Purification and reconstitution of two anion carriers from rat liver mitochondria: the dicarboxylate and the 2-oxoglutarate carrier

Two anion-transporting systems, i.e., the dicarboxylate carrier and the 2-oxoglutarate carrier, have been purified from rat liver mitochondria and functionally identified. The dicarboxylate carrier has been isolated in active form by hydroxyapatite chromatography after partial removal of the solubilizing detergent Triton X-114 from the mitochondrial extract. The SDS gel electrophoresis of this preparation consists mainly of one protein band with an apparent Mr of 28,000, identified as the dicarboxylate carrier. Complete purification of the 28 kDa protein in inactive form has been achieved by sequential chromatography on hydroxyapatite and Celite followed by SDS extraction of the retained protein. The 2-oxoglutarate carrier has been purified by hydroxyapatite chromatography after extensive removal of Triton X-114 from the detergent extract. SDS gel electrophoresis of the purified fraction shows a single band with an apparent Mr of 32,500. When reconstituted into liposomes, the functional properties of the two isolated carrier proteins resemble closely those of the dicarboxylate and the 2-oxoglutarate transport systems characterized in mitochondria.     

Isolation and reconstitution of an N-ethylmaleimide-sensitive phosphate transport protein from rat liver mitochondria

An N-ethylmaleimide-sensitive phosphate transport protein has been isolated from rat liver mitochondria, substantially purified, and reconstituted into phospholipid vesicles. Purified inner mitochondrial membrane vesicles depleted of F1-ATPase by urea treatment proved to be the most satisfactory starting material. Treatment of these membrane vesicles with Triton X-100 resulted in solubilization of the phosphate transport protein. Further purification was achieved using hydroxylapatite powder. Polyacrylamide gel electrophoresis of the purified fraction in sodium dodecyl sulfate indicated the presence of two Coomassie blue-staining bands with apparent Mr's of 30,000 and 35,000. Labeling of the 35,000 Mr band by the Pi transport inhibitor diazobenzene sulfonate was reduced markedly by prior treatment of the mitochondria with the inhibitor N-ethylmaleimide. The purified fraction containing both proteins could be reconstituted into liposomes prepared from purified asolectin. Phosphate efflux from these vesicles was inhibited by N-ethylmaleimide, by the impermeant mercurial agent, p-chloromercuribenzoate, and by diazobenzene sulfonate. Treatment of the purified fraction with N-ethylmaleimide prior to incorporation into liposomes resulted in a reconstituted system incapable of catalyzing Pi efflux. These studies summarize the first detailed attempt to purify the Pi/H+ transport system from rat liver mitochondria and emphasize the need to commence the purification with purified inner membrane vesicles depleted of F1-ATPase. In addition, these studies show that the final fraction contains a reconstitutively active transport system which when incorporated into phospholipid vesicles has its essential sulfhydryl groups oriented outward. Finally, it is shown that the purified fraction also contains a 30,000 Mr component.     

Purification of reconstitutively active alpha-oxoglutarate carrier from pig heart mitochondria

The alpha-oxoglutarate carrier from pig heart mitochondria has been solubilized with Triton X-114 and purified by chromatography on hydroxyapatite and celite in the presence of cardiolipin. When applied to SDS gel electrophoresis, the purified protein consists of only a single protein band with an apparent Mr of 31.5 kDa. It corresponds to band 4 of the five protein bands previously identified in the hydroxyapatite pass-through of Triton X-114 solubilized heart mitochondria (Bisaccia, F. and Palmieri, F. (1984) Biochim. Biophys. Acta 766, 386-394). When reconstituted into liposomes the alpha-oxoglutarate transport protein catalyzes a phthalonate-sensitive alpha-oxoglutarate/alpha-oxoglutarate exchange. It is purified 250-fold with a recovery of 62% and a protein yield of 0.1% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e., the requirements for a counteranion, the substrate specificity and the inhibitor sensitivity, are similar to those described for alpha-oxoglutarate transport in mitochondria.     

Identification and purification of the carnitine carrier from rat liver mitochondria

The carnitine carrier from rat liver mitochondria, solubilized in Triton X-100 and partially purified on hydroxyapatite, was identified and completely purified by specific elution from celite in the presence of cardiolipin. On SDS-gel electrophoresis, the purified celite fraction consisted of a single band with an apparent Mr of 32,500. When reconstituted into liposomes the carnitine transport protein catalyzed an N-ethylmaleimide-sensitive carnitine/carnitine exchange. It was purified 970-fold with a recovery of 43% and a protein yield of 0.04% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e., requirement for a countersubstrate, substrate specificity and inhibitor sensitivity, were similar to those of the carnitine transport system as characterized in intact mitochondria.     

