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Substrate preferences of the heart mitochondria of the horseshoe crab Limulus polyphemus
Comp. Biochem. Physiol. Vol. 93B, No. 4, pp. 883-887, 1989
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SUBSTRATE PREFERENCES OF THE HEART MITOCHONDRIA OF THE HORSESHOE CRAB L I M U L U S POL Y P H E M U S C. DOUMEN and W. R. ELLINGTON* Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA (Tel: 904 644-5406)
(Received 28 November 1988) Abstract--1. Tightly coupled mitochondria were isolated from the longitudinal hearts of Limulus
polyphemus, the horseshoe crab. 2. Succinate and ~-ketoglutarate were oxidized at the highest rate while active malate and fumarate utilization was only observed in the presence of small amounts of pyruvate. 3. The mitochondria showed a relatively high respiratory activity with pyruvate and proline. 4. Palmityol-l-carnitine was a poor substrate. 5. The respiratory data, coupled with the enzyme profile on crude extracts of the hearts, are indicative of a carbohydrate-based metabolism.
INTRODUCTION A measure of the importance of substrates in supplying the needed energy for a tissue may be obtained by studying the rate of oxidation of these substrates by isolated, coupled mitochondria. Most such studies have been performed on mitochondria from vertebrate tissues. The limited amount of work done on invertebrate tissues has concentrated on mitochondria from molluscs and insect flight muscle. Relatively little information is available on respiration and oxidative phosphorylation in mitochondria isolated from tissues of aquatic arthropods. Oxidation of substrate has been investigated in mitochondria isolated from crustacean hepatopancreas and abdominal muscle (Poat and Munday, 1971; Chen and Lehninger, 1973; Skorkowski et al., 1976). N o such studies have been performed on arthropod hearts. The aim of this study was to isolate and characterize mitochondria from the longitudinal heart of Limulus polyphemus, the horseshoe crab. This member of the primitive Merostomata class (subphylum Chelicerata) is known as a "living fossil" and, as such, provides an interesting comparative subject regarding its metabolic machinery. Aspects of anaerobic metabolism have already been partly investigated (Falkowski, 1973; Fields, 1982; G/ide et al., 1986; Carlsson and G/ide, 1986) and this research on the respiratory and enzymatic properties of heart mitochondria will enable us to outline the basic features of oxidative metabolism in horseshoe crab hearts. MATERIALS AND METHODS
Animals Animals were collected on a regular basis during periods of high tides of a full or new moon on the beaches of Mashes Sand, Panacea, Florida. The animals were kept in running *Author to whom correspondence should be addressed. 883
seawater at the Florida State University Marine Laboratory, Turkey Point, until needed for experimentation.
Isolation of mitochondria Before being sacrificed, animals were kept for about 20 hr in a 4°C room to render them immobile. Eight to ten animals were used per isolation experiment. The hearts were exposed by cutting away a dorsal section of the carapace after which the hearts were dissected out. The longitudinal hearts were blotted dry and minced with a single edge razor blade. The minced tissue was diluted into 9 volumes (wt: vol) of isolation medium and this material was forced through a syringe. The isolation medium was developed empirically so as to optimize respiratory properties, and consisted of 600mM sucrose, 150mM KC1, 25mM HEPES, 2mM EGTA, 1 mM EDTA and 0.2% defatted bovine serum albumin (BSA). The solution was adjusted to pH 7.4 with 1 N NaOH. The minced tissue solution was homogenized gently with an Ultra-Turrax tissue grinder after which it was passed three times in a Potter-Elvehjem homogenizer with a loosely fitting, hand driven Teflon pestle. This homogenate was centrifuged for 10min at 750g and the pellet was resuspended in buffer, re-homogenized and re-centrifuged as before. The supernatants were combined and centrifuged for 10min at 2000g and 10min at 12,000g. This final mitochondrial pellet was washed twice and the resulting pellet was resuspended in isolation medium to about 3 mg protein per ml. The average yield was around I mg mitochondrial protein per g wet weight. All manipulations were carried out at 0-4°C.
Measurement of mitochondrial respiration Oxygen uptake by isolated mitochondria was monitored with a Clark-type electrode using a Yellow Springs Instrument Model 53 biological monitor and bath stirrer, interfaced with a Kipp and Zonen BD 40 linear chart recorder. Temperature was maintained at 25°C using a Lauda K/2R constant temperature bath. The respiration medium consisted of the isolation medium supplied with 5 mM potassium phosphate and 2.5 mM magnesium acetate. The final volume in the cell was 2.5 ml with a mitoehondrial protein concentration of 0.2-0.3 mg/ml. Substrates, ADP and inhibitors were added with graduated Glenco micro-syringes. Rates of oxygen consumption, respiratory control ratios
884
C, DOUMEN and W. R. ELLINGTON
and ADP/O ratios were calculated according to Estabrook (1967), using the defined respiratory rates according to Chance and Williams (1956). Oxygen content of the medium was determined using sonicated mitochondria and limiting amounts of NADH while the ADP concentrations were measured spectrophotometrically according to Lowry and Passonneau (1972). Protein was measured by the Lowry method (Lowry et al., 1951). Mitochondria was solubilized with 0.2% deoxycholate and bovine serum albumin was used as a standard. Enzyme assays
