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Biogenesis of the Mitochondrial Matrix Enzyme, Glutamate

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Biogenesis of the Mitochondrial Matrix Enzyme, Glutamate J. Biochem. 82, 1403-1416 (1977) Biogenesis of the Mitochondrial Matrix Enzyme, Glutamate Dehydrogenase, in Rat Liver Cells I. Subcellular Localization, Biosynthesis, and Intracellular Translocation of Glutamate Dehydrogenase Kaname KAWAJIRI, Tomoyuki HARANO, and Tsuneo OMURA Department of Biology, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka, Fukuoka 812 Received for publication, May 21, 1977 1. The presence of glutamate dehydrogenase in the microsomal fraction of rat liver was confirmed. The identities of mitochondrial and microsomal glutamate dehydrogenases were proved by immunochemical methods and by SDS polyacrylamide gel electrophoresis of purified enzymes. 2. Synthesis of glutamate dehydrogenase by the membrane-bound ribosomes of rough endoplasmic reticulum was determined. Newly synthesized enzyme molecules were discharged on the cytoplasmic surface of endoplasmic reticulum membranes. 3. A precursor-product relationship was found between microsomal and mitochondrial glutamate dehydrogenases. About six hours were needed for the transport of glutamate dehydrogenase from the site of synthesis to mitochondria. 4. The half-life of glutamate dehydrogenase was about 5.5 days, which was somewhat longer than that of mitochondrial total protein determined in the same experiment. 5. Mitochondrial-type malate dehydrogenase was also present in the microsomal fraction. Subfractionation of smooth microsomes revealed the existence of particular light microsomal vesicles in which both glutamate dehydrogenase and malate dehydrogenase were concentrated. These vesicles may participate in intracellular transport of matrix enzymes from microsomes to mitochondria. Glutamate dehydrogenase (GDH) is functional of amino acids (1). Therefore the biosynthesis in mitochondria as a key enzyme in the metabolism and intracellular translocation of this enzyme from the site of synthesis to mitochondria are important not only in the biogenesis of intracellular organelles Abbreviations: GDH, glutamate dehydrogenase; but also in the manifestation of the physiological MDH, malate dehydrogenase; AGG, anti-glutamate function of GDH. dehydrogenase-immunoglobulin; AMG, anti-malate de The interdependence between mitochondrial hydrogenase-immunoglobulin; AOG, anti-ovalbumin immunoglobulin; AAG, anti-serum albumin-immuno protein synthesis and cytoplasmic protein synthesis in the biogenesis of mitochondria is now well globulin; AMPase, adenosine monophosphatase; IgG, immunoglobulin. established (2-7). Synthesis of outer membrane Vol. 82, No. 5, 1977 1403 1404 K. KAWAJIRI, T. HARANO, and T. OMURA proteins (8) and some protein components of the genizer to give a 15% homogenate. The homog inner membrane (9) by cytoplasmic ribosomes enate was centrifuged at 600 •~ g for 10 min to has been repeatedly shown. Mitochondria] matrix remove nuclei and cell debris. The supernatant proteins, which constitute about 50% of the total was centrifuged at 5,000 •~ g for 15 min to pre mitochondrial proteins, are also synthesized on cipitate mitochondria. The post-mitochondrial cytoplasmic ribosomes (10-12). In the case of supernatant was centrifuged at 10,000 •~ g for matrix enzymes, they must be transported from 15 min to remove lysosomes. The post-lysosomal the cytoplasm into the mitochondrial inner com supernatant was centrifuged at 170,000 •~ g for partment across two membranes, the outer and 60 min to precipitate microsomes. Both the inner membranes. mitochondria and the microsomes were washed The purpose of this study was to elucidate once with 0.25 M sucrose-10 mm Tris-HCl-1 mm the mechanism of intracellular transfer of GDH EDTA (pH 7.5). To prepare rough and smooth from its site of synthesis to the mitochondria) microsomes, 15 ml of the post-lysosomal super inner compartment. We confirmed the presence natant were layered over 15 ml of 1.3 M sucrose of GDH in the microsomal fraction obtained from 10 mm Tris-HCl-1 mm EDTA (pH 7.5). After rat liver. We also demonstrated the synthesis of 4 h centrifugation at 170,000 •~ g, the layer of GDH by the membrane-bound ribosomes of rough smooth microsomes which formed between the endoplasmic reticulum. The GDH in the micro 0.25 M sucrose and the 1.3 M sucrose was carefully somal fraction was present on the outside surface separated from the tightly packed pellet of rough of microsomal vesicles, which coincided with the microsomes, and diluted five to six fold with observation that the nascent peptides of the enzyme 0.25 M sucrose-10 mm Tris-HCl-1 mm EDTA (pH were discharged from the ribosomes to the cyto 7.5). The pellets were also suspended in the plasmic surface of endoplasmic reticulum. As a same sucrose solution and the suspensions were precursor-product relationship was found between centrifuged at 170,000 •~ g for 60 min to sediment microsomal and mitochondrial GDHs, the enzyme the rough and smooth microsomes. Both micro associated with microsomal particles is a pool