Strophocheilus oblongus heart mitochondria: Respiration and oxidative phosphorylation

EXPERIMENTAL PAFiASI’Q3LoGY 26, 203-208

Strophocheilus

Brandao2

Gregori

Institute de Bioquimica

Heart Mitochondria: Oxidative Phosphorylation’

oblongus

Respiration Dorei

(1969)

and

X. Niculitcheff,

Dinor

0.

Voss3 and

Annibal

P. Campello3

da Unioersidude Federal do Parand and Institute de Biologic Paranli, Brazil

e Pesquisas

Tecnol6gica.s( Divikio de Bioquimica ) , Curitiba, (Submitted

for publication

21 May 1969)

X., Voss, DINOR O., AND CAMPELLO, BRAND~O, DOREI, NICULITCHEFF, GREGOFU respiration and ANNUAL P. 1969. Strophocheilus oblongus heart mitochondria: Experimental Parasitology 26, 203-208. A study of rcspiraoxidative phosphorylation. tory chain of molluscan (Strophoceiks oblongus) heart mitochondria at room temperature was carried out. The concentration of cytochromes was determined. Oxidative phosphorylation was assayed by polarographic technique. It was found that ADP/O ratios were the same in the presence of either glutamate or a-ketoglutamte

(about 2.9), whereas for malate it was about 2.5. By Warburg respirometry the oxidation

of malate was higher

than that of succinate.

The oxidation

of a-glycerophos-

phate was similar to that of succinate. INDEX DESCRIPTORS: Strophocheilus oblongus; mitochondria; respiration; oxidative phosphorylation; respiratory chain difference spectra; cytochromes; polarograph;

oxygen uptake; mollusca; physiology; Respiration. Studies relating to comparative cellular metabolism among a wide variety of organisms are of interest for biochemistry. Although oxidative phosphorylation is readily demonstrable in several mammalian tissues, the occurrence of this process in molluscan tissues has been reported by few authors. The presence of triearboxylic acid cycle enzymes in snail tissues (Rees, 1953; Weinbath, 1953) suggests that one of their possible functions is the coupling of phosphorylation to oxidation. Rees (1953) obtained no evidence of inorganic orthophosphate uptake associated with the oxidation of tricarboxyhc acid cycle intermediates in 1 This research was supported by Conselho de Pesquisas da Universidade Federal do Param and Grants GM-11912 and TW-00223 of National Institutes of Health, U.S. Public Health Service. 2 Present address: Departmento de Bioquimica, Universidade de Campinas, S5.o Paulo, Brazil. 3 Bolsistas Chefe de Pesquisas do Conselho Nacional de Pesquisas.

Helix pomatiu. Similarly, Weinbach ( 1953) was unable to demonstrate oxidative phosphorylation with hepato pancreas of the aquatic snail, Lymnaea stagndis. However, after some experiments, Weinbach observed oxidative phosphorylation in the albumin gland of this snail and Weinbach and Nolan (1956) obtained evidence for the existence of oxidative phosphorylation in the albumin gland of BiomphaZuriu glabrata. Michejda et al. (1964) did a thorough study of this exergonic process in isolated sarcosomes from heart muscle (ventricle) of the snail, Helix pomutiu, with several substrates and in various conditions of incubation. Brandlo et al. (1967) and Brand50 et al. (1968) studied the: oxidative phosphorylation of Strophocheilus: oblongus heart mitochondria. The present paper shows some properties of the respiratory chain of molluscan (Strophocheilus oblongus) heart mitochondria.

BRAND;iO ET AL.