The activities of the enzymes of interest were assayed in crude extracts and isolated mitochondria of L. polyphemus. For crude extracts, freshly dissected hearts were homogenized with a Brinkmann Polytron homogenizer using three bursts of 15 sec each at maximal speed in 1 : 9 (wt : vol) volumes of ice cold 100 mM imidazole/HCl, pH 7.0, 1 mM EDTA, 14raM 2-mercaptoethanol except for citrate synthase (50 mM Tris-HCl, pH 7.6, 1 mM EDTA). After centrifugation at 15,000g for 15min, the supernatant was passed through a Sephadex G-25 column (10 x 1 cm) which had been equilibrated in extraction buffer, and the void volume was subsequently assayed for the various enzyme activities. Citrate synthase was assayed on the crude homogenate. The protocol for all enzyme assays was as outlined by Meinhardus-Hager and G/ide (1986), except for arginine kinase which was assayed as follows: 100 mM Tris-HC1 buffer, pH 8.1, 2 mM ATP, 1.25 mM phosphoenolpyruvate, 0.25 mM NADH, 5 mM magnesium acetate, 75 mM KC1, 5 IU/ml of lactate dehydrogenase and pyruvate kinase; the reaction was started by the addition of 5 mM arginine. The mitochondrial distribution of certain enzymes was investigated in mitochondria isolated and resuspended as described above, but to which 0.5% Triton X-100 and 14 mM 2-mercaptoethanol was added. Materials
Biochemicals were purchased from Sigma Chemical Company (St Louis, MO, USA) and Boehringer Mannheim Biochemicals (Indianapolis, IN, USA). All other chemicals were of reagent grade quality. RESULTS
M i t o c h o n d r i a isolated following the procedures outlined a b o v e were tightly coupled a n d showed
A
typical respiratory responses to added substrates and ADP. Respiratory control ratios ( R C R ) over ten were o b t a i n e d on several occasions a n d addition of exogenous N A D H did n o t result in increased respiration, indicating the functional integrity of these mitochondria. U n u s u a l was the observation t h a t addition of A D P prior to substrates resulted in a stimulation o f state 3 respiration. This state 3 response without substrates, slowed d o w n u p o n subsequent additions of A D P to the point t h a t it equalled the state 4 rate, indicating t h a t e n d o g e n o u s substrate(s) was the basis of this o b s e r v a t i o n (Fig. 1). In order to insure that respiration rates observed were not obscured by this e n d o g e n o u s respiration, substrates were added after the e n d o g e n o u s state 3 had disappeared a n d respiration p a r a m e t e r s were calculated on the next A D P stimulation. All tested intermediates of the Krebs cycle were oxidized to some extent, with the highest rates seen for succinate a n d e - k e t o g l u t a r a t e (Table 1). The state 4 rate for succinate was higher t h a n all other substrates (Fig. 1), resulting in a lower t h a n average R C R . The o t h e r dicarboxylic acids malate and f u m a r a t e showed very low rates of respiration when not " s p a r k e d " by the a d d i t i o n o f a small a m o u n t of pyruvate, in which case b o t h intermediates exhibited similar rates o f oxidation. The m i t o c h o n d r i a were able to oxidize citrate, as well, indicating the presence of the tricarboxylic acid transporter. Pyruvate at 0.1 m M was used to some extent, while 5 m M pyruvate, in the presence o f 0.1 m M malate, was readily oxidized with rates similar to " s p a r k e d " f u m a r a t e a n d malate. G l u t a m a t e was oxidized at intermediate rates, while a s p a r t a t e yielded c o m p a r a b l e results only in the presence of catalytic a m o u n t s of pyruvate. Limulus heart m i t o c h o n d r i a utilized proline at fairly high rates and did n o t require pyruvate as a sparking agent. O r n i t h i n e alone yielded a slow response. Rates of oxidation with c~-glycerophosphate a n d [~h y d r o x y b u t y r a t e were barely detectable as the observed state 4 rate could not be m a d e to increase upon addition of A D P . Palmityol-L-carnitine was observed
C
B
- ~ A ADp DP
PRO 98ng-oxygen
~%in
,
p
gBrig-oxygen~
ADP
ADPi
98 ng-oxygen
~ ~
5 min
Fig. 1. Oxidation of succinate, proline and pyruvate by coupled mitochondria from horseshoe crab hearts. Approximately 0.2 mg mitochondrial protein was added to a final volume of 2.46 ml. State 3 respiration was initiated with 308 nmol ADP. Where indicated, the following substances were injected: (A) 12.5 ~mol of succinate. (B) 12.5 pmol of proline. (C) 12.5 #mol of pyruvate and 0.25 #mol of L-malate.
Horseshoe crab mitochondrial respiration
885
Table 1. Oxidation of substrates by isolated mitochondria from L. polyphemushearts. Assays were run at 25°C. Each value represents a mean _+ 1 SD except where noted Substrate 5.0 mM 5.0 mM 5.0 mM 5.0 mM 5.0 mM 5.0 mM 5.0 mM 0.1 mM 5.0 mM 5.0 mM 5.0 mM 5.0 mM
State 3 respiration (natoms O/min mg protein)
RCR
P:0 Ratio
156.6 + 14.8 109.7 _+3.3 55.2 _+ 11.11 26.9 + 3.7 86.8 _+21.6 16,4 _+5.9 83.3 _+20.2 22.7 + 28.9 89.5 _+8.6 47.2 _+3.3 12.1 - 12.7 58.7 + 10.5
4.6 __.0.4 5.6 + 2.0 6.2 + 3.3 -6.8 + 1.6 -7.6 _+0.8 5.7 - 6.9 6.4 _+0.9 3.6 -I- 1.0 -6.5 - 7.2
1.7 + 0.1 2.6 + 0.2 2.5 _+0.3 -2.8 _+0.1 -2.8 _+0.1 3.0 - 3.0 2.9 _+0.1 2.5 +_0.2 -3.1 - 2.5
95.3 -+ 12.7 26,9 +_5.09
6.4 _+2.0 2.2 - 3.3
2.6 + 0.2 2.4 - 2.6
--
--
3 3 3 3 3 3 4 2 4 3 2 3 (2 for RCR, P:O) 3 3 (2 for RCR, P:O) 3
-----
---2.7 _+0.1
3 2 2 8
Succinate a-Ketoglutarate Citrate Fumarate Fumarate+0.1 mM Pyruvate Malate Malate+0.1 mM Pyruvate Pyruvate Pyruvate+0.1 mM Malate Glutamate Aspartate Aspartate+0.1 mM Pyruvate
5.0 mM Proline 5.0 mM Ornithine 10 #M PalmityoI-L-Carnitine 10/aM Palmityol-L-Carnitine + 0.1 mM Pyruvate 5.0 mM a-Glycerophosphate 5.0 mM ~-Hydroxybutyrate ADP*
12.49 + 2.0 21.5 + 2.1 ND ND 99.3 + 12.4
n
