Fiske and Subbarow (8). ATPase activity was ... soluble Pi released by the Fiske-Subbarow mletlhod. ..... J. T. WX"ISKICH, AND R. N. ROBERT-. SON. 1964. loIo.
Plant Phy0iol. ( 1l9#8) 4, 1108-1114
Characterization of Energy-Dependent Ca2+ Transport in Maize Mitochondrial 0. E. Elzam2 and T. K. Hodges Department of Horticulture, University of Illinois, Urbana, Illinois 61801 Received February 8, 1968.
Abstract. Experimental conditions which optimize both substrate- and ATP-dependent Ca2+ transport in corn (Zea mays) mitoohondria have been determined. It has been found that a substrate (pyruvate + suceintaite) dependent, Pi independent, binding of Ca2+ occurs. This reaction is very rapid and complete in less than 30 6econds. For massive accumutlation of calcium, Pi is essential. Phosphate is accumulated along with the calcium and the ratio of Ca :Pi accumulated is about 1.6:1 indicating the precipitation of hydroxyapatite inside the mitochondria. The activation energies aind Michaelis constants for both the substrate- and ATP-driven reactions have been determined. It has also been shown that the substrate-driven system is more efficient in Ca2' accumulation than the ATP-driven system. This is partialily due to the fact that Mg2+ is essential for the ATP-driven system but not for the substrate-driven system and that Mg2+ acts as a strong competitor of Ca2+ transport. The effect of other inorganic ions on Ca2+ transport energized by both substrate and ATP were examined. The results lend support to the hypothesis that high energy intermediates of oxidative phosphorylation participate directly in Ca2' binding and transport in plant mitochondria.
Plant mitochondria (7, 9, 10, 11, 12, 14, 18, 19) as wvell as animal mitochondria (1, 2, 3,4,5, 22, 23) accumulate a variety of inorganic ions at the expense of metabolic energy. In a previous report we (11) described the basic features of Ca2' accumulation in imlaize mitochondria. In brief, it was shown that either oxidizable substrates or ATP could provide the energy for Ca2' accumulation by these organelles. The addition of ADP inhibited the substrate-driven transport and this inhibition could be partially relieved by oligomycin. Oligomycin had no effect on sutbstrate-driven transport itself but completely abolislhe(d ATP-driven transport. Similar observations have been made with animal mitochondria and these resuilts have led to the postulate that a common high eniergy internmediate of oxidative phosphorylation can 1e uitilized for either ATP formation or Ca2' transport (2, 3, 4). With ATP as the energy source for ('a' transl)ort the highi energy intermediate is apparently forimied via a reversal of the normal phosphorylation sequence. Brierley et al. (2, 3) have depicted the formation and participation of a high energy intermnediate in Ca2+ tralnsport in animal
1 This research was supported by the National Science Foundation (grants GB-2281 anld GB-5549) and by the Universitv of Illinois Graduate Research Board. 2 Present address: Department of Biology, CaseWNTesterni Reserve University, Clevelanfd, Ohio.
mitochondria as follows:
oligomycin
Sutbstrate
>
[.]
