Amino acid sequence of alpha-chain of hemoglobin IV from trout (Salmo irideus)

The amino acid sequence of the alpha-chain of trout hemoglobin (Hb) IV is given, thus completing the primary structure of the hemoglobin component of trout's blood characterized by the Root effect. The trout Hb IV alpha-chain consists of 142 amino acid residues; comparison with the corresponding sequences from human and carp hemoglobins shows differences of 50.0 and 35.9%, respectively. A difference of 39.6% is found with the alpha-chain of trout Hb I, the other major hemoglobin component of trout blood, devoid of heterotropic effects.     

Redox properties of components I and IV of trout hemoglobins: kinetic and potentiometric studies

Redox properties of component I and IV from trout hemoglobin (Salmo irideus) have been studied kinetically and at equilibrium. In the case of component I of trout hemoglobin, the mid-point potential (Eh) is pH independent below the acid-alkaline transition (pKa approximately equal to 8.6) and decreases at higher pH, following the deprotonation of the water molecule. Similarly to human hemoglobin, the mid-point potential of component IV of trout hemoglobin is pH-dependent, but the redox Bohr effect is extended to more acid pH. Moreover, the cooperativity of the redox equilibrium process is higher than in human hemoglobin. These features parallel the oxygen-binding properties of the same hemoglobin components from trout hemolysate. Differently from human hemoglobin, the oxidation kinetics of the two hemoglobins from trout by potassium ferricyanide show markedly biphasic progress curves with pH-independent second-order rate constants. This behavior suggests a different energy barrier for the interaction with ferricyanide in the two types of subunit of both Hb components from trout.     

The influence of social rank on adenohypophysial cell activity in Salmo irideus

Summary In nine cell types of the adenohypophysis in untreated adult rainbow trout, histologically different activity phases, seasonal changes in activity, and the relation between certain cell types and the interrenal gland, thyroid or gonads were investigated by light and, occasionally, by electron microscopy. Special attention was given to the effect of social rank on the synthetic activity in adenohypophysial cells of trout kept in small groups in which a social hierarchy with one (light) dominant and several (dark) submissives is established.Cell types in the rostral pars distalis were azocarminophil (I) or amphiphil (II). Proximal pars distalis cell types were slightly basophil (IV), orangeophil (V), strongly basophil (VI) or chromophobe (VII). In the pars intermedia, cell types were amphiphil (VIII) or very slightly basophil (IX). Type III was a non-secretory supporting (?) cell.Histologically different activity phases abounded in type IV cells, which mainly occurred in the proximal pars distalis but were also found dispersed in the rostral pars distalis, the pars intermedia and the neurohypophysis.Influences of social rank were pronounced in type IV cells. Phases with a high synthetic activity were exclusively found in submissive animals, phases with a low synthetic activity occurred in dominants. As a positive relation existed between type IV cell activity and the social rank dependent activity of the interrenal gland, it was suggested that type IV cells produce ACTH.In (dominant) male trout treated with DOCG or ACTH, colloid-containing type IV cell phases, reflecting accumulation of the secretory product, were found. This supported the earlier suggestion that ACTH in the trout is produced in the basophil type IV cells and not, as reported in the literature, in cells comparable to type II.The author is greatly indebted to Miss M.C. Wentzel, Miss I. Stulen, Mr. J. Veening and Dr. J.G. van Rhijn for their help with histological techniques, interrenal cell measurements and statistical aspects     

Effect of day length on oxidative capacities of mitochondria from red muscle of rainbow trout (Oncorhynchus mykiss)

In nature, seasons may be more reliably announced by changes in photoperiod than in temperature. To evaluate the role of day length in setting oxidative capacities of trout muscle mitochondria, we acclimated trout to summer (15 °C, 16L:8D), winter (5 °C, 8L:16D) and mixed conditions (15 °C, 8L:16D). Maximal oxidative capacities of isolated mitochondria at 5 and 15 °C were higher in mixed than summer conditions and higher again in winter conditions. At 5 °C, state 4 rates changed little with acclimation state whereas at 15 °C state 4 rates were lower in summer than in mixed or winter conditions. Using concentrations of the adenylate nucleotide translocase as the denominator for these rates gave much the same conclusions. By using inhibitors to block flux at specific points in the electron transport chain, we found that flux through Complexes II–IV was lowest in summer acclimated trout, increased upon acclimation to mixed and to winter conditions. Flux through complex IV was similar in trout acclimated to summer and mixed conditions, but increased significantly with acclimation to winter conditions. Flux through complex IV was 1.5 fold higher than state 3 rates for summer-acclimated trout but was similar to state 3 rates in trout acclimated to mixed or winter conditions. Our results indicate that a reduction in day length initiates increases in mitochondrial oxidative capacity typically associated with cold acclimation and that acclimation to both cold temperatures and short day lengths enhanced these changes. The overall similarity of the responses of state 3, of flux through complexes II–IV and of flux through complex IV suggests that a generalised mechanism such as changes in the phospholipid composition of the inner mitochondrial membrane may coordinate these changes.     

