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The Prevention of Swelling of Liver Mitochondria in Vitro

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The Prevention of Swelling of Liver Mitochondria in Vitro Vol. 64 769 The Prevention of Swelling of Liver Mitochondria in vitro BY A. FONNESU* AND R. E. DAVIESt Medical Re8earch Council Unitfor Re8earch in Cell Metaboli8m, Department of Biochemi8try, Univer8ity of Oxford (Received 21 March 1956) Previous work has shown that mitochondria can (Broyeur de Fischer a, Main; Jouan, Paris) to remove most maintain a low water content if incubated under ofthe connective-tissue framework and then forced through conditions favourable to oxidative phosphorylation a Craigie (1949) pressure mincer. The pulp was weighed, (Bartley & Davies, 1952, 1954; Macfarlane & suspended in 9 vol. ofice-cold 0-25M sucrose and centrifuged in a Servall angle centrifuge for 30min. at 300 g to sediment Spencer, 1953; Price & Davies, 1954; Price, unbroken liver cells, nuclei and red cells. The supernatant Fonnesu & Davies, 1956). Harman & Feigelson was removed with a hook-tipped pipette and centrifuged (1952) used the phase-contrast microscope to assess for 30 min. at 6000g to sediment the mitochondria; the the morphological changes of isolated heart-muscle supernatant and the 'fluffy layer' (Schneider & Hogeboom, mitochondria, and concluded that oxidative phos- 1951) were poured off and the well-packed mitochondria phorylation is the essential mechanism for main- resuspended in 0-25M sucrose for washing or use. When tenance ofmitochondrial form. They also found that indicated, the mitochondria were washed once or twice in uncoupling agents, such as 2:4-dinitrophenol, a volume of 0-25M sucrose equal to that of the original Janus green and usnic acid, produce swelling of suspension and isolated by centrifuging for 30min. at isolated mitochondria. It has also been found that 6000g. Incubation. The mitochondria in 0.25M sucrose were added adenosine triphosphate (ATP) inhibits the added to the various media in conical flasks at 280 in a swelling of mitochondria (Raaflaub, 1952, 1953 a, b) Dubnoff Metabolic Shaking Incubator (Precision Instru- and that the 'spontaneous' swelling ofmitochondria ments Co., Chicago). The gas phase was either air or N2. left at room temperature is paralleled by a fall in Complete anaerobiosis was maintained with a stick of the concentration of the intramitochondrial ATP yellow phosphorus placed in the centre well of the flask. (Brenner-Holzach & Raaflaub, 1954). Separation of the mitochondria. After incubation, the The reciprocal relationship between water content reaction mixture was pipetted or poured into a weighed (or swelling) and oxidative phosphorylation (or Pyrex centrifuge tube and centrifuged at room temp. availability of 'high-energy' phosphate bonds) in (approx. 200) in the high-speed head no. 295 of an Inter- national Centrifuge, size 2, under the conditions described mitochondria has been much emphasized, and this later. The supernatant was decanted and the inside of the paper records the results ofan investigation into the tube dried with strips of filter paper. The tube was sealed conditions which prevent the swelling of mito- with a square ofParafilm (Marathon Corporation, Menasha, chondria. Part ofthis work has been communicated Wis., U.S.A.) until it was weighed. The dry weight of the to the Biochemical Society (Fonnesu & Davies, mitochondria was obtained after drying overnight at 1050. 1955). Chromatography. Cold trichloroacetic acid [0.5 ml. of a 25 % (w/v) solution] was added to a sample (3 0 ml.) of the EXPERIMENTAL incubation mixture. The clear supernatant obtained after Abbreviation8. These are as follows: adenosine mono- centrifuging was kept frozen at - 150 until it was chromato- phosphate, AMP; adenosine diphosphate, ADP; adenosine graphed on paper by the method of Krebs & Hems (1953). triphosphate, ATP; 2:4-dinitrophenol, DNP; inosine mono- Marker spots containing adenine, adenosine, AMP, ADP, phosphate, IMP; inosine diphosphate, IDP; inosine ATP, IMP, IDP and ITP were placed on each paper. triphosphate, ITP. Special chemical8. The dibarium salts of ATP and IMP Preparation of mitochondria. The method used was very were prepared by the methods of LePage (1949) and of similar to that of Macfarlane & Spencer (1953). All opera- Ostern (1932), respectively. 2:3-Dimercaptopropanol (BAL) tions were carried out in a cold room at 1° with previously was a gift from Dr L. A. Stocken. The following compounds chilled materials. were commercial preparations: AMP (free acid, Roche Male albino rats were fasted for 12 hr., stunned by a blow Products Ltd., Welwyn Garden City), adenosine 3'- on the head and decapitated. The liver was quickly excised phosphate (free acid, L. Light and Co., Colnbrook), ADP and cooled for 3 min. in partially frozen 0-25M sucrose. The (dibarium salt, Schwarz Lab. Inc., New York), uridine chilled tissue was blotted, passed through a Fischer mincer 5'-phosphate (disodium salt, Pabst Lab., Milwaukee, Wis., U.S.A.; lot 801), flavin mononucleotide (Sigma Chemical * Present address: Istituto di Patologia Generale dell' Co., St Louis, U.S.A.; lot H 62-1) and guanosine 5'- Universita di Milano, Italy. phosphate (Sigma Chemical Co.; lot 144-608). The dibarium t Present address: Department of Biochemistry, salts were converted into free acids by the method of University of Pennsylvania, Philadelphia, Pa., U.S.A. Deutsch & Nilson (1953) with Amberlite Resin IR-120 (H), 49 Bioch. 