The requirement for the presence of phosphate in the grinding medium is apparently associated with the inhibition of some phosphorolytic breakdown since phosphate can in part be replaced by a phosphatase inhibitor, sodium fluoride. Fluoride cannot, however, completely substitute for phosphate, and there appears to be a further specific need for the presence of the phosphate ion in the grinding medium. The principal further requirement for the grinding medium is an osmotic one. The presence of compounds such as sucrose in the grinding medium, by controlling the osmotic environment, aid the maintenance of the integrity of the particles during their isolation. How far do the results reported in this paper together with those
162
ADELE MILLERD
previously described go toward establishing the Krebs cycle as participating in the respiration of higher plants? The isolated cytoplasmic particles of mung bean are able to oxidize the acids of the Krebs cycle. The oxidation of pyruvate by these particles is dependent upon the concomitant oxidation of a catalytic amount to one of the di- or tricarboxylic acids. In this catalytic function the various acids are approximately equivalent. Finally, oxidation of the acids of the cycle is accompanied by a coupled phosphorylation (12). These facts taken together support the view that a system similar to the Krebs cycle is responsible for the respiratory oxidation of pyruvate in higher plants. In what way and to what extent do the mitochondrial oxidations fit into the metabolism of the plant? The evidence which is available, direct as well as indirect, suggests that the bulk if not all of the respiration of the mung bean seedling is mediated by the mitochondria. Let us consider the quant,itative aspects of this matter and inquire how the rate of respiratory oxygen uptake of the living plant compares with the rate of oxygen uptake which can be accomplished by the isolated mitochondria. In a particular case, segments of mung bean hypocotyls were found to consume 150 cu. mm. OJhr./g. fresh weight. Mitochondria isolated from the same material consumed oxygen, in the presence of succinate, at the rate of 45 cu. mm/hr./g. of original fresh weight. Microscopic observation shows that only roughly one-half of the cells of the tissue are disrupted in the grinding process. In addition, many of the mitochondria, perhaps half or more, are lost during the successive centrifugatione as judged by visual observation. The mitochondria alone can therefore oxidize succinate, transfer electrons, and consume oxygen at a rate roughly sufficient to account in this instance for the respiration of the living tissue. SUMMARY
Cytoplasmic particles isolated from mung bean seedlings can oxidize all of the acids of the Krebs cycle. The oxidation of pyruvate is dependent upon the concomitant oxidation of a catalytic amount of one of the di- or tricarboxylic acids, and in this function the various acids are approximately equivalent. Preparational procedures for the isolation of such particles have been defined. Among the most important requirements for full activity of the isolated particles is the presence of phosphate in the grinding medium. It has been shown that the isolated particles oxidize at a rate sufficient to account for all of the respiration of the inta ct tissue.
OXIDATION
OF PYRWATE
163
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
JAMES, G. >I., AND JAMES, W. O., New Phytologist 39, 266 (1940). BONNER, J., AND WILDMAN, S. G., Arch. Biochem. 10, 497 (1946). BONNER, J., Arch. Biochenl. 17, 311 (1948). LATIES, G. G., Arch. Biochem. 22, 8 (1949). KREBS, H. A., AND JOHNSON, W. A., Enzymologiu 4, 148 (1937). LATIES, G. G., Abstracts of Amer. Sot. Plant Physiol., p. 2, June, 1951. MILLERD, A., Proc. Linnean Sot. N. S. Wales 76, 123 (1951). STAFFORD, H. A., Physiol. Pkzntarum 4, 696. (1951). BHAGVAT, Ii., ASD HILL, R., New Phytologist, 60, 112 (1951). MILLERD, A., BONNER, J., AXELROD, B., AND BANDURSKI, R. S., Proc. Natl. rlcud. Sci. I!. S. 37, 855 (1951). 11. HOGEBOOM,G. H., SCHNEIDER, W. C., AND PALLADE, G.&J. Viol. Chem. 173, 619 (1948). 12. BONNER, J., AND MILLERD, A., Arch. Biochem. and Biophys. 43, 135 (1953).