I -
ATP
Ion Accumulation Studies with animal mitochondria have led Chance and associates (4, 22) to the opinion that a non-phosphorylated intermediate of oxidative phosphorylation is the one participating in the transport reaction. However, we (11) could find no evidence for energized Ca2' binding or transport in the absence of phosphate and this led to the suggestion that a phosphorylated intermediate of oxidative phosphorylation was the one involved in Ca2' binding and transport. fHowever, with the further characterization reported here we have been able to distingtilish a small substrate-dependent binding of Ca2 in the abselnce of Pi. Only in the presence of Pi, however, does one observe high rates of Ca 2 transport and accumulation inside the mitochondria. TIn our earlier work, Pi transport was shown to occur in association with Ca2+ transport with a stoichiometry of 1 Ca2+ 1 Pi. With the more optimum conditions, we now find a stoichiometry of about 1.6 Ca2+ :1 Pi which is in accord with results obtained with animal mitochondria and which suggests the Ca 2 is deposited inside the mitochondria as hydroxvapatite (3). The improved general characterization of Ca2+ transport in maize mitochondria along with exami-
1108
ELZAMki ANI) HOD(GES-CA2
nations of the effect of various other ionls and inhibitors and uncouplers of phosphorylation has led us to believe that the energized Ca2+ binding initiallv occurs onto a non-phosphorylated intermediate. Actual transport of Ca2+ into the mitochondria, however, appears to require the intermediate to be phospIhorylated which then breaks down rapidly when in association with Ca2+, releasing Ca2+ and Pi inside the mitochondria. Special attention has also been given to the characterization of ATP-driven Ca2+ transport since no other laboratory has thus far been able to demonstrate this phenomenon in plant mitochondria.
1.109
TRANSPORT IN MITOCUIONI)RIA
pellet withl 5 % trichlioroacetic acid (TrCA) and determining the acid soluble Pi by the miethlod of Fiske and Subbarow (8). ATPase activity was measured by stopping the reaction (lirectly with TCA (final concentration of 5 %) and measuring the soluble Pi released by the Fiske-Subbarow mletlhod. Mitochondrial nitrogen was determined bv aci(d (igestion and nesslerization.
Results and Discussion In our previous report (11) no energy-dependenit binding could be observed in the absence of Pi. However, Kenefick and Hanson (14) have shown with rather complex centrifugation experiments, that a slight substrate-driven Ca2+ binding could occLir in the absence of Pi. Using the conventional reisolation procedure we have also found a substrate dependent, Pi independent, Ca2' binding (fig 1). This Ca2+ binding in the absence of Pi is enhanced somewhat by increasing the concentration of tris (fig 1). The higher tris concentration resulted in both a lowering of the superficial (energy independent) binding of Ca2' and an increase in substrate-dependent binding. The substrate stimulation of Ca2' binding in the absence of Pi is about 2 fold. In the presence of Pi, however, substrate stimulates Ca2+ uptake (presumably transport into the mitochondria) by several fold (fig 1). Thus, oxidizable substrates, in this case pyruvate and succinate, enhance Ca2+ binding in the absence of Pi but in order for large accumulations of Ca2+ to occur, Pi is absolutely essential. The effect of Pi concentrations on Ca2+ transport in both the substrate- and ATP-driven systems is shown in figure 2. In both cases about 5 mm phosphate is optimal. Higher Pi concentrations reduce
CaQ2