--: Not determined; ND: not detected. *Rates represent the state 3 respiration upon the first addition of ADP in the absence of exogenous substrates. to be a p o o r s u b s t r a t e f o r Limulus h e a r t m i t o c h o n dria; v e r y l o w r a t e s o f o x i d a t i o n were o b s e r v e d w h i c h o n l y slightly i n c r e a s e d u p o n t h e a d d i t i o n o f 0.1 m M pyruvate. R e s p i r a t o r y c o n t r o l r a t i o s were o n t h e a v e r a g e a r o u n d six, b u t o c c a s i o n a l l y v a l u e s b e t w e e n 10 a n d 20 c o u l d be o b t a i n e d . T h e l o w e s t v a l u e s were o b t a i n e d w i t h g l u t a m a t e a n d o r n i t h i n e . A D P / O r a t i o s were n e a r the theoretical values expected for most substrates. T h e m i t o c h o n d r i a o f Limulus h e a r t s also e x h i b i t e d t h e t y p i c a l r e s p o n s e s to classical r e s p i r a t o r y inh i b i t o r s . R o t e n o n e i n h i b i t e d o x i d a t i o n o f proline, pyruvate and malate but not succinate; oligomycin inhibited respiration of succinate, pyruvate and m a l a t e ; a t r a c t y l o s i d e b l o c k e d r e s p i r a t i o n , while FCCP uncoupled the mitochondria (data not shown). A profile o f activities o f e n z y m e s i n v o l v e d in e n e r g y m e t a b o l i s m in h o r s e s h o e c r a b h e a r t s is g i v e n in
T a b l e 2. E n z y m e s i n v o l v e d in g l u c o s e c a t a b o l i s m were p r e s e n t in h i g h activities a n d c o m p a r a b l e to the values obtained for the telson levator muscle of L. polyphemus ( C a r l s s o n a n d G/ide, 1986). E n z y m e s i n v o l v e d in a m i n o acid m e t a b o l i s m were p r e s e n t as well, h a v i n g l o w e r t r a n s a m i n a s e b u t h i g h e r g l u t a m a t e d e h y d r o g e n a s e activities w h e n c o m p a r e d w i t h Limulus skeletal m u s c l e . A r g i n i n e p h o s p h o k i n a s e ( A P K ) activity in h e a r t t i s s u e w a s h i g h b u t w a s l o w e r relative to t h e activity in m u s c l e ( C a r l s s o n a n d G/ide, 1986). M i t o c h o n d r i a c o n t a i n e d t h e e n z y m e g l u t a m a t e dehydrogenase (GDH) and the enzymes glutamate oxaloacetate transaminase (GOT) and glutamate p y r u v a t e t r a n s a m i n a s e ( G P T ) in relatively h i g h activities. N o m a l i c e n z y m e activity w a s d e t e c t e d while low activities o f p h o s p h o e n o l p y r u v a t e carb o x y k i n a s e ( P E P C K ) w e r e p r e s e n t in b o t h t h e c y t o p l a s m i c a n d m i t o c h o n d r i a l f r a c t i o n s ( T a b l e 2).
Table 2. Activities of enzymes involved in the energy metabolism in the cytoplasm and mitochondria of the hearts of L. polyphemus. Each value represents a mean _+ 1 SD (n) Crude extract enzyme Hexokinase Pyruvate kinase Lactate dehydrogenase Arginine phosphokinase Citrate synthase Malate dehydrogenase Glutamate dehydrogenase Glutamate oxaloacetate transaminase Glutamate pyruvate transaminase Phosphoenolpyruvate carboxykinase Malic enzyme Mitochondrial extract enzyme Malate dehydrogenase Glutamate dehydrogenase Glutamate oxaloacetate transaminase Glutamate pyruvate transaminase PEPCK Malic enzyme ND: Not detected, no appreciable activity.
#mol/min.g wet wt 3.13_+0.27 (3) 89.85 + 25.30 (3) 71.30+3.51 (3) 91.10 + 14.50 (3) 9.29 _+2.12 (3) 199.80 _+34.2 (3) 3.47 _+0.3 (3) 10.02 _+ 1.15 (3) 1.66 + 0.12 (3) 1.39 _+0.14 (3) ND (3) #mol/min. mg mitochondrial protein 3.83 _+ 1.33 (4) 0.35 + 0.11 (4) 0.75 + 0.10 (3) 0.47 + 0.04 (3) 0.05 + 0.01 (3) ND (3)
886
C. DOUMENand W. R. ELLINGTON DISCUSSION
The results described herein indicate that the mitochondria of the hearts of L. polyphemus were functionally intact and tightly coupled. Based on the observed rates of oxidation of substrates in state 3, this study indicates that these mitochondria are fully able to utilize most Krebs Cycle intermediates. As with most species, succinate was oxidized at the highest rate, being slightly higher than the rates reported for other marine arthropods (Poat and Munday, 1971; Chen and Lehninger, 1973). On the other hand, oxidation of malate and fumarate was relatively poor, unless pyruvate was added at low concentrations. Since the Gibbs free energy for the malate dehydrogenase reaction is extremely positive, the oxidation of malate into oxaloacetate can only proceed if the latter is further metabolized, that is, when it condenses with acetyl-CoA to form citrate. It has been suggested that simultaneous oxidation of endogenous fatty acids is the basis of observed high rates of malate oxidation (Skordowski et al., 1976). Our observation of low rates of oxidation malate (and hence fumarate) and the indication that fatty acids are a poor substrate for Limulus heart mitochondria agree well with this line of reasoning and is substantiated by the fact that these mitochondria need exogenous added pyruvate as a source of acetylCoA due to the absence of malic enzyme. The observation that palmityol-L-carnitine is not oxidized at any significant rate, even in the presence of 0.1 mM pyruvate, is interesting and is in contrast with the vertebrate heart system. Similar results were reported for squid heart mitochondria (Ballantyne et al., 1981; Mommsen and Hochachka, 1981) and other molluscan ventricles (Ballantyne and Storey, 1983; Chih and Ellington, 1987) although hepatopancreas mitochondria from the blue crab seem to readily oxidize fatty acids (Chen and Lehninger, 1973). Since we did not try to initiate fatty acid oxidation in isolated mitochondria by using free fatty acids, the possibility of the existence of a different mechanism of transport cannot be ruled out. Mitochondria failed to oxidize ~-glycerophosphate and fl-hydroxybutyrate at appreciable rates either. The enzyme profile in the cytoplasm of Limulus heart tissue reveals much similarity with that of the telson levator muscle of this species (Table 2). The high activity of citrate synthase suggests a high aerobic capacity, although this appeared to be insufficient to meet enhanced energy demands during exercise in muscle (Carlsson and G/ide, 1986). D-Lactate production and breakdown of arginine phosphate provided the needed ATP synthesis in those muscles, which is believed to be the case as well in these heart tissues considering the high activities of D-lactate dehydrogenase (D-LDH) and APK. Although glycogen phosphorylase was not measured, the comparable high activity of hexokinase and pyruvate kinase in heart versus muscle and the high capacity for pyruvate oxidation are indicative of a similar carbohydrate based metabolism, using glucose rather than endogenous glycogen (Zammit and Newsholme, 1976; Carlsson and G/ide, 1986). The presence of cytoplasmatic and mitochondrial PEPCK activity has been reported earlier by
