Anisakis simplex: Uncoupling of oxidative phosphorylation in the muscle mitochondria of infected fish

EXPERIMENTAL

PARASITOLOGY

Anisakis

61,

270-279

simplex: Uncoupling of Oxidative Phosphorylation Muscle Mitochondria of Infected Fish KRYSTYNA

Division

(1986)

of Microbiology,

BOCZON' ANDJEFFREY W. BICR~

U.S. Food

(Accepted

in the

and Drug

Administrurion,

for publication

Washingion.

DC 20204,

U.S.A.

28 October 198s)

BOCZON, K., AND BIER, J. W. lY86. Anisakis simplex: Uncoupling of oxidative phosphorylation in the muscle mitochondria of infected fish. Exprrimrn~ul f’urusirology, 61, 270-279. Adenosine triphosphatase activity stimulated by Mg’+ was greater in muscle mitochondria of fish infected with lat-val Anisukis simplex nematodes than in uninfected fish. When muscle mitochondria were isolated in a sucrose ethylene-glycol bis(p-aminoethyl ether)N,N’-tetraacetic acid medium from fr-esh uninfected fish, they were loosely coupled, and their adenosine triphosphatase activity was comparable to that of mitochondria from rat tissue. Activity in infected fish was dose dependent, increasing with the number of worms per fish. Excretory secretory products or a cytoplasmic fraction of anisakines, when incubated with coupled rat mitochondria, also caused adenosine triphosphatase activity to increase. Storage of fish flesh caused an increase in adenosine triphosphatase activity, but such aging was not significant until 5 and 10 days after death in refrigerated and froren samples, respectively. The Mg” stimulated adenosine triphosphatase activity of muscle mitochondria can be used to estimate the number of nematodes per market fish. The type of medium used to isolate the mitochondria is crucial in such studies; an ionic medium with Nagarse proteinase was optimal for fish muscle mitochondria. D 1986 Academic Pres,. Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Anisakis simplex; Ascaris SUII~: Nematodes, parasitic; Fish host; Salmon, Pacific, Onchorhynchus spp.; Trout, Salmu Knit-drrrri: Goldfish, Carassius aurafzrs; Mitochondria, fish muscle; Respiration, rate; Respiratory control index (RCI); Adenosine triphosphatase (ATPase) (E.C. 3.6.3); Mg’+ stimulation; Excretory secretory product(s) (ES); Cytoplasmic fraction (CF); Aging of fish flesh; Storage temperature for fish flesh; Phosphate-buffered saline (PBS); Bovine serum albumin (BSA): Nicotinamide adenine nucleotide reduced (NADH); Ethylene glycol bis(B-aminoethyl ether)N,N’-tetraacetic acid (EGTA); Dinitr-ophenol (DNP); Adenosine diphosphate (ADP); Coefficient of variation (CV).

INTRODUCTION

Current methods for digesting fish or shellfish flesh in vitro and identifying morphological features of recovered worms by microscopy are time consuming and labor intensive. The goal of these investigations was to develop biochemical techniques for detecting Anisakis simplex and other anisakine nematodes in seafoods. Previous attempts to identify parasite constituents biot Present address: Department of Biology and Mcdical Parasitology, Poznan Academy of Medicine, Fredry 10, Poznan 61-701, Poland. * To whom requests for reprints should be addressed. 270 0014-4894/86

$3.00

Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.

chemically were unsuccessful. Unlike their terrestrial ascarid relatives, anisakine nematodes do not contain ascarosides or other unique components in quantities practical for assessing parasitism (G. D. Cain 1978, U. of Iowa, Iowa City, IA, USA, unpublished report). We investigated the metabolic changes that occur in hosts invaded by parasites because these changes might amplify the stimulus provided by the parasite. There are precedents for this approach. Raybourne et a/. (1983) demonstrated that the ES products of A. simplex inhibited proliferation of mitogen induced lymphoblasts. In trichinellosis, two major changes

