Comp Bmchem Ph~stol

1976 [ol 54B pp 81 to 92 Perqamon Press Printed in Great Britain

MITOCHONDRIAL ENZYMES OF TROPICAL FISH" A COMPARISON WITH FISH FROM COLD-WATERS D O IRVING AND K WATSON Department of Chemistry and Biochemistry, James Cook Umversity of North Queensland, Townswlle 4811, Austraha

(Recewed 18 February 1975) Abstract--1 Mltochondrla from heart and hver of the following speoes of tropical fish were isolated Plectropoma maculatum, Lethrmus chrysostomus, Acanthrus xanthopterus and Mucdtl cephalus 2 The fatty acid composmon of the tropical fish mltochondrml membranes was found to be more saturated than m prewously reported cold-water fish motochondrlal membranes 3 Temperature effects were observed on the membrane-associated enzymes succmate oxldase and succmate dehydrogenase and compared with the matrix-associated malate dehydrogenase The membrane-associated system was characterized by a sharp temperature profile m comparison with the broader profile for the matrix enzyme 4 All enzyme actwItles were higher m the heart than hver mltochondna The heart mltochondrml enzymes were also more temperature-stable than the corresponding enzymes of the hver 5 Linear Arrhenms plots below about 20-25°C were recorded for all enzymes However, departures from lineanty were observed above these temperatures xn the case of membrane-assocmted enzymes 6 Actwatlon energies for succmate oxldase m the temperature ranges 5-25 and 25-40°C were between 65-120 and 25-55 kJ mol-1 respectwely 7 The results are discussed m relatmn to the physiological slgmficance of hnear and non-hnear Arrhenms plots m polkllothermlc and homeothermlc animals

INTRODUCrlON

cies of fish found in the tropical waters of the Great Barrier Reef, Australia The temperature dependence of the mitochondrial enzymes and the fatty acid composition of the Isolated membranes from these tropical species are compared with swmlar data obtained by other workers with cold-water fish

Non-hnear Arrhemus plots, with increases in activation energy at lower temperatures, have been observed with mltochondrlal membrane-bound enzymes from a number of organisms These Include mitochondria from homeothermlc animals (Rmson et al, 1971a, b, Roberts et al, 1972, Smith, 1973a, McMurchie et al, 1973) and yeast (Alnsworth et al, 1972, Watson et al, 1973, Sklpton et al, 1974) By contrast, the corresponding enzymes of polkllothermic animals have been reported to show linear Arrhenius plots with a constant energy of activation over the entire temperature range studied (Lyons & Raison, 1970, McMurchie et al, 1973, Smith, 1973a) In this respect mitochondrial enzymes from fish, a poikIlotherm, reportedly have a constant energy of activation (Lyons & Raison, 1970, Hazel, 1972, Smith, 1973a, Vandenheede et al, 1973) It must be pointed out, however, that the majority of studies on the effect of temperature on fish mltochondrlal enzymes have been confined to cold-water species This situation IS exemphfied by Arrhenms plots which show constant energies of activation but which rarely include data for temperatures greater than 25-30°C This temperature range cannot be regarded as hmlting for fish in general since the normal environmental temperature for fish from tropical waters is commonly in this range or above The present study is an investigation of the effects of temperature on mltochondnat enzymes of four spe-

MATERIALS *ADP, NADH, BSA (Fraction V, essentially fatty acidfree), DCPIP, PMS, lSocitrate, and oxaloacetate were obtained from Sigma Chemical Company Methyl ester standards of fatty acids and cardiohpm were obtained from Applied Science Laboratories Incorporated, Cahforma, U S A Petroleum ether AR (bolhng point 40-60°C), Chloroform AR, Methanol AR, and n-hexane (u v-grade) were obtained from Ajax Chemicals Ltd, Sydney Silica gel H (type 60) was obtained from Laboratory Supply, Brisbane All other chemicals were Analytical R e a g e n t grade and obtained from B D H Chemicals or Ajax Chemicals Ltd EXPERIMENTAL M E T H O D S

