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Tbamitchodral L Alizaion of the 4Aminobutyrate-2-&Oxoglutarate

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Tbamitchodral L Alizaion of the 4Aminobutyrate-2-&Oxoglutarate 5d.em. J. (lWg77) 161,9O.-307 3O1 Printed in Great Britain Tbamitchodral L alizaion of the 4Aminobutyrate-2-&Oxoglutarate Transminase from Ox Brait By INGER SCHOUSDOE,* BIRGIT 1MO* and ARNE SCHOUSBOEt Department ofBDahemistry At andC*, University ofCopenhagen, 2200 Copenhagen M, Denark (Receved 4 June 1976) In order to determine the intramitochondrial location of 4-aminobutyrate transaminase, mitochondria were prepared from ox brain and freed from myelin and syiaptosomes by using conventional demitygradient-centrifugation techniques, and the purity was checked electron-microscopically. Iner and outer mimbrenes and matrix were prepared from the mitochondria by large-amplitude sweling and subsequent density-gradient centrfugationt The fractions were characterized by using both electron microscopy and differnt marker enzymes. From the specific activity of the 4-aminobutyrate transaminase in the submitochondrial fractions it was concluded that this enzyme is associated with the inner mitochondrial membrane. It is generally agreed that the 4-aminobutyrate-2- pyridoxal phosphate were from Sigma Chemical oxoglutarate transaminase (EC2.6.1.19) from brain is Co., St. Louis, MO, U.S.A. Ficoll was from mainly associated with free mitochondria (Salganicoff Pharmacia, Uppsala, Sweden, and crystallized & De Robertis, 1963, 1965; van den Berget al., 1965; bovine serum albumin was from BDH Biochemicals, van Kempen et at., 1965; Balazs et al., 1966; Poole, Dorset, U.K. 4-Amino[1-'4C]butyrate (sp. Waksman et al., 1968; Reijnierse et al., 1975), radioactivity 50mCi/mmol) and [1-14qtyramine (sp. and a preparation of a crude mitochondrial fraction radioactivity 9mCi/mmol) were obtained from was used by Schousboe et al. (1973) and Maitre et al. New England Nuclear Corp., Boston, MA; U.S,A, (1975) as a powerful purification step in the prepara- All other chemicals were of the purest grade available tion of a homogeneous 4-aminobutyrate trans from regular commercial sources, aminase from mouse or rat brain. No direct evidence seem, however, to be available about the intra. Preparation ofmitochondria andmemlbranefractions mitochondrial localization of this enzyme, but on the Ox brains obtained from the slaughterhouse after basis of experiments in which mitochondria were removal from the skull were placed in ice-cold 0.4M- treated with Triton X-100, Salganicoff & De sucrose containing 20mM-Hepes4 pH7.2, 2mM- Robertis (1965) suggested that it should be a EDTA and 0.02 % (w/v) heparin (medium A). After matrix enzyme. mincing, the tissue was homogenized at 4°C in 5vol, Knowledge about the precise intramitochondrial ofmediumA byusing a Potter-Elvehjem homogenizer localization of the 4-aminobutyrate transaminase is with a motor-driven Teflon pestle (Colora Mesg- of importance not only for the understanding of the tochnik, Lorch, W. Germany). After re-adjustment function of the enzyme (cf. Schousboe et al., 1974) of the pH to 7.2 with 2m-KOH, nedium A was but also for the interpretation ofresultg related to the added to the homogenate to give a total volume of compartmentation ofglutamate and 4-aminobutyrate 1Sml of medium A/g fresh wt. of brain. The mixture metabolism in brain (van den Berg & Garfinkel, was centrifuged for 20min at 4VC and lOOOg and the 1971; Rejinierse et al., 1975). In order to obtain such supernatant was re-centrifuged for 15min at 4VC information, inner- and outer-membrane fractions and 12000g. The pellet was resuspended in 4ml were prepared from purified mitochondria and of 10% (w/v) Ficoll in medium A/g fresh wt. and characterized by using marker enzymes as well as centrifuged for 30min at 4°C and 12000g. The electron microscopy, supernatant was re-centrifuged for 30min at 40C and 35000g and the pellet resuspended in medium A Materias ad Methods (0.5ml/g fresh wt.). This procedure for the prepara- tion of a crude mitochondrial fraction is a slight Chemicals modification of the method of Basford (1967). The Malate dehydrogenase, NAD+, NADH, NADP+ mitochondria were freed from synaptosomes and and cytochrome c were purchased from Boehringer, * Abbreviations Hepes, 2-(N-2-hydroxyethylpiperain- Mannheim, Germany. Heparin (sodium salt) and N'-yl)sulphonio asid, VQl. 162 304 I. SCHOUSBOE, B. BRO AND A. SCHOUSBOE myelin by the method of Gray & Whittaker fumarase (EC 4.2.1.2) activity (27°C) by the method (1962), except that a Ficoll gradient was used instead of Racker (1950). Rotenone-insensitive NADH- of a sucrose gradient. The suspension of the crude cytochrome c reductase (EC 1.6.99.3) activity was mitochondria was layered on top of a discon- assayed at 27°C by the method of Sottocasa et al. tinuous gradient consisting of 20% (w/v) Ficoll in (1967). NADP+-dependent isocitrate dehydrogenase medium A and 