Materials and Methods Corn seeds (Zea inays L., WF9XM14) were germinated and grown as described previously (11). Three and one-half day old shoots were excised and chilled in cold deionized water prior to grinding in an ice jacketed mortar and pestle with 0 25 M sucrose, 5 mM EDTA (Na) and 5.9 mm tris (pH 7.5). The slurry was strained through cheesecloth and the mitochondria were isolated at 10 as the fraction sedimenting between 1500 X g for 15 minutes and 12,000 X g for 15 minutes. The mitochondria were washed twice-first in fresh grinding media and then in 0.25 M sucrose. The final mitochondrial pellet was suspended in 0.25 M sucTose. Calcium uptake was followed using 45Ca (0.001 ,aC 45Ca/,Lmole Ca.) The mitochondria were incubated at 300 for various periods of time (2 min vhen substrate served as the energy source and 10 min when ATP served as the energy source) in pyrex centrifuge tubes in a shaking water bath. The basic reaction system consisted of 0.25 M sucrose, 0.2 mm Ca2+ (45Ca), 20 mm Pi, and 24 mM tris buffer, pH 7.5 in a total volume of 2.5 ml. The oxidizable substrate, when added, consisted of a mixture of pyruvate and succinate (10 /,moles of each) as well as the cofactors NAD+, thiamine pyrophosphate and CoA at concentrations of 80, 40, and 20 ug/ml, respectively. The reaction conditions when ATP (Na+ salt, 1.3 mM) served as the energy source were the same except for the addition of 3 mm MgCl2 and the omission of the cofactors. Reactions were terminated by forcing 2.5 ml of 0.5 M sucrose (10) under the reaction system, chilling the tubes briefly (2 min maximum) in crushed ice and then centrifuging (10) the mitochondria through the layer of 0.5 M sucrose. This procedure serves to separate the mitochondria from the reaction mixture as well as to wash them in 1 operation. The sedimented mitochondria were suspended in water and counted for 45Ca with a Nuclear Chicago gas flow counter. All experiments were repeated at least once and the reliability and consistency of the results were very good as can be seen by the maximum values obtained in the different experiments. Determination of Pi was made by extracting the
0 .C\ z E 0
E
wi
2/
Y-V//
I xTRIS -Pi
-SUB
Ix TRIS -Pi +SUB
2xTRIS -Pi -SUB
2xTRIS -Pi
+SUB
2xTRIS +Pi -SUB
2xTRIS
+Pi + SUB
FIG. 1. Ef fect of substrate, Pi and tris on Ca2+ uptake. Experimental conditions described in text. The 1X and 2X tris concentrations were 10 mm and 20 mm, respectively. The Pi concentration was 20 mM.
1110
PLANT PHYSIOLOGY
1; E
cli
zI E E
z
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E 9
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-
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PHOSPHATE CONG. (mM)
6
5
pH
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4
0.9I
z
z
3.0
E
z
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z ci
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,
t~~~
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0.25
+ Pi
+ SUBSTRATE
I2.2 4
0
3.1
3.2
3.3
34
33
3
2
5
TIME (min)
Mg CONC (m M) T
r
111004 4AZ N
1
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a
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I I
.
OUGOYCIN
+ SUBSTRATE
.a bo
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0
+ SUBSTRATE
60-
DNP
.
z 0
40-
4
3
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-SUBSTRATE
I
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-SUBSTRATE
1,-4 ADP CONC
-6 (M)
62
'-6
l0
10
INHIBITOR
25C CONC
e4 (M)
-3
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10
-B
10
-4
10-5
INHIBITOR CONC
10F3
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FIG. 2. Effect of Pi concentrations on substrate- and ATP-driven Ca2+ utptake. Experimental condition's in this and subsequent figures described in M,aterials and Methods. FIG. 3. Time course of substrate- and ATP-driven Ca2+ uptake. FIG. 4. Effect of pH on substrate- and ATP-driven Ca2+ uptak.e. The triis conc-entration was 24 nim and HCI was added to give the desired pH's. FIG. 5. Arrhenius plots of the effect of temperature on isubstrate- and ATP-driven Ca2+ uptake. FIG. 6. Effect of Mg2+ concentrations on suibstrate- and ATP-driven Ca2+ uptake. FIG. 7. Time course of Ca2+ uptake with sequential additions of mitochondria, substrate and Pi. N,ote change in axis after adding Pi. FIG. 8. Effect of ADP (Na salt) concentrations on substrate-driven Ca2+ uptake. FIG. 9. Effect of varying concentrations of DNP, oligomycin, and sodium azide on substrate-driven Ca2+ uptake. FIG. 10. Effect of varying concentrations of DNP, oligomycin, and !sodium azide on ATP-d'riven Ca2+ uptake.
