Metabolic specialization of mitochondria from scallop ... - Springer Link

Marine Biology (1995) 122:409-416

9 Springer-Verlag 1995

H. E. Guderley 9F. M. Rojas 9 O. A. Nusetti

Metabolic specialization of mitochondria from scallop phasic muscles

Received: 26 September 1994 ! Accepted: 6 December 1994

Abstract In fast, glycolytic muscles, oxidative phosphorylation presumably facilitates recuperation from exhaustive exercise and supports growth and maintenance metabolism. Given the shifts in pH with extensive glycolytic activity, the pH optima of mitochondrial processes should indicate whether mitochondria are adapted for recuperation from exercise or for growth and maintenance. We examined this question using mitochondria from the phasic adductor muscle of the scallop, Euvola (Pecten) ziczac, collected from the Golfo de Cariaco, Venezuela in 1992 and 1993. Scallop muscle mitochondria showed well coupled oxidation of glutamate and pyruvate at pH 7.0 and 6.4. The preferred substrates (glutamate, pyruvate and succinate) were oxidized at approximately 40 nmol 0 2 min -t mg -1 mitochondrial protein at 25 ~ while malate and glutamine were oxidized at - 75% and proline at - 30% of these rates. Neither palmitoyl carnitine nor aspartate were oxidized. Succinate oxidation was not coupled to ADP utilization at pH 7.0 but was somewhat coupled at pH 6.4. Generally, State 3 rates of oxygen uptake were similar at pH 7.0 and 6.4. Maximal rates of oxidation of glutamate and pyruvate showed broad pH optima. For both glutamate and pyruvate, the highest respiratory control ratio (RCR) values were found at pH 6.5. The saturation curves of scallop muscle mitochondria for pyruvate, glutamate and ADP were well described by the Michaelis-Menten equation. The affinity for pyruvate was greater at pH 6.4 (apparent Km, app=0.013 mM) than at pH 7.0 (Km.app=0.026 mM) while the affinity for A D P (Km,app= 0.015 In/I//) and that for glutamate (Km,app=0.55 mM) changed little with pH. The ADP affinity was the same whether pyruvate or glutamate was the carbon substrate. The combination of maintenance of sensitivity to ADP with an enhanced affinity for pyruvate at acidic pH values should facilitate recuperation

Communicated by R. J. Thompson, St. John's H. E. Guderley (N) Ddp. de Biologie, Universit6 Laval, Qu6bec, R Q. Canada H. E. Guderley 9E M. Rojas 9O. A. Nusetti Escuela de Ciencias, Universidad de Orienta, Cumana, Venezuela

from bouts of glycolytic activity. Scallops harvested in September and those harvested in January differed in the maximal rates of glutamate and pyruvate oxidation.

Introduction In fast glycolytic muscles, contractile activity depends upon ATP generation from phosphagen stores and from anaerobic glycolysis. In these muscles, contractile activity is of too high an intensity to be supported solely by mitochondrial ATP production. Recuperation from short bursts of activity ultimately relies upon ATP production by mitochondria. As recharging the adenylate pool is a major role of mitochondria in fast glycolytic muscles, mitochondrial levels and properties in these muscles may be set to facilitate recuperation from burst exercise. Nonetheless, in trout white muscle, the oxidative capacity of mitochondria is far higher than required to support the rates of oxygen consumption encountered during recuperation from exhaustive exercise (Moyes et al. 1992). Mitochondria also support energetically costly processes such as protein synthesis, suggesting that their properties reflect the need for aerobic ATP generation in support of growth. The scallop phasic muscle is used for burst swimming activity during the escape response, while the tonic muscle is used both during catch closure and burst swimming (Livingstone et al. 1981). During burst swimming, contractile activity in these muscles is powered by arginine phosphate utilization followed by glycolytic ATP production during activity and during the initial recuperation period (de Zwaan et al. 1980; Livingstone et al. 1981). This glycolytic activity leads to acidification of the intracellular environment. Subsequently, the levels of arginine and anaerobic end products decline while arginine phosphate level rise during the final recuperation process. The simultaneous return of muscle metabolite levels and blood oxygen levels in the adductor muscle sinus to pre-exercise values suggests that complete recuperation depends upon oxidative processes (Livingstone et al. 1981). Thus, in the