204 MATERIALS AND METHODS

Specimens of Strophocheilus oblongus, with a maximum shell of 8 cm in length, were collected around Porto Alegre, Rio Grande do Sul, Brazil. Preparation of S. oblongus heart mitochondria. S. oblongus heart mitochondria were prepared in a mannitol-sucrose medium (Voss et al., 1961). This medium contained 0.21 M mannitol, 0.075 M sucrose, 0.01 M Tris ( tris-hydroxyaminomethane), and 0.1 mM EDTA (ethylene diamine tetracetate, disodium salt). The final pH was 7.4. After the snail’s shells were broken the hearts were minced finely and homogenized in a Potter-Elvehjem homogenizer with 100 ml of the above-mentioned medium. After homogenization, the whole suspension was centrifuged 10 minutes at 1000 g. The sedimented fraction was discarded and the supernatant portion centrifuged for 10 minutes at 10,000 g. The lastsedimented fraction was washed twice with the medium. The pellet was then resuspended in 2 ml of the same extraction medium. Methods of assay. Oxidative phosphorylation was measured by the oxygen electrode described by Voss et al. (1963a). The P/O ratios were calculated as ADP/O ratios according to the method of Chance and Williams (1955a) for assaying oxidative phosphorylation. The ADP/O ratios were calculated from the uptake of oxygen in muatomsper liter during the active state of respiration and the molar concentration of ADP added. The respiratory control coefficients ( RC ) , were calculated as the ratios of the respiratory rates with ADP to the control respiration after ADP was consumed. Oxygen uptake was measured by two different techniques, namely, the polarographic method with an oxygen electrode and by Warburg respirometry. The first technique expressed the rate of oxygen up-

take in pmoles Oz/second/liter, while by War-burg respirometry the values are expressed as ul 0, per hour. The oxygen uptake measured polarographically was based on the concentration of oxygen from an airsaturated solution of water. Difference spectra of the respiratory chain were recorded at room temperature ( Chance, 1957), with a substrate-reduced preparation against an oxidized preparation. Protein was determined by the method of Lowry et al. (1951). RESULTS

The respiratory chain. Figure 1 shows the room temperature spectra of the respiratory chain after reduction with succinate. Table I shows the cytochrome concentration of the S. oblongus heart mitochondria calculated from the spectra recorded at room temperature (Fig. 1) according to the molar extinction coefficients given by Chance (1952). The relative amounts of the cytochromes were calculated by setting the cytochrome a value equal to 1.0. ( Chance and Williams, 195513.). Respiration rates and oxidative phosphorylation. The values for endogenous respiration, for oxygen uptake during substrate respiration, the ADP/O ratios, and the respiratory control coefficients of S. oblongus heart mitochondria for the substrates used are shown in Table II. The data were taken from polarographic determinations by the oxygen electrode method. Oxygen consumption by Warburg respirometry. Experiments were carried out employing the Warburg respirometer to study the utilization of organic acids known to be substrates for other mitochondrial preparations. Results are shown in Table III. DISCUSSION

The presence of the tricarboxylic acid cycle has been demonstrated in most mam-

S. oblongus HEART MITOCHONDRU

Y

205

I 500

Wavelength

(my

)

FIG. 1. Room temperature difference spectra of Strophocheilus obZongw heart mitochondria. The line represents the difference spectra of succinate reduced minus oxidized preparation. The following peaks were determined: 600 mn (cyt. a + ua), 562 mp (cyt. b), 550 my (cyt, c), 519 rnp (cyt. b + c), 442 rnp (cyt. as), 430 my (cyt. b), 416 mp (cyt. c).

TABLE

I

Cytochrome Concentration of Strophocheilus Cytochrome a3

a b c + Cl

442 600 562 550

my rnp rnp rnv

= = = =

0.52 0.20 0.07 0.25

a 9.8 mg of protein per ml. b Relative amounts of cytochromes:

Heart Mitochondriaa

mM-1

FcM

10-S moles/g protein

Ratiob

91 14 20 19

5.7 14.2 17.5 13.1

0.57 1.44 1.78 1.33

1.3 1.0 0.35 1.2

ECU-l

AOD

oblongus

a = 1.0.

malian tissues investigated (Green et al., 1948), where it appears to be the major pathway for the oxidation of carbohydrate, fat, and protein intermediaries. However, very few examinations have been made in invertebrates like Strophochei1u.s oblongus. The demonstration of oxidative phosphorylation in S. oblongus tissue is of interest for comparative biochemistry since it provides additional evidence of this important exer-

gonic process. Weinbach (1954) showed that pentachlorophenol (PCP) is a powerful uncoupler of oxidative phosphorylation in rat tissues; Weinbach (1956) and Weinbath and Nolan (1956) showed the same effect of pentachlorophenol in Lymnaea stagnulis and Biomphalaria glabrata, respectively. A better knowledge of S. oblongus biology could provide a biochemical basis for the mechanism of action of mol-

206

BBANDiO

Respiratory

AL.