the Kerckhoff
Laboratories
of Biology, California Pasadena, California
Institute
of Technology,
Received June 2, 1952
That pyruvate is an intermediate in the respiratory oxidation of hexose by plant tissue has now been firmly established. The most direct demonstration of the participation of pyruvate in plant respiration is that of James and James (1) who isolated the compound as the 2,4-dinitrophenylhydraxone from barley leaves in which the further metabolism of the material had been inhibited by trapping agents. Other lessdirect experiments have also indicated that pyruvate is produced by the plant. The respiration of spinach leaves was found by Bonner and Wildman (2) to be inhibited by fluoride. Fluoride inhibition of respiration is frequently to be attributed to inhibition of enolase which is responsible for the conversion of 2-phosphoglyceric acid to phosphoenolpyruvic acid. That fluoride inhibition of spinach leaf respiration is actually due to inhibition at this level was indicated by the fact that fluoride-treated leaves were able to respire normally if supplied with pyruvate although they were unable to utilize sucrose or other sugars under these conditions. It was also found that the respiration of spinach leaves is inhibited by malonate and that this inhibition is partially reversed by added pyruvate. It is clear then that pyruvate may be metabolized by plant tisuses in vim. Pyruvate is not only produced by plant tissues but available evidence indicates that it is also oxidatively metabolized by the Krebs cycle. From an examination of the effect of malonate on the accumulation of succinate Bonner (3) concluded that succinic dehydrogenase must func1 Report of work supported by the Herman Frasch Foundation for Agricultural Chemistry. 2 Predoctoral Fellow, Frasch Foundation; recipient of Fulbright travel grant, administered by the Institute of International Education. 149
150
ADELE
MILLERD
tion in the metabolism of pyruvate by sections of the Avena coleoptile. Avena coleopt.iles, partially depleted of endogenous substrates, showed an increased rate of respiration on the addition of pyruvate. This response was inhibited by malonate, and such inhibition could be partially offset by the addition of succinate, indicating the competitive nature of the malonate inhibition. In addition, depleted Avena coleoptiles respiring at the expense of endogenous substrate accumulated succinate in the presence of malonate. The addition of cu-ketoglutarate or fumarate under these conditions causeda marked increase in the extent of succinate accumulation. Similar experiments have been done with other plant tissues. Thus, Laties (4) has demonstrated that accumulation of succinate in the presence of malonate occurred both in spinach leaves and in barley roots. This accumulation was enhanced by feeding pyruvate or fumarate, or especially by feeding both. The interaction of these two compounds indicates that they or their derivatives are together essential to the oxidative formation of succinate. The findings of Bonner and of Laties are interpretable in terms of a Krebs cycle and in fact are quite similar to the observations made by Krebs and Johnson (5) and on which the suggestion of the Krebs cycle was originally based. The participation of the succinoxidase system in the metabolism of higher plants has been established for a number of species,and the earlier work of Laties (6) with cauliflower, of Millerd (7) with potato, of Stafford with pea (8), and of Bhagvat and Hill (9) with various species has established that the succinoxidase activity of plant tissues is associated with the particulate matter of the plant cell. It has also been shown by Millerd et al. (10) that these particles cont.ain additional enzymes and that it is in fact possible to isolate from seedlings of mung bean, particles which can carry out the complete oxidation of pyruvate to carbon dioxide and water by way of an apparent Krebs cycle. It has also been shown that these particles must be considered to be identical with the mitochondria since they stain vitally with Janus Green B. The method adopted for the isolation of particles active in the oxidation of pyruvate is the result of a series of experiments on the effects of varied preparational procedures on subsequent particle activity. Evaluation of the varied factors which affect the oxidative activity of the isolated particles forms the subject matter of the present paper.