410 phasic muscle, contractile activity is directly supported by arginine phosphate b r e a k d o w n and oxidative m e t a b o l i s m only supports the final recovery. G i v e n that recuperation from episodes of intense anaerobic m e t a b o l i s m starts at low pH values (Hochachka and M o m m s e n 1983), specialization of m i t o c h o n d r i a in fast glycolytic muscles for participation in recuperation should be reflected by acidic pH optima for the parameters, controlling the oxidation of anaerobic end products. These parameters i n c l u d e the m a x i m a l oxidative capacity, the coupling of oxidative phosphorylation as well as the affinities of m i t o c h o n d r i a for their carbon and adenylate substrates. As m a n y anaerobic end products yield pyruvate, m i t o c h o n d r i a specialized for facilitating recuperation should favor pyruvate oxidation at low pH values. By contrast, oxidation of a m i n o acids during growth or osmoregulatory processes is less likely to occur at acid pH and could therefore demonstrate a higher pH optimum. Considerable data are available in the literature c o n c e r n i n g the pH d e p e n d e n c e of m a x i m a l rates of substrate oxidation for invertebrate and fish m i t o c h o n d r i a (Ballantyne and Storey 1983, i984, 1985; B a l l a n t y n e and M o o n 1985; M o y e s et al. 1988, 1989). In contrast, few studies have e x a m i n e d the pH sensitivity of the regulatory properties such as mitochondrial sensitivities to carbon and adenylate substrate concentrations (apparent affinities). As it is u n l i k e l y that m i t o c h o n d r i a consistently operate at m a x i m a l rates in vivo, the pH sensitivity of substrate affinities m a y better reflect physiological specialization than pH effects on m a x i m a l rates of substrate oxidation. To e x a m i n e whether mitochondria in fast glycolytic muscles are specialized for participation in recuperation, we e x a m i n e d the pH sensitivity of substrate affinities and preferences of m i t o c h o n d r i a isolated from the phasic adductor muscle of the scallop Euvola (Pecten) ziczac. We chose the phasic adductor muscle since oxidative processes are not i n v o l v e d in l o c o m o tion, but rather in recuperation from locomotion, and since the phasic and tonic portions of the adductor muscle are easily separated.

Mitochondrial isolation Initial experiments established that scallop muscle mitochondria showed the best properties when isolated and tested in sucroseHepes, KC1 containing media, adapted from Ballantyne and Moon (1985) to account for the osmolality of the scallops' habitat. All media were prepared in triple-distilledwater. The isolation medium contained 480 mM sucrose, 30 mM Hepes, 230 mM KCI, 3 mM Na2 EDTA, 6 mM EGTA, 5 mM MgCI> 0.1% bovine serum albumin (BSA), pH 7.0 at 25 ~ Phasic muscles from two to three individuals were rinsed in 10 ml isolation medium and finely minced. The muscle mince was homogenized in 4 to 5 vol ice-cold isolation buffer and centrifuged at 900xg for 10 min. The supernatant was centrifuged at 10000xg for 10 min. The resulting pellet was resuspended in 1 vol of isolation medium without MgC12 and recentrifuged at 10000xg for 10 min. The final pellet was resuspended in a minimal volume of the Mg-free isolation medium containing 0.5% BSA. Mitochondrial preparations contained - 1 0 mg mitochondrial protein ml J.