Anisakis simplex: PHOSPHORYLATION UNCOUPLING

occur in the bioenergetic metabolism of muscle mitochondria in experimentally infected rats (Michejda and Boczon 1972). (1) An uncoupling of oxidative phosphorylation, reversible by adding BSA during the acute phase of the infection (2-4 weeks), becomes irreversible after about 4 weeks. (2) After 4 weeks, all NADH mediated substrate oxidation is inhibited and succinate oxidation is increased. The factor responsible for the uncoupling of mitochondrial phosphorylation, as indicated in vitro, is present in both a cytoplasmic fraction of Trichinella spiralis larvae and, in smaller amounts, in ES products. It acts as an uncoupler of oxidative phosphorylation and as an inhibitor of NADH linked dehydrogenases. The size of the molecule, its temperature sensitivity, and the correlation of activity with the amount of protein in the preparation suggest that the factor is indeed a protein (Michejda and Boczon 1972). The total uncoupling of rat liver mitochondria during invasion by the trematode Fasciola hepatica has been reported for as few as two worms in the host’s liver or bile duct (Van den Bossche et al. 1982). The factor responsible for the uncoupling was heat labile and could not be dialyzed. In looking for such an effect of anisakine nematodes on fish muscle mitochondria, we reasoned that the degree of uncoupling could be correlated with the number of anisakines in a fish to provide a quantitative measure of fish anisakiasis. We quantitated in vivo and in vitro conditions for the uncoupling effects of the ES products of nematodes using the level of mitochondrial ATPase activity as a measure of the “degree” of uncoupling. Reports in the literature on the bioenergetic metabolism of fish tissues deal exclusively with liver tissue, and results with fish liver mitochondria vary greatly from fish species to species (Hochachka 1969; Suarez and Hochachka 1981). Because the classic uncouplers of oxidative phosphorylation reduce respiratory control and stimu-

271

late oligomycin sensitive ATPase, we investigated the effect of anisakines on the bioenergetic characteristics of fish muscle mitochondria in two ways: polarographically, by measuring the rate of respiration and the RCI with succinate, and colorimetrically, as the level of activity of mitochondrial Mg* + stimulated ATPase. The ATPase in properly isolated mitochondria is latent; it can be activated by uncouplers and by such factors as aging or improper handling. MATERIALS AND METHODS Pacific salmon (Unchorhynchus spp.) were used in the infectivity studies. Trout (Salmu gairdneri) and goldfish (Carassius auratus) were used in the aging studies. The salmon were market samples, dead but fresh, air-shipped to Washington, DC, USA from Seattle, WA, USA. Infection of fish with anisakine nematodes was determined by digestion (Jackson et al. 1981). All anisakines recovered by digestion were identified as Anisakis simplex third stage larvae; each larva had a boring tooth, an anterior ventral excretory pore, a ribbon shaped excretory gland attached to the left lateral cord, an elongate glandular esophagus (ventriculus) with an oblique intestinal junction, and a mucron on the tip of the tail. To obtain ES products and the CF, we maintained worms recovered from fish in vitro at 4 C in Hanks’ solution. Four media were tested to isolate intact mitochondria from fish muscle: (1) 0.25 M sucrose + 0.03 mM EGTA (modified from Michejda and Boczon 1972); (2) 0.25 M sucrose + 0.3 nuI4 EGTA; (3) 0.25 M sucrose + 2 nuI4 EGTA + 10 mM Tris-HCl + heparin, 500 U/g fish tissue (Van Hinsberg et al. 1978); (4) 0.1 m&I KC1 + 0.05 M Tris-HCl, pH 7.4, + 1 n&I ATP + 5 r&f MgCl + 1 mM EGTA + 3 mg Nagarse proteinase (Makinen and Lee 1968; Cheah and Cheah 1981). Media 1 and 2, which were developed for isolating rat muscle mitochondria, were used in the earlier experiments; Media 3 and 4 were developed for isolating human muscle mitochondria. Medium 4 was used in our later experiments. To homogenize tissues, we used a Potter-Elvehjem grinder with a Teflon pestle (about 10 strokes) for 2-3 min in an ice bath. (Homogenization with a blender, operated intermittently at 25,000 rpm for a total of 3 min, yielded damaged mitochondria.) For Media l-3, 10 g minced fish muscle tissue was homogenized with 100 ml of the appropriate medium. The homogenate was centrifuged at 600s for 6 min. The second centrifugation of the supematant at 12,000g for 10 min produced a sediment that was the mitochondrial fraction. Mitochondria were suspended in the medium for a protein concentration of 10 mg/ml.

