Comp. Biochem. Physiol., 1978, Vol. 59B, pp. 231 to 237. Pergamon Press. Printed in Great Britain
THE TEMPERATURE DEPENDENCE OF STATE IV RESPIRATION, THE CALCIUM UPTAKE SYSTEM, AND THE ACTIVITY OF THE CALCIUM IONOPHORE A23187 IN MITOCHONDRIA FROM ENDO- AND ECTOTHERMIC ANIMALS C. L. SMITh Department of Zoology, The University of Liverpool, P.O. Box 147, Liverpool L69 3BX, England (Received 3 June 1977)
Abstract--1. Arrhenius plots of State IV respiratory activity of liver mitochondria from both rainbow trout and rat were linear over the temperature range 5--35°C. 2. Calcium uptake was monitored by stimulation of oxygen consumption and by calcium electrode recording, with quite comparable results. Rainbow trout gave the usual linear Arrhenius plot but this plot for rat mitochondria exhibited two well-defined inflections or discontinuities. 3. The temperature dependence of the activity of the ionophore A23187 was investigated by measuring the increase in oxygen uptake following a sub-maximal dose of this drug. Again a linear relation was found for rainbow trout, but in this case the rat curves showed only a single inflection point. 4. These results are discussed in relation to other work on the effects of lipid phase transitions on mitochondrial membrane-associated systems.
Fairly recently a new ionophorous antibiotic, A23187, which apparently functions as a mobile It has been shown that breaks or inflections occur transmembrane carrier for divalent ions such as Ca 2÷ in the Arrhenius plots of the activities of various has become available. A23187 causes the release of membrane-bound enzyme systems in mitochondria Ca 2+ ions from the mitochondrial matrix to the isolated from endothermic animals. Similar plots for external medium but in the presence of an oxidizable preparations made from ectothermic animals, how- substrate and inorganic phosphate the calcium ever, are generally linear over the temperature range released is rapidly taken up again by the calcium used (Raison, 1973; Smith, 1973). I have also de- pump. This cycling of Ca 2 + ions leads to a continued scribed an essentially similar state of affairs for the stimulation of oxygen uptake by the mitochondrial temperature dependence of the stimulation of oxygen suspension (Reed & Lardy, 1972; Wong et al., 1973). uptake by intact mitochondria which can be effected In the present work it was, therefore, decided to by the ionophorous antibiotics valinomycin and gra- monitor the transient stimulation of oxygen uptake micidin (Smith, 1974). There is general agreement that by mitochondria from both endo- and eeto-thermic these inflected Arrhenius plots are a reflection of a animals following Ca 2 ÷ addition to the medium and phase change in the membrane lipids in the endother- also its maintained stimulation after a subsequent mic preparations which alters the kinetics of the sys- sub-maximal dose of A23187. Observations were tem under investigation (Raison, 1973). It is, however, planned over a wide range of temperature so that still of considerable interest to extend this type of the temperature dependence of both the calcium enquiry to other mitochondrial membrane-associated uptake and the exogenous ionophore carrier systems systems, and one such is the Ca 2 + transport system could be studied on the same preparation. associated with the inner mitochondrial membrane in Arrhenius plots of data obtained in some laboramost animal species (Lehninger et al., 1967). In the tories for State IV oxygen uptake by mitochondrial presence of metabolic energy, inorganic phosphate preparations from endotherms show an inflection and ATP considerable amounts of Ca 2+ are taken essentially similar to plots of enzyme activity and of up from the external medium by a high affinity trans- other membrane-dependent phenomena (Lee & Gear, locase system, accompanied by a stoichiometric ejec- 1974; Lyons & Raison, 1970; Pye et al., 1976). In tion of protons and stimulation of oxygen uptake. sharp contrast with this I, myself, have always found Glycoproteins have been implicated as possible Ca 2 + that liver mitochondri a from ~t variety of endothermic carriers in the mitochondrial membrane but it is by vertebrates show a linear relationship for State IV no means certain that a mobile or immobile carrier respiration with overall E A values very similar to rather than a specific superiieial receptor is the basis those for ectothermic material (Smith, 1973, 19741 In of this transport mechanism (Carafoli & Crompton, the present experiments the rate of State IV respir1976). It is, therefore, very pertinent to investigate the ation was always recorded prior to Ca 2+ addition effect of temperature-induced transitions of the memand so the data was available for a re-examination brane lipids on the kinetics of calcium uptake. of its temperature dependence. 231 INTRODUCTION
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MATERIAL AND METHODS Rainbow trout (S. oairdneri) were obtained from a hatchery and kept under 8 hr day-length conditions at 10°C. Rats were taken from general laboratory stocks as required.
M itochondria The procedure for isolation of mitochondria from liver tissue was basically the same as that described previously (Smith, !973~ The isolation medium contained 250raM sucrose, 10 mM Tris-HCl and 0.5 mM EDTA and the mitochondrial pellet was washed once in the same medium. The mitochondria were then washed again in 250raM sucrose plus 10 mM Tris-HCI only and the final suspension was made in the same solution in the proportions of 1.0 or 0.5 ml per gram of original tissue for rat or trout respectively.
rate evoked by A23187. It was found that rainbow trout mitochondria became increasingly susceptible to damage by the Ca 2+ load at higher temperatures as the preparation aged. This was manifested by a decline in the State IV rate after Ca 2+ addition and failure to respond fully or even at all to subsequent addition of A23187, ADP or DNP. However, using two oxygen electrodes a full temperature profile could be obtained before there was any evidence of this increase in fragility. It is perhaps of interest to note that when increased susceptibility of Ca 2 + damage was already very evident parallel observations on the same preparation showed no change in either acceptor control or P : O ratios as determined by ADP addition.