Identification and purification of the tricarboxylate carrier from rat liver mitochondria

The tricarboxylate carrier from rat liver mitochondria was solubilized with Triton X-100 and purified by chromatography on hydroxyapatite and celite. SDS-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent Mr of 30,000. When reconstituted into liposomes, the tricarboxylate transport protein catalyzed a 1,2,3-benzenetricarboxylate-sensitive citrate/citrate exchange. We obtained a 1070-fold purification with respect to the mitochondrial extract, the recovery was 22% and the protein yield 0.02%. The properties of the reconstituted carrier, i.e., requirement for a counteranion, substrate specificity and inhibitor sensitivity, were similar to those of the tricarboxylate transport system as characterized in intact mitochondria.     

Sensitivity and variability of the Bradford protein assay in the presence of detergents

The effects of Triton X-100, sodium dodecyl sulfate (SDS), and urea on the response of Coomassie blue G to 16 different proteins and peptides of Mr 1140 to 146,000 were studied to assess the significance of protein conformation and of ionic and nonionic interactions for the dye response to individual proteins. Triton X-100 at a final concentration of 0.008% (v/v) increased the sensitivity of the Bradford assay toward all proteins of Mr 5700 or higher by an average 33%. Increases ranged from +11% with myelin basic protein to +128% with aprotinin. The relative range of absorbance of proteins and deviations from bovine serum albumin decreased by approximately 25%. Triton X-100 appears to facilitate nonionic interactions of the dye with proteins of limited capacity for ionic binding. Conformation of proteins also seemed to be of some significance because the chaotropic agent urea (0.16 M final concentration) increased sensitivity of the assay by 14%. Sensitivity of the assay was lowered by SDS (0.004% final concentration, w/v) by an average 75% from that of the control assay. The results indicate that the incorporation of low concentrations of a nonionic detergent may be useful in improving sensitivity and variability of the Bradford assay.     

Oxidation of protoporphyrinogen to protoporphyrin, a step in chlorophyll and haem biosynthesis. Purification and partial characterization of the enzyme from barley organelles.

The protoporphyrinogen-oxidizing enzyme from Triton X-100 extracts of the mitochondrial and etioplast fractions of etiolated barley was purified by using ion-exchange and hydroxyapatite chromatography. The purified enzyme from both organelle fractions exhibited a Km of 5 microM and was labile to mild heat and acidification. The pH optimum (5-6) and the substrate-specificity (mesoporphyrinogen was oxidized as rapidly as protoporphyrinogen) revealed properties very different from the protoporphyrinogen-oxidizing enzyme of rat liver or yeast mitochondria, which is specific for protoporphyrinogen as substrate. The purest fractions showed a polypeptide band corresponding to an Mr of approx. 36,000 on SDS/polyacrylamide-gel electrophoresis. This is the first purification and characterization of the enzyme from a plant, and indicates no readily detectable differences between the enzyme isolated from mitochondrial or etioplast fractions, although only the latter organelle has the capacity for both haem and chlorophyll synthesis.     

Isolation of protein components from rat lung lamellar bodies

Lamellar bodies isolated from 10% (w/v) rat lung homogenates by discontinuous sucrose gradient centrifugation were shown to contain variable amounts of adhering proteins. These contaminating proteins could be removed by either Sepharose 4B gel filtration or precipitation of the crude preparation at pH 11.5. Both purification methods yielded membrane preparations with a phospholipid-to-protein ratio of 10.0 μmol/mg. Nearly complete separation of lamellar body phospholipid and protein could be achieved upon application of the purified membranes to DEAE-cellulose in the presence of 0.2% (v/v) Triton X-100. Phospholipid analyses showed that 83% of total lipid phosphorus was recovered in phosphatidylcholine. In phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine and phosphatidylinositol recoveries amounted to 4, 8, 2 and 2%, respectively. Molecular mass determinations of the isolated protein component of lamellar bodies by means of SDS polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue revealed the presence of three protein bands with molecular masses of 64, 33 and 31 kDa. Upon staining with silver a 16 kDa protein was also visible. Sephadex G-100 gel filtration showed only one protein peak corresponding to a molecular mass of 64 kDa when protein was assayed with Coomassie brilliant blue.     