272

BOCZON AND BIER

For Medium 4, 10 g minced fish muscle tissue was mixed with a stirring bar for 2-3 min; the mixture was adjusted to pH 7.3 with a 0.1 N HCI solution and then homogenized for l-2 min. The homogenate was centrifuged at 14,OOOg for 8 min, the supernatant was discarded, and the sediment was dispersed in the initial volume of Medium 4 without proteinase. A second centrifugation, at IOOOgfor IO min, served to sediment the cellular debris and cell nuclei that were then discarded. The third and final centrifugation, at 10,OOOg for 10 min, yielded a sediment of both heavy and light mitochondria. These were resuspended in Medium 4 without proteinase for a protein concentration of 10 mgiml The enzymatic activity of ATPase (an index of the bioenergetic state of mitochondria) was measured using a micromethod (Muszbek et al. 1977). Mitochondria (equivalent to IO-20 pg protein) were incubated for 10 min at 13 C with 0.4 mM ATP solution (in 0.06 M KC1 buffered to pH 7.4). The activity of Mg’stimulated ATPase was calculated as the differrence between activity measured in the presence and absence of 0.4 mM MgCI,. Total volume was OS ml. The reaction was started by adding the mitochondrial preparation and stopped by adding 0.25 ml 15% trichloroacetic acid to the reaction mixture. The amount of inorganic phosphate released after IO min was measured calorimetrically. Another estimate of the bioenergetic state of mitochondria, the RCI, was determined polarographically by measuring oxygen uptake with a Clark electrode at 0.6 V (Estabrook 1967) in an oxygraph (Yellow Springs Instruments, Yellow Springs, OH, USA), modified using a 13 x I5 mm vial containing 1.5 ml of the following medium: 125 mM KCI, 5 mM potassium phosphate, 20 mM Tris buffer, 0.1 mM EGTA, 3 mM MgCI,, 27 mgiml BSA free of fatty acids, and 10 mM succinate or 10 mM NADH. Mitochondria (0.7-1.5 mg) were added to the equilibrated reaction medium followed by substrate and 2.50-300 p,M ADP Oxygen content in the medium at 37 and 16 C was estimated using heart muscle mitochondria and spectrophotometrically standardized NADH. The parasitic worms used for production of ES and CF were recovered from fish muscle or viscera just before in vitro incubation. To inhibit bacterial and fungal growth in the incubation medium, we prewashed the worms for 30 min in PBS with a I-ml addition of a penicillin-streptomycin solution (penicillin 10,000 U/ml; streptomycin 10,000 kg/ml) and 1 ml of kanamycin (10,000 pgiml) per 100 ml. ATPase activity of mitochondrial preparations was measured after 45 min incubation with the appropriate nematode fraction at 37 C (rat tissue) or 13 C (salmon muscle). The crude ES product was obtained after 90hr incubation of parasite larvae in PBS at 30 C; the ES fraction was dialyzed in Spectrapor membrane tubing (molecular weight cutoff 12,000-14,000 for 2 hr

against polyethylene glycol at +4 C). The CF supernatant was obtained after differential centrifugation of 20% sucrose homogenate of parasite larvae (0.25 M sucrose, 20 mM Tris buffer, 1 mM EGTA; first centrifugation at 8OOgfor 6 min; second centrifugation at 13,OOOgfor 15 min). The ratio of nematode product’s protein content to mitochondrial protein content during common incubation was kept at about I:1 (or 2: I for the dialyzed ES fraction). Rat liver mitochondria were isolated according to Weinbach (1961); rat and salmon muscle mitochondria were isolated in ionic medium according to Makinen and Lee (1968), with the addition of Nagarese proteinase (Cheah and Cheah 1981). ATPase activity in coupled rat mitochondria was 5-7 umole of P,lmin/mg protein, 37-43 in loosely coupled rat liver, 13-15 in coupled rat muscle mitochondria, and about 30 umole P,lmin/mg protein in salmon muscle mitochondria (market sample). Protein estimates were made by the Bradford method (1976).

RESULTS

To correlate ATPase activity (measured enzymatically) and the RCI (measured polarographically), a proper medium is crucial for isolating intact mitochondria. The intact state of mitochondria is characterized by an RCI of 6-9 and little mitochondrial ATPase sensitivity to oligomycin. To obtain the best medium, we compared four media that seemed potentially useful. Measurements of Mg2+ stimulated mitochondrial ATPase in the muscle mitrochondria of two fish species for Media 1-4, respectively, were: trout, 48.0, 27.4, 19.5, and 24.4; goldfish 47.0, 67.0,--, and 2.7 (Medium 3 was not determined). The least contaminated mitochondrial preparation (as judged by oligomycin sensitivity of ATPase and RCI), therefore, was obtained in Medium 4. This was the medium of choice for continued experimentation, although preparations with similar Mg2+ stimulated ATPase activity were obtained in Media 2 and 3. Use of Medium 1 and homogenization with a blender were discontinued after preliminary experiments because they caused severe damage to mitochondria (as judged by a multiple increase of ATPase activity and a high respiration rate with exogenous NADH).