Sample collectwn The four species of fish used in this study were Plectropoma maculatum (Bloch), Lethrmus chrysostomus Richardson, Acanthurus xanthopterus Valenciennes and Mugd cephalus Linnaeus These fish are commonly known as coral trout, sweethp-emperor, ring-tailed surgeon-fish, and the sea mullet respectively M cephalus was netted in the estuaries and inshore waters of Cleveland Bay by a local professional fisherman while the other three species were caught by hne or spearfishmg on the Great Barrier Reef in the vlcimty of Townsvllle The suitability of a particular fishmg method depends on the feeding and behavlour patterns of the fish in question A xanthopterus and L chrysostomus both display

*Abbrewatwns ADP, Adenosine-5'-&phosphate, NADH, Nlcotmamlde adenine dlnucleotIde (reduced form), BSA, Bowne serum albumin, DCPIP, 2,6 Dichlorophenohndophenol, PMS, Phenazlne methosulphate, EDTA, ethylenedlamlne tetraaeetate ,,~,(,,~

~l.

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81

82

D O IRVING AND K WATSON

similar behavlour and swimming activities They are usually found swimming as Individuals around coral bombies or together in loosely formed schools when they range over large areas of reef Acanthurus are herbivorous, feedIng on encrusting corrahne algae (Vine, 1974), while Lethrmus have a varied diet of small fish. molluscs, and crustaceans (Grant, 1972) Consequently the herbivore IS almost exclusively caught by spearfishmg while Lethrmus is frequently snared by line-fishermen P macalatum, which feeds mainly on small fish. is usually a solitary organism which is found in or near crevices in coral outcrops (Grant, 1972) As such, this species is equally susceptible to both hne and spearfishing M cephalus is m u c h more active than the three species of reef fish and Is found m schools containing large numbers of individuals These fast swimming fish inhabit coastal and estuarlne environments where they feed on bottom living micro-crustaceans, benthic and epiphytic micro-algae, a n d decaying plant detritus (Odum, 1970) M cephalus has an almost world-wide distribution extending between 40JN and 40°S and is found in marine, estuarine and fresh water habitats In the present study, specimens of M cephalus were obtained during the winter m o n t h s when water temperatures were between 22 25°C, while the other reef fish were caught from warmer waters (25-30~C) With all four species of fish, the heart and liver were removed immediately following capture and stored separately in polythene bags on dry ice The samples were maretamed at - 2 3 ° C in the laboratory until used, which was m general within 3 weeks of capture

Homogenization oJ tissue and preparation of mltochondria The stored samples were allowed to thaw slowly at 4°C prior to use and all subsequent procedures were carried out at this temperature The thawed organs were blotted free of any excess blood, weighed, and washed several times In Ice-cold 0 2 5 M sucrose solution containing 1 m M EDTA, 2 0 m M Trls-HC1, pH 72, and 0 2 % BSA (SET Buffer) The tissues were sliced into approximately 0 5 g segments In the case of liver, the whole organ was homogenized while with hearts, only the most metabolically active chambers, that is the atrium and ventricle, were used The small pieces of tissue were homogenized in a Potter Elvenhjem-type homogenizer using a loose-fitting teflon pestle so as not to disrupt the mitochondrial membranes The homogenization was performed in 10 vol (w/v) of Ice-cold SET buffer MItochondria were isolated from the homogenate by dlfferentml centrifugation using a Sorval RC-2B refrigerated centrifuge Nuclei and cell debris were removed by centrifuging the homogenate at 3000 g for 5 min using the SS-34 rotor The supernatant was then centrifuged at 20,000 g for 20 min and the tightly packed mitochondrial pellet was subsequently suspended in a small volume of SET buffer to give a final protein concentration of 25-30 m g m l - i for hver preparations and 10-15 m g m l - 1 for heart preparations The mitochondrlal suspensions were stored in an ice bucket during the experimental procedures