Potarographic measurements Mitochondrial respiration was studied using chambers and oxymeters from Yellow Springs Instruments (Yellow Springs, Ohio, USA). Temperature was controlled at 25 -+0.5 ~ using a Haake circulating water bath. The reaction medium contained 480 mM sucrose, 70 mM Hepes, 158 mM KC1, 10 mM KHaPO4, 50 mM tanrine, 50 mM [3-alanine, isolation medium and that the volumes of mitochondria added to the reaction chamber differed slightly among experiments, the final concentrations in the reaction medium were approximately 480 mM sucrose, 66.3 mM Hepes, 165 mM KC1, 9.07 mM KH2PO4, 45.4 mM tanrine, 45.4 mM ~-alanine, 0.28 mM Na2EDTA, 0.56 mM EGTA, 0.5% BSA, pH 7.0 at 25 ~ The variation in exact concentrations due to differing volumes of mitochondria was at most 2%. The respiratory control ratios (RCR, the ratio of the rate of oxygen uptake in the presence of 600 gM ADP, State 3, relative to the rate of oxygen consumption when this ADP was depleted, State 4) were calculated according to Estabrook (1967) and were used to evaluate mitochondrial quality. Only mitochondrial preparations with RCR values _>4 with glutamate at pH 7.0 were used. Preliminary experiments established that NADH (0.2 raM) did not stimulate oxygen uptake with these mitochondrial preparations. For each carbon substrate, maximal rates of oxygen uptake were determined after the addition of 600 gM ADR Preliminary experiments established the substrate concentrations required for maximal rates of oxidation of the different substrates. To attain maximal rates of pyruvate oxidation, "sparking" levels of malate, which by themselves did not support significantrates of oxygen uptake, were required. Malate was not required to attain maximal rates of glutamate oxidation, pH values were adjusted at 25 ~ For pH curves, the exact pH was measured after the reaction.

Materials and methods Substrate affinities Specimen collection and acclimatization conditions Scallops were harvested from their natural beds in the Golfo de Cariaco, Venezuela using SCUBA in September 1992 (-25 ~ and in January 1993 (-23 ~ Wild scallops harvested in September were suspended at 30 m near the Marine Laboratory at Turpialito, Venezuela (mean temperatures -25 ~ to avoid the higher temperatures nearer the surface. This group of individuals was studied from September 1992 to January 1993. In January, the freshly harvested scallops were suspended at 16 m as, at this time, upwelling maintained temperatures at 23 to 25 ~ at this depth (Lodeiros and Himmelman 1994). These specimens were studied from January to March 1993. Salinity was 35 + 1%0 throughout the study. Before their use for experiments, the scallops were removed from the long-lines, transported by truck to the University laboratory and maintained in filtered seawater at 25 to 26 ~ for 2 to 3 d.

To determine the mitochondrial affinity for pyruvate, individual oxygen uptake curves were carried out for at least eight pyruvate concentrations in the presence of saturating levels of ADP. Oxygen uptake rates were followed for the least 5 rain. ADP-stimulated rates of oxygen uptake were corrected for the State 2 rates of substrate oxidation to evaluate mitochondrial affinity during coupled pyruvate oxidation. Pyruvate solutions were calibrated spectrophotometrically, using the lactate dehydrogenase reaction (Bergmeyer 1983). To determine the mitochondrial affinity for ADP, we followed a polarographic approach using the hexokinase reaction to maintain constant ADP levels. The above reaction medium was supplemented with 20 mM glucose, 10 mM MgC12 and 2 units hexokinase (yeast, Boehringer) m1-1. ADP saturation curves were determined starting with the lowest concentration of ADR followed by gradual additions of ADP to attain higher concentrations. A minimum of 10 ADP concentrations was used. For each concentration, oxygen