Anisakis simplex: PHOSPHORYLATIONUNCOUPLING The oligomycin sensitivity of ATPase in the uninfected fish muscle mitochondria isolated in Medium 2 was low in most fish muscle preparations; however, for mitochondria isolated from fresh trout (known death time) in Medium 4, the ATPase sensitivity to oligomycin was high (68%), as shown in Table I. Neither infected nor uninfected fish muscle preparations exhibited DNP stimulated ATPase activity. However, the ATPase sensitivity to oligomycin increased in mitochondria of infected fish muscle. To protect fish muscle mitochondria for storage in liquid nitrogen, we treated them with dimethyl sulfoxide and 10% BSA. Because the ATPase activity of these mitochondria increased more than it did after storage at 4 C for 2 days, only fresh mitochondrial preparations were used. An advantage of the calorimetric estimation of ATPase activity (Muszbek et al. 1977) over other calorimetric methods of phosphorus estimation (Fiske and Subbarow 1925), is greater sensitivity (nanomoles rather than micromoles of Pi are measured), thus requiring lo-20 times less mitochondrial protein and ATP for each test. The reproducibility of this ATPase assay adapted to fish muscle mitochondria was satisfactory (CV,

273

8%), and the results were comparable (49.1 and 57.4, respectively) to those obtained with the same rockfish preparation by the modified Fiske-Subbarow test (Sigma Colorimetric Test Kit 670). In 1960, Pullman used beef heart muscle mitochondria to demonstrate the proportionality in an ATP regenerating system between activity and the amount of enzyme protein; we constructed such a curve for fish muscle mitochondria (Fig. 1) and adjusted our protein concentration to the linear portion. The effect of enzyme protein concentration on enzymatic activity in two different fish species is shown in Fig. 1. Proportionality was observed only in a range of O-15 Fg with salmon muscle ATPase and to about 50 p.g with trout muscle ATPase; in all other assays, the protein concentration of the sample was between 10 and 20 kg/ml. The specific activity of mitochondrial Mg* + stimulated ATPase in mitochondria isolated in Medium 2 was similar for different fish species (Table II) and higher than the activity of control rat muscle mitochondria isolated in Medium 4. The oxygen uptake (with succinate as substrate) and the respiratory control ratios that were obtained are close to the theoretical values for

TABLE1 Effect of Specific Inhibitor (Oligomycin) and Uncoupler (2,Cdinitrophenol) on Mitochondrial Mg2+ Stimulated ATPase in Muscle of Fish Infected with Anisnkis simplex and in Uninfected Fish Oligomycin” % inhibition Fish Trout Cod Salmon

Medium 2 3 4 2 2

Uninfected fish 10 (2)b 49 (2)

68 (2) 0 (3)

12(3

2,4-Dinitrophenol % stimulation Infected fish NDc ND ND 73 (3) 73 (8)

Uninfected fish 0 0 4-l (2) 0 0 (4)

Infected fish ND ND ND 3-l (2) 0 (6)

(1Oligomycin (l-2 p.g/mg mitochondrial protein) or 2,4-dinitrophenol (0.8 mkf) were added 2-3 min before incubation of mitochondrial preparations for estimation of Pi release in ATPase assay. b Number of experiments shown in parentheses. c ND = not determined.

274

BOCZON

0.0

-‘1 100

mg0‘ RoteIn FIG. 1. Effect of enzyme concentration (kg protein) on Mgz+ stimulated ATPase activity in fish muscle mitochondria. 0, Trout; 0, goldfish; Pi = inorganic phosphorus.

rat liver mitochondria (RCI = 3). In a few cases, we worked with fish of known death time (goldfish, trout, and two salmon specimens). Mitochondria with relatively low

AND

BIER

ATPase activity were isolated from fresh trout. No substantial differences were found between Mg2+ stimulated ATPase activity in mitochondria isolated from uninfected market fish and those from muscles of fish with a known death time. Pooled data of ATPase measurements of uninfected fish muscle mitochondria were compared with those of anisakine infected fish. In 19 experiments, ATPase activity was 21.1% (CV, 26%) in uninfected fish as compared with 69.6% in 15 experiments on infected fish (P = 0.0062). ATPase activity in uninfected salmon (six experiments) was 21.2% (CV, 26%) as compared with 71.5% in 11 experiments on infected salmon (P = 0.0370). The Student I test indicated that the means were significantly different (P > 0.05). The separated means for uninfected

TABLE II Normal Fish Muscle Mitochondria: Enzymatic Activity of ATPase and Polarographic Measurements of Bioenergetics (Oxygen Uptake and Respiratory Control Index with Succinate) QOz (in nmole OJminlmg protein)”

Host tissue Rockfish Cod Haddock spot Flounder Salmon Trout Goldfish Rat control (Loosely coupled liver mitochondria) (Coupled muscle mitochondria) (Uncoupled muscle mitochondria)