Infrared stretching frequencies of CO in carbomonoxyhemoglobin from trout

The infrared spectra of the carbomonoxy derivatives of the hemoglobin components I and IV from trout have been measured in the CO stretching frequency region using a high resolution infrared spectrometer. The CO stretching frequency of Hb I CO is very close to that of carbomonoxy human hemoglobin and is pH-independent. In contrast, the CO stretching frequency of Hb IV CO is higher and shows a small but significant pH dependence in the range 6.2-7.8. These results point to a decreased strength of the iron-CO bond in Hb IV CO at low pH, in agreement with the conclusions drawn from the reported difference spectra of Hb IV CO as a function of pH.     

The temperature dependence of State IV respiration, the calcium uptake system, and the activity of the calcium ionophore A23187 in mitochondria from endo- and ectothermic animals.

1. Arrhenius plots of State IV respiratory activity of liver mitochondria from both rainbow trout and rat were linear over the temperature range 5-35 degrees C. 2. Calcium uptake was monitored by stimulation of oxygen consumption and by calcium electrode recording, with quite comparable results. Rainbow trout gave the usual linear Arrhenius plot but this plot for rat mitochondria exhibited two well-defined inflections or discontinuities. 3. The temperature dependence of the activity of the ionophore A23187 was investigated by measuring the increase in oxygen uptake following a sub-maximal dose of this drug. Again a linear relation was found for rainbow trout, but in this case the rat curves showed only a single inflection point. 4. These results are discussed in relation to other work on the effects of lipid phase transitions on mitochondrial membrane-associated systems.     

Partial amino acid sequences of several globin chains from the sockeye salmon, Oncorhynchus nerka

1. Partial amino acid sequences for several sockeye salmon hemoglobin beta-chains have been determined and compared to several other fish beta-chain sequences. 2. A 90% homology exists between the sockeye cathodal (C1) beta-chain and the trout Hb I beta-chain for residues 1-19. 3. The sockeye salmon anodal (A1-3) beta-chain is virtually identical to the trout HB IV beta-chain for the first 55 amino acid residues. 4. The alpha-chains of the sockeye salmon appear to be acetylated at the N-terminal position and about 0.6% of the sockeye hemoglobin is glycosylated.     

Structural analysis of fish versus mammalian hemoglobins: effect of the heme pocket environment on autooxidation and hemin loss

The underlying stereochemical mechanisms for the dramatic differences in autooxidation and hemin loss rates of fish versus mammalian hemoglobins (Hb) have been examined by determining the crystal structures of perch, trout IV, and bovine Hb at high and low pH. The fish Hbs autooxidize and release hemin approximately 50- to 100-fold more rapidly than bovine Hb. Five specific amino acid replacements in the CD corner and along the E helix appear to cause the increased susceptibility of fish Hbs to oxidative degradation compared with mammalian Hbs. Ile is present at the E11 helical position in most fish Hb chains whereas a smaller Val residue is present in all mammalian alpha and beta chains. The larger IleE11 side chain sterically hinders bound O(2) and facilitates dissociation of the neutral superoxide radical, enhancing autooxidation. Lys(E10) is found in most mammalian Hb and forms favorable electrostatic and hydrogen bonding interactions with the heme-7-propionate. In contrast, Thr(E10) is present in most fish Hbs and is too short to stabilize bound heme, and causes increased rates of hemin dissociation. Especially high rates of hemin loss in perch Hb are also due to a lack of electrostatic interaction between His(CE3) and the heme-6 propionate in alpha subunits whereas this interaction does occur in trout IV and bovine Hb. There is also a larger gap for solvent entry into the heme crevice near beta CD3 in the perch Hb (approximately 8 A) compared with trout IV Hb (approximately 6 A) which in turn is significantly higher than that in bovine Hb (approximately 4 A) at low pH. The amino acids at CD4 and E14 differ between bovine and the fish Hbs and have the potential to modulate oxidative degradation by altering the orientation of the distal histidine and the stability of the E-helix. Generally rapid rates of lipid oxidation in fish muscle can be partly attributed to the fact that fish Hbs are highly susceptible to oxidative degradation.     