Direct measurement of calcium uptake Madeira (1975) has described the application of a Radiometer calcium electrode to the monitoring of Ca 2+ uptake by sarcoplasmic reticulum membranes, showing that the response time of this model was fast enough to follow the rapid kinetics of the system. As a check on Measurement of oxygen uptake my earlier measurements of respiratory stimulation during Oxygen uptake was recorded polarographically using calcium uptake I have used this electrode (F 2112 Ca SelecYellow Springs Instrument Co. oxygen electrodes. Chance trode) to follow the removal of added calcium from the (1965) pointed out that the response time of a Teflonexternal medium by mitochondria. The method was essencovered Clark type oxygen electrode may be too slow to tially the same as that described by Madeira (1975) except register accurately the very rapid and short lasting stimuthat a Pye Dynacap pH meter switched to pH input was lation of oxygen uptake following the addition of low conused. As this meter requires a low impedance connection centrations of calcium to a mitochondrial suspension. In to the output a 2 kt) multi-turn potentiometer, as well as the present work the electrodes were thoroughly cleaned the circuitry for the backing off voltage, was interposed with dentifrice and a thin Teflon membrane (12.5#m) between the meter and a Beckman Log/Linear recorder. lightly stretched over them and secured in the usual way. A calibration curve relating pH meter readings to the This procedure gave a very sensitive and fast responding concentration of free C a 2+ in the system was established electrode system and at the relatively high Ca 2 + concen- by means of an EGTA calcium buffer series (Portzehl et trations used there was no reason to believe that a true al., 1964) over the range 3.30-7.06 pCa. This enabled the estimate of oxygen-uptake stimulation was not being free Ca 2 + concentration of the reaction mixtures presented obtained. This is borne out by the experimental records~ to the witochondria to be estimated. Calcium binding to shown in Fig. 1 where the trace very rapidly changes tot various constituents of the mixture, and in particular to a steeper, linear slope after Ca 2+ addition. The reaction 1 mixture for Ca 2 + uptake experiments was based on that of Chance (1965) and contained 80raM KC1; 20raM Tris-HCl; 5 mM inorganic phosphate; 7 mM sodium glu(a) A tamate; 7 mM sodium succinate; 3.5~t.0 rag witochondrial protein; the pH was 7.4 and the final volume 3.0 ml. In most of the experiments 0.55 mM ATP was included in the reaction mixture (Rossi & Lehninger, 1954; Drahota et al., 1965). Details of other reaction mixtures are given in the legends to the appropriate figures. The general experimental procedure is best illustrated by the records of actual experiments with rat and rainbow trout mitochondria which have been combined in Fig. 1. After incubating the mitochondria in State IV for 2.0 rain an amount of Ca 2 + (432 nmoles) was added from a Repette (Jencons) which preliminary trials had shown to be required for approximately half maximal stimulation of oxygen uptake. Both preparations responded with a marked increase in respiratory rate and the traces show that there was an initial linear phase lasting some 5 sec at 25°C, after which they began to level off and regained the initial State IV rate about 10-12 sec after the calcium load was presented. In other experiments it was shown that both kinds of mitochondria would respond to a Time second addition of calcium in a quantitatively similar manFig. 1. Nimulation of mitochondrlal respiration by calner indicating that the concentration used was not so high that it damaged the witochondria. After a further cium and by the ionophore A23187. Record Ca) is from an experiment with rat and (b) from one with rainbow 1.5-2.0win of State IV respiration the ionophore A23187 trout liver mitochondria. At the points marked 'A' (0.48 nmoles) was added in t h e same way using a stock solution prepared in ethanol. Again the dose was selected 432 nmoles Ca 2 + were added while points 'B' denote the addition of 0.48 nmoles of A23187. The basic reaction mixby preliminary trials so that the consequent increase in ture contained 80 mM KCI, 20 mM Tris--HCl, 5 mM inoroxygen uptake was sub-maximal, as can be seen from the ganic phosphate, 7 mM Na glutamate, 7 m M Na succinate, further increase elicited by a second addition made to the 0.55 mM ATP, 3.5-3.7 m& witochondrial protein, pH 7.4 rat preparation (Fig. la), and total volume was 3.0 nil. In both cases the temperature Thus at the conclusion of each run measurements could was 25°C and the figures alongside each section give the be made of the respiratory rates in State IV, the maximum oxygen uptake rates in ng.atoms win- i mg P - t. initial rates after Ca 2 + addition, and the maintained steady
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Temperature dependence of mitochondrial membrane systems the 0.55 mM ATP used in most of these experiments, leads to the free calcium being' very much lower than the total calcium concentration (Reed & Bygrave, 1975). For example addition of 72/aM calcium (total concentration) to the reaction mixture complete with ATP and substrates yielded an electrode output coi:responding to 13/aM free Ca 2 +. When estimating the rate of calcium uptake by mitochondria the calcium and calomel electrodes were immersed in 3.0 ml of the same basic reaction mixture used for oxygen-uptake stimulation measurements which was contained in an oxygen electrode reaction chamber and was continuously and rapidly stirred by the magnetic follower. By initial trials the sensitivity of the system was adjusted so that the desired final concentration of calcium gave an approximately full scale deflection on the recorder. At the start of each determination successive additions (usually 108 nmoles) of a standard CaCl 2 solution were made until the desired final total concentration was attained. The electrode response was linear for equal increments of calcium over the concentration range used and the system was quite stable for the whole experimental period. Repetition of this procedure for each determination provided a calibration trace for the electrode response at the particular operating temperature, which was used for calculating the subsequent rate of calcium uptake. Calcium uptake was initiated by the rapid addition of mitochondria to the reaction system. The response was immediate and very fast at the higher temperatures used, but tests made by adding EDTA instead of mitochondria showed that the electrode system was certainly not the rate limiting step. The slope of the initial, linear, part of the trace after mitochondrial addition was, therefore, assumed to indicate the maximum initial rate of calcium uptake by the particles. For rat mitochondrial preparations the total Ca2 + concen, tration in the medium before addition of mitochondria was 144/aM and the free Ca 2 + concentration 25 #M (from the electrode calibration curve)~ Again the rainbow trout preparations were more unstable and apparently even more so than when the oxygen uptake was being monitored in the earlier experiments. When freshly isolated they would take up and hold 144/d~i Ca 2+ but within 1 hr after isolation records at temperatures above 20°C showed an initial rapid uptake which gave way to a rapid elflux of the ion. This indicated irreversible damage to the mitochondrial organization and made estimation of initial uptake rates very dubious. It was found that trout preparations would maintain their ability to take up and retain a 72 ~ total Ca 2+ load (13 p.M free Ca 2+) for at least 3hr after isolation which allowed adequate time to cover the full temperature range. Replicate determinations at one of the upper temperatures were made at the beginning and end of each series and showed that Ca2 + uptake activity was unimpaired. Protein content
The protein content of all preparations was determined by a biuret method (Smith, 1973). Chemicals
Standard chemicals used were B.D.H. Analar grade when this was available. ATP was obtained from Sigma Chemical Co. Ltd., and the ionophore A23187 was a generous gift from Dr. Robert Hamili, Eli Lilly Co., Indianapolis, U.S.A. to Prof. C. J. Duncan. RESULTS
Arrhenius plots of the oxygen uptake by liver mitochondria from both rat and rainbow trout when in State IV are shown in Fig. 2. The trout data (A) and that for one of the rat curves (B) were obtained in
the course of Ca 2+ uptake and release experiments in which t h e KCI-Tfis-PO4 medium was used (see Methods~ Clearly in neither case is there any suggestion of an inflection or break within the temperature range 5°-35°C, nor d o the E,4 values differ significantly. In view of this confirmation of my previous experience some further experiments were made using rat mitochondria suspended in the reaction mixture described by Raison et al. (1971). In the present context perhaps the most significant feature of this mixture is that it contains Mg 2+ which could arguably affect the temperature dependence of State IV respiration by stimulating endogenous ATP-ase activity. In Fig. 2 the two upper lines are based on the data for the rates of oxygen uptake by a typical preparation before (C) and after (D) the phosphorylation of exogenous A D P (204/aM). The rate after added A D P had been phosphorylated was significantly higher than that recorded before any A D P was added, as is apparent from Fig. 2 where both these sets of data are plotted on the same ordinate. This increased respiration after phosphorylation of A D P was almost completely inhibited when N a F (12raM) was included in the reaction mixture indicating that it is indeed largely attributable to ATP-ase activity. It should be borne in mind that the KC1-Tris-PO4 mixture contained 0.55 m M A T P so that the initial State IV rates of lines A and B could be considered to be more comparable to the post-ADP rates for the other reaction mixture (i.e. line D). However, comparison of the actual rates of oxygen uptake when parallel observations were made on the same preparation indicated that in the absence of M g 2+ the ATP-ase activity was low. F r o m Fig. 2 it is quite
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Fig. 2. The temperature dependence of the State IV respiration of liver mitochondria. A, • • (rainbow trout), and B, 0 - - - - 0 (rat) are based on data obtained with the basic reaction mixture described in Fig. 1. C, • --" and D, • • are for the same rat preparation and show the mean rates of State IV oxygen uptake before (C) and after (D) phosphorylation of added ADP (204 #M). In this case the reaction mixture contained 250raM sucrose, 10raM Tris-HCl, 10raM inorganic phosphate, 5raM MgCI2, 0.5 mM EDTA, 5 mM succinate, 3 #g rotenone, 1.5 mg bovine serum albumen, 3.7 mg protein, pH 7.4 and total volume was 3.0ml (Raison et al., 1971). Activation energies (LI mole- 1 + S.E.) are shown alongside each line" with the number of observations in parentheses.