State III’

State IV

ATPase activity”

+ ADP

-ADP

20.2 (3)d 11.5 (3) 19.5 (2) 21.7 (2) 27.5 (1) 21.2 (6) CV, 26%’ 27.4 (2) 67.0 (1)

20.6 (3) ND 7.2 (2) ND 20.4 (4) 64.8 (2) 4.6 (1)

ND 9.8 (3) ND 5.5 (2) ND 11.4 (4) 18.5 (2) 1.6 (1)

ND (3) 2.1 ND 1.3 (2) ND 2.4 (4) 2.9 (2) 2.9 (1)

5.6

56.5 (5)

22.6 (5)

2.8 (5)

9.1

15.8 (2)

8.8 (2)

1.8 (2)

29.6 (1)

ND'

ND

ND

RCI

1.0

a ATPase activity of mitochondria was isolated in Medium 2 at 13 C and measured in pH 7.2-7.4 buffer (expressed in nmoles P,/min/mg protein. b QO, was measured with succinate as substrate at 16 C for fish mitochondria and at 30 C for rat tissue mitochondria. c State III respiration was measured after addition of 30 (*M ADP Control rat liver mitochondria was isolated according to Weinbach (1961). d Number of experiments in parentheses. e ND = not determined. f CV = coefficient of variation.

Anisakis simplex: PHOSPHORYLATIONUNCOUPLING and infected salmon also appeared to differ significantly. The relatively high (25%) CVs in all uninfected fish samples and in the salmon samples might be due to the age differences of the fish meat rather than basic differences in the ATPase activities of different fish species. Intensity of infection and the level of Mg2+ stimulated ATPase activity (Table III) appear to be related. In a group of moderately infected salmon, cod, or rockfish (5-39 worms/kg), the increase of ATPase activity was 2.5-4.0 times; in a sample of heavily infected salmon (85 worms/kg), the increase was more than sixfold. Because ATPase is activated during the aging of mitochondria, we performed a series of experiments to determine the influence of refrigerated storage on the ATPase activity of fish muscle mitochondria within fish flesh and in an isolated preparation. Aging for 2-4 days activated the latent ATPase 1.3- to 1.5-fold, depending on the type of medium used. However, activation was 1.6- to 4.4-fold between 5 and 14 days of storage (Table IV). In two experiments, the possible cumulaTABLE III Dependence of Mitochondrial Mg2+ Stimulated ATPase Activity in Fish Muscle Mitochondria on Number of Anisakis simplex Nematodes per Fish Fish species

Intensity of infection’

Salmon

0 S- 17 worms/kg 29-39 worms/kg 85 worms 0 2-3 worms/kg 10 worms 15 worms 0 7-28 worms/kg

275

tive effect of parasitic nematodes and aging at 4 C were investigated. There was little aging effect after 2 days (1.1 x stimulation) in uninfected and lightly infected (2 worms/ kg) salmon; however, in moderately infected salmon, the effect of aging resulted in a 2.9 x increase of ATPase activity. To confirm that the increase in mitochondrial ATPase activity of fish muscle was caused by anisakine infection, we performed the following in vitro experiments. The ES product or the CF of Anisakis simplex larvae and one fraction marked Ascaris suum were incubated in vitro with isolated rat liver or salmon muscle mitochondria. In uncoupled or loosely coupled mitochondria (RCI = l-2), there was almost no change in ATPase level. In coupled preparations (RCI = 5-6), ES induced a strong stimulation (Table V). A similar, less pronounced effect was observed when the CF isolated from homogenized parasites was used. In some experiments, dialyzed ES product (a fivefold concentration of protein) was even more effective in stimulating ATPase activity, suggesting the involvement of a high molecular weight substance(s). The ratio of ES product or cytoplasmic fraction to mitochondrial protein in these incubations should be 1: 1 on the basis of 80

ATPase activityb

70 60 1

Cod

Rockfish

21.2 (6) 50.0 (8) -c 15.1

50 40 1

65.0 (3)

130.0 (1) 17.5 (3) 17.0 (2) 43.7 (1) 72.8 (1) 20.2 (3) 70.6 (2)

0 Intensity of infection in fish was estimated by digestion. b ATPase activity was measured at pH 7.2-7.4 after 10 min of incubation at 13 C. Protein content in ATPase assay was between 10 and 20 pg.

30 6 B c

1 20 10 ri oc6

2

FIG. 2. pH dependence of Mg?+ stimulated ATPase activity. Average values are given for two mitochondrial preparations isolated in Medium 4. Protein content was lo-20 pg per test vial; buffer range, pH 5.4-8.2; ratio of Mg2+ to ATP was 1:l. n , Cod: Cl, haddock; 0, goldfish; 0, salmon; Pi = inorganic phosphorus.