Mltochondrml protein determination Protein was estimated using the Hartree modification of the Lowry method (Hartree, 1972)

Cytochrome spectra Cytochrome compositions of turbid mltochondrial suspensions were determined by difference spectroscopy Mitochondria were suspended in SET buffer to a concentration of about 10mg protein ml t The contents of the sample cell were reduced with a few grams of sodium dithlonite and the reference oxidized with 10#1 of 100vol H 2 0 2 Difference spectra between these suspensions, an anaerobic and aerobic states respectively, were recorded spectrophotometncally in 1 cm light path cells (final vol

2 5-3 0ml) using the hght scattering position (slit width of 0 10 mm). In a Unican SP800 spectrophotometer Spectra were recorded on a U n i c a m SP20 flat-bed recorder equipped with scale expansion facilities The difference spectra were analysed using the following wavelength pairs and extinction coefficients cytochrome c 55(~540nm emM = 19 (Wilson & Epel, 1968), cytochrome h 560-540 nm, emM = 22 (Wilson & Epel, 1968). c}tochromes a + a 3 605 630nm, e.M = 24 (van Ge[der, 1966)

Enzyme assays (a) Succlnate oxldase Succinate oxldase activity was measured polarographically using a Rank oxygen electrode with a glass reaction chamber Temperature was controlled by a Haake thermostat and heating unit connected to a refrigerated thermocirculator A Honeywell Electronlk 15 recorder (1 mV range) was used to record the oxygen uptake The temperature of the reaction mixture was measured by means of an Atklns stainless steel immersion probe (0-100°C + 0 5°C) which allowed the temperature of the reaction medium to be continuously monitored The assay m e d m m contamned, in a final volume of from 215 to 2 5 m l , 0 2 5 M sucrose, l m M EDTA, 10raM TrIs-HC1, pH 7 2, 0 2°0 BSA and 1-15 mg protein dependlng on whether heart or hver mItochondria were being assayed After temperature equilibration, sodium succmate (final concentration 20 raM) was introduced, with a syringe, through a small orifice in the cell cap thereby ensuring minimal Introduction of atmospheric oxygen to the system The rate of oxygen uptake by the mitochondrla was recorded for 15-20min at each temperature tested The concentration of oxygen in the reaction m e d m m at different temperatures was calculated from standard tables (Hodgman, 1961) (b) Succlnate dehydrogenase and malate deh~drogenase Succinate dehydrogenase (succinate-(acceptor) oxtdoreductase, E C 1 399 1) and malate dehydrogenase (L-malat~ N A D oxldoreductase, E C 1 1 1 37) activities were determined spectrophotometrically using a Unlcam SP800 spectrophotometer fitted with a refrigerated thermocirculator The temperature was controlled by means of a Haake heatIng unit and circulator and was monitored c o n t m u o u s b in the reaction cuvette by means of an lmmersible Atklns temperature probe A l cm light path cell and a final volume of 2 8 m l was used In all assays Both enzyme assays were recorded against a reference cell containing 2 8 ml of 50 m M phosphate buffer, pH 7 4 Succinate dehydrogenase was assayed using a modification of the method of Arrtgoni & Singer (1962) The reaction cuvette contained 50 m M potassium phosphate buffer. p H 74, l m M K C N , 2 0 m M sodium succinate and 0 2 - 0 4 m g mltochondrIal protein The mixture was incubated for 5 min and the reaction initiated by rapid addition of 0 8 m M D C P I P and 0 5 m M P M S The decrease m absorbance of D C P I P at 6 0 0 n m was recorded and an extraction coefficient of 21 m M - 1 c m - ~ was used when calculating the specific activity of succlnate dehydrogenase at 600 n m (King & Howard, 1967) The concentrations of P M S and D C P I P were varied at a low and high temperature in order to eliminate an.~ possiblhty of these electron carriers being rate hmltmg in the enzyme assays Malate dehydrogenase was determined by the method of Ochoa (1955) The reaction medium contained 50 m M potassium phosphate buffer, pH 7 4 and 50-100#g mltochondrial protein The mixture was equilibrated for 5 mln after which 0 15 m M N A D H was added and absorbance at 340 n m was followed When necessary, a correction was applied for N A D H oxIdase activity in the mltochondrlal preparations The absorbance decrease at 340 n m was then followed after the addition of 1 m M oxaloacetate An extinction coefficient of 6 2 2 m M 1 c m - I for N A D H at