Falkowski (1973) in muscle and hepatopancreas tissue of Limulus polyphemus and was explained as an indication for the capacity for facultative anaerobiosis. Fields (1982) and Carlsson and G~ide (1986) found no evidence to support the existence of a succinate/propionate pathway as no significant increases in succinate or propionate were detected in heart and muscle tissue, respectively, during anaerobic conditions. The observation of PEPCK activity in heart tissue in the same levels as in skeletal muscle must therefore indicate that this enzyme functions in the gluconeogenic direction for which evidence has been provided recently in muscle (G~ide et al., 1986). The potential contribution of amino acids in mitochondrial metabolism is believed to be low. In contrast to other euryhaline invertebrates, the tissues of the horseshoe crab contain only low levels of free amino acids, making up 10% or less of the osmotically active substances. Cell volume is regulated mostly by Na +, CI- and glycine betaine (Warren and Pierce, 1982). Glutamate, proline and arginine are the most abundant free amino acids (+_ 5 ~ mol/g wet wt; Warren and Pierce, 1982) albeit relatively low when compared with other marine invertebrates. With respect to this, it is interesting to note that proline showed fairly high rates of oxidation in horseshoe crab heart mitochondria. Proline utilization has been reported for some marine invertebrae tissues such as cephalopod (Ballantyne et al., 1981; Mommsen and Hochachka, 1981) and other molluscan hearts (Ballantyne and Storey, 1983; Chih and Ellington, 1987) and crab hepatopancreas (Chen and Lehninger, 1973), although no such oxidation was observed in bivalve hepatopancreas (Ballantyne and Storey, 1984; Ballantyne and Moon, 1985). In accordance with its function in flying insects, the utilization of proline as a mitochondrial substrate in squid hearts has been explained as a "flare-up" substance (Ballantyne et al., 1981). Considering the carbohydrate character of mitochondrial metabolism in Limulus heart mitochondria, the function of proline utilization must be viewed in a similar manner, to augment Krebs cycle intermediates and thus increase the flux of glucose derived acetyl-CoA through the Krebs cycle. The oxidation of glutamate at intermediate rates underlies the possible minor contribution of amino acids to mitochondrial metabolism considering the fact that glutamate is an intermediate in the oxidation of several amino acids and that the respective enzymes such as GDH, GOT and GPT are present in mitochondrial preparations. Arginine was not utilized and its relatively high concentration is a consequence of its participation in the APK reaction. The failure of Limulus heart mitochondria to oxidize ct-glycerophosphate probably rules out the existence of ~-glycerophosphate shuttle for transport of reducing equivalents from the cytoplasm to the mitochondria. The ability to oxidize aspartate, ~-ketoglutarate and malate, and the presence of both mitochondrial and cytoplasmic M D H and GOT activities, support the idea that these mitochondria rely on the malate-aspartate shuttle to transfer reducing equivalents into the matrix. Finally, it should be emphasized that during every mitochondrial isolation according to the procedures outlined in the method section, ADP was able to
Horseshoe crab mitochondrial respiration stimulate state 3 respiration prior to the addition of substrates. It could be argued that our results are incorrect since they represent a superimposed image of exogenous and endogenous substrates. Furthermore, the addition of small amounts of pyruvate might have "sparked" endogenous substrates. However, we determined our respiration parameters only after the "endogenous" state 3 response had vanished following a number of state 3 to state 4-cycles. Also, when using low concentrations of pyruvate as a "sparking" agent, pyruvate was added first prior to A D P stimulation, after which the substrate of interest was added before the next A D P stimulated response. The state 3 rates thus obtained with 0.1 m M pyruvate, 5.0 m M malate and fumarate and paimityol-Lcarnitine are indicative that the contribution by endogenous substrates was minor, if not absent, when compared with the initial endogenous state 3 rate (see Table 1). A similar endogenous state 3 rate was reported by Ballantyne and M o o n 0985). Unfortunately, this observation was not further elaborated upon, and no explanation was given as to how this problem was dealt with in the analysis of their results. Acknowledgements--This research was supported by a grant from the U.S. National Science Foundation (Grant No. DCB-8710108) to W.R.E. REFERENCES
Ballantyne J. S., Hochachka P. W. and Mommsen T. P. (198 I) Studies on the metabolism of the migratory squid, Loligo opalescens: enzymes of tissues and heart mitochondria. Mar. Biol. Left. 2, 7545. Ballantyne J. S. and Storey K. B. 0983) Mitochondria from the ventricle of the marine clam, Mercenaria mercenaria: substrate preferences and effect of pH and salt concentration on proline oxidation. Comp. Biochem. Physiol. 7613, 133-138. Ballantyne J. S. and Storey K. B. (1984) Mitochondria from the hepatopancreas of the marine clam, Mercenaria mercenaria: substrate preferences and salt and pH effects on the oxidation of palmityol-L-carnitine and succinate. J. exp. Zool. 230, 165-174. Ballantyne J. S. and Moon T. W. (1985) Hepatopancreas mitochondria from Mytilus edulis: substrate preferences and effects of pH and osmolarity. Mar. Biol. 87, 239-244. Carlsson K. H. and G/ide G. (1986) Metabolic adaptation of the horseshoe crab, Limuluspolyphemus during exercise and environmental hypoxia and subsequent recovery. Biol. Bull. 171, 217-235. Chance B. and Williams G. R. (1956) The respiratory chain and oxidative phosphorylation. Adv. Enzymol. 17, 65 134.