C. L. SMITH
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Fig. 3. Arrhenius plots of calcium uptake by liver mitochondria from rainbow trout. O-----O, increase in oxygen uptake rates following addition of Ca 2÷ to State IV mitochondria (mean data for three preparations). I - - I , initial rates of Ca2+ uptake (nmolesmin-lmg P-~) as recorded by a calcium electrode on adding mitochondria to the complete reaction mixture (means for a single preparation). Basic reaction mixture was the same as in Fig. 1. Total Ca 2+ added was 216nmoles and the free Ca2+ concentration was 13#M (see text). Activation energies (kJ mole- 1 + S.E.) are given alongside each curve with the number of observations in parentheses. clear that the stimulation of ATP-ase activity by Mg 2+ does not affect the temperature dependence as neither set of data shows any significant departure from linearity (C and D). The two EA values are also virtually the same and they do not differ significantly from those found for the rat and trout mitochondria in the KCI-Tris--PO4 mixture (B and A). In Figs. 3 and 4 Arrhenius plots illustrate the temperature dependence of calcium uptake by rainbow trout and rat mitochondria respectively. In each figure there are separate plots for oxygen uptake following Ca 2÷ addition and for the initial rate of Ca 2÷
uptake as measured with the calcium electrode. The oxygen uptake rates were calculated by subtracting the initial State IV rate from the maximum rate recorded after Ca 2÷ addition, that is they represent the increase in respiration, which should be entirely attributable to Ca 2÷ uptake. Considering Fig. 3 first, the two methods of monitoring Ca 2÷ uptake show an essentially similar temperature dependence as both are linear over the experimental temperature range and the E,~ values do not differ significantly. In Fig. 4 the temperature relationship for rat mitochondria is more complex as the Arrhenius plots for both methods show two breaks, one occurring at approximately 11°C and the other at 23°C. Regression analysis shows that the differences in slope of the sections of the curves are statistically significant at the 0.001% level for the electrode readings and at approximately the 0.02% level for increase in oxygen uptake. The actual rate of Ca 2÷ uptake at 25°C by rat mitochondria ( 9 3 4 n m o l e s m i n - l m g P-1) is somewhat faster than that by the trout preparation at the same temperature (759 nmoles min- 1 mg P - ~) but this would be at least partly accounted for by the lower Ca :÷ concentration used with the more unstable trout organelles. However, owing to the marked difference in temperature dependence this situation is reversed at 5°C where the corresponding rates for rat and trout are 117 and 309nmoles min-~ mg P-1 respectively. This could be an important feature of Ca 2÷ sequestration in the intact cells of ectothermic animals if the mitochondria play a major role in the homeostatic maintenance of the cytoplasmic Ca 2÷ concentration (Carafoli & Crompton, 1976; Bode & Anderson, 1976). Figure 5 shows Arrhenius plots for the maintained stimulation of oxygen uptake when a sub-maximal dose of A23187 (0.48 nmoles) was added to suspensions of either rat or rainbow trout mitochondria which had been pre-loaded with Ca: ÷ (432 nmoles). As in Figs. 3 and 4 the oxygen uptake rates plotted
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Fig. 4. Arrhenius plots of calcium uptake by rat liver mitochondria. O------O,increase in oxygen uptake rates following addition of Ca2÷ to State IV mitochondria (means for a single preparation). I - ~ - I , initial rates of Ca2÷ uptake as recorded by a Ca2÷ electrode on adding mitochondria to the complete reaction mixture (mean data for two preparations). Basic reaction mixture as in Fig. 1. Total Ca2÷ added was 432 nmoles and the free Ca 2÷ concentration was 25 #M. The number of observations on which the activation energies (kJ mole- 1 ___S.E.) are based are given in parentheses.
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Fig. 5. The effect of temperature on the stimulation of mitochondrial oxygen uptake by the ionophore A23187. The mitochondria were loaded with 432 nmoles Ca 2* prior to addition of 0.48 nmoles of A23187. Other conditions as in Fig. 1. V - - V , rat liver mitochondria (means for a single preparation); e - - - - ~ , rainbow trout liver mitochondria (mean data for three preparations). Activation energies (kJ mole- 1 + S.E.) are given alongside the curves with the number of observations in parentheses.
Temperature dependence of mitochondrial membrane systems were derived by subtracting the post-Ca 2+ State IV rates from those recorded after A23187 addition (cf. Fig. 1). The mean values for three trout preparations show a linear relationship between 3 ° and 30°C, but there is a sharp decrease in oxygen uptake at 35°C which probably indicates that mitochondrial efficiency is becoming impaired by the combination of the Ca 2+ load and the high temperature. The data shown for stimulation of rat mitochondrial respiration during Ca 2+ uptake (Fig. 4) and following A23187 addition (Fig. 5) are from the same experimental series based on a single preparation. Despite this the temperature dependence of the two systems is clearly different as the response to A23187 has only a single inflection, at 18°C, in the Arrhenius plot. This difference has also been seen in analogous experiments with other rat preparations. The activation energy (E4) for rat mitochondria above the inflection temperature is very similar to that for trout mitochondria over the whole range, but is more akin to the EA for calcium uptake over the middle rather than the upper part of the temperature range (Fig. 4). DISCUSSION The discrepancy between my data for State IV respiration by rat mitochondria and that reported by other workers (Lee & Gear, 1974; Lyons & Raison, 1970; Pye et al., 1976) remains unexplained. Yet the question whether such oxygen uptake does or does not give an inflected Arrhenius plot is not an unimportant one. In State IV the mitochondria are supplied with oxygen, inorganic phosphate and an oxidizable substrate but lack ADP or other suitable energy acceptor and it is generally thought that the low level of respiration which does occur is due to energy dissipation processes operating across or within the inner membrane (NichoUs, 1974; Stucki, 1976). A i g 2+ activated ATP-ase acting on either endogenous or exogenous ATP can certainly increase State IV respiration and I thought that this might account for the above discrepancy. However, Fig. 2 (D) clearly shows that this is not the ease as I still obtained a linear relationship despite clear evidence of ATP-ase activity. Re-cycling of Ca 2+ ions across the inner membrane could also contribute to State IV oxygen uptake, but Fig. 4 shows that Ca 2+ uptake by rat liver mitochondria has a quite complex temperature dependence and is therefore unlikely to be a major contributor to the respiration recorded in my systems. Probably the major cause of energy dissipation under State IV conditions is either recycling of H + ions across the coupling membrane or spontaneous hydrolysis of groups such as X ~ I, according to which theory of energy transduction is favoured (Greville, 1969). Lee & Gear (1974) who found two discontinuities in the temperature dependence of State IV, and also of uncoupler-stimulated, respiration concluded that they reflected a general temperature effect on the coupling membrane. If, however, as I have found, such discontinuities do not occur in the State IV temperature relation then it indicates that the energy dissipating processes taking place are passive in nature and, unlike the activities of membrane-bound enzymes and mobile carriers, are not affected by perturbations in the lipid milieu. The similarity of the
235