276

BOCZON

AND

BIER

TABLE IV Effect of Storage at 4 C on Mgr+ Stimulated ATPase Activity in Fish Muscle Mitochondria” Days of storage Medium

0

l-5

5-14

Isolated fish muscle mitochondria in medium Uninfected fish (including salmon) Infected salmonc (2 worms/kg) Infected salmon (15 worms/kg)

27.3 (5)b

31.0 (5)

33.4 (2)

27.3 (1)

29.8 (1)

ND“

26.0 (1)

75.4 (I)

ND

Fish muscle mitochondria in fish meat Trout (refrigerated) with Medium with Medium with Medium Salmon (frozen) with Medium

2 3 4

21.6 (2) 19.5 (2) 24.4 (2)

29.6 (5) 29.7 (5) 37.0 (5)

2

19.0 (2)

ND

45.3 (6) CV, 3lW 86.5 (4) 50.1 (5) 29.6 (2)

u Specific ATPase activity was measured at pH 7.2-7.4; mitochondrial protein content was IO-20 pg. b Number of experiments shown in parentheses. c Infected with Anisakis simplex. d ND = not determined. c CV = coefficient of variation.

protein content to produce significant results. We also have preliminary evidence of a similar but less pronounced effect produced by the ES product of A. suum migratory larvae (stages 2-3) on rat liver mitochondria. DISCUSSION The bioenergetic features of fish muscle mitochondria were investigated polarographically and calorimetrically (enzymatitally). Fish muscle mitochondria isolated in sucrose EGTA medium oxidized succinate in State IV, i.e., ADP-limited respiration (Chance and Williams 1955), at a rate similar to that of our rat muscle mitochondria. However, the rate of succinate oxidation in our experiments was lower than that found by Makinen and Lee (1968) or by Max et al. (1972) in rat muscle isolated in sucrose medium with heparin. Mitochondria from fish (trout) muscle isolated immediately after killing oxidized succinate at

only a slightly lower rate than that obtained by us with fresh rat muscle or by Weinbach (1961) with coupled rat liver mitochondria. These rates were within the range of values obtained by Smith (1973) in studies on the temperature-dependent State III, i.e., ADP-stimulated respiration (Chance and Williams 1955), in rainbow trout. Most of our fish samples were market aged (time of death unknown); however, for fish kept at 4 C, this aging had little effect on mitochondrial ATPase activity, i.e., 27.4 in fresh trout mitochondria (RCI = 2.9), 21.1 in all other fish species (RCI = 1.9). Our mitochondrial fraction from fish muscle (isolated in ionic medium obtained after differential centrifugation and final sedimentation at 10,OOOg)was a mixture of heavy and light subfractions. The latter (sedimented for rat muscle mitochondria at 14,OOOg/lOmin) was characterized by poor respiratory control, high Mg* + stimulated

Anisakis simplex: PHOSPHORYLATION UNCOUPLING

277

TABLE V In Vitro Effect of Anisakis

simplex Excretory Secretory Product or A. simplex Cytoplasmic Fraction on Mg*+

Stimulated ATPase Activity of Rat and Fish Muscle Mitochondria Nematode fraction”

Tissue Tightly coupled rat liver mitochondria Loosely coupled rat liver mitochondria Tightly coupled rat muscle mitochondria Salmon muscle (market sample)

RCIb

Stimulation

ESc CF

7.0 (2) 7.0 (2)

6.2x (2) 3.5x (3)

ESc ESc Ascaris suum ESc ESd CF ESc ESd

2.0 (2) 2.0 (2) 6.0 6.0 6.0 ND“ ND

1.6x 1.3x 3.7x 5.9x 2.5x 1.3x 2.9x

(2) (2) (2) (1) (2) (2) (1)

0 ESc = crude exeretory secretory product; CF = cytoplasmic fraction; ESd = dialyzed excretory secretory product. b RCI = respiratory control index. c Number of experiments shown in parentheses. d ND = not determined.