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Chen C. H. and Lehninger A. L. (1973) Respiration and phosphorylation by mitochondria from the hepatopancreas of the blue crab (Callinectes sapidus). Arch. Biochem. Biophys. 154, 449-459. Chih C. P. and Ellington W. R. (1987) Studies of the mitochondria and enzymes from the ventricle of the whelk, Busycon contrarium. Comp. Biochem. Physiol. 86B, 541-545. Estabrook R. W. (1967) Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. In Methods in Enzymology (Edited by Estabrook R. W. and Pullman M. E.), Vol. X, pp. 41-47. Academic Press, N.Y. Falkowski P. G. (1973) Facultative anaerobiosis in Limulus polyphemus: phosphoenolpyruvate carboxykinase and heart activities. Comp. Biochem. Physiol. 49B, 749-459. Fields J. H. A. (1982) Anaerobiosis in Limulus. In Physiology and Biology of Horseshoe Crabs (Edited by Bonaventura J., Bonaventura C. and Tesh S.), pp. 125-131. Alan R. Liss, New York. G/ide G., Graham R. A. and Ellington W. R. (1986) Metabolic disposition of lactate in the horseshoe crab Limulus polyphemus and the stone crab Menippe mercenaria. Mar. Biol. 91, 473 479. Lowry O. H., Rosenbrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Lowry O. H. and Passonneau J. Y. (1972) A Flexible System of Enzymatic Analysis. Academic Press, N.Y. Meinhardus-Hager G. and G/ide G. (1986) The pyruvate branchpoint in the anaerobic energy metabolism of the jumping cockle Cardium tuberculatum L.: D-lactate formation during environmental anaerobiosis versus octopine during exercise. Exp. Biol. 45, 91-110. Mommsen T. P. and Hochachka P. W. 0981) Respiratory and enzymatic properties of squid heart mitochondria. Eur. J. Biochem 120, 345-350. Poat P. C. and Munday K. A. (1971) Respiratory control in mitochondria from the hepatopancreas of Carcinus maenus. Int. J. Biochem. 2, 49-54. Skorkowski E. F., Aleksandrowicz Z., Wrzolkowa T. and Swierczynski J. (1976) Isolation and some properties of mitochondria from the abdomen muscle of the crayfish Orconectes limosus. Comp. Biochem. Physiol. 55B, 493 500. Warren M. K. and Pierce S. K. (1982) Two cell volume regulatory systems in the Limulus myocardium: an interaction of ions and quarternary ammonium compounds. Biol. Bull. 163, 504-516. Zammit Y. A. and Newsholme E. A. (1976) The maximum activities of hexokinase, phosphorylase, phosphofructokinase, glycerol phosphate dehydrogenase, lactate dehydrogenase, octopine dehydrogenase, phosphoenolpyruvate carboxykinase, nucleoside diphosphatekinase, glutamate-oxaloacetate transaminase and arginine kinase in relation to carbohydrate utilization in muscles from marine invertebrates. Biochem. J. 160, 447-462.
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SUBSTRATE PREFERENCES OF THE HEART MITOCHONDRIA OF THE HORSESHOE CRAB L I M U L U S POL Y P H E M U S C. DOUMEN and W. R. ELLINGTON* Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA (Tel: 904 644-5406)
(Received 28 November 1988) Abstract--1. Tightly coupled mitochondria were isolated from the longitudinal hearts of Limulus
polyphemus, the horseshoe crab. 2. Succinate and ~-ketoglutarate were oxidized at the highest rate while active malate and fumarate utilization was only observed in the presence of small amounts of pyruvate. 3. The mitochondria showed a relatively high respiratory activity with pyruvate and proline. 4. Palmityol-l-carnitine was a poor substrate. 5. The respiratory data, coupled with the enzyme profile on crude extracts of the hearts, are indicative of a carbohydrate-based metabolism.
INTRODUCTION A measure of the importance of substrates in supplying the needed energy for a tissue may be obtained by studying the rate of oxidation of these substrates by isolated, coupled mitochondria. Most such studies have been performed on mitochondria from vertebrate tissues. The limited amount of work done on invertebrate tissues has concentrated on mitochondria from molluscs and insect flight muscle. Relatively little information is available on respiration and oxidative phosphorylation in mitochondria isolated from tissues of aquatic arthropods. Oxidation of substrate has been investigated in mitochondria isolated from crustacean hepatopancreas and abdominal muscle (Poat and Munday, 1971; Chen and Lehninger, 1973; Skorkowski et al., 1976). N o such studies have been performed on arthropod hearts. The aim of this study was to isolate and characterize mitochondria from the longitudinal heart of Limulus polyphemus, the horseshoe crab. This member of the primitive Merostomata class (subphylum Chelicerata) is known as a "living fossil" and, as such, provides an interesting comparative subject regarding its metabolic machinery. Aspects of anaerobic metabolism have already been partly investigated (Falkowski, 1973; Fields, 1982; G/ide et al., 1986; Carlsson and G/ide, 1986) and this research on the respiratory and enzymatic properties of heart mitochondria will enable us to outline the basic features of oxidative metabolism in horseshoe crab hearts. MATERIALS AND METHODS
Animals Animals were collected on a regular basis during periods of high tides of a full or new moon on the beaches of Mashes Sand, Panacea, Florida. The animals were kept in running *Author to whom correspondence should be addressed. 883
seawater at the Florida State University Marine Laboratory, Turkey Point, until needed for experimentation.
Isolation of mitochondria Before being sacrificed, animals were kept for about 20 hr in a 4°C room to render them immobile. Eight to ten animals were used per isolation experiment. The hearts were exposed by cutting away a dorsal section of the carapace after which the hearts were dissected out. The longitudinal hearts were blotted dry and minced with a single edge razor blade. The minced tissue was diluted into 9 volumes (wt: vol) of isolation medium and this material was forced through a syringe. The isolation medium was developed empirically so as to optimize respiratory properties, and consisted of 600mM sucrose, 150mM KC1, 25mM HEPES, 2mM EGTA, 1 mM EDTA and 0.2% defatted bovine serum albumin (BSA). The solution was adjusted to pH 7.4 with 1 N NaOH. The minced tissue solution was homogenized gently with an Ultra-Turrax tissue grinder after which it was passed three times in a Potter-Elvehjem homogenizer with a loosely fitting, hand driven Teflon pestle. This homogenate was centrifuged for 10min at 750g and the pellet was resuspended in buffer, re-homogenized and re-centrifuged as before. The supernatants were combined and centrifuged for 10min at 2000g and 10min at 12,000g. This final mitochondrial pellet was washed twice and the resulting pellet was resuspended in isolation medium to about 3 mg protein per ml. The average yield was around I mg mitochondrial protein per g wet weight. All manipulations were carried out at 0-4°C.