411 uptake rates were followed for at least 90 s before the addition of the next aliquot of ADR Preliminary experiments established that the oxygen uptake rate at a given concentration of ADP were linear for the duration of the determination of the saturation curves, as long as oxygen saturation levels remained above 5%. Therefore, no measurements were taken below 10% saturation. The ADP affinities determined using this incremental approach did not differ from those determined using individualcurves for each ADP concentration. The incremental approach gave Km app values with a higher coefficient of determination (iterative fitting of the Michaelis-Menten equation with the Marquardt algorithm, Regression, Blackwell Scientific Publications) than the use of individual curves for each ADP concentration. When pyruvate was the carbon substrate, its concentration was adjusted to 0.925 mM and malate was added to a final concentration of 0.6 mM. When glutamate was the carbon substrate, its concentration was 29 mM. ADP solutions were calibrated spectrophotometrically using the pyruvate kinase and lactate dehydrogenase reactions (Bergmeyer 1983). Glutamate saturation curves were carried out at 0.6 mM ADP with the additon of 20 mM glucose, 10 mM MgC12 and 2 units mlq hexokinase to maintain this ADP concentration. Saturation curves were determined starting with the lowest concentration of glutamate, followed by gradual additions of higher concentrations. A minimum of ten concentrations was used. For each concentration, oxygen uptake rates were followed for 90 s before the addition of the next aliquot. We calculated the glutamate concentrations on the basis of the amount added since depletion of glutamate by mitochondrial oxidation was negligible compared to the amount added. For calculation of glutamate affinities, ADP-stimulated rates were corrected for the oxygen uptake at the same glutamate concentration in the absence of ADR As for the ADP saturation curves, the solutions to the Michaelis-Menten equation determined using this approach had higher coefficients of determination than those determined using individual curves for each glutamate concentration. However, the Vmax values obtained by the incremental method were lower than those obtained in individual curves, possibly due to an inhibition of succinate dehydrogenase due to intramitochondrial accumulation of oxaloacetate (Bouwer and Van Den Thillart 1984) Protein concentrations Protein concentrations were determined using the biuret method with 10% deoxycholate to solubilize mitochondrial membranes (Robytt and White 1987). BSA was used as a standard. Mitochondrial protein concentrations were obtained by subtracting the concentration of BSA in the resuspension medium. Calculations and statistical analysis Oxygen uptake rates were calculated using the oxygen solubility values (x given in Graham (1987) for dextran-containingmedium. Since couloximetry indicates that BSA does not decrease the solubility of oxygen in KCl-based media (Johnston et al. 1994), we used the coefficients given by Graham without correcting for an effect of albumin. For all substrates, mitochondriat Km.appvalues were calculated by iterative fitting of the data for the substrate concentrations and oxygen uptake rates to the Michaelis-Menten equation using the Marquardt algorithm (Regression, Blackwell Scientific Publications). Statistical comparisons were carried out using Statview (Abacus Concepts, Brainpower Inc. Calabasas, California, USA).

Results

M i t o c h o n d r i a l isolation To isolate coupled m i t o c h o n d r i a from scallop muscle, we modified the procedure g i v e n by B a l l a n t y n e and M o o n

9 State3, pH 7.0 70

[]

State4, pH 7.0

60

[]

State3, pH 6.4

"g o 50

[] State 4, pH 6.4

0

L t

e0

0

,

glutamate malate

pyruvate glutamine suecinate

proline

Fig. 1 Euvola ziczac. Oxidation rates of various substrates by phasic muscle mitochondria isolated from scallops harvested in January 1993. Oxygen uptake measured polarographically as described in "Materials and methods". State 3 rates obtained at saturating levels of ADP, while State 4 rates were obtained after depletion of ADR Values expressed as nmol 02 min-1 mg t mitochondrial protein and given as means _+S.E. Statistical differences between values obtained at neutral and acidic pH values were evaluated with Mann-Whitney U-tests

(1985) by increasing the osmolarity of the isolation and reaction media, and i n c l u d i n g 5 m M MgC12 in the initial extraction m e d i u m . A second high-speed centrifugation was used to remove most of the MgC12. The m i t o c h o n d r i a isolated by this procedure were well coupled and m a i n t a i n e d respiratory control ratios, RCR, _>4.0 for a m i n i m u m of 6 h. All our experiments were completed in this timeframe.

Substrate preferences Substrate preferences studies, carried out on scallops collected in January, indicate that glutamate (29 raM), pyruvate (0.9 raM, in the presence of "sparking" levels of realate, 0.56 raM) and succinate (24 raM) were the substrates which led to the highest rates of A D P - s t i m u l a t e d oxygen uptake ( - 4 0 n m o l O z rain -1 mg -a m i t o c h o n d r i a l protein) (Fig. 1). G l u t a m i n e (12 raM) and malate (5.6 raM) were oxidized at approximately 75% of the rates found for glutamate, pyruvate and succinate, while proline (9.0 raM) was oxidized at roughly 35% o f these rates. Aspartate (18.1 raM) and palmitoyl carnitine (0.08 raM) did not stimulate respiration. For all substrates, State 3 rates of oxygen uptake were similar at pH 6.4 and 7.0. State 4 rates differed little b e t w e e n pH 6.4 and 7.0, except in the case of succinate, where State 4 rates were c o n s i d e r a b l y higher at neutral than at acidic pH. R C R values were highest with glutamate and pyruvate, intermediate for glutamine, realate and proline and lowest for succinate (Fig. 2). The effect of pH on the R C R values was only significant for succinate (Fig. 2).