1956, 64 770 A. FONNESU AND R. E. DAVIES I956 analytical grade, manufactured by the Rhom & Haas Co., Resinous Products Division, Philadelphia, U.S.A., and Investigation of the action of the components obtained from British Drug Houses Ltd., Poole. All the of the Macfarlane & Spencer system acids were neutralized to pH 7 with dilute NaOH before use. A column containing a mixture of 10 ml. of Dowex 1 x 10 Cytochrome c. Table 1 shows that added cyto- in the hydroxyl form and 10 ml. of Dowex 50 x 8 in the acid chrome c is not necessary to prevent swelling of the form was used, when indicated, to de-ionize 31. of 0825m mitochondria. The results obtained here with A.R. sucrose (British Drug Houses Ltd.). 5 x 10-6M concentrations and in the complete absence of cytochrome c are identical with those of RESULTS Macfarlane & Spencer who used 2 x 10-5M cyto- chrome c. The starting point for these experiments was the Time of centrifuging. Macfarlane & Spencer work of Macfarlane & Spencer (1953) who found (1953) separated the mitochondria after the incuba- that the swelling of rat-liver mitochondria which tion at 6000 g for 20 min. at room temperature and occurs during incubation at 280 in the presence of pointed out that the conditions of oxygenation were oxygen, glutamate, phosphate buffer, cytochrome c, obviously not optimum. The same results were sucrose and Mg2+ ions was largely prevented by obtained whether the centrifuging after incubation AMP. They suggested that 'the prevention of was for 20 min. at 6000 g or for 4 min. at 25 000 g at swelling is associated with the capacity ofthe system room temperature (cf. Bartley & Davies, 1954). The for oxidative phosphorylation or, more accurately, results are given in detail in Table 7 of Price et al. with the presence of an adenine nucleotide'. (1956). Table 1. Effect of cytochrome c and AMP on water content of mitochondria Incubation for 30 min. at 280 in a Dubnoff Shaker; gas phase, air. Medium (final concentrations): 0-02M sodium L-glutamate; 0-002M-MgSO4; 001M potassium phosphate buffer, pH 7-4; 0-083M sucrose (introduced with the added mitochondria) and, where indicated, 5 x 10-sr cytochrome c. AMP, when present, was 0 001m. Rat-liver mitochondria (unwashed) were incubated at a concentration of 5 mg. dry wt./ml. After incubation the mitochondria were separated at 6000g for 20 min. at room temp. Relative water content Dry wt. (ml. of water/g. Conditions (%) of dry solids) Mitochondria incubated in the medium: 13-1 6-6 (a) With cytochrome c { +AMP 20-1 4'0 12-4 7-1 (b) Without cytochrome c I +AMP 20-2 4-0 Table 2. Effect of substrate and comparison of the effects of AMP and ATP on water content of mitochondria Incubation at 28 in a Dubnoff Shaker; gas phase, air. Medium (final concentrations): 0-002M-MgSOj; 0OO1M potassium phosphate buffer, pH 7-4; 0-083m sucrose and, when indicated, 0-001 M AMP or ATP. Substrate, when present, was 0-02m sodium L-glutamate. Rat-liver mitochondria (washed twice): 5 mg. dry wt./ml. After incubation the mitochondria were separated at 25000g for 4 min. Relative Period of water content incubation Dry wt. (ml. of water/g. Conditions (min.) (%) of dry solids) Mitochondria in 0-25m sucrose separated at 0° 0 30-5 2-3 Mitochondria separated at room temp. after incubation in the medium: 30 15-7 5-4 With +AMP 30 23-5 3.3 (a) substrate t 24-5 3X1 (+ATP 13030 23-0 3.3 30 12.4 7X1 Without I +AMP 30 22-8 3.4 (b) substrate 23-9 3-2 I +ATP l30 22-1 3.5 Vol. 64 PREVENTION OF SWELLING OF LIVER MITOCHONDRIA 771 Table 3. Effects of AMP, cyanide, DNP and anaerobiosi8 on water content of mitochondrta Incubation for 30 min. at 280 in a Dubnoff Shaker. Medium (final concentrations): 0-002M-MgSO,; 0-08M-NaCl; O-O1M potassium phosphate buffer, pH 7-4; 0-021M sucrose (introduced with added mitochondria) and, where indicated, -OOO1MY AMP. Rat-liver mitochondria (washed once): 5 mg. dry wt./ml. After incubation the mitochondria were separated at 25000g for 4 min. at room temp. neiatuve water content Dry wt. (ml. of water/g. Conditions (%) of dry solids) Mitochondria in 0-25m sucrose 32-2 2-1 (separated at 0° without incubation) Mitochondria incubated in Gas phase Additions to medium *~~~ - 15-9 5-3 Air-Alr 1 ~+AMP 28-2 2-5 23-3 3-3 Air 4 mM KCN +AMP 21-9 3-6 20-8 3-8 Air 0.01mMDNP { +AMP 23-4 3-3 24-4 3-1 Air 0-1 mm DNP {+AMIP 23-4 3-3 21-1 3-7 Air mM DNP +AMP 19-9 4-0 19-6 4-1 Ns +AMP 23-0 3-3 Substrate and metabolism.
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