Reproductive biology of brown trout, Salmo trutta macrostigma Dumeril 1858, in a tributary of the Ceyhan River which flows into the eastern Mediterranean Sea

The study describes some key elements of the reproductive biology, including spawning season, age at sexual maturity, fecundity and egg diameter of the native brown trout, Salmo trutta macrostigma, in a tributary of the Ceyhan River. A total of 197 brown trout (118 females and 79 males) were captured in 2000–2001 by electric fishing. In observations on monthly changes, the gonadosomatic index (GSI) and the monthly frequency distribution of egg diameter confirmed that spawning lasted from November to January. Some 27.7% of the females and 62.5% of the males attained sexual maturity in their second year. The smallest fork length (FL) of brown trout attaining sexual maturity was 17.4 cm for males and 17.8 cm for females. Mean fecundity in age groups II, III, IV and V were 360, 452, 693 and 1283 eggs per female, respectively. One 9‐year‐old female had a unique 3232 egg count. The mean fecundity of the sampled population was 554 eggs per fish, positively correlated with the FL (mm) (R = 0.8227 ) and body weight (R = 0.8130). The diameter of mature eggs in the spawning season ranged from 3.250 to 5.930 mm, with a 4.146 mm average. Mean egg diameter in age groups II, III, IV and V in the spawning season were 0.813, 3.799, 4.663 and 5.243 mm, respectively. Fecundity, egg weight and diameter were statistically different in all age groups.     

Genomic cloning of novel isotypes of the rainbow trout interleukin-8

A cDNA clone, designated IL-8nL, was obtained by suppression subtractive hybridisation between lipopolysaccharide-stimulated and non-stimulated populations of the rainbow trout macrophage-like cell line, RTS11. IL-8nL was similar but not identical to a recently published sequence of the gene encoding rainbow trout interleukin-8 (IL-8). Amplification of genomic DNA by the polymerase chain reaction (genomic PCR) using a single outbred trout with common primers in the 5' and 3' untranslated regions gave six distinct genomic sequences, including one ( IL-8A) almost identical to that of the published IL-8 gene and another identical to IL-8nL. The other four clones were termed IL-8B, IL-8C, IL-8D and IL-8E. The deduced amino acid sequences of IL-8A through IL-8E are all identical to the published IL-8, while the IL-8nL protein has a substitution of Arg87 to Lys. Analysis of ten outbred trout by genomic PCR of a repeat region in exon 4, which has three different sizes in the above alleles, revealed a shorter, fourth fragment termed IL-8X and another of the same size as IL-8nL, but with a different single nucleotide replacement, called IL-8nL2. These results, together with a Southern blot of the same ten individuals showing up to five bands, indicate that rainbow trout has at least four copies of the IL-8 gene. Like IL-8nL, IL-8X lacks the repeat sequence in exon 4 and encodes a protein identical to IL-8nL protein. Polymerase chain reaction of the repeat region was useful for typing rainbow trout into four categories, and the type III and IV fish have a new allele, IL-8F, which lacks one repeat unit compared with IL-8A.     

Intestinal carbonic anhydrase, bicarbonate, and proton carriers play a role in the acclimation of rainbow trout to seawater