412 40-

7 o

9

RCR, pH 7.0

[]

RCR, pH 6.4

30

eY

,0 1~,

~s 0

State 3 State 4

.E

"0 '0~

4

1

glutamate

m

/hlJ

pyruvate glutamine succinate

,= 2 0

204 0

10 E

I

I

proline e- 3 0

Fig. 2 Euvola ziczac. Respiratory control ratios (RCR; State 3/State 4 rates) for the oxidation of various substrates by scallop muscle mitochondria. Values expressed as means _+S.E. Statistical differences between values obtained at neutral and acidic pH values were evaluated with Mann-Whitney U-tests

I

I

13----

State 3

9

State 4

20

~T "~,_ E E 4J N ~0 10

The pH optima for maximal (State 3) rates of pyruvate and glutamate oxidation were broad (Fig. 3). Pyruvate oxidation declined more steeply at alkaline pH values than glutamate oxidation. State 4 of glutamate oxidation increased at alkaline pH values, whereas State 4 rates of pyruvate oxidation changes less with pH. As a result, the RCR values for pyruvate and glutamate were low at alkaline pH, increased with decreasing pH to a maximum near pH 6.5 and then decreased again with decreasing pH (Fig. 4).

Seasonal differences in mitochondrial properties Scallops harvested in September and those harvested in January differed in the maximal ADP-stimulated rates of oxidation of glutamate and pyruvate (p < 0.05) (Fig. 5). The pH sensitivity of State 3 and 4 rates of glutamate oxidation, and thus of the RCR values, was the same in mitochondria isolated from these two groups of scallops (Fig. 6). In contrast, the RCR values for pyruvate oxidation were more pH sensitive in September than in January scallops. This primarily reflected a drop in State 4 rates with a decrease in pH for mitochondria isolated from September specimens. Accordingly, the pH curves for the RCR values of mitochondria isolated from September scallops showed a somewhat steeper pH dependence than those for January scallops (data not shown). However, as the tendency for increases in RCR values with decreases in pH was clear in both groups of scallops, we show the combined data (Fig. 3).

pH sensitivity of apparent K m values for substrates Saturation curves for State 3 rates of pyruvate, glutamate and ADP oxidation were well described by the Michaelis-

E t-

I

;

7

i

;

9

pH

Fig. 3 Euvola ziczac, pH sensitivity of State 3 and 4 rates of glutamate and pyruvate oxidation by scallop muscle mitochondria. Values expressed as nmol 02 min- L mg- 1 mitochondrial protein and given as means -+SE

Menten equation as judged by the high coefficients of determination and the low standard deviations for catalytic constants obtained by iterative fitting of the substrate concentration and oxygen uptake data to this equation. In the case of glutamate oxidation, State 2 rates of oxygen uptake rose with increasing glutamate concentration and were saturated at lower glutamate concentrations ( - 0 . 5 mM) than State 3 rates (- 1.5 raM). State 2 and 4 rates of pyruvate oxidation were saturated at low pyruvate concentrations, with a Km, apv lower than the lowest pyruvate concentration tested (5 gM). The mitochondrial Kin, app for glutamate was approximately 0.5 mM and changed little with pH (Table 1). The Kin, app values of scallop muscle mitochondria for ADP (- 10 gM) also did not differ between pH 7.0 and 6.4. The Km, app for ADP was similar whether glutamate or pyruvate was the carbon substrate. The mitochondrial Km, app for pyruvate was also quite low (13 to 26 gM). In contrast with the other substrates, the Kin, app for pyruvate decreased markedly (p < 0.05) with a drop in pH. Mitochondrial sensitivites to ADP and glutamate were determined with scallops harvested in January. Pyruvate Kin, app values were determined both for September and January scallops; similar values and pH sensitivities were obtained for both groups.