The purification of a detergent-soluble glucose-6-phosphatase from rat liver.

A highly active and soluble glucose-6-phosphatase has been purified to near homogeneity from rat liver. Successful purification has been initiated by covalent labeling of the enzyme in native rat liver microsomes with pyridoxal 5'-phosphate and NaBH4, followed by solubilization of the microsomes with Triton X-100, chromatography on phenyl-Sepharose, hydroxyapatite, DEAE-Sephacel and a second chromatography step on hydroxyapatite. The final enzyme preparation obtained was approximately 700-fold purified over the activity of starting microsomes. As judged by SDS/PAGE the purified glucose-6-phosphatase is composed of a single protein with a molecular mass of 35 kDa. The present work demonstrates that the purified glucose-6-phosphatase must be arranged in the native microsomal membrane so that it is accessible to pyridoxal 5'-phosphate from the cytoplasmic side.     

Purification and partial characterization of squalene epoxidase from rat liver microsomes

Squalene epoxidase (EC 1.14.99.7, squalene 2,3-monooxygenase (epoxidizing) was purified to an apparent homogeneity from rat liver microsomes. The purification was carried out by solubilization of microsomes by Triton X-100, fractionation with ion exchangers, hydroxyapatite, Cibacron Blue Sepharose 4B, and chromatofocusing column chromatography. A total purification of 143-fold over the first DEAE-cellulose fraction was achieved. The purified enzyme gave a single major band on SDS-polyacrylamide gel electrophoresis and the Mr was estimated to be 51 000 as a single polypeptide chain. The enzyme showed no distinct absorption spectrum in the visible regions. The squalene epoxidase activity was reconstituted with the purified enzyme, NADPH-cytochrome P-450 reductase (EC 1.6.2.4), FAD, NADPH and molecular oxygen in the presence of Triton X-100. The apparent Michaelis constants for squalene and FAD were 13 microM and 5 microM, respectively. The Vmax was about 186 nmol per mg protein per 30 min for 2,3-oxidosqualene. The enzyme activity was not inhibited by potent inhibitors of cytochrome P-450. It is suggested that squalene epoxidase is distinct from cytochrome P-450 isozymes.     

Purification and characterization of membrane-bound phosphatidylinositol kinase from rat brain

A membrane-bound phosphatidylinositol (PI) kinase was purified from rat brain. The enzyme was solubilized with Triton X-100 from salt-washed membrane and purified 11,183-fold, with a final specific activity of 150 nmol/min/mg of protein. Purification steps included several chromatography using Q-Sepharose Fast Flow, cellulose phosphate, Toyopearl HW 55 and Affi-Gel Blue. The purified PI kinase had an estimated molecular weight of 80,000 by gel filtration and 76,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified kinase phosphorylated only PI and did not phosphorylate phosphatidylinositol 4-phosphate or diacylglycerol. Km values for PI and ATP were found to be 115 and 150 microM, respectively. The enzyme required Mg2+ (5-20 mM) or Mn2+ (1-2 mM) for activity, was stimulated by 0.1-1.0% (w/v) Triton X-100, and completely inhibited by 0.05% sodium dodecyl sulfate. The enzyme activity showed a broad pH optimum at around 7.4. The enzyme utilized ATP and not GTP as phosphate donor. Nucleoside triphosphates other than ATP and diphosphates significantly inhibited the kinase activity. However, inhibitory effects of adenosine, cAMP, and quercetin were weak.     

Purification of the mitochondrial carnitine carrier by chromatography on hydroxyapatite and celite

The carnitine carrier from rat liver mitochondria has been extracted with Triton X-100 ad partially purified by chromatography on hydroxyapatite and celite. During purification the activity of the carrier was monitored by functional reconstitution into liposomes. The purified fraction is 250-fold enriched with respect to the N-ethylmaleimide-sensitive carnitine/carnitine transport activity. The substrate specificity and the inhibitor sensitivity of carnitine transport in liposomes resemble closely those described for the transport of carnitine in mitochondria.     