Arrhenius plots and EA values for rat and trout mitochondria (Fig. 2) is also consistent with this view as the respective membrane lipids certainly have different properties. There have been recent reports of breaks in the temperature dependence curves for the activity of membrane-bound enzymes in mitochondria from tench muscle (Pye et al., 1976) and from liver and muscle of carp (Wodtke, 1976). My data for Ca 2+ uptake by rainbow trout liver mitochondria (Fig. 3) show no departure from linearity between 3 ° and 35°C. In this respect this system conforms with others I have investigated in trout mitochondria, but the E A value, while similar to that for NADH oxidase (31.86 kJ mole- t), is lower than that for succinoxidase (49.57kJmole-1)~ for valinomycin stimulation of oxygen uptake (40.32 kJ mole-t), for State III respiration (58.11 kJ mole-1)and for the respiratory stimulation by A23187 shown in Fig. 5 (Smith, 1977, 1974, 1973). The Ca 2+ uptake system in rat mitochondria (Fig. 4) is the only one for which I have found changes in the slope of the Arrhenius plot at more than one temperature. Raison & McMurchie (1974), however, have described Arrhenius plots for State III respiration by both sheep and rat liver mitochondria which showed breaks or inflections at two temperatures which were coincident with changes in the molecular ordering of the membrane lipids as detected by spin labelling. They concluded that there was a continued shift in the equilibrium between gel and liquid-crystalline phases of the lipids in the temperature zone between the inflections. Lee & Gear (1974) confirmed the presence of double inflections in the Arrhenius plots for several systems in rat liver mitochondria including that for respiration-driven Ca 2+ uptake as monitored by proton ejection. For the latter the lower break was at 12.5°C (Tt) and the upper one at 26.5°C (7"2) with corresponding EA values of 105.9, 65.7, and 23.9 kJ mole -~. This position of T1 and T 2 on the temperature scale shows an upward displacement of 2-3 ° compared with my data in Fig. 4, while the EA values though not identical, are of a similar magnitude. The presence of two points where temperature dependent changes affect the kinetics of the C a 2+ uptake system seems, therefore, to be confirmed. However, this is not the case for other systems as my earlier data for State III respiration, transport of ions by exogenous ionophores, and succinoxidase activity showed only one inflection (Smith, 1977, 1974, 1973). Figure 5 is of particular interest in this context as the rates of A23187-stimulated respiration were recorded from the same mitochondrial suspensions as those for increase in oxygen uptake when a Ca 2+ load was being taken up (Fig. 1)~ Despite this identity of the experimental conditions the temperature dependence curve for Ca 2+ transport by the exogenous ionophore has only one inflection at the intermediate temperature of 18°C. This suggests that the endogenous Ca 2 + uptake mechanism shows a change in apparent EA at the beginning and end of the phase transition zone of the membrane lipids, while perhaps other systems are less susceptible and may respond in an all or none manner only when the phase change in the lipids is nearer completion. Alternatively if a glycoprotein is involved in the molecular system for
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236
Ca 2÷ uptake then it may be that one of the observed inflections is due to temperature-induced conformational changes in the protein moiety which may or may not be related to the lipid phase change (see also Smith, 1976). It is worthy of note that the present data for rat and rainbow trout show there are no significant differences between these species when EA values for either the Ca 2 + uptake system or for A23187 translocation of Ca 2+ are compared at the normal operating temperatures of the host cells (Figs. 3-5). This would suggest at least a measure of identity between the kinetics of both these translocases in ecto- and endothermic cells when the membrane lipids are in the liquid-crystalline phase. I have previously pointed out a similar situation for other mitochondrial systems though this is not entirely supported by EA values recorded in the literature (see Smith, 1977, 1973). The ionophore A23187 stimulates mitochondrial oxygen uptake by virtue of its ability to create a cyclic, energy-dissipating flux of mitochondrial calcium (Reed & Lardy, 1972). In the present work it has been assumed that, provided a sub-maximal amount is used, the rate limiting process in the stimulation of oxygen uptake will be the outward movement of Ca 2 + ions across the mitochondrial membrane by the ionophore. Therefore the Arrhenius plot should depict the temperature dependence of this translocase rather than that of the Ca 2+ uptake system. I have previously used a similar approach to investigate the effect of temperature on the activity of the ionophorous antibiotics valinomycin and gramicidin. Valinomycin is generally regarded as a mobile cartier which transports K + ions across the membrane while gramicidin, which is less specific, forms pores in t h e membrane through which ions diffuse. A number of features of the action of A23187 suggest that it operates as a mobile carrier (Case et al., 1974) so that it is interesting to find that its temperature dependence (Fig. 5) is much more similar to that of valinomycin than that of gramicidin. This is particularly the case for the apparent EA values above the inflection point which were 46.84 and 13.19 kJ molerespectively for these two antibiotics (Smith, 1974). Case et al. (1974) examined the effect of temperature on the rotational relaxation rate of A23187 in rat liver mitochondrial membranes and found a marked inflection at 25°C with an Ea value over the upper temperature range very similar to that of Fig. 5. However, they also stated that unpublished results indicated that the kinetics of Ca 2+ effiux induced by A23187 were not affected by this apparent transition in the membrane lipids, Although the transition temperature from my data is lower than that found by Case et al. they do nevertheless suggest, contrary to their conclusion, that the physical state of the membrane affects translational as well as rotational diffusion.
REFERENCES BORLE A. B. & ANDV-RSONJ. H. (1976) A cybernetic view of cell calcium metabolism. In Calcium in Biological Systems (Edited by DUNCANC. J.), Symposia of the Society for Experimental Biology No. 30, pp. 141-160. University Press, Cambridge.
CARAFOLI E. ,~ CROMPTONM. (1976) Calcium ions and mitochondria. In Calcium in Biological Systems (Edited by Dtmc^N C. J.), Symposia of the Society for Experimental Biology No. 30, pp. 89-115. University Press, Cambridge. CASEG. D., VANDERKOOXJ. M. & SCARPAA. (1974) Physical properties of biological membranes determined by the fluorescenceof the calcium ionophore A23187. Archs Biochem. Biophys. 162, 174-185. CHANCE B. (1975) The energy-linked reaction of calcium with mitochondria. J. biol. Chem. 240, 2729-2748. DRAHOTAZ., CARAFOUE., ROSS1C. S., GAMBLER. L. & L~NINGER A. L. (1965) The steady state maintenance of accumulated Ca z+ in rat liver mitochondria. J. biol. Chem. 240, 2712-2720. GREWLLE G. D. (1969) A scrutiny of Mitchell's chemiosmotic hypothesis of respiratory chain and photosynthetic phosphorylation. In Current Topics in Bio-eneroetics (Edited by SA~ADID. R.), Vol. 5, pp. 1-78. Academic Press, New York. LEE M. P. & Go~R A. R. L. (1974) The effect of temperature on mitechondrial membrane-linked reactions. J. biol. Chent 249, 7541-7549. LEnNI~OERA. L., CAaAEOLIE., & ROSS1C. S. (1967) Energy-linked ion movements in mitochondrial systems. Adv. Enzymol. 29, 259-320. LYONSJ. M. & RAISONJ. K. (1970) A temperature-induced transition in mitochondriai oxidation: contrasts between cold and warm-blooded animals. Comp. Biochem. Physiol. 37, 405-411. MAOEmA V. M. C. (1975) A rapid and ultrasensitive method to measure Ca 2+ movements across biological membranes. Biochem. biophys. Res. Commun. 64, 870-876. NICHOLLS D. G. (1974) The influence of respiration and ATP hydrolysis on the proton-electrochemical gradient across the inner membrane of rat-liver mitochondria as determined by ion distribution. Eur. J. Biochem. ~0, 305-315. PORTZ~L H., CALDWELLP. C. & RuEc_~ J. C. (1964) The dependence of contraction and relaxation of muscle fibres from the crab Maia squinado on the internal concentration of free calcium ions. Biochim. biophys. Acta 79, 581-591. PYE V. I., WidER W. & ZEeH M. (1976) The effect of season and experimental temperature on the rates of oxidative phosphorylation of liver and muscle mitochondria from the tenth Tinca tinca. Comp. Biochem, Physiol 54B, 13-20. RAISONJ. K. (1973) The influence of temperature-induced changes on the kinetics of respiratory and other membrane-associated enzyme systems. Bioenergetics 4, 285-309. RAISONJ. K., LYONSJ. M. & THOMSONW. W. (1971) The influence of membranes on the temperature-induced changes in the kinetics of some respiratory enzymes of mitochondria. Archs Biochem. Biophys. 142, 83-90. RAISONJ. K. & McMuRcHn~E, J. (1974) Two temperatureinduced changes in mitochondrial membranes detected by spin labelling and enzyme kinetics. Biochim. biophys. Acta 363, 135-140. REED K. C. & BYGRAVEF. L. (1975) A kinetic study of mitochondrial calcium transport. Eur. J. Biochem. 55, 497-504. REED P. W. & LARDY H. A. (1972) A23187: a divalent cation ionophore. J. biol. Chem. 2,47, 6970--6977. Rossl C. S. & L~HNIt~GERA. L. (1964) Stoichiometry of respiratory stimulation, accumulation of Ca2 ÷ and phosphate, and oxidative phosphorylation in rat liver mitochondria. J. biol. Chem. 239, 3971-3980. S~llrd C. L. (1973) The temperature dependence of oxidative phosphorylation and of the activity of various enzyme systems in .liver mitochondria from cold- and
Temperature dependence of mitochondrial membrane systems warm-blooded animals. Comp. Biochem. PhysioL 46B, 445--461. SMrtH C. L. (1974) The temperature dependence of the response to valinomycin and gramicidin by isolated liver mitochondria from warm- and cold-blooded animals. Comp. Biochem. Physiol. 49B, 761-773. SMITHC. L. (1976) The temperature dependence of the respiratory activity of mitochondria. In Perspectives in Experimental Biology (Edited by SPENCERDAVIESP.), Vol. 1, pp. 209-218. Pergamon Press, Oxford. SMITH C. L. (1977) Temperature and the regulation of activity of some mitochondrial enzyme systems in ectoand endothermic animals. J. Thermobiol. 2, 215221.