OXIDATION MATERIALS
OF PYRUVATE
151
AND METHODS
Etiolated seedlings of Phaseolus aureus were grom7-n at 26°C. and 90% humidity under a low intensity red light (less than 1 ft.-candle). The seeds were first sterilized by immersion for 10 min. in 0.5% NaOCl, soaked in distilled water for 60 min., and then planted in vermiculite (heat-expanded mica) which had been previously saturated with water. The seedlings were harvested 90 hr. later at which stage they were approximately 8 cm. in length. For the isolation of active cytoplasmic particles from mung bean, 30 g. tissue representing the complete aerial portion of the plant were first ground in a mortar with 10 g. sand and 40 ml. of medium of the desired composition. The brei was strained through muslin, elarified by low-speed centrifugation (500 X g for 5 min.), and the residual suspension recentrifuged at 10,000 X g for 15 min. The supernatant was then removed by suction and the residue resuspended in 3.0 ml. of the grinding medium. One-half milliter of the preparation was used as the enzyme in the reaction mixture which in all cases had a total volume of 1.5 ml. All steps in the preparation wefe carried out at 2°C. or lower and all apparatus (mortars, etc.) was chilled &fore use. Oxygen consumption was estimated by standard Warburg technique at. 30°C. and measurements were made over a I-hr. period. Total nitrogen was estimated by micro-Kjeldahl digestion, followed by nesslerization and calorimetric determination against known standards. RESULTS
E$‘ect of Composition of Grinding Medium The activity and characteristics of the mitochondria isolated from mung bean depend greatly upon the medium in which the tissue is initially ground, a factor which apparently determines the integrity of the particles. Such integrity may be assessed by the degree to which their oxidative capacity is independent of added cofactors, in particular cytochrome c. Thus, as is shown in Table I, particles prepared from seedlings ground in 0.1 M phosphate buffer, pH 7.1, are capable of oxidizing cr-ketoglutarate without the addition of cytochrome c. Particles prepared from the same batch of seedlings but with 0.3 M sucrose as the grinding medium are unable to oxidize cu-ketoglutarate but possess the ability to oxidize succinate. This latter ability is markedly increased by the addition of exogenous cytochrome c. By the use of grinding medium containing both sucrose and phosphate, particles may be obtained which show greater activity toward cu-ketoglutarate than do particles obtained by grinding in phosphate alone. The data of Table I illustrate these relationships, and they show that the addition of sucrose to the phosphate-containing grinding medium results
152
ADELE
MILLERD
in particles of greater activity than particles isolated by grinding in either medium alone. The composition of the medium in which the plants are ground exerts a marked influence not only on the ability of the enzyme to oxidize substrate but also on the autonomy of the particles. Thus particles prepared by grinding tissue in 0.1 M phosphate buffer, pH 7.1, containing 0.3 M sucrose not only oxidize cu-ketoglutarate and succinate at a rapid rate, but this rate is very little affected by the addition of cytochrome c (Table I). These facts may be taken to indicate that the use of phosphate TABLE Oxidative
I
Activity of Cytoplasmic Particles of Mung Bean as a Function of Composition of Medium Used to Grind Tissue
I Substrate= None.................
a-Ketoglutarate. a-Ketoglutarate + cytochrome c.. . Succinate.. . Succinate + cytochrome 0.. . . . . . . .
01 consumption, cu. m.lhr.fmg. Ground in 0.1 Y phosphate
Ground in 0.3 Y sucrose
N Ground in 0.1 dl phosphate + 0.3 4f sucrose
3 1
3 220
-
0 15
153
-
64
164
10 69
0 Substrate concentration, 3 X 10-* M. Reaction mixture also contains phosphate buffer, pH 7.1, 10-e M; sucrose, 0.1 M; ATP, 3 X lo-4 M; and MgSOd, 10-* M; cytochrome c where added, 10m6M.