ELZAM AND
HODGES-CA2+
Ca2+ uptake onily slightly antd we routinelv used a concentration of 20 mm in further experiments. The optimum Pi concentration of 5 mm of course pertains to an external Ca2+ concentration of 0.2 mM and might be different with other Ca2+ concentrations. It is again apparent in figure 2 that in the absence of Pi some substrate dependent binding of Ca'2 occurs. It is also apparent that ATP, in the absence of added Pi, will support Ca2+ uptake. In this case, the term-inal phosphate of ATP is presnmablv hydrolvzed and the liberated Pi eniables s0oile transport of a2l ( anld Pi) ii1to the ilitochotidria to occur. The tiimie course of Ca2'tranisport for botlh the substrate- aild ATJ'-driven systeimis is shown in figure 3. Substrate-driven tranisport is essentially linear over anl absorptionl period of 2 minutes. \Vith loniger absorption periods the rate of transport decreases as a result of depletion of the exogenious Ca2+ concentrations. Longer periods of linear absorption could be obtained using higlher Ca2+ concentrations but these were not used because of possible deleterious side effects on the mitochondria. The ATP-driven Ca2+ transport is not as rapid as the substrate-driven system (note change in axis) anid foll-owing an initial rapid burst in uptake, the rate of uptake is nearly coinstant over a 10 minute absorption period. Further results on ATP-driven Ca'2 transport are all based on 10 minute absorption periods. Althoulgh this should be satisfactory for comparisons of other variables such as pH, temperature, etc., it should be noted that slightly greater absorption values would have been obtained if only the initial rate was considered. The effect of pH on energy-depeildent Ca2+ trainsport is shownl in figure 4. The optimum pH is about 7.5 and 8.0 for the ATP- and substratedriven systems, respectively. Both systems also show similar temperature optimums of 30 to 350. The energies of activation deternmined on the basis of Arhenius plots (fig 5) indicate the substrate system to be 14.6 Kcal/mole and the ATP-driven system to be 10.97 Kcal/mole. These values correspond quite closely to the energies of activation for the active transport of K+ (12 Kcal/mole) and Na+ (20 Kcal/mole) in erythrocytes (25) as well as K+ transport (9.8 Kcal/mole for substrate-driven and 10.8 Kcal/mole for AT,P-driven transport) in rat liver mitochondria (6). The energies of activation for K+ and Na+ diffusion was reported to be 5 Kcal/mole by Solomen (25). The effect of Ca2+ concentrations on absorption (data not shown) showed typical saturation type kinetics for both energized systems. Oni the basis of linear Lineweaver-Burk plots the Ku;t's for the substrate- and ATP-driven systems were 0.37 mni and 1.0 mM, respectively, and the Vmax's were 4.15 jmnioles/nmgN/nmin and 0.50 unmoles/mgN/min, respectively. The differences in the 2 systems is niot too sturprising since eaclh of the eniergy transducing systemiis undoubtedly represenits many reactionis and
1111
TRANSPORT IN MITOCHONDRIA
the all)parent affinities mlust be a comlplex funiction of several rate constanits. Figure 6 illustrates that Mg2 markedly depresses Ca2+ uptake when substrate is the energy source. A similar competition must exist when ATP is the energy source but this is masked by the fact that Mg2+ is essential for the ATP-supported reaction. The optimum IMg2+ concenitration is about 4 mM which represenits a concelntrationi yielding about 50 % ilnhibitioni of the substrate-driven Ca2+ tranisport. This undoubtedlv represents one of tlle miiajor reasons whv the ATP-driven svstemi does not appear to he as effective as the substrate-driven system in powering Ca2+ tranlsport. That is. Mg2+ reduces Ca2' ul)take, presulmiably by a comipetitive reaction, yet it is absolutelv essenitial for the demonstrationi of ATP-driveln Ca2+ transport. A lack of appreciation for this particular phenomiienia may contribute to the absence of other reports concerning ATP-driven ion transport in plant mitochondria. Using the optimum conditions found in the preceding experiments, several experiments were conducted to ascertain the stoichiometry between Ca2+ and phosphate accumulation when either substrate or ATP served as the energy source. Table I shows the average of 2 such experiments in which botlh Ca2+ and phosphate uptake were determined. When the blank values are subtracted (i.e. uptake in the absence of an energy source) the Ca :Pi ratio for both systems approximates 1.6:1 which is similar to that found for animal mitochondria and which suggests the deposition of hydroxyapatite inside the mitochondria (3). Our previous report of Ca :Pi ratios of 1:1 (11) were partially in error because the superficial binding of Ca2+ and Pi were not taken into consideration. However, certain conditions (particularly the amount of sucrose) do exist which cause the ratio of Ca :Pi to vary over wide linmits and this particular phenomeina is under current
investigation.
