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extramatrix concentrations of ATP, ADP and Pi and the matrix volume being determined in parallel. As is shown in Fig. I , the thermodynamic relation is consistent with a mean stoicheiometry of 2.66+0.01-+ H+/ATP obtained by linear regression forced through the origin. As any systematic error in matrix volume or in activity coefficients for the isotopic indicators of membrane potential or pH gradient would affect the apparent magnitude of A&+ equally for each point, it is significant that the slope of Fig. 1 (i.e. not forced through the origin) is 2.61 k0.02 H+/ATP. Thus the apparent stoicheiometry remains constant as A,&,+ is decreased, and it was not possible to confirm the increase in stoicheiometry observed, under these circumstances, by Weichmann et al. (1975). In conclusion, two independent lines of evidence suggest that not less than 5.4 protons cycle across the membrane to cause the synthesis of 2mol of ATP during the transfer of two electrons from a flavoprotein-linked substrate to oxygen. This work was supported by a grant from the Medical Research Council. Brand, M. D. & Lehninger, A. L. (1975) J. Biol. Chem. 250, 7958-7960 Brand, M. D., Chen, C.-H. & Lehninger, A. L. (1976) J. Biol. Chem. 251, 968-974 Garland, P. B., Clegg, R. A., Boxer, D., Downie, J. A. & Haddock, R. A. (1975) in Electron Transfer Chains and Oxidative Phosphorylation (Quagliarello, E., Papa, S., Palmieri, F., Slater, E. C. & Siliprandi, N., eds.), pp. 351-358, North-Holland, Amsterdam Heaton, G. M. & Nicholls, D. G. (1976) Biochem. J. 156, 635-646 Mitchell, P. (1975a) FEBSLett. 56, 1-6 Mitchell, P. (19756) FEBS Letr. 59, 137-139 Mitchell, P. (1976) Biochem. SOC.Trans. 4,399430 Nicholls, D. G. (1974) Eur. J . Biochem. 50,305-315 Reed, K. C. & Bygrave, F. L. (1974) Biochem. J. 142, 555-566 Rosing, J. & Slater, E. C. (1972) Biochim. Biophys. Acta 267, 275-290 Rottenberg, H. & Scarpa, A. (1974) Biochemistry 13,4811-4817 Weichmann,A.H. C.A.,Beem,E.P. &vanDam,K.(1975)in EIectronTransfer ChainsandOxidatiuePhosp~orylation(Quagliarello, E., Papa, S., Palmieri, F., Slater, E. C. & Siliprandi, N., eds.), pp. 335-342, North-Holland, Amsterdam

Inhibition of Mitochondrial Potassium Ion Flux by Thallous Ions JOYCE JOHNSON DIWAN and PATRICIA HARRINGTON LEHRER Department of Biology, Rensselaer Polytechnic Institute, Troy, N Y 12181, U.S.A.

TI+ ions have been shown to interact with a number of mechanisms which selectively transport K+ across cellular membranes (Cornelius et al., 1974; Cavieres &Ellory, 1974; Neher, 1975). It has been reported that T1+ inhibits the net leakage of endogenous K+ from mitochondria and additionally inhibits net K+ uptake by K+-depleted mitochondria (Barrera & Gomez-Puyou, 1975). However, a detailed kinetic analysis of these effects of T1+ on K+ flux was not carried out. Uptake of 2oT by mitochondria has also been demonstrated (Barrera & Gbmez-Puyou, 1975). Such an uptake is consistent with the swelling and stimulation of mitochondria1 respiration, which result from TI+ addition (Melnick et al., 1976). The unidirectional flux of K+ into rat liver mitochondria requires a supply of metabolic energy and is stimulated by an alkaline external pH (Diwan, 1973; Diwan & Harrington, 1975). Saturability of the K+-uptake mechanism is indicated by the Iinear relationship between the reciprocal of the initial K+-influx rate and the reciprocal of the external K+ concentration (Diwan & Harrington, 1975). That the unidirectional efflux of endogenous K+ also occurs via the energy-linked mechanism is indicated by the finding that respiratory inhibitors slow the K+-effluxrate (Diwan & Tedeschi, 1975). The present experiments have examined, by use of the radioisotope 42K,the effect of T1+ on unidirectional Kf fluxes across the membranes of isolated rat liver mitochondria. Techniques used were the same as in previous studies (Johnson & Pressman, 1969; Diwan, 1973). The mitochondria (4.2-7.4mg of protein/ml in different experiments)

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1/tK+lb-7 Fig. 1. Efect of Z7+on the dependence of K + influx on external K + concentration The reciprocal of the initial K+-influx rate [in units of (p-equiv. of K+/min per g of protein)-'] is plotted against the reciprocal of the external molar K+concentration. The lines drawn are calculated from the data points by the method of least squares. 0 , Control samples; 0 , the medium included 7.5m-T1+.