and sucrose together in the grinding medium contributes to the maintenance of the integrity of the particles during their isolation. Cytoplasmic particles prepared in the phosphate-sucrose medium are not only able to oxidize cr-ketoglutarate and succinate but are also capable of oxidizing pyruvate, provided only that a catalytic amount of a Krebs cycle acid such as malate is present. The data of Table II show that neither pyruvate alone nor malate alone in the low concentration used is oxidized at any considerable rate. Only when the two are present together does appreciable oxidation take place. The particles prepared by grinding in and centrifugation from phosphate-sucrose medium may be further purified by a second high-speed centrifugation. The data of Table II indicate that washing the particles
OXIDATION
153
OF PYRUVATE
(obtained by high-speed centrifugation) by resuspension in grinding medium and recentrifugation does indeed result in a lowered endogenous oxygen consumption and higher activity toward pyruvate. Optimal Composition of Grinding Medium It has already been shown (Table I) that the addition of sucrose to the grinding medium has a marked effect on the subsequent activity of isolated particles. In a series of experiments, the concentration and proportions of phosphate buffer and sucrose were varied for determination of TABLE Oxidation
of Pyruvic
II
Acid by Cytoplasmic
Reaction mixtw?
Particles of Mung Bean
Steps used in preparation
02 consumption cu. mm.fhr./mg.
Enzyme Enzyme + pyruvateb Enzyme + L-malateb Enzyme + pyruvate + L-malateb Enzyme Enzyme + pyruvateb
Ground, clarified, used
centrifuged,
I\;
19 24 27 115
Ground, clarified, centrifuged, resuspended,” recentrifuged, used
0
168 ____-
0 Also contains phosphate buffer pH, 7.1,O.l M; and sucrose, 0.1 M. b Pyruvate concentration 3 X low2 M. Also contains ATP 3 X 10e4M, MgSO, 10-a M, and L-malate 1.7 X 1OV M. In all cases where malate is used, nL-malate was added and the concentration given is that of the L-isomer only. c Resuspended in 20 ml. of original grinding medium.
the optimal composition of the grinding medium. It was found that grinding in a sucrose concentration of 0.4 M and a phosphate concentration of 0.1 M yielded particles of higher activity toward pyruvate than any other combination tested. The function of the sucrose in the grinding medium is no doubt osmotic since the osmotic value of the suspending medium is important in the maintenance of particle integrity (11). It has also been shown by Laties (6) that other osmotic agents such as mannitol apparently serve this function as well as sucrose and that the optimal phosphate concentration is relatively high because of several factors. Thus the phosphat’e serves
154
ADELE
MILLERD
in part to satisfy the osmolar requirement. In addition the activity of the particles possessesa requirement for the phosphate ion itself (10). It is apparent that the requirement of the mung bean particles for phosphate ions is twofold. Thus a relatively high phosphate concentration in the grinding medium is necessary for the isolation of cytoplasmic particles possessingmaximal oxidat.ive activity. In addition (lo), the presence of phosphate ion is also necessary during the course of the reaction itself if the maximal rate of substrate oxidation is to be attained. Let us consider the first aspect and examine more closely the nature of the requirement for phosphate in the grinding medium. The experiments described below indicate that the requirement for phosphate in the grinding medium is associated with the inhibition of a phosphorolytic breakdown of someessential substance or substances. In this function the phosphate ion may be replaced by other phosphatase inhibitors such as sodium fluoride. Fluoride cannot, however, completely replace phosphate, and some phosphate must be contained in the grinding medium. The data of Table III show that fluoride cannot completely replace phosphate in the grinding medium, since particles prepared from tissue ground in 0.1 M