By way of summary of the general characteristics of the substrate-driven Ca2+ binding and transport into maize mitochondria a time course of Ca2+ uptake involving sequential additions of substrate and Pi Table I. Ca2+ and Pi Uptake in the Presence and Absence of Energxy Source and the Net Ca:Pi Ratios
in Mai_.ve Mitochondria, Experimental conditions are described in the text. Additives
+ Substrate - Substrate
Net
Ion tptak-e Ca2+ Pi gI..iolcs mgN-l v2 mun 4.961 4.201 0.381 1.329
4.580 /Jjfloles migN
+ ATP - ATP Net
0.868 0.868 0.699
2.872
Ca :Pi ratio 1
1.595
1 10 1w1in-
1.432'
1.013
0.419
1.668
1112
I'LANT PHYSIOLOGY
are showni in figure /7. The actual absorption times involved may be slightly longer than indicated because of the chilling technique used for stopping the reaction (see Methods), nevertheless, they serve to illustrate the relative magnitude of the various types of binding and transport described here. In the absence of an energy supply a rapid superficial binding of Ca2+ occurs with the addition of mitochondria (the shortest reasonable reaction time to use was 30 sec). Upon addition of substrate a further increase in binding occurs which is also quite rapid and is essentially completed within 30 seconds. With the addition of Pi, Ca2+ transport into the mitochondria occurs at an extremely rapid rate (note change in ordinate) and presumably would continue at this rate until the Ca2+ or Pi concentrations became limiting.
Effect of Other Ionis and Inhibitors o01 Ca 2 Tranisport. In order to further characterize the Ca92 transport process the effect of various ions and inhibitors were examined. As already mentioned, Mg2' acts as a strong competitor of Ca2+ transport. Both Mn2' and Baa2+ inhibited Ca2~ transport similarly. The effects of Li', Cs', K+, Na', and NH4+ (all provided with Cl- as the counter anion) onl substrate-driven Ca2+ transport was also determined. At concentrations of 20 mm, anmnonium ions caused the greater inhibition of Ca2+ transport (about 40 %). All other ions inhibited by about 20 %. Since the Ca2+ concentration was only 0.2 mM these iolns must not be acting as competitive inhibitors as suggested for their effect on Mg2+ accumulation in mitochondria from red beet root (19). Potassium and Na+ at concentrations of 20 mni- inhibited ATPdriven Caa2+ transport by only 10 % anid 20 %, respectivelv. One of the major findings w\,hich had earlier led to the hypothesis that a high energy intermediate of oxidative phosphorylation participated in Ca2+ transport was that the presence of ADP would inhibit the substrate-driven transport reaction. The effect of various levels of ADP are shown in figure 8. The progressively increasing inhibitions of Ca'2 transport with increasing concentrations of ADP suggests, as pointed out previously (3, 11), that ATP formation from ADP and Pi are competitive reactions with Ca2+ and Pi transport. The effects of 3 inhibitors, 24-dinitrophenol (DNP), azide and oligomycin on both the substrateaind ATP-driven Ca2+ transport reactions are shown in figures 9 and 10, respectively. It has beeni reported previously (2, 3,1 1 ) that oligomycin has no effect on the substrate-driven reaction. However, if high concentrations of oligomycin are used, this generalization is incorrect as the data in figure 9 indicate. lOn a molar basis the relative effectiveness of these inhibitors on substrate-driven transport is DNP>oligomycin>azide. For the ATP-driven systenm the relative effectiveness of the inhibitors is oligomycin>azide>DNP (fig 10). Both the substrate- and ATP-driven systems are equally sensitive
DNP; 10-5 M yielding 2U to 25 % inhibition and yielding a 70 to 75 % inhibition. Both oligomycin and azide are more inhibitory to the
to
10-4 M