413 60 6

~- 50

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State 3, pH 7.0 [] State 4, pH 7,0 []

State 3, pH 6.4

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o,, r

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State 3, State 4, State 3, State 4,

pH pH pH pH

7.0 7.0 6.4 6.4

~ ~.40

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30

20

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=El0 0 7 pH

8

9

fall

winter

Discussion

Fig. 5 Euvolaziczac. Seasonal differences in the State 3 and 4 rates of pyruvate and glutamate oxidation by scallop muscle mitochondria. Values expressed as nmol 02 min i mg-1 mitochondrial protein and given as means +S.E. Fall scallops harvested in their natural beds in September and suspended at 30 m in the Golfo de Cariaco until studied (November-mid-January). Winter scallops harvested in mid-January and suspended at 16 m until studied in Feburary and March. Statistical differences (p < 0.05) between seasons determined with a Mann-Whitney U-test and are shown by an *

The properties of scallop phasic muscle mitochondria should facilitate the muscle's recuperation from bouts of intense contractile activity. The decrease in the pyruvate Km,a p p value of scallop muscle mitochondria with a drop in pH should enhance pyruvate oxidation at the low pH values which can be encountered during recuperation from burst activity. Similarly, the increased RCR's for pyruvate and succinate oxidation at acidic pH values should facilitate oxidation of anaerobic end products. These mitochondrial properties should accelerate recovery and enhance the scallop's capacity to resume escape responses. The broad pH optimum for glutamate oxidation and the pH independence of the mitochondrial affinity for glutamate and ADP suggest that this apparent facilitation of recovery metabolism does not decrease the regulatory sensitivity or the capacity for oxidation of amino acids during growth and maintenance processes. The pH optima of maximal rates of substrate oxidation in a variety of invertebrate tissus are generally more alkaline than those we found for scallop phasic muscle mitochondria. In Mercenaria rnercenaria, mitochondria from the ventricle exhibit pH optima between 7.2 and 7.5 for

State 3 rates of oxygen uptake and RCR values at 15 ~ (Ballantyne and Storey 1983), whereas slightly lower pH values are optimal for mitochondria from the hepatopancreas (Ballantyne and Storey 1984). For Mytilus edulis, mitochondria from the hepatopancreas oxidizing glutamate show highest State 3 rates and RCR values at pH 7.5 to 7.8 at 5 ~ As mitochondria from carp, Cyprinus carpio, red muscle have highest values of State 3 oxygen uptake and RCR at pH 6.4 at 30 ~ (Moyes et al. 1988), their pH sensitivity is similar to that of scallop phasic muscle. This may reflect the carp's tolerance of hypoxic conditions which could lead to intraceltular acidification, even in the normally aerobic red muscle. The substrate preferences of scallop phasic muscle mitochondria suggest that the most important substrates for mitochondrial oxidation are derived from glycogen and proteins. While these mitochondria were only tested with palmitoyl carnitine and may be capable of oxidizing other acyl carnitines, the absence of acyl carnitine oxidation would not be surprising given the low lipid levels in this muscle (Brea and Nusetti unpublished data). Marked shifts in glycogen and protein contents of the adductor muscle

Fig. 4 Euvola ziczac, pH sensitivity of respiratory control ratios (RCR) of scallop muscle mitochondria oxidizing glutamate and pyruvate. Values expressed as means + SE

414 Table 1 Euvola ziczac. pH sensitivity of the apparent Km of mitochondria from the scallop phasic adductor muscle for pyruvate, glutamate and ADR Values given in mM and shown as the mean +-SE with the number of samples given in parentheses

8

.2

[] [] [] []

pyruvate glutamate Kin,app ADP Cosubstrate, pyruvate Cosubstrate, glutamate Kin, app K m , app

glutamate RCR,pH 7.0 glutamate RCR,pH 6,4 pyruvate RCR, pH 7.0 pyruvate RCR, pH 6.4

er 6 '