Mltochondrlal enzymes of tropical fish 340 nm was used when calculating malate dehydrogenase specific actlwty (Dawson et al, 1972)

83

Table 1 Cytochrome content of fish liver mltOChondrm Source

Cytochromes

(nmoles mg proteln-l|

of

Lipid analysis Llplds were extracted from the mitochondria or from whole tissues of liver and heart samples according to the method of Folch et al (1957) The fatty acids of the isolated hplds were estenfied by refluxmg for 2 hr m methanohcHC1 (0 5 M) and the methyl esters were extracted into 5 ml of u v-grade n-hexane The samples were evaporated to a small volume (0 5-1 0 ml) under reduced pressure m a rotary evaporator Samples were stored under nitrogen gas at -23°C until analysed by gas-hqmd chromatography Analysis was carried out using a Schlmadzu GC-4APF gas chromatograph fitted w~th a dual flame lomzatlon detector A column of charcoal was used as the reference column Two columns with different stationary phases were used to analyse the methyl esters In early experiments, a stainless steel column (2 m m length and 3 mm o d ) containing 10~o polyethyleneglycol succlnate on 100/200 mesh Gaschrom-Q, operating at 185°C was used The mjectmn and detection ports were fixed at 230°C A carrier gas (nitrogen) flow rate of 40 ml mm- 1 was employed Under these assay conditions, good separation of methyl esters of fatty acids of chain length up to CIS was obtained However, fatty acid methyl esters of chain length greater than C1, were poorly resolved and approximately 60mln was required to elute long chain fatty acids More recent assays were carried out using a stainless steel column (2 m in length and 3 mm o d ) containing 10~o Sdar-10C on 100/200 mesh Gaschrome-Q The column temperature was 210°C The carrier gas (mtrogen) flow rate was 40 ml mln- 1 The high polarity and temperature stabihty of the Silar-10C stationary phase enabled the rapid analysis of methyl esters of fatty acids For example, methyl esters of fatty aods of chain length C22 containing six double bonds were eluted m less than 10 mln

RESULTS

1 General properties of the isolated mttochondna (a) Respzratory actwtty The oxidative activity of mullet liver mltochondria at 25°C was examined by measuring oxygen uptake with various substrates The only substrates to produce any appreciable oxygen uptake were succinate, lSOCltrate, N A D H and ~-oxoglutarate with specific activities of 1 2, 0 9 and 0 2 nmoles 0 2 m l n - t mg protein- ~ respectively Other substrates which were tested (pyruvate, malate, citrate, fumarate and glutamate) gave no significant increases in respiratory rate over the rate of endogenous respiration Succlnate and N A D H were also found to be the most active substrates when mltochondrIa from the other fish species were examined The isolated mltochondria did not exhibit respiratory control although a direct analysis of oxidative phosphorylatlon was not carried out The addition of A D P (0 1 mM) to mltochondrla oxidizing succlnate in the standard reaction medium supplemented with 5 m M MgC12 and 1 0 m M potassium phosphate, pH 7 4, did not result in any significant increase in oxygen uptake (b) Cytochromes Difference spectra of the fish liver mltochondrla were quahtatlvely similar to those found in rat liver (Chance & Williams, 1955) with the positions of the absorbance maxima for the ct bands of cytochromes c, b and a + a3 occurring at 550, 560 and 598 nm respectively

b

a + a3

Mltochondrla

c

Coral trout

0 2

0 3

0 1

0 5

0 5

0.2

0 7

0 7

0.2

0 5

0 6

0 2

(P macuZa~w.) Sweet-llp emperor

(L ehrysosto,~us ) Rlng-talled surgeon

(.4 zc~thopterus) Mullet

(M

cephalu,~)