ATP-driven system than the substrate-driven systems (12 and figs 9 and 10). Thus at 10-6 M oligomycini has no effect on the substrate-driven Ca2+ transport (fig 10) but inhibits the ATP-driven system (fig 10) by 100 %. This phenomenon is responsible for the earlier claims, and rightly so at the appropriate concentration, that oligomycin had no effect on substrate-driven transport but did abolish ATP-driven transport. The effect of higher concentrations of oligomycin on substrate-driven Ca2+ transport are probably an indirect effect of this antibiotic on electron transport. Azide appears to function about like oligomycin. Thus at 10-4 M, azide has practicallv no effect on substrate-driven Ca2+ transport but reduces ATP-driven transport by about 60 %. This illustrates that azide has an effect oIn the intermediate reactions involved in phosphorylationi as suggested first by Loomis and Lipman (17) as well as indicating that these reactions are more sensitive to azide than the reactions of electron transport (presumably cytochrome oxidase). This is also evident from the studies of Wilson and Clhance (27) whlichl show that state 3 respirationl is more sensitive to azide thain state 4 respirationi. The inihibitioni of ATP-driven Ca2 transport by azide aind oligomvcin apl)ears to be due to an inhibitioni of the utilizationi of ATP sinice both substances block ATPase activity (fig 11). The mitochondria exhibit somiie inhibitor resistant ATPase but at inhibitor concentrations effective in blocking Ca" transport they also have anI appreciable effect oIn the ATPase activity.
1000..._
OLIGOMYCIN
NoN3
-~~~~~~ ~
60
0
cr
z0
040
0
20
-7 l-6 00 1010
-5 10
-4
10-3
102-2
INHIBITOR CONC (M)
FIG. 1 1. Effect lof oligomycin and sodium azide on ATPase activity. Reaction mixture consisted of 4 mNi ATP (tris), 4 mAI MgCI2, and 50 mm sucrose. Control ATPase activity N-as 3 /Lmoles/mgN/15 min.
ELZAM AND IIODGES-CA 2 TRANSPORT IN MITOCHONDRIA
General Discussion The present report provides a description of the experimental conditions necessary for optimizing both the substrate- and ATP-driven Ca2+ transport reaction in maize mitochondria. The work has shown some energy-dependent Ca2' binding in the absence of Pi to occur, but for massive accumulation of Ca2+, phosphate is essential. It was also shown that Ca2+ and Pi are accumulated in the mitochondria in a ratio of 1.6:1 indicating the precipitation of calcium phosphate (3, 4, 22). We have previously presented a scheme (12) of the phosphorylation reactions associated with either substrate oxidation or ATP utilization involving 1 nonphosphorylated and 2 phosphorylated intermediates which accounts for these and previous observations (10). Under normal phosphorylation conditions (i.e., ATP formation) it is envisioned that Mg2+ ions bind to the various high energy intermediates and tend to stabilize them, especially the phosphorylated intermediate (9). In the presence of Ca2+ (no exogenous Mg2+) the substrateinduced formation of the non-phosphorylated intermediate could lead to a small but significant binding of Ca2+ (such as shown in figs 1 and 7). With the addition of Pi the non-phosphorylated intermediate becomes phosphorylated and this intermediate then presumably breaks down, in the presence of Ca2 , with the directional release of Pi and Ca 2 inside the mitochondria. In the presence of Mg2+ the initial phosphorylated intermediate appears to be more stable (9) and in the presence of ADP, an ADPphosphorylated intermediate appears to immediately precede ATIP formation (12). Thus the substrate energized Ca2+ transport in mitochondria could be thought of as a diversion or aborted reaction. When ATP serves as the energy source the reverse reactions are thought to occur. The intermediates are phosphorylated and in the presence of Ca2' breakdown directionally releasing Pi and Ca2+ inside the mitochondria. The suggested sites of action of azide and oligomycin (12) are in keeping with the hypothesis that the initial phosphorylated intermediate (when substrate is the energy source) is the one involved in Ca2+ transport. That is, at low concentrations, neither