Measurement of mitochondrial respiration Oxygen uptake by isolated mitochondria was monitored with a Clark-type electrode using a Yellow Springs Instrument Model 53 biological monitor and bath stirrer, interfaced with a Kipp and Zonen BD 40 linear chart recorder. Temperature was maintained at 25°C using a Lauda K/2R constant temperature bath. The respiration medium consisted of the isolation medium supplied with 5 mM potassium phosphate and 2.5 mM magnesium acetate. The final volume in the cell was 2.5 ml with a mitoehondrial protein concentration of 0.2-0.3 mg/ml. Substrates, ADP and inhibitors were added with graduated Glenco micro-syringes. Rates of oxygen consumption, respiratory control ratios
884
C, DOUMEN and W. R. ELLINGTON
and ADP/O ratios were calculated according to Estabrook (1967), using the defined respiratory rates according to Chance and Williams (1956). Oxygen content of the medium was determined using sonicated mitochondria and limiting amounts of NADH while the ADP concentrations were measured spectrophotometrically according to Lowry and Passonneau (1972). Protein was measured by the Lowry method (Lowry et al., 1951). Mitochondria was solubilized with 0.2% deoxycholate and bovine serum albumin was used as a standard. Enzyme assays
The activities of the enzymes of interest were assayed in crude extracts and isolated mitochondria of L. polyphemus. For crude extracts, freshly dissected hearts were homogenized with a Brinkmann Polytron homogenizer using three bursts of 15 sec each at maximal speed in 1 : 9 (wt : vol) volumes of ice cold 100 mM imidazole/HCl, pH 7.0, 1 mM EDTA, 14raM 2-mercaptoethanol except for citrate synthase (50 mM Tris-HCl, pH 7.6, 1 mM EDTA). After centrifugation at 15,000g for 15min, the supernatant was passed through a Sephadex G-25 column (10 x 1 cm) which had been equilibrated in extraction buffer, and the void volume was subsequently assayed for the various enzyme activities. Citrate synthase was assayed on the crude homogenate. The protocol for all enzyme assays was as outlined by Meinhardus-Hager and G/ide (1986), except for arginine kinase which was assayed as follows: 100 mM Tris-HC1 buffer, pH 8.1, 2 mM ATP, 1.25 mM phosphoenolpyruvate, 0.25 mM NADH, 5 mM magnesium acetate, 75 mM KC1, 5 IU/ml of lactate dehydrogenase and pyruvate kinase; the reaction was started by the addition of 5 mM arginine. The mitochondrial distribution of certain enzymes was investigated in mitochondria isolated and resuspended as described above, but to which 0.5% Triton X-100 and 14 mM 2-mercaptoethanol was added. Materials
Biochemicals were purchased from Sigma Chemical Company (St Louis, MO, USA) and Boehringer Mannheim Biochemicals (Indianapolis, IN, USA). All other chemicals were of reagent grade quality. RESULTS
M i t o c h o n d r i a isolated following the procedures outlined a b o v e were tightly coupled a n d showed
A
typical respiratory responses to added substrates and ADP. Respiratory control ratios ( R C R ) over ten were o b t a i n e d on several occasions a n d addition of exogenous N A D H did n o t result in increased respiration, indicating the functional integrity of these mitochondria. U n u s u a l was the observation t h a t addition of A D P prior to substrates resulted in a stimulation o f state 3 respiration. This state 3 response without substrates, slowed d o w n u p o n subsequent additions of A D P to the point t h a t it equalled the state 4 rate, indicating t h a t e n d o g e n o u s substrate(s) was the basis of this o b s e r v a t i o n (Fig. 1). In order to insure that respiration rates observed were not obscured by this e n d o g e n o u s respiration, substrates were added after the e n d o g e n o u s state 3 had disappeared a n d respiration p a r a m e t e r s were calculated on the next A D P stimulation. All tested intermediates of the Krebs cycle were oxidized to some extent, with the highest rates seen for succinate a n d e - k e t o g l u t a r a t e (Table 1). The state 4 rate for succinate was higher t h a n all other substrates (Fig. 1), resulting in a lower t h a n average R C R . The o t h e r dicarboxylic acids malate and f u m a r a t e showed very low rates of respiration when not " s p a r k e d " by the a d d i t i o n o f a small a m o u n t of pyruvate, in which case b o t h intermediates exhibited similar rates o f oxidation. The m i t o c h o n d r i a were able to oxidize citrate, as well, indicating the presence of the tricarboxylic acid transporter. Pyruvate at 0.1 m M was used to some extent, while 5 m M pyruvate, in the presence o f 0.1 m M malate, was readily oxidized with rates similar to " s p a r k e d " f u m a r a t e a n d malate. G l u t a m a t e was oxidized at intermediate rates, while a s p a r t a t e yielded c o m p a r a b l e results only in the presence of catalytic a m o u n t s of pyruvate. Limulus heart m i t o c h o n d r i a utilized proline at fairly high rates and did n o t require pyruvate as a sparking agent. O r n i t h i n e alone yielded a slow response. Rates of oxidation with c~-glycerophosphate a n d [~h y d r o x y b u t y r a t e were barely detectable as the observed state 4 rate could not be m a d e to increase upon addition of A D P . Palmityol-L-carnitine was observed
C
B
- ~ A ADp DP
PRO 98ng-oxygen
~%in
,
p
gBrig-oxygen~
ADP
ADPi
98 ng-oxygen
~ ~
5 min
Fig. 1. Oxidation of succinate, proline and pyruvate by coupled mitochondria from horseshoe crab hearts. Approximately 0.2 mg mitochondrial protein was added to a final volume of 2.46 ml. State 3 respiration was initiated with 308 nmol ADP. Where indicated, the following substances were injected: (A) 12.5 ~mol of succinate. (B) 12.5 pmol of proline. (C) 12.5 #mol of pyruvate and 0.25 #mol of L-malate.