ATPase activity, weak sensitivity to stimulation by uncoupler DNP, and inhibition by oligomycin (Hedman 1965). The ATPase activity of heavy subfraction (sedimented for rat muscle at 35OOg/8min) is stimulated by DNP. Our mitochondrial preparation isolated in ionic Medium 4 had more intact mitochondria than those isolated in sucrose-EGTA media, as indicated by the 6times greater inhibition by oligomycin and stimulation (1.6~) by DNP (Table I). The pH may influence the uncoupler-stimulated ATPase activity; our preliminary experiments on uninfected haddock showed higher activity for DNP-stimulated ATPase activity measured at pH 8.2 than at pH 7.2. Cheah and Cheah (1981), using a modified Nagarse preparation, obtained pig muscle mitochondria that had an RCI of 6. Despite higher activity levels of Mg*+ stimulated ATPase in the pig than we obtained with fish mitochondria, their values for oligomycin inhibition were similar to ours for Media 3 and 4. The lack of oligomycin sensitivity of trout muscle mitochondria indicates that the preparation was contaminated. Therefore, we recommend that Me-

dium 3 or 4 be used or modified for further studies on the bioenergetic metabolism for fish muscle mitochondria. In future experiments, submitochondrial contaminants should be eliminated and the fish mitochondrial preparations monitored by electron microscopy. No detailed data on the pH dependence of DNP-stimulated ATPase were obtained. The lack of significant uncoupler stimulated ATPase activity (Table I) may be due to masking by an endogenous inhibitor, such as that isolated from beef heart mitochondria by Pullman and Monroy (1963). Full expression of uncoupler stimulated ATPase is clearly dependent on a number of parameters related to isolation and assay procedures (Chefurka 1981). However, a faster method for the isolation of undamaged and unaged fish muscle mitochondria is still needed for detailed work. Despite the limitations of extant methodology, differences in the ATPase level activity of muscle mitochondria in Anisakis simplex infected and uninfected fish are significant. The calorimetric micromethod of ATPase estimation is simple and rapid

278

BOCZON

enough to be considered for the practical diagnosis of fish anisakiasis. The uncoupling effect of the presence of Fasciola hepatica in rat liver was measured polarographically and cytochemically by Van den Bossche et a/. (1982). We carried out similar dual estimations but concluded that the simpler calorimetric (enzymatic) test should be used as the basis for routine comparisons between infected and uninfected fish. The difference between the ATPase level in the muscle mitochondria of infected and uninfected fish is statistically significant and depends on the intensity of infection. This observation is important because all our infected fish were market samples. The difference between the ATPase level in moderately infected fish muscle (5-39 worms/kg) ranged from 2.5 to 3.5 times that of uninfected fish. Assuming the age of our market samples to be 1 week or less, aging was probably not a significant influence. Like the in vitro effect of a Trichinella spirafis ES product or CF on rat muscle mitochondria, the products of fishborne anisakine nematodes uncoupled rat and salmon mitochondria (Table V). Tightly coupled rat liver mitochondria that we obtained by using a sucrose medium (Weinbath 1961) had the same bioenergetic features as those in work on reserpine uncoupling (Weinbach et al. 1983): RCI (7.0 and 5.6 nmole PJminlmg protein, respectively), succinate oxidation rate (20.6 and 20.0 nmole Pilminimg protein, respectively), and mitochondrial ATPase activity (5.6 and 9.0 nmole of Piiminimg protein, respectively). The intensity (Table V) of the in vitro stimulation of ATPase by crude anisakine ES products (using tightly coupled rat liver mitochondria) or dialyzed ES products (using tightly coupled rat muscle mitochondria) was strong (6 x ), similar to stimulation exhibited by the potent classic uncoupler, carbonyl cyanide m-chlorophenylhydrazone (Weinbach et al. 1983). Dialysis

AND

BIER

did not remove the active factor(s) present in parasite ES products and, according to our preliminary tests, boiling at 100 C for 15 min did not destroy this factor either (data not shown). This implies that a heat stable substance(s) of higher molecular weight is involved or that two substances are involved. We did not have enough ES to test more than one parameter. It is important that a similar degree of mitochondrial ATPase stimulation (6. lfold) was observed in tests with fish muscle heavily infected with Anisakis simplex. The results of the in vitro experiments prove that parasites cause an increase in the ATPase activity of infected fish muscle. ACKNOWLEDGMENTS Some of the data in this paper were first presented at the 1983 Annual Meeting of the American Society of Parasitologists in San Antonio, Texas. We thank Dr. J. E. Urban, Jr. and Dr. F. W. Douvres, Animal Parasitology Institute, Beltsville Agricultural Research Center. U.S. Department of Agriculture, Behsville, MD, U.S.A., for donating the Ascuris SL~U~excretory secretory product, and Dr. W. E. Hill, U.S. Food and Drug Administration, for help in preparing the figures. REFERENCES

M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.

BRADFORD,

Analytical

Biochemistry

12, 248-354.

CHANCE,B.. AND WILLIAMS. G. R. 1955. A method for localization of sites for oxidative phosphorylation. Nature (London)

176, 250-255.