Abrupt transfer of rainbow trout from freshwater to 65% seawater caused transient disturbances in extracellular fluid ionic composition, but homeostasis was reestablished 48 h posttransfer. Intestinal fluid chemistry revealed early onset of drinking and slightly delayed intestinal water absorption that coincided with initiation of NaCl absorption and HCO(3)(-) secretion. Suggestive of involvement in osmoregulation, relative mRNA levels for vacuolar H(+)-ATPase (V-ATPase), Na(+)-K(+)-ATPase, Na(+)/H(+) exchanger 3 (NHE3), Na(+)-HCO(3)(-) cotransporter 1, and two carbonic anhydrase (CA) isoforms [a general cytosolic isoform trout cytoplasmic CA (tCAc) and an extracellular isoform trout membrane-bound CA type IV (tCAIV)], were increased transiently in the intestine following exposure to 65% seawater. Both tCAc and tCAIV proteins were localized to apical regions of the intestinal epithelium and exhibited elevated enzymatic activity after acclimation to 65% seawater. The V-ATPase was localized to both basolateral and apical regions and exhibited a 10-fold increase in enzymatic activity in fish acclimated to 65% seawater, suggesting a role in marine osmoregulation. The intestinal epithelium of rainbow trout acclimated to 65% seawater appears to be capable of both basolateral and apical H(+) extrusion, likely depending on osmoregulatory status and intestinal fluid chemistry.     

Pharmacokinetics of morphine in fish: winter flounder (Pseudopleuronectes americanus) and seawater-acclimated rainbow trout (Oncorhynchus mykiss)

We made a single intraperitoneal (IP) injection of morphine sulfate (40 mg/kg) into winter flounder and seawater acclimated rainbow trout at 10 degrees C and then followed its disposition by measuring the change in plasma morphine concentration for 100 h using a morphine specific ELISA. Disposition also was followed for 6h after a single IV injection of 7.5mg morphine sulfate in winter flounder. Plasma morphine reached a maximum within an hour post-injection IP and then decreased in a bi-exponential fashion with a rapid distribution phase followed by a slower elimination phase. The disposition was slower in flounder than in trout even though the fish were held at the same temperature. For example, plasma clearance was 76 mL h(-)(1) kg(-)(1) in the flounder but was almost twice as much in the trout (153 mL h(-)(1) kg(-)(1)) and mean residence time was 27.9h in the flounder but was 7.0 h in the trout. The present study is the first comprehensive pharmacokinetic analysis for any analgesic in an ectotherm, and our results show that: 1) significant intra-specific variation exists between fishes: and 2) the disposition of morphine in fish is approximately one order of magnitude slower than it is in mammals. These differences may be due in part to mass specific differences in cardiac output.     

Steady-state fluorescence and circular dichroism of trout hemoglobins I and IV interacting with tributyltin

The effect of tributyltin chloride (TBTC) on rainbow trout (Salmo irideus) hemoglobin I (HbI) and hemoglobin IV (HbIV) was characterized by the steady-state fluorescence of intrinsic and extrinsic fluorescent probes. The fluorescence emission spectrum (lambdaex 280 nm) is greatly increased in intensity by the presence of the organotin in both proteins. Circular dichroism spectra in the same samples show a small decrease in theta222, a measure correlated with the percentage of the alpha-helical content. Morever, important changes in near-UV, Soret, and visible regions of CD were induced by TBTC. The correlation of data obtained with trout hemoglobins (HbI and HbIV) with similar measurements on globins suggests that the presence of heme is necessary for the interaction of the organotin compound with the proteins.     

Effect of anions on the oxygen binding properties of the hemoglobin components from trout (Salmo irideus).

The effect of several anions on the oxygen equilibrium of hemoglobin components (Hb Trout I, II, and IV) from trout has been investigated.The functional properties of Hb Trout I and II are very slightly affected by organic phosphates (ATP, IHP) and pyridoxal phosphate. On the other hand the oxygen affinity of both components is affected, to the same extent, by the presence of sodium chloride; this effect seems to be pH and temperature independent. For Hb Trout I experiments on the effect of orthophosphate, pyrophosphate and pyridoxal phosphate point to a certain degree of correlation between the size of the phosphate and its effect on the functional behavior of the protein.In the case of Hb Trout I and II the differences in the effect of the various organic and inorganic phosphates may be interpreted, at a molecular level, in terms of loss of charge complementarity and (or) steric hindrance effects.On the other hand, as in the case of human hemoglobin, organic or inorganic phosphates decrease the oxygen affinity of Hb Trout IV. In addition various phosphates shift the region where the Root effect is operative toward higher pH values, thereby acting as allosteric effectors. For pyridoxal phosphate, kinetic experiments have shown that the rate of binding to Hb trout IV is several orders of magnitude smaller than that for other organic phosphates, similarly to what has been reported for human hemoglobin.     

Properties of trout hemoglobins reconstituted with unnatural hemes.