sodium fluoride containing 0.4 M sucrose are unable to oxidize cu-ketoglutarate or pyruvate, and under the same conditions particles prepared in phosphate-sucrose medium show marked activity toward these substrates. Although fluoride alone is thus unable to replace completely phosphate in the grinding medium, it does seem that fluoride may be substituted for the high concentration of phosphate otherwise necessary. This is also shown by the experiment of Table III in which one preparation was ground in 0.011cI phosphate buffer containing 0.1 M sodium fluoride and 0.4 M sucrose.The particles prepared in the high-fluoride, low-phosphate medium are as active toward a-ketoglutarate as the standard particles but are less active toward pyruvate. The preparation of particles highly active in the oxidation of pyruvate requires that phosphate, in low concentrations at least, be present continuously throughout the successive steps of the procedure. Thus phosphate cannot be omitted during the grinding step even though it is present during the washing and subsequent steps. The results summarized in the experiment of Table III are not to be attributed only to the use of unbuffered grinding media since preparations made by grinding tissue in 0.1 M trishydroxymethylaminomethane (THAM) buffer containing 0.4 M sucrose yielded particles which were
OXIDATION
OF
155
PYRWATE
incapable of oxidizing pyruvate. Tissue was ground in 0.1 M THAM containing 0.4 M sucrose, and the residue, after high-speed centrifugation, was resuspended in this medium. Such a preparation, with phosphate present in, or absent from, the reaction medium, shows no capacity to oxidize pyruvate. The experiments described above have defined certain conditions as optimal or near optimal for isolation of active cytoplasmic particles from TABLE
III
For the Isolation
of Active Cytoplasmic Particles from Mung Bean, Fluoride Partially but Not Completely Replace the Requirement for a High Concentration of Phosphate in the Grinding Mixture Reaction mixture”
Composition of grinding medium
Can
02 consumption w. nim.lhr.lmg.
Phosphate, 0.1 Mb and sucrose, 0.4 M
Enzyme
Enzyme + wketoglutarate Enzyme + pyruvateb Enzyme
157 92 Phosphate, fluoride, 0.4 M
0.01 ;Irl; sodium 0.1 M; sucrose,
Enzyme + cu-ketoglutarate Enzyme + pyruvateb Enzyme
N
0
0
151 36 Sodium fluoride, sucrose, 0.4 M
0.1 111; and
Enzyme + a-ketoglutarate Enzyme + pyruvateb
10 15 5
B Also contains phosphate buffer, pH 7.1, 0.05 Jf; sucrose, 0.3 M; ATP, 5 1OWM; and MgSOd, 10-S M. Substrate concentration, 2 X 1OWM. b Also contains r,-malate, 1.7 X 10-S M.
X
mung bean. On the basis of these experiments, the method previously described (10) was selected as the standard procedure and has been employed in all further experiments. E#ect of Concentration of Sucrose in the Reaction Mixture Since the concentration of sucrose employed in the grinding medium has such a marked effect on the subsequent activity of the particles toward pyruvate, it is of interest to determine whether the concentration of sucrose present in the reaction mixture also influences the rate
156
ADELE
MILLERD
of pyruvate oxidation. The data of Fig. 1 show that the concentration of sucrose during the course of the reaction does indeed affect the rate of pyruvate oxidation. The optimal concentration appears to be 0.3 M, a concentration used in all further experiments. Oxidative Capacity of Isolated Cytoplasmic Particles. Oxidation of the Krebs Cycle
of Acids
It has already been reported (10) that isolated cytoplasmic particles are able to oxidize all of the principal acids of the Krebs cycle. The relation of substrate concentration to rate of oxygen consumption is ex-
01
1 0.1
I
, 0.5
0.3 Concentration
of Sucrose : M
1. Effect of concentration of sucrose in the reaction mixture upon the rate of oxidation of pyruvate by cytoplasmic particles of mung bean. The reaction mixture also contains enzyme; phosphate buffer, pH 7.1, 3 X 10-p M; ATP, 3 X 10-d M; MgSO+ lO+ .V; L-malate, 1.7 X lO+ M; and pyruvate, 10-a M. FIG.