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STUCm J. W. (1976) Efficiency of oxidative phosphorylation and energy dissipation by H ÷ ion recycling in ratliver mitochondria metabolizing pyruvate. Eur. J. Biochem. 6S, 551-562. WoD'rr~ E, (1976) Discontinuities in the Arrhenius plots of mitochondrial membrane-bound enzyme systems from a poikilotherm: acclimation temperature of carp affects transition temperatures. J. comp. Physiol. !10, 145--157. WoNo D. T., WILKINSON J. R., HAM_ILLR. L. & HORNG J.-S. (1973) Effects of antibiotic ionophore, A23187, on oxidative phosphorylation and calcium transport of liver mitochondria. Archs Biochem. BMphys. 156, 578-585.
THE TEMPERATURE DEPENDENCE OF STATE IV RESPIRATION, THE CALCIUM UPTAKE SYSTEM, AND THE ACTIVITY OF THE CALCIUM IONOPHORE A23187 IN MITOCHONDRIA FROM ENDO- AND ECTOTHERMIC ANIMALS C. L. SMITh Department of Zoology, The University of Liverpool, P.O. Box 147, Liverpool L69 3BX, England (Received 3 June 1977)
Abstract--1. Arrhenius plots of State IV respiratory activity of liver mitochondria from both rainbow trout and rat were linear over the temperature range 5--35°C. 2. Calcium uptake was monitored by stimulation of oxygen consumption and by calcium electrode recording, with quite comparable results. Rainbow trout gave the usual linear Arrhenius plot but this plot for rat mitochondria exhibited two well-defined inflections or discontinuities. 3. The temperature dependence of the activity of the ionophore A23187 was investigated by measuring the increase in oxygen uptake following a sub-maximal dose of this drug. Again a linear relation was found for rainbow trout, but in this case the rat curves showed only a single inflection point. 4. These results are discussed in relation to other work on the effects of lipid phase transitions on mitochondrial membrane-associated systems.
Fairly recently a new ionophorous antibiotic, A23187, which apparently functions as a mobile It has been shown that breaks or inflections occur transmembrane carrier for divalent ions such as Ca 2÷ in the Arrhenius plots of the activities of various has become available. A23187 causes the release of membrane-bound enzyme systems in mitochondria Ca 2+ ions from the mitochondrial matrix to the isolated from endothermic animals. Similar plots for external medium but in the presence of an oxidizable preparations made from ectothermic animals, how- substrate and inorganic phosphate the calcium ever, are generally linear over the temperature range released is rapidly taken up again by the calcium used (Raison, 1973; Smith, 1973). I have also de- pump. This cycling of Ca 2 + ions leads to a continued scribed an essentially similar state of affairs for the stimulation of oxygen uptake by the mitochondrial temperature dependence of the stimulation of oxygen suspension (Reed & Lardy, 1972; Wong et al., 1973). uptake by intact mitochondria which can be effected In the present work it was, therefore, decided to by the ionophorous antibiotics valinomycin and gra- monitor the transient stimulation of oxygen uptake micidin (Smith, 1974). There is general agreement that by mitochondria from both endo- and eeto-thermic these inflected Arrhenius plots are a reflection of a animals following Ca 2 ÷ addition to the medium and phase change in the membrane lipids in the endother- also its maintained stimulation after a subsequent mic preparations which alters the kinetics of the sys- sub-maximal dose of A23187. Observations were tem under investigation (Raison, 1973). It is, however, planned over a wide range of temperature so that still of considerable interest to extend this type of the temperature dependence of both the calcium enquiry to other mitochondrial membrane-associated uptake and the exogenous ionophore carrier systems systems, and one such is the Ca 2 + transport system could be studied on the same preparation. associated with the inner mitochondrial membrane in Arrhenius plots of data obtained in some laboramost animal species (Lehninger et al., 1967). In the tories for State IV oxygen uptake by mitochondrial presence of metabolic energy, inorganic phosphate preparations from endotherms show an inflection and ATP considerable amounts of Ca 2+ are taken essentially similar to plots of enzyme activity and of up from the external medium by a high affinity trans- other membrane-dependent phenomena (Lee & Gear, locase system, accompanied by a stoichiometric ejec- 1974; Lyons & Raison, 1970; Pye et al., 1976). In tion of protons and stimulation of oxygen uptake. sharp contrast with this I, myself, have always found Glycoproteins have been implicated as possible Ca 2 + that liver mitochondri a from ~t variety of endothermic carriers in the mitochondrial membrane but it is by vertebrates show a linear relationship for State IV no means certain that a mobile or immobile carrier respiration with overall E A values very similar to rather than a specific superiieial receptor is the basis those for ectothermic material (Smith, 1973, 19741 In of this transport mechanism (Carafoli & Crompton, the present experiments the rate of State IV respir1976). It is, therefore, very pertinent to investigate the ation was always recorded prior to Ca 2+ addition effect of temperature-induced transitions of the memand so the data was available for a re-examination brane lipids on the kinetics of calcium uptake. of its temperature dependence. 231 INTRODUCTION
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C. L. SMI~
MATERIAL AND METHODS Rainbow trout (S. oairdneri) were obtained from a hatchery and kept under 8 hr day-length conditions at 10°C. Rats were taken from general laboratory stocks as required.
M itochondria The procedure for isolation of mitochondria from liver tissue was basically the same as that described previously (Smith, !973~ The isolation medium contained 250raM sucrose, 10 mM Tris-HCl and 0.5 mM EDTA and the mitochondrial pellet was washed once in the same medium. The mitochondria were then washed again in 250raM sucrose plus 10 mM Tris-HCI only and the final suspension was made in the same solution in the proportions of 1.0 or 0.5 ml per gram of original tissue for rat or trout respectively.
rate evoked by A23187. It was found that rainbow trout mitochondria became increasingly susceptible to damage by the Ca 2+ load at higher temperatures as the preparation aged. This was manifested by a decline in the State IV rate after Ca 2+ addition and failure to respond fully or even at all to subsequent addition of A23187, ADP or DNP. However, using two oxygen electrodes a full temperature profile could be obtained before there was any evidence of this increase in fragility. It is perhaps of interest to note that when increased susceptibility of Ca 2 + damage was already very evident parallel observations on the same preparation showed no change in either acceptor control or P : O ratios as determined by ADP addition.