were incubated in media (adjusted to pH 8.0 with HZSO4) containing 200m~-sucrose, 30m~-Tris,8 mM-succinic acid and variable concentrations of KCI and T12S04, plus trace amounts of 3H20, ['4C]sucrose and 42K.After 0.75min and 7min of incubation, samples of mitochondria were separated from the medium by rapid centrifugation through silicone. Mitochondria1 fluid compartments and the amounts of total and labelled K+ in the mitochondria1 samples were calculated from radioactive counts and atomic-absorption measurements as previously described (Johnson & Pressman, 1969). The unidirectional K+ influx is calculated as the difference in labelled K+ content between the 0.75min and 7min samples. The net K+ flux is calculated as the change in total mitochondria1 K+ between 0.75 and 7min. The unidirectional K+ efflux is calculated as the difference between influx and net flux values. The effect of TI+on the initial rate of K+ influx at varied K + concentrations is depicted in Fig. 1. The Lineweaver-Burk plots, calculated from the data by the method of least squares, intersect near the vertical axis, indicating that TI+ causes a marked increase in the apparent K, for K+ and little change in Vmax..This pattern is characteristic of competitive inhibition. A summary of kinetic constants, determined similarly in several different experiments, is given in Table 1, which also includes values of K , calculated from the change in slope of the double-reciprocal plots. Other experiments show an inhibitory effect of T1+ on the rate of unidirectional K+ efflux. For example, pooled data from four experiments, in each of which the external K+ concentration was held constant at some value within the range from 2.6 to 4 . 9 m ~ , show that the addition of 5 m ~ - T 1 +resulted in a change in K+-efflux rate from 2.5k0.3 to 1.5f0.3pmoI of K+/min per g of protein (meansks.D.). In the same experiments the K+ influx averaged 1.520.2 in the absence and 0.7k0.1 pmol of K+/min per g of protein in the presence of 5.0m~-T1+. These results support the conclusion that TI+ competitively inhibits the flux of K+ into rat liver mitochondria. The mechanism by which externally added TI+ inhibits K+ efflux is less clear. Perhaps sufficient TI+is taken up during the time-course of the present experiments to result in competition between K+ and TI+ for binding to transport sites On the imer surface of the mitochondria1 semipermeable membrane. 1977

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Table 1. Apparent kinetic constants for K + influx in the presence and absence of TI+ In experiments similar to that of Fig. 1, linear plots of 1/K+ influx against the reciprocal of the external K+ concentration were fitted to the data by the method of least squares. Values of K, and VmaX.were calculated from these plots according to the usual relationships: 1/Vmax.equals 1/K+ influx when l/[K+] equals zero, and -1/K, equals l/[K+] when 1/K+ influx equals zero. The values of K, were calculated from the double-reciprocal plots according to the relationship that the ratio of the slope in the presence of TI+ to the slope in the absence of TI+ equals 1+(T1+ concentration)/K,. Each value shown is the mean of values from four experimentsfs.~. vmax.

Experimental series Conditions A Control +3.75m~-T1+ B Control +7.5~-Tl+

K, (mM-K+) 4.8f 0.5 14.7+ 5.7 4.75 1.3 35.6-1-11.8

@equiv./min per g of protein) 3.7 f 0.5 4.3f1.8 3.4 2 0.7 6.6 f 3.2

K, (mM-Tl+) 2.3 k 0.2 2.4 & 0.3

This research was supported by grant no. GM-20726 from the National Institute of General Medical Sciences, U.S.A.

Barrera, H. & G6mez-Puyou, A. (1975) J. Biol. Chem. 250, 5370-5374 Cavieres, J. D. & Ellory, J. C. (1974) J. Physiol. (London) 243,243-266 Cornelius, G., Giirtner, W. & Haynes, D. H. (1974) Biochemistry 13,3052-3057 Diwan, J. J. (1973) Biochem. Biophys. Res. Commun. 50, 384-391 Diwan, J. J. & Hamngton, P. (1975) Fed. Proc. Fed. Am. SOC.Exp. Biol. 34, 518 Diwan, J. J. & Tedeschi, H. (1975) FEBSLett. 60, 176-179 Johnson, J. H. & Pressman, B. C. (1969) Arch. Biochern. Biophys. 132, 139-145 Melnick, R. L., Monti, L. G.& Motzin, S. M. (1976) Biochem. Biophys. Res. Commun.69,68-73 Neher, E. (1975) Biochim. Biophys. Actu 401, 540-544

Oxidative Phosphorylation:A New Biological Function for Lipoic Acid DAVID E. GRIFFITHS, KELVIN CAIN and ROBERT L. HYAMS Department of Molecular Sciences, University of Warwick, Coventry CV4 7AL, U.K.

This paper will summarize the evidence that lipoic acid plays a key role in mitochondrial oxidative phosphorylation. In contrast with substrate-levelphosphorylation, lipoic acid is the active functional group in the ATP synthase complex and is a mobile component of the mitochondria1inner membrane, which functions as an energy-coupling factor that can link the electron-transfer chain and the ATP synthase compIex. The demonstration of this function of lipoic acid thus fulfils the basic requirements of any chemical hypothesis of oxidative phosphorylation. Materials and methodr

Dibutylchloromethyltin chloride and dibutylchlor~[~H]rnethyltinchloride were synthesized from dibutyltin chloride and diazomethane or dia~o[~H]methane respectively (Seyferth & Rochow 1955; K. Cain & D. E. Griffiths, unpublished work). chloride to mitochondria, submitochondrial Binding of dibutylchlor~[~HJmethyltin particles and chloroplasts was measured by addition of an excess of dib~tylchloro[~H]methyltin chloride (1&20nmol/mg of protein) to membrane preparations and washing four times with suspending medium after high-speed centrifugation. Free lipoic acid was assayed by bioassay of sterile protein fractions or acid extracts, by using a lipoic acidVOl. 5