pressed in Table IV in terms of the K, for each of the Krebs cycle acids. In these experiments, the rates of oxidation of the varied substrates are determined under conditions previously described (10) for maximal pyruvate oxidation. In each case, substrate saturation is reached or essentially reached in the region of 2 X lOA M, and this concentration is used in all later experiments. E$ect of Added Cofactors on the Oxidatioe Capacity of Isolated Cytoplasmic Particles As has already been reported, the only supplement to the reaction mixture required for the oxidation of pyruvate by mung bean particles
OXIDATION
OF
157
PYRUVATE
is a catalytic amount of one of the acids of the Krebs cycle. However, the addition of adenosine triphosphate (ATP), adenosine di- or monophosphate, or of magnesium ions to the reaction mixture markedly inincreases the rate. The effect on the rate of oxidation of other acids of the cycle by the addition of these substances is expressed in Table V. The data show that in each case the addition of magnesium ions causes con-
Substrate-Afinity
TABLE IV Constants (K,) for the Oxidation of Krebs Cycle Acids by Cytoplasmic Particles of Mung Bean
Substrate
K&O-0
Citrate a-Ketoglutarate Succinate Fumarate L-Malate Pyruvate
3.4 4.1 7.9 7.9 3.6 5.4 TABLE
V
Effect of Catalytic Amounts of ATP and Mg Ions on the Rate of Oxidation of Krebs Cycle Acids by Cytoplasmic Particles of Mung Bean (01 consumption in cu. (mm./hr./mg. Substrate (2 X lo-’ M)”
Citrate ................. a-Ketoglutarate ......... Succinate. ............. Fumarate. ............. Malate ....... ........ a Reaction mixture and eucrose, 0.3 M.
Plus ATP 6 x lo-w)
NO addition
67 72 122 2 33
142 113 218 25 74
110 57 79 1150 124
87 89 137 2 28
Per cent increase
30 24 12 0 -15
-
N)) p1us Mg Per cent bns ATPad increase
172 181 262 31 71
157 152 115 1450 115
also contains enzyme; phosphate buffer, pH 7.1, 0.05 M;
siderable increase in the rate of oxygen consumption. The addition of ATP causes small increases in the rate of oxidation of citrate, cY-ketoglutarate, and succinate but not of malate or fumarate. In those cases in which both magnesium ions and ATP individually increase the rate of oxidation, the increase observed in the presence of both together is more than additive.
158
ADELE
MILLERD
E$ect of Concentration of Factors Injluencing
Pyruvate Oxiohtion
In the experiments reported earlier on the oxidation of pyruvate, the concentrations of malate, ATP, and magnesium ions used were those known to be optimal. The relation between the concentration of each of these substances and the rate of oxidation is expressed in Figs. 2 to 5. It is of particular interest that while the concentration of pyruvate needed for maximal rate is approximately the same as for the other Krebs cycle acids, the concentration of L-malate needed to give maximal 120
100
20
0 Pyruvole
10-O Concentrolion
:M
2. Effect of concentration of pyruvate on rate of oxidation by cytoplasmic particles of mung bean. The reaction mixture also contains enzyme; phosphate buffer, pH 7.1, 0.05 M; sucrose, 0.3 M; ATP, 5 X IO-’ M; and MgSOd, lO+ M. Malate concentration, 1.7 X lWa M. FIQ.
rate of pyruvate oxidation is much less, 1.7 X 1e3 M (2.5 pmoles in the reaction mixture). The concentration of magnesium ions needed for maximal rate of pyruvate oxidation is, as is illustrated in Fig. 4, in the range of 2 x 10-s to 1W3 M. A concentration of Ws M was employed in all experiments recorded. Figure 5 illustrates the effect of varied concentration of ATP on the rate of oxidation of pyruvate. The increase in rate brought about by ATP although real is not striking. A concentration of 5 X 10-a M which is
OXIDATION
159
OF PYRUVATE
100
2
00
i? L
; 2 ;
60
“, 0
40
i E 20
0 L-Malale
Cancentratlon
:M
FIG. 3. Effect of concentration of L-malate on the rate of oxidation of pyruvate by cytoplasmic particles from mung bean. The reaction mixture also contains enzyme; phosphate buffer, pH 7.1, 0.05 M; sucrose, 0.3 M; MgSOa, 1W3 M; ATP, 5 X KY4 .lil; and pyruvate, 2 X 10-* M.