Direct measurement of calcium uptake Madeira (1975) has described the application of a Radiometer calcium electrode to the monitoring of Ca 2+ uptake by sarcoplasmic reticulum membranes, showing that the response time of this model was fast enough to follow the rapid kinetics of the system. As a check on Measurement of oxygen uptake my earlier measurements of respiratory stimulation during Oxygen uptake was recorded polarographically using calcium uptake I have used this electrode (F 2112 Ca SelecYellow Springs Instrument Co. oxygen electrodes. Chance trode) to follow the removal of added calcium from the (1965) pointed out that the response time of a Teflonexternal medium by mitochondria. The method was essencovered Clark type oxygen electrode may be too slow to tially the same as that described by Madeira (1975) except register accurately the very rapid and short lasting stimuthat a Pye Dynacap pH meter switched to pH input was lation of oxygen uptake following the addition of low conused. As this meter requires a low impedance connection centrations of calcium to a mitochondrial suspension. In to the output a 2 kt) multi-turn potentiometer, as well as the present work the electrodes were thoroughly cleaned the circuitry for the backing off voltage, was interposed with dentifrice and a thin Teflon membrane (12.5#m) between the meter and a Beckman Log/Linear recorder. lightly stretched over them and secured in the usual way. A calibration curve relating pH meter readings to the This procedure gave a very sensitive and fast responding concentration of free C a 2+ in the system was established electrode system and at the relatively high Ca 2 + concen- by means of an EGTA calcium buffer series (Portzehl et trations used there was no reason to believe that a true al., 1964) over the range 3.30-7.06 pCa. This enabled the estimate of oxygen-uptake stimulation was not being free Ca 2 + concentration of the reaction mixtures presented obtained. This is borne out by the experimental records~ to the witochondria to be estimated. Calcium binding to shown in Fig. 1 where the trace very rapidly changes tot various constituents of the mixture, and in particular to a steeper, linear slope after Ca 2+ addition. The reaction 1 mixture for Ca 2 + uptake experiments was based on that of Chance (1965) and contained 80raM KC1; 20raM Tris-HCl; 5 mM inorganic phosphate; 7 mM sodium glu(a) A tamate; 7 mM sodium succinate; 3.5~t.0 rag witochondrial protein; the pH was 7.4 and the final volume 3.0 ml. In most of the experiments 0.55 mM ATP was included in the reaction mixture (Rossi & Lehninger, 1954; Drahota et al., 1965). Details of other reaction mixtures are given in the legends to the appropriate figures. The general experimental procedure is best illustrated by the records of actual experiments with rat and rainbow trout mitochondria which have been combined in Fig. 1. After incubating the mitochondria in State IV for 2.0 rain an amount of Ca 2 + (432 nmoles) was added from a Repette (Jencons) which preliminary trials had shown to be required for approximately half maximal stimulation of oxygen uptake. Both preparations responded with a marked increase in respiratory rate and the traces show that there was an initial linear phase lasting some 5 sec at 25°C, after which they began to level off and regained the initial State IV rate about 10-12 sec after the calcium load was presented. In other experiments it was shown that both kinds of mitochondria would respond to a Time second addition of calcium in a quantitatively similar manFig. 1. Nimulation of mitochondrlal respiration by calner indicating that the concentration used was not so high that it damaged the witochondria. After a further cium and by the ionophore A23187. Record Ca) is from an experiment with rat and (b) from one with rainbow 1.5-2.0win of State IV respiration the ionophore A23187 trout liver mitochondria. At the points marked 'A' (0.48 nmoles) was added in t h e same way using a stock solution prepared in ethanol. Again the dose was selected 432 nmoles Ca 2 + were added while points 'B' denote the addition of 0.48 nmoles of A23187. The basic reaction mixby preliminary trials so that the consequent increase in ture contained 80 mM KCI, 20 mM Tris--HCl, 5 mM inoroxygen uptake was sub-maximal, as can be seen from the ganic phosphate, 7 mM Na glutamate, 7 m M Na succinate, further increase elicited by a second addition made to the 0.55 mM ATP, 3.5-3.7 m& witochondrial protein, pH 7.4 rat preparation (Fig. la), and total volume was 3.0 nil. In both cases the temperature Thus at the conclusion of each run measurements could was 25°C and the figures alongside each section give the be made of the respiratory rates in State IV, the maximum oxygen uptake rates in ng.atoms win- i mg P - t. initial rates after Ca 2 + addition, and the maintained steady
(b)37A~212 ~
233
Temperature dependence of mitochondrial membrane systems the 0.55 mM ATP used in most of these experiments, leads to the free calcium being' very much lower than the total calcium concentration (Reed & Bygrave, 1975). For example addition of 72/aM calcium (total concentration) to the reaction mixture complete with ATP and substrates yielded an electrode output coi:responding to 13/aM free Ca 2 +. When estimating the rate of calcium uptake by mitochondria the calcium and calomel electrodes were immersed in 3.0 ml of the same basic reaction mixture used for oxygen-uptake stimulation measurements which was contained in an oxygen electrode reaction chamber and was continuously and rapidly stirred by the magnetic follower. By initial trials the sensitivity of the system was adjusted so that the desired final concentration of calcium gave an approximately full scale deflection on the recorder. At the start of each determination successive additions (usually 108 nmoles) of a standard CaCl 2 solution were made until the desired final total concentration was attained. The electrode response was linear for equal increments of calcium over the concentration range used and the system was quite stable for the whole experimental period. Repetition of this procedure for each determination provided a calibration trace for the electrode response at the particular operating temperature, which was used for calculating the subsequent rate of calcium uptake. Calcium uptake was initiated by the rapid addition of mitochondria to the reaction system. The response was immediate and very fast at the higher temperatures used, but tests made by adding EDTA instead of mitochondria showed that the electrode system was certainly not the rate limiting step. The slope of the initial, linear, part of the trace after mitochondrial addition was, therefore, assumed to indicate the maximum initial rate of calcium uptake by the particles. For rat mitochondrial preparations the total Ca2 + concen, tration in the medium before addition of mitochondria was 144/aM and the free Ca 2 + concentration 25 #M (from the electrode calibration curve)~ Again the rainbow trout preparations were more unstable and apparently even more so than when the oxygen uptake was being monitored in the earlier experiments. When freshly isolated they would take up and hold 144/d~i Ca 2+ but within 1 hr after isolation records at temperatures above 20°C showed an initial rapid uptake which gave way to a rapid elflux of the ion. This indicated irreversible damage to the mitochondrial organization and made estimation of initial uptake rates very dubious. It was found that trout preparations would maintain their ability to take up and retain a 72 ~ total Ca 2+ load (13 p.M free Ca 2+) for at least 3hr after isolation which allowed adequate time to cover the full temperature range. Replicate determinations at one of the upper temperatures were made at the beginning and end of each series and showed that Ca2 + uptake activity was unimpaired. Protein content
The protein content of all preparations was determined by a biuret method (Smith, 1973). Chemicals
Standard chemicals used were B.D.H. Analar grade when this was available. ATP was obtained from Sigma Chemical Co. Ltd., and the ionophore A23187 was a generous gift from Dr. Robert Hamili, Eli Lilly Co., Indianapolis, U.S.A. to Prof. C. J. Duncan. RESULTS
Arrhenius plots of the oxygen uptake by liver mitochondria from both rat and rainbow trout when in State IV are shown in Fig. 2. The trout data (A) and that for one of the rat curves (B) were obtained in
the course of Ca 2+ uptake and release experiments in which t h e KCI-Tfis-PO4 medium was used (see Methods~ Clearly in neither case is there any suggestion of an inflection or break within the temperature range 5°-35°C, nor d o the E,4 values differ significantly. In view of this confirmation of my previous experience some further experiments were made using rat mitochondria suspended in the reaction mixture described by Raison et al. (1971). In the present context perhaps the most significant feature of this mixture is that it contains Mg 2+ which could arguably affect the temperature dependence of State IV respiration by stimulating endogenous ATP-ase activity. In Fig. 2 the two upper lines are based on the data for the rates of oxygen uptake by a typical preparation before (C) and after (D) the phosphorylation of exogenous A D P (204/aM). The rate after added A D P had been phosphorylated was significantly higher than that recorded before any A D P was added, as is apparent from Fig. 2 where both these sets of data are plotted on the same ordinate. This increased respiration after phosphorylation of A D P was almost completely inhibited when N a F (12raM) was included in the reaction mixture indicating that it is indeed largely attributable to ATP-ase activity. It should be borne in mind that the KC1-Tris-PO4 mixture contained 0.55 m M A T P so that the initial State IV rates of lines A and B could be considered to be more comparable to the post-ADP rates for the other reaction mixture (i.e. line D). However, comparison of the actual rates of oxygen uptake when parallel observations were made on the same preparation indicated that in the absence of M g 2+ the ATP-ase activity was low. F r o m Fig. 2 it is quite