O

~- 2' ev,

fall

winter

Fig. 6 Euvola ziczac. Seasonal differences in the respiratory control (RCR, State 3/State 4) for scallop muscle mitochondria oxidizing pyruvate and malate. Values expressed as means + S.E. Statistical differences (p < 0.05) between seasons determined with a MannWhitney U-test and are shown by an *

during short (5-d) periods of starvation and refeeding (Marcano and Nusetti unpublished data) indicate rapid substrate mobilisation in this muscle as found in Argopecten irradians concentricus (Barber and Blake 1981) and A. irradians irradians (Epp et al. 1988). These substrates could be oxidized in situ in the muscle or could be exported to support the metabolic requirements of other tissues. The maximal rates of oxygen consumption by scallop muscle mitochondria, during glutamate, pyruvate or succinate oxidation, are similar to those obtained for muscle mitochondria from invertebrate and fish species. For mitochondria isolated from the ventricle of Mercenaria mercenaria and respiring proline at 15 ~ Ballantyne and Storey (1983) obtained maximal rates of 2 0 . 0 n m o l 02 min q mg -1 protein. Mitochondria from carp red muscle oxidize pyruvate plus malate at a rate of 27.3 __+3.4 nmol 02 min - a m g -1 protein at 15 ~ (Moyes et al. 1989). For mitochondria from trout (Oncorhynchus mykiss) white muscle, the equivalent rate was 33.25 +__2.45 (Moyes et al. 1992). Mitochondria from trout red muscle oxidize pyruvate at 33 nmol 02 rain - l m g -1 protein at 15 ~ (Blier and Guderley 1993a). Mitochondria from red muscle of Oreochromis atcalicus grahami and O. andersoni oxidize pyruvate at 50 nmol 02 min -a mg q protein at 25 ~ (Johnston et al. 1994). Nonetheless, the maximal capacities of muscle mitochondria from ectothermal animals are generally lower than those obtained for mitochondria from rat red muscle (Davies et al. 1981; Gollnick et al. 1985).

pH 6.4

pH 7.0

0.013 + 0.006 (4) 0.584+0.212 (3)

0.026 _+0.008 (4) 0.551 +0.178 (3)

p < 0.001 NS

0.0136_+0.0023 (4) 0.0115 _+0.0024 (4)

0.0152+0.0025 (4) 0.0144+0.0019 (4)

NS NS

The Km, app for pyruvate for scallop phasic muscle mitochondria was somewhat lower than those found for trout red and white muscle mitochondria (46 and 37 gM, respectively) (Moyes et al. 1992; Blier and Guderley 1993a;) and considerably lower than that found for goldfish (Carassius auratus) red muscle (1.17 raM) (Mourik 1983). The value obtained for goldfish muscle mitochondria seems higher than the physiological range of pyruvate concentrations. The apparent K m for pyruvate of scallop mitochondria was somewhat higher than that of rat red muscle mitochondria (Km, app 5 to 9 ~M) (Gollnick et al. 1985) and of carp red muscle mitochondria (Km, app < 5 ~ m ) (Moyes et al. 1992). Moyes et al. (1992) established that the energetic conditions under which mitochondria respire affect the apparent K m for pyruvate. In State 3, trout white muscle mitochondria the apparent K m for pyruvate is 37 ~M, whereas in State 4 this value drops to < 5 gM, a concentration 10-fold below physiological pyruvate levels. At the ATP/ADP ratios typical of recovering white muscle (Schultes et al. 1992), such a high affinity would be expected. Evaluation of the pyruvate, ATP and ADP concentrations in scallop muscle following burst locomotion as well as of the oxidation rates and substrate affinities of the mitochondria under these conditions is required to fully understand the means by which mitochondria participate in metabolic recovery from exercise. The apparent K m of mitochondria for pyruvate may be set by properties of the pyruvate transporter or of pyruvate dehydrogenase. In trout red muscle, the Kin,app for pyruvate of mitochondria and that of isolated pyruvate dehydrogenase have a similar temperature and pH sensitivity. Thus, decreases in temperature at constant pH reduce the Km, app for pyruvate of pyruvate dehydrogenase (PDH) from 23 ~tM at 22 ~ to 9 g M at 8 ~ When pH covaries with temperature (ApH/~ = - 0.03 ~ the Kin, app does not change with temperature (14 gM) (Blier and Guderley 1993a). The Km, app for pyruvate of isolated mitochondria from trout red muscle is higher than that of PDH, but shows the same dependence upon temperature and pH (Blier and Guderley 1993a). This suggests that a gradient in pyruvate concentration between the intra- and extra-mitochondrial compartments may be sustained by PDH kinetics. However, the thermal sensitivity of maximal rates of pyruvate oxidation by mitochondria and by PDH differ, indicating that PDH is not the sole determinant of pyruvate kinetics of trout red muscle mitochondria (Blier and Guderley 1993a). For mitochondria from carp red muscle, a decrease in external pH enhances the mitochondrial pH gradient (Moyes et al. 1988). Such an increased pH gradient would