Native globins isolated from trout hemoglobin compoents I and IV have been reconstituted with proto-, meso-, and deuteroheme, and the spectral and functional properties of the reconstituted hemoglobins have been investigated. Equilibrium and kinetic studies allow the following conclusions. (a) The properties of the proto-reconstituted hemoglobins are very similar, or indistinguishable, from those of the native Hb's I and IV. (B) The CO binding kinetics for both proteins were found to be consistent with the equilibrium data: the overall association rate constant increases (and the autocatalytic character of the reaction decreases) in the order proto, meso, deutero. (c) A marked pH dependence of both ligand affinity and cooperativity is maintained in the reconstituted Hb's IV: at pH 6 the fractional saturation with oxygen in air (Root effect) is lower for proto- than for meso- and deutero-Hb IV. The results obtained, including partial photodissociation experiments at different pH values, can be considered, to a first approximation, consistent with the basic features of a simple two-states model.     

Roles of cortisol and carbonic anhydrase in acid–base compensation in rainbow trout, <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

Fish compensate for acid–base disturbances primarily by modulating the branchial excretion of acid–base equivalents, with a supporting role played by adjustment of urinary acid excretion. The present study used metabolic acid–base disturbances in rainbow trout, Oncorhynchus mykiss, to evaluate the role played by cortisol in stimulating compensatory responses. Trout infused with acid (an iso-osmotic solution of 70 mmol L⁻¹ HCl), base (140 mmol L⁻¹ NaHCO₃) or saline (140 mmol L⁻¹ NaCl) for 24 h exhibited significant elevation of circulating cortisol concentrations. Acid infusion significantly increased both branchial (by 328 μmol kg⁻¹ h⁻¹) and urinary (by 5.9 μmol kg⁻¹ h⁻¹) net acid excretion, compensatory responses that were eliminated by pre-treatment of trout with the cortisol synthesis inhibitor metyrapone (2-methyl-1,2-di-3-pyridyl-1-propanone). The significant decrease in net acid excretion (equivalent to enhanced base excretion) of 203 μmol kg⁻¹ h⁻¹ detected in base-infused trout was unaffected by metyrapone treatment. Acid- and base-infusions also were associated with significant changes in the relative mRNA expression of branchial and renal cytosolic carbonic anhydrase (tCAc) and renal membrane-linked CA IV (tCA IV). Cortisol treatment caused changes in CA gene expression that tended to parallel those observed with acid but not base infusion. For example, significant increases in renal relative tCA IV mRNA expression were detected in both acid-infused (~2x) and cortisol-treated (~10x) trout, whereas tCA IV mRNA expression was significantly reduced (~5x) in base-infused fish. Despite changes in CA gene expression in acid- or base-infused fish, neither acid nor base infusion affected CAc protein levels in the gill, but both caused significant increases in branchial CA activity. Cortisol treatment similarly increased branchial CA activity in the absence of an effect on branchial CAc protein expression. Taken together, these findings provide support for the hypothesis that in rainbow trout, cortisol is involved in mediating acid–base compensatory responses to a metabolic acidosis, and that cortisol exerts its effects at least in part through modulation of CA.     

Crystallization of the rainbow trout (Salmo gairdneri) haemoglobin IV

Crystals of rainbow trout (Salmo gairdneri) haemoglobin IV were grown in mini batches from a solution of ammonium sulphate. Large single crystals grew over five days and were up to 2 mm in length. X-ray diffraction experiments indicated a space group of C222(1) with unit cell dimensions of a = 85.3 A, b = 94.6 A and c = 105.7 A. The crystals diffract to better than 2.5 A but exhibit some mosaicity along the c axis.     

Nuclear Magnetic resonance quadrupole relaxation studies of chloride binding to the isolated hemoglobins from trout (Salmo irideus).

NMR studies of chloride binding to the main components of trout blood, Hb Trout I and Hb Trout IV, indicate that although the affinity of chloride is high for both hemoglobins, the characteristics of the binding process are markedly differnet. In Hb Trout IV chemical exchange at the chloride binding site(s) is fast and quadrupole effects determine the linewidth; chloride binding has a definite pH dependence, but there is no significant oxygen linkage. In contrast Hb Trout I represents a unique case of slow chemical exchange, which may depend on unusual stereoche mical characteristics of the chloride binding site; chloride binding is pH independent, but shows a significant oxygen linkage, which may be attributed to changes of the lifetime of chloride at the binding site. The chloride binding properties displayed by Hb Trout I and IV have been compared with those of normal and modified human hemoglobins and discussed in terms of the structural differences in the C- and N-terminal regions of the alpha- and beta-chains.     