An immunological study of the uncoupling protein of brown adipose tissue mitochondria

1. Ewes were injected with purified 32,000-Mr uncoupling protein from mitochondria of brown adipose tissue of cold-adapted rats in order to raise antibodies. 2. The existence of antibodies in the plasma of ewes and the cross-reactivity of plasmas were demonstrated and studied by 125I-labelled antigen-antibody reaction, double immunodiffusion, the inhibition of GDP binding to the 32,000 Mr protein and by immunohistochemistry. 3. The antibodies raised against the homogeneous protein yielded a single immunoprecipitation band with detergent-solubilized mitochondrial membranes of brown adipose tissue from rat, hamster, guinea-pig, rabbit and with the purified uncoupling protein of these animals. No immunoprecipitation was obtained with the protein purified from brown adipose tissue of term lamb foetus. 4. The GDP-binding activity of the uncoupling protein (isolated or in solubilized membranes) was largely inhibited by the antiserum. 5. The anti-(rat uncoupling protein) could not cross-react with solubilized membranes from liver or muscle, nor with the purified beef heart or rat liver ADP/ATP translocator.     

Purification of reconstitutively active α-oxoglutarate carrier from pig heart mitochondria

The α-oxoglutarate carrier from pig heart mitochondria has been solubilized with Triton X-114 and purified by chromatography on hydroxyapatite and celite in the presence of cardiolipin. When applied to SDS gel electrophoresis, the purified protein consists of only a single protein band with an apparent M_r of 31.5 kDa. It corresponds to band 4 of the five protein bands previously identified in the hydroxyapatite pass-through of Triton X-114 solubilized heart mitochondria (Bisaccia, F. and Palmieri, F. (1984) Biochim. Biophys. Acta 766, 386–394). When reconstituted into liposomes the α-oxoglutarate transport protein catalyzes a phthalonate-sensitive α-oxoglutarate / α-oxoglutarate exchange. It is purified 250-fold with a recovery of 62% and a protein yield of 0.1% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e., the requirements for a counteranion, the substrate specificity and the inhibitor sensitivity, are similar to those described for α-oxoglutarate transport in mitochondria.     

Calmodulin binding proteins in rat liver mitochondria

Calmodulin binding proteins have been found in submitochondrial fractions obtained from highly purified rat liver mitochondria. The matrix fraction contains two major calmodulin binding proteins: one, having Mr of 145,000, apparently is carbamoyl-phosphate synthetase. Another has a Mr of 58,000 and has not been associated with enzyme activities. A major calmodulin binding protein of unknown function and having Mr of 32,000 has been found in the Triton X-100 solubilizate of the inner membrane. Minor amounts of two calmodulin binding proteins having Mr of about 37,000 and 56,000 have been found in the outer membrane.     

Electrophoretic and immunochemical characterization of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenases of rat tissues.

The properties of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from Sprague-Dawley rat liver cytosol have been re-examined in light of several reports which suggest that multiple forms of the enzyme may exist in this tissue. During enzyme purification, chromatography on DE-52 cellulose and chromatofocusing columns indicated the existence of only one form of the protein. Re-chromatography of the purified enzyme by either of these techniques failed to resolve the protein into additional forms. When the purified enzyme was subjected to SDS/polyacrylamide-gel electrophoresis a single band corresponding to Mr 34,000 was detected. Two-dimensional gels showed one predominant protein with a pI of 5.9. Using the homogeneous enzyme as antigen, high-titre polyclonal antibody was raised in rabbits. Western-blot analysis of cytosolic proteins prepared from male and female Sprague-Dawley rat liver indicated the presence of a single immunoreactive band with an Mr of 34,000 in both sexes. All of the 3 alpha-hydroxysteroid dehydrogenase activity present in rat liver cytosol could be immunotitrated with the antibody and the resulting titration curve was superimposable on the titration curve obtained with the purified enzyme. Western-blot analysis of cytosolic proteins prepared from livers of male Wistar and Fischer rats also revealed the presence of a single immunoreactive protein with an Mr of 34,000. These data indicate that, contrary to previous reports, only one form of the dehydrogenase may exist in liver cytosols prepared from a variety of rat strains. Although 3 alpha-hydroxysteroid dehydrogenase activity is known to be widely distributed in male Sprague-Dawley rat tissues, Western blots indicate that only the liver, lung, testis and small intestine contain immunoreactive protein with an Mr of 34,000. The levels of immunoreactive protein in these tissues follow the distribution of dihydrodiol dehydrogenase.     

Isolation of insecticide resistance-related forms of cytochrome P-450 from Drosophila melanogaster.