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C. L. SMITH
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Fig. 3. Arrhenius plots of calcium uptake by liver mitochondria from rainbow trout. O-----O, increase in oxygen uptake rates following addition of Ca 2÷ to State IV mitochondria (mean data for three preparations). I - - I , initial rates of Ca2+ uptake (nmolesmin-lmg P-~) as recorded by a calcium electrode on adding mitochondria to the complete reaction mixture (means for a single preparation). Basic reaction mixture was the same as in Fig. 1. Total Ca 2+ added was 216nmoles and the free Ca2+ concentration was 13#M (see text). Activation energies (kJ mole- 1 + S.E.) are given alongside each curve with the number of observations in parentheses. clear that the stimulation of ATP-ase activity by Mg 2+ does not affect the temperature dependence as neither set of data shows any significant departure from linearity (C and D). The two EA values are also virtually the same and they do not differ significantly from those found for the rat and trout mitochondria in the KCI-Tris--PO4 mixture (B and A). In Figs. 3 and 4 Arrhenius plots illustrate the temperature dependence of calcium uptake by rainbow trout and rat mitochondria respectively. In each figure there are separate plots for oxygen uptake following Ca 2÷ addition and for the initial rate of Ca 2÷
uptake as measured with the calcium electrode. The oxygen uptake rates were calculated by subtracting the initial State IV rate from the maximum rate recorded after Ca 2÷ addition, that is they represent the increase in respiration, which should be entirely attributable to Ca 2÷ uptake. Considering Fig. 3 first, the two methods of monitoring Ca 2÷ uptake show an essentially similar temperature dependence as both are linear over the experimental temperature range and the E,~ values do not differ significantly. In Fig. 4 the temperature relationship for rat mitochondria is more complex as the Arrhenius plots for both methods show two breaks, one occurring at approximately 11°C and the other at 23°C. Regression analysis shows that the differences in slope of the sections of the curves are statistically significant at the 0.001% level for the electrode readings and at approximately the 0.02% level for increase in oxygen uptake. The actual rate of Ca 2÷ uptake at 25°C by rat mitochondria ( 9 3 4 n m o l e s m i n - l m g P-1) is somewhat faster than that by the trout preparation at the same temperature (759 nmoles min- 1 mg P - ~) but this would be at least partly accounted for by the lower Ca :÷ concentration used with the more unstable trout organelles. However, owing to the marked difference in temperature dependence this situation is reversed at 5°C where the corresponding rates for rat and trout are 117 and 309nmoles min-~ mg P-1 respectively. This could be an important feature of Ca 2÷ sequestration in the intact cells of ectothermic animals if the mitochondria play a major role in the homeostatic maintenance of the cytoplasmic Ca 2÷ concentration (Carafoli & Crompton, 1976; Bode & Anderson, 1976). Figure 5 shows Arrhenius plots for the maintained stimulation of oxygen uptake when a sub-maximal dose of A23187 (0.48 nmoles) was added to suspensions of either rat or rainbow trout mitochondria which had been pre-loaded with Ca: ÷ (432 nmoles). As in Figs. 3 and 4 the oxygen uptake rates plotted
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Fig. 4. Arrhenius plots of calcium uptake by rat liver mitochondria. O------O,increase in oxygen uptake rates following addition of Ca2÷ to State IV mitochondria (means for a single preparation). I - ~ - I , initial rates of Ca2÷ uptake as recorded by a Ca2÷ electrode on adding mitochondria to the complete reaction mixture (mean data for two preparations). Basic reaction mixture as in Fig. 1. Total Ca2÷ added was 432 nmoles and the free Ca 2÷ concentration was 25 #M. The number of observations on which the activation energies (kJ mole- 1 ___S.E.) are based are given in parentheses.
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Fig. 5. The effect of temperature on the stimulation of mitochondrial oxygen uptake by the ionophore A23187. The mitochondria were loaded with 432 nmoles Ca 2* prior to addition of 0.48 nmoles of A23187. Other conditions as in Fig. 1. V - - V , rat liver mitochondria (means for a single preparation); e - - - - ~ , rainbow trout liver mitochondria (mean data for three preparations). Activation energies (kJ mole- 1 + S.E.) are given alongside the curves with the number of observations in parentheses.
Temperature dependence of mitochondrial membrane systems were derived by subtracting the post-Ca 2+ State IV rates from those recorded after A23187 addition (cf. Fig. 1). The mean values for three trout preparations show a linear relationship between 3 ° and 30°C, but there is a sharp decrease in oxygen uptake at 35°C which probably indicates that mitochondrial efficiency is becoming impaired by the combination of the Ca 2+ load and the high temperature. The data shown for stimulation of rat mitochondrial respiration during Ca 2+ uptake (Fig. 4) and following A23187 addition (Fig. 5) are from the same experimental series based on a single preparation. Despite this the temperature dependence of the two systems is clearly different as the response to A23187 has only a single inflection, at 18°C, in the Arrhenius plot. This difference has also been seen in analogous experiments with other rat preparations. The activation energy (E4) for rat mitochondria above the inflection temperature is very similar to that for trout mitochondria over the whole range, but is more akin to the EA for calcium uptake over the middle rather than the upper part of the temperature range (Fig. 4). DISCUSSION The discrepancy between my data for State IV respiration by rat mitochondria and that reported by other workers (Lee & Gear, 1974; Lyons & Raison, 1970; Pye et al., 1976) remains unexplained. Yet the question whether such oxygen uptake does or does not give an inflected Arrhenius plot is not an unimportant one. In State IV the mitochondria are supplied with oxygen, inorganic phosphate and an oxidizable substrate but lack ADP or other suitable energy acceptor and it is generally thought that the low level of respiration which does occur is due to energy dissipation processes operating across or within the inner membrane (NichoUs, 1974; Stucki, 1976). A i g 2+ activated ATP-ase acting on either endogenous or exogenous ATP can certainly increase State IV respiration and I thought that this might account for the above discrepancy. However, Fig. 2 (D) clearly shows that this is not the ease as I still obtained a linear relationship despite clear evidence of ATP-ase activity. Re-cycling of Ca 2+ ions across the inner membrane could also contribute to State IV oxygen uptake, but Fig. 4 shows that Ca 2+ uptake by rat liver mitochondria has a quite complex temperature dependence and is therefore unlikely to be a major contributor to the respiration recorded in my systems. Probably the major cause of energy dissipation under State IV conditions is either recycling of H + ions across the coupling membrane or spontaneous hydrolysis of groups such as X ~ I, according to which theory of energy transduction is favoured (Greville, 1969). Lee & Gear (1974) who found two discontinuities in the temperature dependence of State IV, and also of uncoupler-stimulated, respiration concluded that they reflected a general temperature effect on the coupling membrane. If, however, as I have found, such discontinuities do not occur in the State IV temperature relation then it indicates that the energy dissipating processes taking place are passive in nature and, unlike the activities of membrane-bound enzymes and mobile carriers, are not affected by perturbations in the lipid milieu. The similarity of the
235
