182813a0Nature1824638195809208138140028-0836195810.1038/182813a0ukNatureNatureNATUREnatureNature is a weekly international journal publishing the finest peer-reviewed research in all fields of science and technology on the basis of its originality, importance, interdisciplinary interest, timeliness, accessibility, elegance and surprising conclusions. Nature also provides rapid, authoritative, insightful and arresting news and interpretation of topical and coming trends affecting science, scientists and the wider public./nature/journal/v182/n4638issueJournal homeArchiveCurrent issueAdvance online publicationPrivacy policySubscribeNature Publishing GroupCurrent issue182813a0Dependence of Mitochondrial Swelling on Oxidizable Substrates
AU  - CHAPPELL, J. B.
AU  - GREVILLE, G. D.Department of Biochemistry, University of Cambridge. Aug. 11.THAT the swelling of isolated mitochondria induced by various agents fails to occur under anaerobic conditions1,2 suggested to us that swelling may be dependent on respiration. The probability of this is increased by the finding3,4 that malonate prevents the swelling induced by succinate of mitochondria suspended in mannitol or sucrose solutions. We have therefore studied the effects of addition of substrates and inhibitors of oxidative metabolism on the swelling of mitochondria under various conditions. Mitochondria from livers of male rats (180-280 gm. body-weight) were prepared, suspended and stored in 0.44 M sucrose at 0[deg] C. 0.05 ml. of mitochondrial suspension (25 mgm.-equivalents; 0.1 mgm. nitrogen) was added to 2.95 ml. of medium containing 2-amino-2-hydroxymethylpropane-1 : 3-diol (tris) chloride buffer, pH 7.5, and sucrose (final concentrations 25 mM and 0.3 M respectively). The optical density at 520 m[micro]. (D520) was recorded for periods up to 1 hr., the temperature being thermostatically controlled at 20[deg]. Decreases in D520were taken as a measure of swelling. Substrates were used in 1 mM concentration.In contrast to the experience of previous workers2,4, negligible swelling occurred during 1 hr. incubation in standard medium containing no further additions, even at 25  C. or after storage of the mitochondria at 0  C. for 26 hr.
With fresh mitochondria, the swelling caused by phosphate (10 mM) was increased in extent by addition of D- -hydroxybutyrate, succinate, L-gluta-mate, L-malate or L-proline. These substrates, in absence of phosphate, produced negligible swelling. The effect of phosphate alone decreased as the age of the mitochondria increased. It was negligible after 18 hr., and was then restored by addition of the above substrates, which again had no effect when added alone. Pyruvate and -oxoglutarate were, however, ineffective under these conditions.
Fig. 1. Inhibitory effects of malonate and amytal on substrate -phosphate swelling in aged (23 hr.) mitochondria. Each cell contained 0.3 M sucrose and 25 mM tris chloride buffer, pH 7.52, with the following further additions, the substrate being 1 mM succinate in (a) and 1 mM D- -hydroxybutyrate in (b): I, none ; II, 10 mM phosphate; III, substrate; IV, substrate + phosphate ; V, substrate + 1 mM malonate ; VI, substrate (succinate only) + 2 mM amytal; VII, substrate + phosphate + malonate ; VIII, substrate + phosphate + amytal
The swelling of day-old mitochondria in the presence of succinate and phosphate (succinate -phosphate swelling ) was inhibited by malonate (1 mM) (Fig. 1), whereas the D- -hydroxybutyrate -phosphate and L-glutamate - phosphate swelling was not. l-8mM amytal (5-ethyl-5-isoamylbarbiturate) prevents the oxidation by mitochondria of substrates with diphosphopyridine nucleotide-linked dehydro-genases, but has no effect on the oxidation of suc-cinate5. It is therefore of interest that 1-8 or 2 mM aniytal inhibited the swelling caused by D- -hydroxy-butyrate, L-glutamate, L-malate or L-proline when added together with phosphate, but not the succinate - phosphate swelling (Fig. 1). Antimycin A (0.05 [micro]gm./ml.), which blocks the respiratory chain6, inhibited by 50-100 per cent the swelling induced by all the above substrates, including succinate, in presence of phosphate. Cyanide (1 mM) abolished the D- -hydroxybutyrate - phosphate swelling of fresh and aged mitochondria.
Studies of the swelling induced by thyroxine have provided similar evidence that swelling may be dependent on the presence of oxidizable substrates. Thus, with fresh mitochondria, D- -hydroxybutyrate enhanced, and with aged mitochondria restored, the thyroxine effect. D- -Hydroxybutyrate - thyroxine swelling was inhibited by amytal and antimycin A. Still further evidence has been provided by the observation that with day-old particles in 0.2 M sucrose buffered with 25 mM tris, swelling was induced by succinate, D- -hydroxybutyrate and L-glutamate. Malonate strongly inhibited the effect of succinate but accelerated that of the other two substrates.
With fresh mitochondria, replacing the tris buffer by glyoxaline or raising the sucrose concentration from 0.3 M to 0.5 M prevented swelling due to phosphate. This could then be restored by addition of D- -hydroxybutyrate but not of L-glutamate, L-proline or succinate. With 0.45 M sucrose, however, L-proline and succinate were effective. In this connexion the recent finding7 that high concentrations of sucrose inhibit the oxidation of different substrates in different degrees may be significant.
To summarize, under various conditions addition of certain oxidizable substrates promotes swelling. The effect of these substrates is diminished by substances known to inhibit their oxidation. We therefore suggest that in certain circumstances the swelling of mitochondria is dependent on their respiration. Since cyanide, amytal and high concentrations of sucrose inhibit the swelling of fresh mitochondria in the presence of phosphate or thyroxine, it may be that such swelling is supported by the oxidation of intrinsic substrates. Ageing might deplete the particles of substrates, and swelling then only occur if these are supplied. The stabilizing action of 2 : 4-dinitrophenol4, which we have observed under many conditions, indicates that any effect of respiration on swelling may be exerted through coupled phosphorylation.
We thank Mr. Colin Taylor for his technical assistance.Hunter, , F. E., Davis, , J., and Carlat, , L., Biochim. Biophys. Acta, 20, 237 (1956).PubMedISIChemPortLehninger, , A. L., and Ray, , B. L., Biochim. Biophys. Acta, 26, 643 (1957).ArticlePubMedISIChemPortRaaflaub, , J., Helv. Physiol. Pharmacol. Acta, 11, 142 (1953).PubMedISIChemPortTapley, , D. F., J. Biol. Chem., 222, 325 (1956).PubMedISIChemPortErnster, , L., Jalling, , O., Low, , H., and Lindberg, , O., Exp. Cell Res., Supp. 3, 124 (1955).ChemPortPotter, , V. R., and Reif, , A. E., J. Biol. Chem., 194, 287 (1952).PubMedISIChemPortJohnson, , D., and Lardy, , H. A., Nature, 181, 701 (1958).PubMedISIChemPort