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase was purified from rat brain by Blue-Sepharose, phosphocellulose and calmodulin (CaM)-Sepharose affinity chromatography. The purified enzyme was stimulated by Ca2+/CaM by 3-6-fold as compared with the activity measured in the presence of EGTA. Rat brain InsP3 3-kinase activity was associated with two silver-stained bands of about equal activity which migrated with an apparent Mr of 50,000 on SDS/polyacrylamide gels. InsP3 3-kinase activity from rat brain could be immunoprecipitated by an antiserum against the SDS/PAGE-purified 50,000-Mr protein doublet. InsP3 kinase activity from bovine brain and the InsP3 5-phosphatase activity from rat brain were not immunoprecipitated. On Western blot, the human brain crude InsP3 3-kinase reacted specifically, but less strongly than the rat brain enzyme, with the antiserum.     

Endocytosis of a small dermatan sulphate proteoglycan. Identification of binding proteins.

Endosomal preparations from human osteosarcoma cells and from fibroblasts contain 51,000- and 26,000-Mr proteins which bind a small dermatan sulphate proteoglycan after SDS/polyacrylamide-gel electrophoresis and Western blotting. Binding can be inhibited by unlabelled proteoglycan core protein. The proteins co-precipitate with a proteoglycan core protein-antibody complex. Scatchard analysis of immobilized endosomal proteins yielded a KD of about 37 nM for the proteoglycan. In intact cells proteins of the same size can be found. They are sensitive to trypsinization. A 51,000-Mr protein is the predominant membrane protein with strong binding to immobilized dermatan sulphate proteoglycan. There are additional proteoglycan-binding proteins with Mr values of around 30,000 and 14,000 which are insensitive to trypsin treatment. In contrast with the 51,000- and 26,000-Mr proteins, they resist deoxycholate/Triton X-100 extraction several days after subcultivation.     

A major polypeptide component of rat liver mitochondria: carbamyl phosphate synthetase.

One of the major components of rat liver mitochondria detected by gel electrophoresis in sodium dodecyl sulfate is a 165,000 molecular weight polypeptide that makes up 15 to 20% of the total mitochondrial protein. This component appears to be a single molecular species. Evidence is presented here for the identification of this protein with the polypeptide chain of a urea cycle enzyme, carbamoylphosphate synthetase I (EC 2.7.2.5). The 165,000 molecular weight polypeptide was solubilized from mitochondria with Triton X-100 and purified to 90% homogeneity by DEAE-cellulose chromatography. This component co-migrated with carbamyl phosphate synthetase activity when mitochondrial proteins were separated by gel filtration or sucrose gradient centifugation. The identification of the 165,000 molecular weight polypeptide with this activity was also supported by the presence or absence of this protein in a variety of rat tissue mitochondria, in liver and kidney mitochondria from various ureotelic and nonureotelic species, and in fetal rat liver mitochondria.     

Characterization of the mitochondrial carnitine palmitoyltransferase enzyme system. II. Use of detergents and antibodies

Exposure of rat liver mitochondrial membranes to octyl glucoside, Triton X-100, or Tween 20 solubilized an active and tetradecylglycidyl-CoA (TG-CoA)-insensitive carnitine palmitoyltransferase (presumed to be carnitine palmitoyltransferase II). The residual membranes after octyl glucoside or Triton X-100 treatment were devoid of all transferase activity. By contrast, Tween 20-extracted membranes were still rich in transferase; this was completely blocked by TG-CoA and thus was presumed to be carnitine palmitoyltransferase I. The residual carnitine palmitoyltransferase activity disappeared from the membranes upon subsequent addition of octyl glucoside or Triton X-100 and could not be recovered in the supernatant fraction. Antibody raised against purified rat liver transferase II (Mr 80,000) recognized only this protein in immunoblots from untreated liver mitochondrial membranes containing both transferases I and II. Tween 20-extracted membranes, which contained only transferase I, did not react with the antibody. Purified transferase II from skeletal muscle (also of Mr 80,000) was readily recognized by the antiserum, suggesting antigenic similarity with the liver enzyme. These and other studies on the effects of detergents on the mitochondrial [3H]TG-CoA binding protein provide further support for the model of carnitine palmitoyltransferase proposed in the preceding paper. They suggest that: 1) carnitine palmitoyltransferases I and II in rat liver are immunologically distinct proteins; 2) transferase I is more firmly anchored into its membrane environment than transferase II; 3) association of carnitine palmitoyltransferase I with a membrane component(s) is necessary for catalytic activity. While carnitine palmitoyltransferase I is a different protein in liver and muscle, it seems likely that both tissues share the same transferase II.