inhibitor blocks substrate-driven transport but both are potent inhibitors of ATP-driven Ca2+ transport. Some investigators feel oligomycin prevents the initial entry of Pi into organic combination (16, 20, 24, 26), however, others prefer to place its site of action beyond the phosphorylated intermediate ( 10, 11, 12, 13, 15). In the case of maize mitochondria, all evidence indicates that oligomycin functions beyond the phosphorylated intermediate (10, 11, 12, 13). Azide must block beyond oligomvcin (coming from the substrate side) since it does not alleviate the ADP inhibition of substrate-driven Ca2+ transport, whereas oligomycin does (12). In order to
1113 second phosphorylated-
account for these results a ADP bound intermediate appears to be necessary (12). This would enable the ADP to continue to be effective in reducing substrate-driven transport in the presence of azide. Some investigators of Ca2+ transport in animal mitochondria feel that a non-phosphorylated intermediate, rather than a phosphorylated intermediate as suggested here, is the one directly involved in the transport reaction (4, 32). The Pi requirement for massive Ca2' accumulation to occur is thought by these workers to merely represent the need for a permeating anion to balance the Ca2+ transported and they have found other anions such as acetate to substitute for the phosphate. Thus in our experiments, the substrate dependent, Pi independent binding of Ca2+ could be interpreted as an actual transport with sufficient endogenous Pi or organic acids to act as trapping or holding agents for the Ca.2+. However, this does not seem likely because of the readily exchangeable nature of this Ca2+ (14). Furthermore, with 1 exception, we have been unable to find any substance, such as acetate, which will substitute for phosphate. The 1 exception was noted by Kenefick and Hanson (14) who found small accumulation of Ca2+ in the presence of arsenate, however, this substitution could be explicable in terms of its direct-'substitution for Pi on the phosphorylated intermediate. Another feature pertinent to this difference in interpretation concerns the respiratory response of animal and plant mitochondria to Ca2+ alone and to Ca2+ and Pi. With animal mitochondria, Ca2' elicits a stimulated rate of oxvgen consumption in the absence of Pi (4, 22, 23). This does not occur to any appreciable extent in corn mitochondria (13). However, in the presence of phosphate, corn mitochondria do exhibit a released respiration upon exposure to Ca2+ (13). Because of the lack of respiratory response to Ca2+ in plant mitochondria, Rasmussen (21) was prompted to conclude, "plant mitochondria, in contrast to animal mitochondria, do not accumulate Ca2+, although they do respond to Mg2+ or Mn2+ (W. Bonner, personal communication) ." Although the respiratory response to Ca2+ by plant mitochondria may differ from that exhibited by animal mitochondria, stuch a conclusion, in view of the present as well as previous work (10) is simply not justified. It is very possible, however, that plant and animal mitochondria may differ in their mechanism of salt transport, such as the participation of different high energy intermediates, however, this must await further experimentation.
Literature Cited 1. BIELAWAKI, J. AND A. L. LEHNINGER. 1966. Steichiometric relationship in mitochondrial accumulation of calcium and phosphate supported bv hydrolysis of adenosine triphosphate. J. Biol Chem. 241: 4316-22.
1114
PLANT PHYSIOLOGY
2. BRIERLEY, G. P., E. BACtIAIANN, AND D. E. GREEN. 1962. Active trainsport of inorganic phosphate and magniesiumii ions by beef heart mitochondria. Proc. Nati. Acad. Sci. U. S. 48: 1928-35. 3. BRIERLEY, G. P., E. MURER, AND E. BACHMIANN. 1964. Studies in ion transport. III. The accumulation of calcium and inorganic phosphate by
4. 5.
6.
7. 8. 9.
10.
11. 12.
13. 14.
15.
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