Change of proton gradient in mitochondria at various energy states.

Changes of H+ gradient at various energy states of mitochondria were studied. There was a close relation between the extent of H+ gradient and the level of ATP formation; the former decreased as a result of ATP synthesis but was not completely abolished. A partial depression of H+ gradient was also observed in the presence of uncouplers of oxidative phosphorylation. The H+ gradient seemed to be more closely related to the ion translocation than ATP formation. In the presence of Ca++ the energy of H+ gradient was utilized in translocating Ca++ rather than synthesizing ATP. These findings further substantiate the chemiosmotic theory of MITCHELL on mitochondrial electron and energy transfer. ∗PMID: 4264429 [PubMed indexed for MEDLINE] Copyright c ©OKAYAMA UNIVERSITY MEDICAL SCHOOL Acta Med. Okayama 25, 493-504 (1971) CHANGE OF PROTON GRADIENT IN MITOCHONDRIA AT VARIOUS ENERGY STATES Kozo UTSUMI, ]. TORRES.PEREIRAE*, MOHAMMAD G. MUSTAFA** and Takuzo ODA Department of Biochemistry, Cancer Institute, Okayama University Medical School, Okayama 700, Japan Received for Publication, September 9, 1971 According to the chemiosmotic hypothesis of MITCHELL (1-3), there must exist a potential difference between the mitochondrial membranes, or a gradient of concentration in H+ or some other ions between inside and outside of mitochondria in order to accomplish ATP formation. In a previous paper (4) we have described that there are three kinds of H+ translocations between inside and outside of mitochondria, namely: (a) H+ displacement dependent on ATP formation, (b) H+ transfer dependent on electron transport, and (c) H+ change dependent on oxidation.reduction of respiratory chain components. Of these H+ transfer processes, the second one is related to chemi. osmotic H+ transfer reaction, but to obtain data similar to those observed by MITCHELL (1-3) rat liver mitochondria require treatment with a small amount of Triton X-IOO or Ca++. Under the experimental conditions of Mitchell, this H+ gradient would be discharged without ATP formation due to anaerobiosis. This is because under these conditions mitochondrial Ca++, which accumulated at the expense of respiratory energy, would be released to external medium in exchange of H+ uptake by mitochondria during the aerobic-anaerobic transition. Introduction of an oxygen pulse would reverse the process, i. e., Ca++ would be taken up by mitochondria in exchange of H+ release. Treatment of mitochondria with Triton X-IOO also caused a discharge of H+ gradient, but the gradient was restored upon introduction of an oxygen pulse (4). This phenomenon is difficult to explain, but it is possible that in mitochondria treated with a small amount of Triton X100 the respiration-dependent H+ transfer may be related to translocation * Department of Botany, Faculty of Sciences, University of Luanda, Luanda, Angola, Portuguese West Africa. ** Departments of Biological Chemistry and Internal Medicine, University of California School of Medicine, Davis, California 95616, U. S. A. 493 1 Utsumi et al.: Change of proton gradient in mitochondria at various energy Produced by The Berkeley Electronic Press, 1971 494 K. UTSUMI, J. TORRE~-PEREIRAE, MOHAMMAD G. MUSTAFA and T.ODA of K + or some other ions. Therefore, it is considered that the oxygen. dependent H+ gradient in both cases (i. e., mitochondria treated either Ca++ and Triton) is closely related to ion translocation. Even though such a correlation exists, an H+ gradient, as suggested by the chemiosmotic hypothesis, must occur between the inside and outside of mitochondria in order to achieve ATP formation. In this study we undertook to determine the extent of H+ gradient between the inside and outside of mitochondria at various energy states, so as to clarify whether H+ gradient, indeed, is required to carry out ATP formation. In this report we describe that there is a correlation between ATP formation and H+ gradient, and that in order to generate ATP the mitochondrial membranes must be intact and capable of maintaining an H+ gradient between the inside and outside without an energy supply, e. g. from respiration or ATP hydrolysis. MATERIALS AND METHODS Rat liver mitochondria were isolated in a medium containing 0.33 M sucrose and 1 mM Tris-EDTA (pH 7.4) as decribed previously (5). Proton translocation in mitochondria was studied in a medium (unless otherwise described) containing 0.15 M choline chloride adjusted to pH 7.4 by using a minimum amount of NaOH or HCl. Reactions were carried out at 25° in an 8-ml volume. Oxygen pulse was introduced by injection of either an aliquot of fresh reaction mixture or hydrogen peroxide into the anaerobic incubation mixture; in the latter case catalase was pre-added. Protein was determined by the method of LOWRY et al. (6). Hexokinase, catalase and oligomycin were obtained from Sigma Chemical Company. Other reagents used were of reagent grade. RESULTS Respiration dependent proton movement: As shown in Fig. I, three kinds of proton movements may be recognized: (a) under anaerobic conditions fresh mitochondria took up H+ through ATP formation; fresh mitochondria also took up a large amount of H+ upon addition of a small amount of Triton X-lOa, (b) in the presence of a small amount of Triton X-lOa, incubated mitochondria released a large amount of H+, but these protons were readily taken up by mitochondria at the aerobic-anaerobic transitions, and (c) in the presence of a high concentration of Triton X-lOa, respiration.dependent H+ uptake was observed. These H+ changes were dependent on oxidation.reduction states of respiratory chain components, e. g. pyridine nucleotides, flavins and cytochromes. 2 Acta Medica Okayama, Vol. 25 [1971], Iss. 5, Art. 1 http://escholarship.lib.okayama-u.ac.jp/amo/vol25/iss5/1 Mitochondrial Proton Gradient and Energy States 495