10% (w/v) Ficoll in medium A and (EC 1.1.1.42) activity was determined at 37°C as centrifuged for 120min at 4°C and 75000g in a described by Bernt & Bergmeyer (1974). Aspartate- Beckman ultracentrifuge equipped with a SW 27.1 2-oxoglutarate transaminase (EC 2.6.1.1) activity rotor. The purified mitochondria obtained as the was determined at 37°C by the enzymic assay of pellet after this centrifugation were used for the Bergmeyer & Bernt (1974), by using 0.1 M-Tris/HCl preparation of inner and outer membranes as buffer, pH 8.0, containing 20mM-L-aspartate, 4mM-2- described by Marks (1974) and for electron oxoglutarate, 0.02niM-pyridoxal phosphate, 0.1 mM- microscopy (see below). The purified mitochondria NADH and 10units of malate dehydrogenase were suspended (2-3mg of protein/ml) in a hypo- (EC 1.1.1.37)/ml. Monoamine oxidase (EC 1.4.3.4) osmotic medium consisting of 20mM-potassium activity was measured at 37°C as described by phosphate, pH7.2, 0.02% (w/v) bovine serum McCaman et al. (1965) by using 2.5mM-[14C]- albumin and 0.02% (w/v) heparin (medium B) and tyramine (sp. radioactivity 40-50d.p.s./nmol) as kept at 40C for 20h. Then it was layered on top of the substrate. 4-Aminobutyrate transaminase activity 10% (w/v) Ficoll in medium A and centrifuged for was determined at 37°C by using a modification of 30min at 40C and 30000g in a Beckman ultracentri- the assay described by Hall & Kravitz (1967). The fuge (SW 60 rotor), giving three fractions: the assay mixture consisted of 0.1 M-Tris/HC1, pH8.0, clear supernatant on top of the 10% (w/v) Ficoll was 1 mM-aminoethylisothiouronium bromide, 0.02mM- taken as the mitochondrial matrix, the layer on top pyridoxal phosphate, 4mM-2-oxoglutarate, 1 mm- ofthe 10% Ficoll as outer mitochondrial membranes succmate, 3mM-NAD+ and 25mM-4-amino[14C]- and the pellet as inner mitochondrial membranes. butyrate (sp. radioactivity 5-8 d.p.s./nmol). Blanks Inner and outer membranes were resuspended in were run in the same buffer minus 2-oxoglutarate. small volumes ofmedium B (4-6mg ofprotein/ml). The '4C-labelled reaction products (succinic semi- Mitochondria from rat liver were prepared as aldehyde and succinate) were separated from described by Johnson & Lardy (1967). The large- 4-amino-[14C]butyrate on Dowex 50 (X2; 50- amplitude swelling of these mitochondria in hypo- 100mesh) columns (0.7cmx7cm). Specific enzyme osmotic medium was performed as described above activities are expressed as units/mg of protein, where for the brain mitochondria. The swollen mitochon- 1 unit is defined as that activity catalysing the dria were centrifuged for 60min at 40C and 1000OOg conversion of 1 pmol of substrate/min at the in a Beckman ultracentrifuge equipped with a SW 60 temperature of the assay. Radioactivity was deter- rotor. The supernatant was taken as the matrix mined as described by Schousboe & Hertz (1971) fraction and the pellet, which represents the in a Packard Tri-Carb liquid-scintillation spectro- mitochondrial-membrane fraction, was resuspended meter. Protein was measured by the method of in a small volume of medium B (6-8mg of Lowry et al. (1951) as modified by Miller (1959), by protein/ml). using bovine albumin as the standard. Statistical analyses were performed by using Student's t test. Electron microscopy Suspensions of purified brain mitochondria and Results inner and outer membranes were centrifuged at high speed (30min at 4°C and 100000g) and the resulting Electron microscopy pellets fixed in 3% (v/v) glutaraldehyde (16h) and Plate 1 shows an electron micrograph of the post-fixed in 1 % (w/v) OSO4 (2h). Both solutions were purified brain mitochondria. They appear relatively buffered with 0.1 M-sodium cacodylate, pH6.8. The homogeneous, with only a few synaptosome-like pellets were then stained in 0.5% (w/v) uranyl acetate bodies, and have an intact double-membrane and embedded in Araldite. structure. The subfractions obtained after large- The blocks were cut on an LKB ultramicrotome amplitude swelling are shown in Plates 2 and 3, and and the sections post-stained in 2% (w/v) uranyl this treatment has disrupted the mitochondria, which acetate and Reynold's lead citrate. The stained appear as 'ghosts' or empty vesicles. It has, however, sections were examined in a Philips 201 C electron not been adequate for a complete separation of the microscope. inner and outer membrane. Fully detached inner membranes are only seen in some instances (Plate 2), Enzyme assays and the outer-membrane fraction (Plate 3) is Cytochrome c oxidase (EC 1.9.3.1) activity was contaminated with inner membranes still in contact measured at 270C as described by Smith (1955) and with outer maembranes. 1977 The Biochemical Journal, Vol. 162, No. 2 Plate 1 EXPLANATION OF PLATE I Electron micrograph ofmitochondria (M)purifiedfrom ox brain The mitochondria appear intact, with double-membrane structures. A few synaptosome-like bodies (S) are seen. I. SCHOUSBOE, B.
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