lo-’
10-S
Cor~centroiion
of Mg ions
M
10-Z
FIG. 4. Effect of concentration of magnesium ions on the rate of oxidation of pyruvate by cytoplasmic particles. The reaction mixture also contains enzyme; phosphate buffer, pH 7.1, 0.05 I4; sucrose, 0.3 M; ATP, 5 X W4 111;L-malate, 1.7 X lC8 M; and pyruvate 2 X 10-* M.
160
ADELE
within the range needed for experiments.
MILLERD
maximal
activity
Speczj% Activity of Mituchondrial
was used in
all
Preparations
It is of interest to consider the maximal oxidative capacity obtainable with isolated mung bean mitochondria. The highest rates of succinate oxidation as yet attained have been with mitochondria made not by the standard procedure outlined above but prepared from the hypocotyl tissue alone rather than the whole shoot. Particularly high rates of succinate oxidation have been obtained when such hypocotyl tissue is
100z so z % 605 e 2 400” “E E 20-
”
lo-*
ATP Concentration:
M
10-3
FIG. 5. Effect of concentration
of ATP on rate of oxidation of pyruvate by cytoplasmic particles. The reaction mixture also contains enzyme; phosphate buffer, pH 7.1,0.05 M; sucrose, 0.2 M; MgSO,, 10-a M; malate, 1.7 X lO+ M; and pyruvate, 2 X lo-* M.
ground in 0.5 M sucrose rather than in the standard sucrose-phosphate buffer mixture. The activities of mung bean mitochondria prepared in these different ways are compared in Table VI. It seems probable that the relatively low Qo,(N) of preparations made in the presence of phosphate ion is in some part due to effects on the aggregation and subsequent centrifugal separation of extraneous and inert cytoplasmic proteins. Even though particles prepared in the absence of phosphate ions are highly active in the oxidation of succinate, they have nonetheless been damaged in some way since their ability to oxidize pyruvate has not been correspondingly increased and, in addition, they have in some measure lost their ability to carry out oxidative phosphorylations (12).
OXIDATION
OF
161
PYRWATE
The data of Table VI include a comparison of the specific activity of mung bean mitochondria with that of rat liver at the same stage of purification (11). It is apparent that the specific activities, on a C&,(N) basis, are essentially similar if the comparison is made at a common temperature and that the activity of the plant mitochondria measured at 30” compares favorably with that of the liver particles measured at 38’ if allowance is made for the difference in temperatures. TABLE VI Comparison of Oxidative Activity of Cytoplasmic various
Particles
Isolated
by
PTOCeduTeS
(Succinate as substrate) Material
Grinding medium
02 consumption cu. mm.lhr. Imp. N
Mung beana complete aerial portion hypocotyl hypocotyl Ratb liver 0 Activity b Activity
0.4 M sucrose, 0.1 M phosphate 0.4 M sucrose, 0.1 M phosphate 0.5 M sucrose 0.88 M sucrose
266 270 773 2010
measured at 30°C. measured at 38°C. DISCXJSSION
It has been shown above that from seedlings of the mung bean, particles may be isolated which, in the absence of added cofactors, can oxidize the acids of the Krebs cycle. Among the preparational procedures which must be observed in the isolation of these particles, perhaps the most interesting is a requirement for the presence of phosphate in the medium in which the seedlings are ground. The requirement for the presence of phosphate in the grinding medium is apparently associated with the inhibition of some phosphorolytic breakdown since phosphate can in part be replaced by a phosphatase inhibitor, sodium fluoride. Fluoride cannot, however, completely substitute for phosphate, and there appears to be a further specific need for the presence of the phosphate ion in the grinding medium. The principal further requirement for the grinding medium is an osmotic one. The presence of compounds such as sucrose in the grinding medium, by controlling the osmotic environment, aid the maintenance of the integrity of the particles during their isolation. How far do the results reported in this paper together with those