Extlnctlon coefflclents for cytochrcme dlfference spectra are descrlbed in 'Methods'

A quantitative analysis of the fish mltochondrm cytochromes is presented m Table 1 Apart from the relatively low content of cytochromes b and c in coral trout, no significant lnterspeclfiC variations m the cytochromes were observed

2 The influence of temperature on enzyme activity (a) Temperature profiles Figure 1 shows the effect of temperature on suecmate oxldase activity m heart and liver mltochondrm isolated from mullet In the case of the heart mltochondrla, a relatively sharp decrease in activity was noted at temperatures beyond about 45°C A similar phenomenon was observed with liver mitochondria except that inactivation occurred at a lower temperature, namely around 35-38°C It was further noted that the specific activity of the sueclnate oxldase system was about 100-fold higher with heart as compared with hver mitochondria The effect of temperature on succlnate oxldase activity of cor~tl trout heart and liver mitochondrla (Fig 2) displayed characteristics similar to those noted with mullet There was a rapid decrease in enzyme activity at temperatures around 50°C for heart and around 40°C for liver mitochondrIa The specific activity of the heart mitochondria in this case was about 10-fold higher than that of the liver mitochondrla The effect of temperature on malate dehydrogenase, an enzyme associated with the mitochondrlal matrix, was examined with liver and heart mltochondria isolated from mullet and coral trout A somewhat broader temperature profile, as compared with succinate oxldase, was observed With mullet, mltochondria from the heart (Fig 3) again exhibited a higher temperature of maximal activity (approximately 45°C) in comparison with mitochondria from the liver as shown In Fig 3 (approximately 35°C) A slightly higher specific activity of heart mitochondrial malate dehydrogenase was observed in relation to that of liver mltochondrla Broad temperature profiles were also noted with malate dehydrogenase In coral trout heart and liver mltoctmndria (Fig 4) No marked differences in specific actwlty were observed between liver and heart malate dehydrogenase from this species The temperature profile of sueclnate dehydrogenase activity in coral trout heart mitochondria was also examined In general, the temperature of inactivation

84

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Fig 1 Succmate oxldase temperature profiles for mullet mItochondrm Specific actlwty is expressed as nmoles 02 consumed rain 1 (rag protein)was not as sharp as that for succmate oxldase, with maximal actw~ty centred at about 40°C All three enzyme systems, that is succanate oxadase, malate dehydrogenase, and succmate dehydrogenase, were completely anactwated at temperatures greater than 55-60°C This was true for laver and heart matochondrm isolated from all four species of fish exammed (b) Arrhenlus plots The effects of temperature on enzyme actw|ty may be exammed an the form of Arrhenms plots m which the logarithm of specific activity is plotted against the reclprlcol of the absolute temperature Arrhenlus plots for succmate oxldase actwlty in mullet heart and laver matochondrla are lllus-

trated in Fig 5 The experimental points show a deviation from hneanty, particularly at temperatures above about 20~C, and a curve appears to be the best fit At temperatures less than about 20°C an essentially hnear Arrhenms plot, with a constant energy of actwatlon, was observed Average Arrhenlus activation energies were calculated by tangential extrapolation of experimental points as illustrated in Fig 5 There was an approximate 2- to 3-fold increase in activation energ~¢ at temperatures below the inflection or "break" in the Arrhenlus plots The actwatlon energies for succlnate oxldase from heart mltochondrla were slgnLficantly higher, both above and below this dlscontmmty, than

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Mitochondrial enzymes of tropical fish: a comparison with fish from cold-waters.

Comp Bmchem Ph~stol 1976 [ol 54B pp 81 to 92 Perqamon Press Printed in Great Britain MITOCHONDRIAL ENZYMES OF TROPICAL FISH" A COMPARISON WITH FISH...
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