of somal fractions were washed once with the same newly synthesized molecules to be transported into sucrose solution. mitochondria. Subfractionation of Smooth Microsomes by Butow and Kellems reported the existence of Sucrose Density Gradient Centrifugation-The cytoplasmic polyribosomes in association with the smooth microsomes (about 16 mg protein) were cytoplasmic face of the outer mitochondria) layered over a continuous concentration gradient membrane in yeast cells, and they suggested the of sucrose from 0.7 M to 1.25 M containing 10 mm vectorial discharge of nascent peptides from the Tris-HCl (pH 7.5). Centrifugation was performed ribosomes, which ensures their transfer into the with a Hitachi SW 25 rotor at 25,000 rpm for mitochondrial inner compartment (13-16). Our 3 h. One ml fractions were collected from the results indicate, however, the principal role of bottom of tube. membrane-bound ribosomes of rough endoplasmic Purification of GDH from Rat Liver Mito reticulum in the biosynthesis of mitochondria) chondria. -GDH was purified from rat liver mito matrix proteins. chondria by the following procedure. The enzyme was solubilized by sonic oscillation of washed mitochondria at 4•Ž for 60 s. The sonicated MATERIALS AND METHODS suspension was centrifuged at 105,000 •~ g for Preparation Mitochondria, Microsomes, Rough, 60 min. The supernatant containing about 3,000 and Smooth Microsomes from Rat Liver-Sprague mg of protein was concentrated by freeze-drying Dawley male rats weighing 200g to 300g were and applied to a column (3.7 •~ 80 cm) of Sepharose used. They were fasted overnight and then 4B equilibrated with 10 mm phosphate-1 mm killed. Their livers were excised, perfused thor EDTA-20 ƒÊM ADP (pH 7.5). ADP was added oughly with ice-cold 0.9 % NaCl, and homogenized in this step to stabilize the enzyme. The column in 0.25 M sucrose-10 mm Tris-HCl-1 mm EDTA was eluted with the same buffer, and the eluate (pH 7.5) with the aid of a Potter glass-Teflon homo- was collected in 15 ml fractions. The GDH J. Biochem. BIOGENESIS OF GLUTAMATE DEHYDROGENASE IN RAT LIVER CELLS (1) 1405 rich fractions were combined, and applied to a column (2.7 •~ 20 cm) of DEAE-Sephadex A-50 equilibrated with 10 mm phosphate-1 mm EDTA (pH 7.0). The column was washed with the same buffer, and then eluted with an increasing linear concentration gradient of KCl from 0 to 0.4 M in the same buffer system. The eluate was col lected in 10 ml fractions. The GDH-rich fractions were combined and dialyzed against 10 mm phosphate-1 mm EDTA (pH 7.0) overnight. The dialyzed solution was then applied to a column (1.4 •~ 15 cm) of hydroxylapatite equilibrated with 10 mm phosphate (pH 7.0). After washing with the same buffer, the enzyme was eluted with an increasing linear concentration gradient of phos Fig. 1. SDS polyacrylamide gel electrophoresis of phate from 10 mm to 0.4 M (pH 7.0). The eluted GDH was concentrated with a collodion bag. purified mitochondrial and microsomal glutamate de hydrogenases. (A) Purified GDH from rat liver mito About 20 mg of purified enzyme was recovered chondria was analyzed by electrophoresis with 5% and its activity was about 170 units per mg of polyacrylamide gel of pH 8.5. Three ƒÊg of enzyme protein. The purification ratio and the recovery protein were applied on the column. (B) A mixture of were about 150 and 34% from whole mitochondria, mitochondrial and microsomal GDH was analyzed as respectively. The purified GDH gave a single described in (A). A mixture of 2 ƒÊg of mitochondrial band when examined by SDS polyacrylamide gel enzyme and 1ƒÊg of microsomal enzyme was applied electrophoresis (Fig. 1). on the column. (C) Purified GDH from rat liver micro Purification of GDH from Microsomes somes was analyzed in the same way. One ƒÊg of enzyme was applied on the column. Purification of GDH from rat liver microsomes was carried out as follows. Microsomes were suspended in 0.15 M KCl to release the microsomal immunoglobulin fraction was prepared by frac GDH. The purification procedure of GDH tionation of the serum with ammonium sulfate was almost the same as that for the mitochondrial (25-45 %) and then dialyzed against 50 mm phos enzyme except DEAE-Sephadex column chro phate buffer (pH 7.5). The quantitative precipita matography was replaced by chromatography on tion reaction was carried out as described in the a phospho-cellulose column, which was equilibrated legend of Fig. 3. Antisera against ovalbumin and with 10 mm phosphate-1 mm EDTA (pH 7.0) and mitochondrial malate dehydrogenase were pre eluted with a concentration gradient of KCl from pared in the same way. 0 to 0.4 M in the same buffer system. Purified Preparation of Antibody-Sepharose Gel-The GDH from microsomes gave a single band when conjugation of immunoglobulin with Sepharose examined by SDS polyacrylamide gel electro 4B was carried out according to Porath et al. (17) phoresis (Fig. 1). with some modifications. Six grams of cyanogen Preparation of Antibody against GDH-Male bromide were added to 60 g (wet weight) of white rabbits, which were generously supplied by Sepharose 4B.
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