TABLE II Phosphorylation of Strophocheilus

Rates and Oxidative

Substrates

ET

Respiration rates before ADP

Respiration rates with ADP (A)

Respiration rates after ADP (B)

0.49 0.71 0.36 0.26 0.11 0.17 0.12 0.28

1.31 1.31 0.78 0.26 0.11 0.17 0.12 0.28

0.49 0.71 0.44 0.26 0.11 0.17 0.12 0.28

Glutamate a-Ketoglutarate Malate Succinate Pyruvate Isocitrate Citrate a-Glycerophosphate

oblongus Respiration control coefficients (A/B) 2.67 1.84 1.77 1.00 1.00 1.00 1.00 1.00

Heart

Mitochondriaa

Protein ADP/O 2.9 2.9 2.5 -

mg/ml

56.5 38.4 39.3 13.1 38.4 38.4 39.3 38.4

a The system contained 2.2 ml of aerobic medium (mannitol 0.25 M, Tris 0.01 M, EDTA 0.2 mM, KC1 0.01 M, and inorganic phosphate 0.1 M (final pH 7.4); 0.2 ml mitochondrial suspension, 10 PM substrates; and 285 pmoles of ADP. Rates of oxygen uptake are expressed in pmoles O,/second/liter.

Oxygen

TABLE III Uptake of Strophocheilus Heart Mitochondriaa

Substrate Endogenous Citrate a-Glycerophosphate Isocitrate Succinate a-Ketoglutarate Glutamate Malate a The system contained medium (mannitol 0.25 M, 0.2 mM, KC1 0.01 M, and 0.1 M final pH 7.4); 0.5 ml pension; 100 pmoles of 0.15 ml of 20% KOH (center 37”C, 100 strokes per minute.

oblongus

0, uptake FLJ.0, per hour 23 118 135 89 137 47 58 153 2.0 ml of reaction Tris 0.01 M, EDTA inorganic phosphate of mitochondrial susindicated substrate; well). Temperature Protein: 21.6 mg/ml.

and for its toxicity to vertebrates. The room temperature difference spectra of S. oblongus heart mitochondria (Fig. I. and Table I) follow the general pattern of mammalian mitochondria (Klingenberg and Biicher, 1960). The relative amounts of cytochromes calculated on the basis of the cytochrome a content is similar to that one obtained for dog (Campello et al., 1966) and for human heart mitochondria luscicides

(Cat et al., 1963). The relative amounts of cytochromes reported here is markedly different from those found by Voss et al. (196313) for Biomphduriu glabrutu albumin gland mitochondria. With the system and method used in our investigations the ADP/O ratios indicated glutamate and a-ketoglutarate were of the same order as those reported in the literature and were extremely close to the theoretical ratios. The ADP/O ratios for glutamate, a-ketoglutarate, and malate showed in the present paper (Table II) are higher than those obtained in heart muscle of Helix pomatiu (Michejda et al., 1964). However, very stable oxidative phosphorylation was obtained, by these authors, with succinate (P/O 1.32-1.75), while we were unable to demonstrate this process when succinate, pyruvate, isocitrate, citrate, or a-glycerophosphate were the substrates. According to Sacktor and Cochran ( 1958) the oxidation of a-glycerophosphate occupies a predominant position in insect flight-muscle mitochondria. The experiments described demonstrate the extraordinary rate of a-glycerophosphate oxidation, almost twice that obtained for succinate. A similar observation was made

S.

oblongus HEART MITOCHONDRIA

by Sacktor et al. (1959) for rat brain mitochondria. The present paper shows a rate for a-glycerophosphate oxidation in S. oblongus heart mitochondria (Table III) which is of the same order of succinate oxidation, and higher than in rat, dog, or human heart mitochondria. The most actively oxidized were the following substrates (in decreasing order) : malate, sucand citrate. cinate, a-glycerophosphate, The substrates oxidized more slowly were a-ketoglutarate and glutamate.

207

tion. II. Difference spectra. The Journal of Biological Chemistry 217, 395407. CAT, I., CAMPELLO, A. P., Voss, D. O., BRAGA, H., AND BACILA, M. 1963. Respiration rates and oxidative phosphorylation of heart sarcosomes in Kwashiorkor. The Lancet, August 24, 415416. GREEN, D. E., LOOMIS, W. F., AND AUERBACH, V. 1948. Studies on the cyclophorase system. I. The complete oxidation of pyruvic acid to carbon dioxide and water. The Journal of Biological Chemisty 172, 389-403. KLINGENBERG, M., AND BUTCHER, T. 1960. Annual Review of logical oxidations.