415 facilitate the transport of pyruvate and phosphate into the mitochondria and could account for the pH effect upon the gm, app for pyruvate of scallop muscle mitochondria. Interspecific differences in mitochondrial pyruvate affinity may reflect differences in the properties of PDH or the pyruvate transporter, the methods used to assess pyruvate affinity or differences in the purity of the mitochondrial preparations. The little information available concerning the pH sensitivity of mitochondrial substrate affinities suggests that decreases in pH favor the uptake of carbon substrates. At physiological pCO 2 values, a drop in pH enhances the succinate affinity of hepatic mitochondria from the desert iguana, Dipsosaurus dorsalis (Yacoe 1986). For mitochondria from trout red muscle, pH has little effect upon the pyruvate affinity, except at low temperatures (8 ~ when a decrease in pH from 7.6 to 7.4 enhances Km, app values (Blier and Guderley 1993 a). At 8 ~ trout red muscle mitochondria also lose their sensitivity to ADP, with Km, app values for ADP rising markedly (Blier and Guderley 1993 b), The loss of sensitivity to ADP may lead this normally aerobic muscle to activate glycolysis. The enhanced mitochondrial affinity for pyruvate at acid pH could facilitate mitochondrial competition for pyruvate. For scallop phasic muscle mitochondria, a decrease in pH did not modify the K,~, ~pp for glutamate but significantly decreased that for pyruvate. We speculate that the decrease of the Kin, app of scallop mitochondria for pyruvate at acidic pH values may facilitate their participation in the oxidative phase of recuperation from bouts of intense locomotor activity. Interestingly, only the oxidation of metabolites generated during burst locomotion seems facilitated by such a pH decrease. In contrast to the Km, app for pyruvate, the sensitivity to ADP of scallop muscle mitochondria was little affected by pH. A similar lack o f p H sensitivity characterizes the ADP sensitivity of trout red muscle mitochondria (Blier and Guderley 1993 b). The ADP Km, app values we obtained are close to that of the mammalian mitochondrial adenine nucleotide translocator and to those of trout red muscle mitochondria. The hyperbolic ADP saturation kinetics of scallop and trout muscle mitochondria are similar to those of ADP/ATP transport in mammalian mitochondria (Pfaff and Klingenberg 1968; Du~e and Vignais 1969) with a K'm,app between 1 and i0 ~M of total ADP for mitochondria and submitochondrial particles (Pfaff et al. 1969; Klingenberg 1976; 1977; Brandolin et al. 1980), As the adenine nucleotide translocator assures a significant part of the control of mitochondrial respiration (Groen et al. 1982; Gellerich et al. 1983; Westerhoff et al. 1987), it is likely that the sensitivity of scallop muscle mitochondria to ADP plays a significant role in the control of mitochondrial respiration. Seasonal differences in the maximal oxidative capacities of the mitochondrial preparations may reflect shifts in the physiology of the scallop's phasic muscle due to the reproductive and feeding cycles. These cycles may change the relative abundance or the functional properties of mitochondria and may be related to mobilisation of muscle

energy reserves in support of gametogenesis. Clearly, further studies are required to elucidate the basis of these changes as well as their functional significance. Acknowledgements This research was supported by a grant from Consejo de Investigacion de la Universidad de Oriente of Venezuela to O. Nusetti, as well as by an International Research Travel Grant from the NSERC of Canada to H. Guderley. Discussions with and assistance from C. Lodeiros were indispensable to the success of this study. The excellent facilities and technical support provided by the personal of the Universidad de Oriente, Cumana, Venezuela and in particular by the technicians for the Marine Laboratory at Turpialito are highly appreciated.

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