Central ventilatory and cardiovascular actions of angiotensin peptides in trout

In the brains of teleosts, angiotensin II (ANG II), one of the main effector peptides of the renin-angiotensin system, is implicated in various physiological functions notably body fluid and electrolyte homeostasis and cardiovascular regulation, but nothing is known regarding the potential action of ANG II and other angiotensin derivatives on ventilation. Consequently, the goal of the present study was to determine possible ventilatory and cardiovascular effects of intracerebroventricular injection of picomole doses (5-100 pmol) of trout [Asn(1)]-ANG II, [Asp(1)]-ANG II, ANG III, ANG IV, and ANG 1-7 into the third ventricle of unanesthetized trout. The central actions of these peptides were also compared with their ventilatory and cardiovascular actions when injected peripherally. Finally, we examined the presence of [Asn(1)]-ANG II, [Asp(1)]-ANG II, ANG III, and ANG IV in the brain and plasma using radioimmunoassay coupled with high-performance liquid chromatography. After intracerebroventricular injection, [Asn(1)]-ANG II and [Asp(1)]-ANG II two ANG IIs, elevated the total ventilation through a selective stimulatory action on the ventilation amplitude. However, the hyperventilatory effect of [Asn(1)]-ANG II was threefold higher than the effect of [Asp(1)]-ANG II at the 50-pmol dose. ANG III, ANG IV, and ANG 1-7 were without effect. In addition, ANG IIs and ANG III increased dorsal aortic blood pressure (P(DA)) and heart rate (HR). After intra-arterial injections, none of the ANG II peptides affected the ventilation but [Asn(1)]-ANG II, [Asp(1)]-ANG II, and ANG III elevated P(DA) (50 pmol: +80%, +58% and +48%, respectively) without significant decrease in HR. In brain tissue, comparable amounts of [Asn(1)]-ANG II and [Asp(1)]-ANG II were detected (ca. 40 fmol/mg brain tissue), but ANG III was not detected, and the amount of ANG IV was about eightfold lower than the content of the ANG IIs. In plasma, ANG IIs were also the major angiotensins (ca. 110 fmol/ml plasma), while significant but lower amounts of ANG III and ANG IV were present in plasma. In conclusion, our study suggests that the two ANG II isoforms produced within the brain may act as a neurotransmitter and/or neuromodulator to regulate the cardioventilatory functions in trout. In the periphery, two ANG IIs and their COOH-terminal peptides may act as a circulating hormone preferentially involved in cardiovascular regulations.     

The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) uncouples mitochondrial oxidative phosphorylation in both sea lamprey (Petromyzon marinus) and TFM-tolerant rainbow trout (Oncorhynchus mykiss)

The toxicity of 3-trifluoromethyl-4-nitrophenol (TFM) appears to be due to a mismatch between ATP supply and demand in lamprey, depleting glycogen stores and starving the nervous system of ATP. The cause of this TFM-induced ATP deficit is unclear. One possibility is that TFM uncouples mitochondrial oxidative phosphorylation, thus impairing ATP production. To test this hypothesis, mitochondria were isolated from the livers of sea lamprey and rainbow trout, and O(2) consumption rates were measured in the presence of TFM or 2,4-dinitrophenol (2,4-DNP), a known uncoupler of oxidative phosphorylation. TFM and 2,4-DNP markedly increased State IV respiration in a dose-dependent fashion, but had no effect on State III respiration, which is consistent with uncoupling of oxidative phosphorylation. To determine how TFM uncoupled oxidative phosphorylation, the mitochondrial transmembrane potential (TMP) was recorded using the mitochondria-specific dye rhodamine 123. Mitochondrial TMP decreased by 22% in sea lamprey, and by 28% in trout following treatment with 50μmolL(-1) TFM. These findings suggest that TFM acted as a protonophore, dissipating the proton motive force needed to drive ATP synthesis. We conclude that the mode of TFM toxicity in sea lamprey and rainbow trout is via uncoupling of oxidative phosphorylation, leading to impaired ATP production.