Bio-

Bio-

chemistry 29, 669-708. ACKNOWLEDGMENTS The authors thank Prof. Celso Paulo Jaeger and Prof. Ema Magalhges Leboute from the Universidade Federal do Rio Grande do Sul, Brazil, for supplying the specimens of Strophocheilus obkmgus. REFERENCES BRANDXO, D., Voss, D. O., AND CAMPELLO, A. P. 196’7. Algumas propriedades da cadeia rede mitokndria de corafio de spiratbria

Strophocheilus

oblongus. Cilncia

e Cultura

19, 388. BRANDLO, D., Voss, D. O., NICULITCHEFF, G. X., AND CAMPELLO, A. P. 1968. Velocidade respiratbria e fosforilatio oxidativa de mitocBndria de coracZo de Strophocheilus ob-

longus. CiBncia e Cultura 20, 390. CAMPELLO, A. P., BACILA, M., BRAND~O, D., AND Voss, D. 0. 1966. Algumas caracteristicas da cadeia respiratbria de sarcossoma de corago de do. Revista da Escola de Agrono-

mia e Veterindria, Para& 2, 33-39.

Universidade

Federal do

CHANCE, B. 1952. Spectra and reaction kinetics of respiratory pigments of homogenized and intact cells. Nature 169, 21%230. CHANCE, B. 1957. Techniques for the assay of the respiratory enzymes. In “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds. ), Vol. 4, pp. 273-329. Academic Press, New York. CHANCE, B., AND WILLMMS, G. R. 1955a. A simple and rapid assay of oxidative phosphorylation. Nature 175, 1120-1124. CHANCE, B., AND WILLIAMS, G. R. 1955b. Respiratory enzymes in oxidative phosphoryla-

LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. 1951. Protein measurement with the folin phenol reagent. The lournul of Biological Chemisty 193, 265-275. MICHEJDA, T., KASPRZAK, L., OBUCHOWICZ, L., AND ZERBE, T. 1964. Respiratory metabolism in the snail, Helix pomutiu. III. Oxidative phosphorylation in heart muscle. Bulletin de .!u Sock& des Amis des Sciences et des Lettres de Poznan, s&e D, 115-134. REES, K. R. 1953. Aerobic metabolism of the hepatopancreas of Helix pomatia. The Biochemical Iournal 55, 478-484. SACKTOR, B., AND COGHRAN, D. G. 1958. The respiratory metabolism of insect flight muscle. I. Manometric studies of oxidation and concomitant phosphorylation with sarcosomes. Archives of Biochemisty and Bio-

physics 74, 266-276. SACKTOR, B., PACKER, L., AND ESTABROOK, R. W. 1959. Respiratory activity of brain mitochondria. Archives of Biochemistry and Biophysics 80, 68-71. Voss, D. O., BRANDXO, D., C~~wso, T. J. T., AND BACILA, M. 1963a. Cadeia respiratbria e microscopia eletrbnica de mitoc&&ia de Australorbis glubratus. II. Simp&io sdbre

Bioquimica de Plunorbideos (Recife, Bra.&), 4548. Voss, D. O., CAMPELLO, A. P., AND BACILA, M. 1961. The respiratory chain and the oxidative phosphorylation of rat brain mitochondria. Biochemical and Biophysical Re-

search Communications 4, 48-51. Voss,

D. O., CO~LES, J. C., AND BACILA, M. 1963b. A new oxygen electrode model for the polarographic assay of cellular and mitochondrial respiration. Analytical Biochemisty 6, 211-222.

208

BRAND20

WEINBACH,

mediary

E. C. 1953. metabolism

Studies of the

on the interaquatic snail

Australorbis glubfatus. Archives of Biochemistry and Biophysics 42, 231-244. E. C. 1954. The chlorophenol on oxidative

effect of pentaphosphorylation. The Journal of Biological Chemistry 210, 545-

WEINBACH,

550.

ET

AL.

1956. The influence of pentaon oxidative and glycolytic phosin snail tissue. Archives of Biochemistry and Biophysics 64, 129-143. WEINBACH, E. C., AND NOLAN, M. 0. 1956. The effect of pentachlorophenol on the metabolism of the snail Australorbis glubratus.

WEINBACH,

E. C.

chlorophenol phorylation

Experimental

Parasitology

5, 276-284.