CHEAH,K. S., ANDCHEAH,A. M. 1981. Mitochondrial calcium transport and calcium-activated phospholipase in porcine malignant hyperthermia. Biochimica et Biophysics

Actu 634, 70-84.

CHEFURKA,W. 1981. ATPase inhibitor and apparent deficiency of uncoupler-stimulated ATPase activity in mitochondria of houseflies (Mltscu domesticu). Compurutive

Biochemistry

und Physiology

69B,

371-376. ESTABROOK,R. W. 1967. Mitochondrial respiratory control and the polarographic measurements of ADP:O ratios. In “Methods in Enzymology,” “Oxidation and Phosphorylation” (R. W. Estabrook, ed.), pp. 41-47. Academic Press, New York. FISKE, C. H., AND SUBBAROW, Y. 1925. The colorimetric determination of phosphorus. Journal ofBiological Chemistry

66, 375-400.

Anisakis simplex:

PHOSPHORYLATION

HEDMAN, R. 1965. Properties of isolated skeletal muscle mitochondria from the rat. Experimental Cell Research 38, 1-12. HOCHACHKA, P. W. 1969. Intermediary metabolism in fishes. In “Fish Physiology” (W. G. Hoar and D. J. Randall, eds.), Vol. 1, pp. 351-390. Academic Press, New York. JACKSON, G. J., BIER, J. W., PAYNE, W. L., AND MCCLURE, F. D. 1981. Recovery of parasitic nematodes from fish by digestion or elution. Applied and Environmental Microbiology 41, 912-914. MAKINEN, M. W., AND LEE, C. P. 1968. Biochemical studies of skeletal muscle mitochondria. Microanalysis of cytochrome content, oxidative and phosphorylative activities of mammalian skeletal muscle mitochondria. Archives of Biochemistry and Biophysics 126, 75-82. MAX, S. R., GARBUS, J., AND WEHMAN, H. 3. 1972. Simple procedure for rapid isolation of functionally intact mitochondria from human and rat skeletal muscle. Analytical Biochemistry 46, 576-584. MICHEJDA, J. W., AND BOCZON, K. 1972. Changes in bioenergetics of skeletal muscle mitochondria during experimental trichinellosis in rats. Expcrimental Parasirology 31, 161-171. MUSZBEK, L., SZABO, T., AND FESUS, L. 1977. A highly sensitive method for the measurement of ATPase activity. Analytical Biochemistry 77, 286-288. PULLMAN, M. E. 1960. Partial resolution of the enzymes catalyzing oxidative phosphorylation. I. Purification and properties of soluble, dinitrophenolstimulated adenosine triphosphatase. ~ooumal ofBiological Chemistry 235, 3322-3329.

UNCOUPLING

279

PULLMAN, M. E., AND MONROY, G. C. 1963. A naturally occurring inhibitor of mitochondrial adenosine triphosphatase. Journal of Biological Chemistry 238, 3762-3769. RAYBOURNE,R., DESOWITZ,R. S., KLICKS, M. M., AND DEARDORFF,T. L. 1983. Anisakis simplex and Terrunova sp. inhibition by larval E-S products of mitogen-induced lymphoblast proliferation. Experimental Parasitology 55, 289-298. SMITH, C. L. 1973. The temperature dependence of oxidative phosphorylation and the activity of various enzyme systems in liver mitochondria from cold- and warm-blooded animals. Comparative Biochemistry and Physiology 46B, 445-461. SUAREZ, R. K., AND HOCHACHKA, P. W. 1981. Preparation and properties of rainbow trout liver mitochondria. Journal of Comparative Physiology 143, 269-273. VAN DEN BOSSCHE,H., VERHEYEN, A., VERHOEVEN,H., AND ARNOUTS, D. 1982. Alterations in rat liver mitochondria caused by Fasciolo hepatica. Contributions to Microbiology and Immunology 7, 30-38. VAN HINSBERG, V. W. M., VEERKAMP, J. H., AND VAN MOERKERK, H. T. B. 1978. Palmitate oxidation by rat skeletal muscle mitochondria. Archives of Biochemistry and Biophysics 190,762-771. WEINBACH, E. C. 1961. A procedure for isolating stable mitochondria from rat liver and kidney. Analytical Biochemistry 2, 335-343. WEINBACH, E. C., COSTA, J. L., CLAFGETT, C. E., FAY, D. D., AND HUNDAL, T. 1983. Reserpine as an uncoupler of oxidative phosphorylation and the relevance of its psychoactive properties. Biochemical Pharmacology 32, 137 l- 1377.