Horseshoe crab mitochondrial respiration
885
Table 1. Oxidation of substrates by isolated mitochondria from L. polyphemushearts. Assays were run at 25°C. Each value represents a mean _+ 1 SD except where noted Substrate 5.0 mM 5.0 mM 5.0 mM 5.0 mM 5.0 mM 5.0 mM 5.0 mM 0.1 mM 5.0 mM 5.0 mM 5.0 mM 5.0 mM
State 3 respiration (natoms O/min mg protein)
RCR
P:0 Ratio
156.6 + 14.8 109.7 _+3.3 55.2 _+ 11.11 26.9 + 3.7 86.8 _+21.6 16,4 _+5.9 83.3 _+20.2 22.7 + 28.9 89.5 _+8.6 47.2 _+3.3 12.1 - 12.7 58.7 + 10.5
4.6 __.0.4 5.6 + 2.0 6.2 + 3.3 -6.8 + 1.6 -7.6 _+0.8 5.7 - 6.9 6.4 _+0.9 3.6 -I- 1.0 -6.5 - 7.2
1.7 + 0.1 2.6 + 0.2 2.5 _+0.3 -2.8 _+0.1 -2.8 _+0.1 3.0 - 3.0 2.9 _+0.1 2.5 +_0.2 -3.1 - 2.5
95.3 -+ 12.7 26,9 +_5.09
6.4 _+2.0 2.2 - 3.3
2.6 + 0.2 2.4 - 2.6
--
--
3 3 3 3 3 3 4 2 4 3 2 3 (2 for RCR, P:O) 3 3 (2 for RCR, P:O) 3
-----
---2.7 _+0.1
3 2 2 8
Succinate a-Ketoglutarate Citrate Fumarate Fumarate+0.1 mM Pyruvate Malate Malate+0.1 mM Pyruvate Pyruvate Pyruvate+0.1 mM Malate Glutamate Aspartate Aspartate+0.1 mM Pyruvate
5.0 mM Proline 5.0 mM Ornithine 10 #M PalmityoI-L-Carnitine 10/aM Palmityol-L-Carnitine + 0.1 mM Pyruvate 5.0 mM a-Glycerophosphate 5.0 mM ~-Hydroxybutyrate ADP*
12.49 + 2.0 21.5 + 2.1 ND ND 99.3 + 12.4
n
--: Not determined; ND: not detected. *Rates represent the state 3 respiration upon the first addition of ADP in the absence of exogenous substrates. to be a p o o r s u b s t r a t e f o r Limulus h e a r t m i t o c h o n dria; v e r y l o w r a t e s o f o x i d a t i o n were o b s e r v e d w h i c h o n l y slightly i n c r e a s e d u p o n t h e a d d i t i o n o f 0.1 m M pyruvate. R e s p i r a t o r y c o n t r o l r a t i o s were o n t h e a v e r a g e a r o u n d six, b u t o c c a s i o n a l l y v a l u e s b e t w e e n 10 a n d 20 c o u l d be o b t a i n e d . T h e l o w e s t v a l u e s were o b t a i n e d w i t h g l u t a m a t e a n d o r n i t h i n e . A D P / O r a t i o s were n e a r the theoretical values expected for most substrates. T h e m i t o c h o n d r i a o f Limulus h e a r t s also e x h i b i t e d t h e t y p i c a l r e s p o n s e s to classical r e s p i r a t o r y inh i b i t o r s . R o t e n o n e i n h i b i t e d o x i d a t i o n o f proline, pyruvate and malate but not succinate; oligomycin inhibited respiration of succinate, pyruvate and m a l a t e ; a t r a c t y l o s i d e b l o c k e d r e s p i r a t i o n , while FCCP uncoupled the mitochondria (data not shown). A profile o f activities o f e n z y m e s i n v o l v e d in e n e r g y m e t a b o l i s m in h o r s e s h o e c r a b h e a r t s is g i v e n in
T a b l e 2. E n z y m e s i n v o l v e d in g l u c o s e c a t a b o l i s m were p r e s e n t in h i g h activities a n d c o m p a r a b l e to the values obtained for the telson levator muscle of L. polyphemus ( C a r l s s o n a n d G/ide, 1986). E n z y m e s i n v o l v e d in a m i n o acid m e t a b o l i s m were p r e s e n t as well, h a v i n g l o w e r t r a n s a m i n a s e b u t h i g h e r g l u t a m a t e d e h y d r o g e n a s e activities w h e n c o m p a r e d w i t h Limulus skeletal m u s c l e . A r g i n i n e p h o s p h o k i n a s e ( A P K ) activity in h e a r t t i s s u e w a s h i g h b u t w a s l o w e r relative to t h e activity in m u s c l e ( C a r l s s o n a n d G/ide, 1986). M i t o c h o n d r i a c o n t a i n e d t h e e n z y m e g l u t a m a t e dehydrogenase (GDH) and the enzymes glutamate oxaloacetate transaminase (GOT) and glutamate p y r u v a t e t r a n s a m i n a s e ( G P T ) in relatively h i g h activities. N o m a l i c e n z y m e activity w a s d e t e c t e d while low activities o f p h o s p h o e n o l p y r u v a t e carb o x y k i n a s e ( P E P C K ) w e r e p r e s e n t in b o t h t h e c y t o p l a s m i c a n d m i t o c h o n d r i a l f r a c t i o n s ( T a b l e 2).
Table 2. Activities of enzymes involved in the energy metabolism in the cytoplasm and mitochondria of the hearts of L. polyphemus. Each value represents a mean _+ 1 SD (n) Crude extract enzyme Hexokinase Pyruvate kinase Lactate dehydrogenase Arginine phosphokinase Citrate synthase Malate dehydrogenase Glutamate dehydrogenase Glutamate oxaloacetate transaminase Glutamate pyruvate transaminase Phosphoenolpyruvate carboxykinase Malic enzyme Mitochondrial extract enzyme Malate dehydrogenase Glutamate dehydrogenase Glutamate oxaloacetate transaminase Glutamate pyruvate transaminase PEPCK Malic enzyme ND: Not detected, no appreciable activity.