162
ADELE MILLERD
previously described go toward establishing the Krebs cycle as participating in the respiration of higher plants? The isolated cytoplasmic particles of mung bean are able to oxidize the acids of the Krebs cycle. The oxidation of pyruvate by these particles is dependent upon the concomitant oxidation of a catalytic amount to one of the di- or tricarboxylic acids. In this catalytic function the various acids are approximately equivalent. Finally, oxidation of the acids of the cycle is accompanied by a coupled phosphorylation (12). These facts taken together support the view that a system similar to the Krebs cycle is responsible for the respiratory oxidation of pyruvate in higher plants. In what way and to what extent do the mitochondrial oxidations fit into the metabolism of the plant? The evidence which is available, direct as well as indirect, suggests that the bulk if not all of the respiration of the mung bean seedling is mediated by the mitochondria. Let us consider the quant,itative aspects of this matter and inquire how the rate of respiratory oxygen uptake of the living plant compares with the rate of oxygen uptake which can be accomplished by the isolated mitochondria. In a particular case, segments of mung bean hypocotyls were found to consume 150 cu. mm. OJhr./g. fresh weight. Mitochondria isolated from the same material consumed oxygen, in the presence of succinate, at the rate of 45 cu. mm/hr./g. of original fresh weight. Microscopic observation shows that only roughly one-half of the cells of the tissue are disrupted in the grinding process. In addition, many of the mitochondria, perhaps half or more, are lost during the successive centrifugatione as judged by visual observation. The mitochondria alone can therefore oxidize succinate, transfer electrons, and consume oxygen at a rate roughly sufficient to account in this instance for the respiration of the living tissue. SUMMARY
Cytoplasmic particles isolated from mung bean seedlings can oxidize all of the acids of the Krebs cycle. The oxidation of pyruvate is dependent upon the concomitant oxidation of a catalytic amount of one of the di- or tricarboxylic acids, and in this function the various acids are approximately equivalent. Preparational procedures for the isolation of such particles have been defined. Among the most important requirements for full activity of the isolated particles is the presence of phosphate in the grinding medium. It has been shown that the isolated particles oxidize at a rate sufficient to account for all of the respiration of the inta ct tissue.
OXIDATION
OF PYRWATE
163
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
JAMES, G. >I., AND JAMES, W. O., New Phytologist 39, 266 (1940). BONNER, J., AND WILDMAN, S. G., Arch. Biochem. 10, 497 (1946). BONNER, J., Arch. Biochenl. 17, 311 (1948). LATIES, G. G., Arch. Biochem. 22, 8 (1949). KREBS, H. A., AND JOHNSON, W. A., Enzymologiu 4, 148 (1937). LATIES, G. G., Abstracts of Amer. Sot. Plant Physiol., p. 2, June, 1951. MILLERD, A., Proc. Linnean Sot. N. S. Wales 76, 123 (1951). STAFFORD, H. A., Physiol. Pkzntarum 4, 696. (1951). BHAGVAT, Ii., ASD HILL, R., New Phytologist, 60, 112 (1951). MILLERD, A., BONNER, J., AXELROD, B., AND BANDURSKI, R. S., Proc. Natl. rlcud. Sci. I!. S. 37, 855 (1951). 11. HOGEBOOM,G. H., SCHNEIDER, W. C., AND PALLADE, G.&J. Viol. Chem. 173, 619 (1948). 12. BONNER, J., AND MILLERD, A., Arch. Biochem. and Biophys. 43, 135 (1953).