Arrhenius plots and EA values for rat and trout mitochondria (Fig. 2) is also consistent with this view as the respective membrane lipids certainly have different properties. There have been recent reports of breaks in the temperature dependence curves for the activity of membrane-bound enzymes in mitochondria from tench muscle (Pye et al., 1976) and from liver and muscle of carp (Wodtke, 1976). My data for Ca 2+ uptake by rainbow trout liver mitochondria (Fig. 3) show no departure from linearity between 3 ° and 35°C. In this respect this system conforms with others I have investigated in trout mitochondria, but the E A value, while similar to that for NADH oxidase (31.86 kJ mole- t), is lower than that for succinoxidase (49.57kJmole-1)~ for valinomycin stimulation of oxygen uptake (40.32 kJ mole-t), for State III respiration (58.11 kJ mole-1)and for the respiratory stimulation by A23187 shown in Fig. 5 (Smith, 1977, 1974, 1973). The Ca 2+ uptake system in rat mitochondria (Fig. 4) is the only one for which I have found changes in the slope of the Arrhenius plot at more than one temperature. Raison & McMurchie (1974), however, have described Arrhenius plots for State III respiration by both sheep and rat liver mitochondria which showed breaks or inflections at two temperatures which were coincident with changes in the molecular ordering of the membrane lipids as detected by spin labelling. They concluded that there was a continued shift in the equilibrium between gel and liquid-crystalline phases of the lipids in the temperature zone between the inflections. Lee & Gear (1974) confirmed the presence of double inflections in the Arrhenius plots for several systems in rat liver mitochondria including that for respiration-driven Ca 2+ uptake as monitored by proton ejection. For the latter the lower break was at 12.5°C (Tt) and the upper one at 26.5°C (7"2) with corresponding EA values of 105.9, 65.7, and 23.9 kJ mole -~. This position of T1 and T 2 on the temperature scale shows an upward displacement of 2-3 ° compared with my data in Fig. 4, while the EA values though not identical, are of a similar magnitude. The presence of two points where temperature dependent changes affect the kinetics of the C a 2+ uptake system seems, therefore, to be confirmed. However, this is not the case for other systems as my earlier data for State III respiration, transport of ions by exogenous ionophores, and succinoxidase activity showed only one inflection (Smith, 1977, 1974, 1973). Figure 5 is of particular interest in this context as the rates of A23187-stimulated respiration were recorded from the same mitochondrial suspensions as those for increase in oxygen uptake when a Ca 2+ load was being taken up (Fig. 1)~ Despite this identity of the experimental conditions the temperature dependence curve for Ca 2+ transport by the exogenous ionophore has only one inflection at the intermediate temperature of 18°C. This suggests that the endogenous Ca 2 + uptake mechanism shows a change in apparent EA at the beginning and end of the phase transition zone of the membrane lipids, while perhaps other systems are less susceptible and may respond in an all or none manner only when the phase change in the lipids is nearer completion. Alternatively if a glycoprotein is involved in the molecular system for
C. L. S~lrn
236
Ca 2÷ uptake then it may be that one of the observed inflections is due to temperature-induced conformational changes in the protein moiety which may or may not be related to the lipid phase change (see also Smith, 1976). It is worthy of note that the present data for rat and rainbow trout show there are no significant differences between these species when EA values for either the Ca 2 + uptake system or for A23187 translocation of Ca 2+ are compared at the normal operating temperatures of the host cells (Figs. 3-5). This would suggest at least a measure of identity between the kinetics of both these translocases in ecto- and endothermic cells when the membrane lipids are in the liquid-crystalline phase. I have previously pointed out a similar situation for other mitochondrial systems though this is not entirely supported by EA values recorded in the literature (see Smith, 1977, 1973). The ionophore A23187 stimulates mitochondrial oxygen uptake by virtue of its ability to create a cyclic, energy-dissipating flux of mitochondrial calcium (Reed & Lardy, 1972). In the present work it has been assumed that, provided a sub-maximal amount is used, the rate limiting process in the stimulation of oxygen uptake will be the outward movement of Ca 2 + ions across the mitochondrial membrane by the ionophore. Therefore the Arrhenius plot should depict the temperature dependence of this translocase rather than that of the Ca 2+ uptake system. I have previously used a similar approach to investigate the effect of temperature on the activity of the ionophorous antibiotics valinomycin and gramicidin. Valinomycin is generally regarded as a mobile cartier which transports K + ions across the membrane while gramicidin, which is less specific, forms pores in t h e membrane through which ions diffuse. A number of features of the action of A23187 suggest that it operates as a mobile carrier (Case et al., 1974) so that it is interesting to find that its temperature dependence (Fig. 5) is much more similar to that of valinomycin than that of gramicidin. This is particularly the case for the apparent EA values above the inflection point which were 46.84 and 13.19 kJ molerespectively for these two antibiotics (Smith, 1974). Case et al. (1974) examined the effect of temperature on the rotational relaxation rate of A23187 in rat liver mitochondrial membranes and found a marked inflection at 25°C with an Ea value over the upper temperature range very similar to that of Fig. 5. However, they also stated that unpublished results indicated that the kinetics of Ca 2+ effiux induced by A23187 were not affected by this apparent transition in the membrane lipids, Although the transition temperature from my data is lower than that found by Case et al. they do nevertheless suggest, contrary to their conclusion, that the physical state of the membrane affects translational as well as rotational diffusion.
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CARAFOLI E. ,~ CROMPTONM. (1976) Calcium ions and mitochondria. In Calcium in Biological Systems (Edited by Dtmc^N C. J.), Symposia of the Society for Experimental Biology No. 30, pp. 89-115. University Press, Cambridge. CASEG. D., VANDERKOOXJ. M. & SCARPAA. (1974) Physical properties of biological membranes determined by the fluorescenceof the calcium ionophore A23187. Archs Biochem. Biophys. 162, 174-185. CHANCE B. (1975) The energy-linked reaction of calcium with mitochondria. J. biol. Chem. 240, 2729-2748. DRAHOTAZ., CARAFOUE., ROSS1C. S., GAMBLER. L. & L~NINGER A. L. (1965) The steady state maintenance of accumulated Ca z+ in rat liver mitochondria. J. biol. Chem. 240, 2712-2720. GREWLLE G. D. (1969) A scrutiny of Mitchell's chemiosmotic hypothesis of respiratory chain and photosynthetic phosphorylation. In Current Topics in Bio-eneroetics (Edited by SA~ADID. R.), Vol. 5, pp. 1-78. Academic Press, New York. LEE M. P. & Go~R A. R. L. (1974) The effect of temperature on mitechondrial membrane-linked reactions. J. biol. Chent 249, 7541-7549. LEnNI~OERA. L., CAaAEOLIE., & ROSS1C. S. (1967) Energy-linked ion movements in mitochondrial systems. Adv. Enzymol. 29, 259-320. LYONSJ. M. & RAISONJ. K. (1970) A temperature-induced transition in mitochondriai oxidation: contrasts between cold and warm-blooded animals. Comp. Biochem. Physiol. 37, 405-411. MAOEmA V. M. C. (1975) A rapid and ultrasensitive method to measure Ca 2+ movements across biological membranes. Biochem. biophys. Res. Commun. 64, 870-876. NICHOLLS D. G. (1974) The influence of respiration and ATP hydrolysis on the proton-electrochemical gradient across the inner membrane of rat-liver mitochondria as determined by ion distribution. Eur. J. Biochem. ~0, 305-315. PORTZ~L H., CALDWELLP. C. & RuEc_~ J. C. (1964) The dependence of contraction and relaxation of muscle fibres from the crab Maia squinado on the internal concentration of free calcium ions. Biochim. biophys. Acta 79, 581-591. PYE V. I., WidER W. & ZEeH M. (1976) The effect of season and experimental temperature on the rates of oxidative phosphorylation of liver and muscle mitochondria from the tenth Tinca tinca. Comp. Biochem, Physiol 54B, 13-20. RAISONJ. K. (1973) The influence of temperature-induced changes on the kinetics of respiratory and other membrane-associated enzyme systems. Bioenergetics 4, 285-309. RAISONJ. K., LYONSJ. M. & THOMSONW. W. (1971) The influence of membranes on the temperature-induced changes in the kinetics of some respiratory enzymes of mitochondria. Archs Biochem. Biophys. 142, 83-90. RAISONJ. K. & McMuRcHn~E, J. (1974) Two temperatureinduced changes in mitochondrial membranes detected by spin labelling and enzyme kinetics. Biochim. biophys. Acta 363, 135-140. REED K. C. & BYGRAVEF. L. (1975) A kinetic study of mitochondrial calcium transport. Eur. J. Biochem. 55, 497-504. REED P. W. & LARDY H. A. (1972) A23187: a divalent cation ionophore. J. biol. Chem. 2,47, 6970--6977. Rossl C. S. & L~HNIt~GERA. L. (1964) Stoichiometry of respiratory stimulation, accumulation of Ca2 ÷ and phosphate, and oxidative phosphorylation in rat liver mitochondria. J. biol. Chem. 239, 3971-3980. S~llrd C. L. (1973) The temperature dependence of oxidative phosphorylation and of the activity of various enzyme systems in .liver mitochondria from cold- and
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STUCm J. W. (1976) Efficiency of oxidative phosphorylation and energy dissipation by H ÷ ion recycling in ratliver mitochondria metabolizing pyruvate. Eur. J. Biochem. 6S, 551-562. WoD'rr~ E, (1976) Discontinuities in the Arrhenius plots of mitochondrial membrane-bound enzyme systems from a poikilotherm: acclimation temperature of carp affects transition temperatures. J. comp. Physiol. !10, 145--157. WoNo D. T., WILKINSON J. R., HAM_ILLR. L. & HORNG J.-S. (1973) Effects of antibiotic ionophore, A23187, on oxidative phosphorylation and calcium transport of liver mitochondria. Archs Biochem. BMphys. 156, 578-585.