#mol/min.g wet wt 3.13_+0.27 (3) 89.85 + 25.30 (3) 71.30+3.51 (3) 91.10 + 14.50 (3) 9.29 _+2.12 (3) 199.80 _+34.2 (3) 3.47 _+0.3 (3) 10.02 _+ 1.15 (3) 1.66 + 0.12 (3) 1.39 _+0.14 (3) ND (3) #mol/min. mg mitochondrial protein 3.83 _+ 1.33 (4) 0.35 + 0.11 (4) 0.75 + 0.10 (3) 0.47 + 0.04 (3) 0.05 + 0.01 (3) ND (3)
886
C. DOUMENand W. R. ELLINGTON DISCUSSION
The results described herein indicate that the mitochondria of the hearts of L. polyphemus were functionally intact and tightly coupled. Based on the observed rates of oxidation of substrates in state 3, this study indicates that these mitochondria are fully able to utilize most Krebs Cycle intermediates. As with most species, succinate was oxidized at the highest rate, being slightly higher than the rates reported for other marine arthropods (Poat and Munday, 1971; Chen and Lehninger, 1973). On the other hand, oxidation of malate and fumarate was relatively poor, unless pyruvate was added at low concentrations. Since the Gibbs free energy for the malate dehydrogenase reaction is extremely positive, the oxidation of malate into oxaloacetate can only proceed if the latter is further metabolized, that is, when it condenses with acetyl-CoA to form citrate. It has been suggested that simultaneous oxidation of endogenous fatty acids is the basis of observed high rates of malate oxidation (Skordowski et al., 1976). Our observation of low rates of oxidation malate (and hence fumarate) and the indication that fatty acids are a poor substrate for Limulus heart mitochondria agree well with this line of reasoning and is substantiated by the fact that these mitochondria need exogenous added pyruvate as a source of acetylCoA due to the absence of malic enzyme. The observation that palmityol-L-carnitine is not oxidized at any significant rate, even in the presence of 0.1 mM pyruvate, is interesting and is in contrast with the vertebrate heart system. Similar results were reported for squid heart mitochondria (Ballantyne et al., 1981; Mommsen and Hochachka, 1981) and other molluscan ventricles (Ballantyne and Storey, 1983; Chih and Ellington, 1987) although hepatopancreas mitochondria from the blue crab seem to readily oxidize fatty acids (Chen and Lehninger, 1973). Since we did not try to initiate fatty acid oxidation in isolated mitochondria by using free fatty acids, the possibility of the existence of a different mechanism of transport cannot be ruled out. Mitochondria failed to oxidize ~-glycerophosphate and fl-hydroxybutyrate at appreciable rates either. The enzyme profile in the cytoplasm of Limulus heart tissue reveals much similarity with that of the telson levator muscle of this species (Table 2). The high activity of citrate synthase suggests a high aerobic capacity, although this appeared to be insufficient to meet enhanced energy demands during exercise in muscle (Carlsson and G/ide, 1986). D-Lactate production and breakdown of arginine phosphate provided the needed ATP synthesis in those muscles, which is believed to be the case as well in these heart tissues considering the high activities of D-lactate dehydrogenase (D-LDH) and APK. Although glycogen phosphorylase was not measured, the comparable high activity of hexokinase and pyruvate kinase in heart versus muscle and the high capacity for pyruvate oxidation are indicative of a similar carbohydrate based metabolism, using glucose rather than endogenous glycogen (Zammit and Newsholme, 1976; Carlsson and G/ide, 1986). The presence of cytoplasmatic and mitochondrial PEPCK activity has been reported earlier by
Falkowski (1973) in muscle and hepatopancreas tissue of Limulus polyphemus and was explained as an indication for the capacity for facultative anaerobiosis. Fields (1982) and Carlsson and G~ide (1986) found no evidence to support the existence of a succinate/propionate pathway as no significant increases in succinate or propionate were detected in heart and muscle tissue, respectively, during anaerobic conditions. The observation of PEPCK activity in heart tissue in the same levels as in skeletal muscle must therefore indicate that this enzyme functions in the gluconeogenic direction for which evidence has been provided recently in muscle (G~ide et al., 1986). The potential contribution of amino acids in mitochondrial metabolism is believed to be low. In contrast to other euryhaline invertebrates, the tissues of the horseshoe crab contain only low levels of free amino acids, making up 10% or less of the osmotically active substances. Cell volume is regulated mostly by Na +, CI- and glycine betaine (Warren and Pierce, 1982). Glutamate, proline and arginine are the most abundant free amino acids (+_ 5 ~ mol/g wet wt; Warren and Pierce, 1982) albeit relatively low when compared with other marine invertebrates. With respect to this, it is interesting to note that proline showed fairly high rates of oxidation in horseshoe crab heart mitochondria. Proline utilization has been reported for some marine invertebrae tissues such as cephalopod (Ballantyne et al., 1981; Mommsen and Hochachka, 1981) and other molluscan hearts (Ballantyne and Storey, 1983; Chih and Ellington, 1987) and crab hepatopancreas (Chen and Lehninger, 1973), although no such oxidation was observed in bivalve hepatopancreas (Ballantyne and Storey, 1984; Ballantyne and Moon, 1985). In accordance with its function in flying insects, the utilization of proline as a mitochondrial substrate in squid hearts has been explained as a "flare-up" substance (Ballantyne et al., 1981). Considering the carbohydrate character of mitochondrial metabolism in Limulus heart mitochondria, the function of proline utilization must be viewed in a similar manner, to augment Krebs cycle intermediates and thus increase the flux of glucose derived acetyl-CoA through the Krebs cycle. The oxidation of glutamate at intermediate rates underlies the possible minor contribution of amino acids to mitochondrial metabolism considering the fact that glutamate is an intermediate in the oxidation of several amino acids and that the respective enzymes such as GDH, GOT and GPT are present in mitochondrial preparations. Arginine was not utilized and its relatively high concentration is a consequence of its participation in the APK reaction. The failure of Limulus heart mitochondria to oxidize ct-glycerophosphate probably rules out the existence of ~-glycerophosphate shuttle for transport of reducing equivalents from the cytoplasm to the mitochondria. The ability to oxidize aspartate, ~-ketoglutarate and malate, and the presence of both mitochondrial and cytoplasmic M D H and GOT activities, support the idea that these mitochondria rely on the malate-aspartate shuttle to transfer reducing equivalents into the matrix. Finally, it should be emphasized that during every mitochondrial isolation according to the procedures outlined in the method section, ADP was able to
Horseshoe crab mitochondrial respiration stimulate state 3 respiration prior to the addition of substrates. It could be argued that our results are incorrect since they represent a superimposed image of exogenous and endogenous substrates. Furthermore, the addition of small amounts of pyruvate might have "sparked" endogenous substrates. However, we determined our respiration parameters only after the "endogenous" state 3 response had vanished following a number of state 3 to state 4-cycles. Also, when using low concentrations of pyruvate as a "sparking" agent, pyruvate was added first prior to A D P stimulation, after which the substrate of interest was added before the next A D P stimulated response. The state 3 rates thus obtained with 0.1 m M pyruvate, 5.0 m M malate and fumarate and paimityol-Lcarnitine are indicative that the contribution by endogenous substrates was minor, if not absent, when compared with the initial endogenous state 3 rate (see Table 1). A similar endogenous state 3 rate was reported by Ballantyne and M o o n 0985). Unfortunately, this observation was not further elaborated upon, and no explanation was given as to how this problem was dealt with in the analysis of their results. Acknowledgements--This research was supported by a grant from the U.S. National Science Foundation (Grant No. DCB-8710108) to W.R.E. REFERENCES
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