Reconstitution of C27-steroid 26-hydroxylase activity from bovine brain mitochondria

We have previously demonstrated the presence of cytochrome P-450 in a soluble preparation of bovine brain mitochondria (Oftebro, H., St?rmer, F.C., and Pedersen, J.I. (1979) J. Biol. Chem. 254, 4331). In the present work we show that this preparation, in the presence of NADPH, adrenodoxin and adrenodoxin reductase catalyzes omega-hydroxylation of a number of C27-steroids that are intermediates in bile acid biosynthesis. The rates of hydroxylation were 1-2 order of magnitudes lower than reported for similar preparations from rat and human liver. No significant activity was detected with cholesterol as substrate. The physiological significance of brain mitochondrial cytochrome P-450 is discussed.     

The C27-steroid hydroxylating system from bovine liver mitochondria. Isolation of ferredoxin (hepatoredoxin) by affinity chromatography on cytochrome-c-sepharose

A technique for the isolation of highly purified hepatoredoxin involving the DE-32 cellulose chromatography of post-mitochondrial supernatant, ammonium-sulfate fractionation, Sephadex G-75 gel chromatography, 1-amino-2-hydroxypropyl-Sepharose ion-exchange chromatography and cytochrome-c-Sepharose affinity chromatography is described. The protein was purified 160-fold with a yield of 19%. The synthesis of cytochrome-c-Sepharose was carried out in a way preventing modification of the lysine-containing binding domain of the cytochrome c molecule. To achieve this, free carboxyl groups were modified with histamine to introduce imidazole residues in cytochrome c and the modified protein was immobilized on bromoacetyl-Sepharose.     

Comparative studies of purified and reconstituted monoamine oxidase from bovine liver mitochondria

Monoamine oxidase, a strictly membrane-bound flavoenzyme, has been purified using a modified procedure recently developed. Probably similarly to other preparations known from the literature, the enzyme solubilizes to a clear suspension, which represents large clusters ranging in size from 5 to 50 nm containing appreciable amounts of residual lipids. The purified and reconstituted enzymes are inhibited differently by deoxycholate. In contrast to deoxycholate, Triton X-100 does not inhibit the purified enzyme, but rather disintegrates the lipid-enzyme clusters to the smallest active units. However, removal of the detergent leads to reconglomeration to larger lipid-enzyme aggregates. Using the irreversible destruction of the enzyme by deoxycholate as assay, reconstitution of the enzyme with exogeneous lipids has been studied. All basic enzyme properties, such as stability, maximal activity ($\text{V}$), Michaelis constant ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}$), pH- and temperature-dependence of the purified and reconstituted systems, are significantly different.     

Primary structure of hepatoredoxin from bovine liver mitochondria

The primary structure of hepatoredoxin from bovine liver mitochondria was established. It consists of 117 amino acid residues. The identity of the amino acid sequences of bovine hepatoredoxin and adrenodoxin from bovine adrenal cortex mitochondria was shown. It is assumed that the role of a ferredoxin component in mitochondrial steroid-hydroxylating systems from different organs is played by the same [2Fe-2S]-protein.     

Isolation of glyoxalase II from bovine liver mitochondria

Bovine liver mitochondria contain about 10% of the total glyoxalase II activity in the homogenate. Electrophoresis and isoelectric focussing of either crude mitochondrial extract or the purified mitochondrial glyoxalase II resolved the enzyme activity into five forms (pl 6.3, 6.7, 7.1, 7.7, and 7.9). Since bovine liver cytosol contains a single form of glyoxalase II (pl 7.5), at least four forms are exclusively mitochondrial with no counterpart in the cytosol. The relative molecular mass of mitochondrial glyoxalase II is about 23-24 kDa, similar to the cytosolic form. The kinetic constants obtained using S-D-lactoyl, S-acetyl-, S-acetoacetyl-, and S-succinyl-glutathione as substrates are similar to those reported for glyoxalase II from rat liver mitochondria. S-D-Lactoyl- and S-acetoacetyl-glutathione are the best substrates. S-Acetylglutathione is the poorest substrate with respect to both Vmax and Km values.     

Various properties of purified hepatoredoxin from bovine liver mitochondria

Hepatoredoxin purified to homogeneity from bovine liver mitochondria has been characterized for the first time in terms of its most important physico-chemical properties. The protein was found to contain in its active center a [2Fe-2S] cluster and has in the oxidized state an absorption maxima at 280, 320, 415 and 455 nm. The spectrophotometric index of purity, A415/A280 of the homogeneous native preparation is 0.84; extinction coefficient, epsilon 415, is 9800 M-1cm-1. The Mr of hepatoredoxin as evidenced by data from SDS gel electrophoresis is 12 500 Da; pI is 4.2. Hepatoredoxin is necessary for the reconstitution of the C27-steroid hydroxylase activity and can be substituted for by a related protein, adrenodoxin. All the above parameters as well as the circular dichroism spectra, immunochemical properties and sequence of the initial five N-terminal amino acids of hepatoredoxin and adrenodoxin are either coincident or very close. At the same time, the amino acid composition of these ferredoxins, apart from some common features, has individual peculiarities.     

Effect of C27-steroids on lipid peroxidation in rat liver mitochondria

The influence of C27-steroids--vitamin D3 and 20-hydroxyecdysone in free-radical reactions on lipid peroxidation in the mitochondrial fraction of rat's liver has been studied. Antioxidant activity of superoxide dismutase and catalase and the content of lipid components--cholesterol and phospholipids has been determined. The assumption was put forward that antioxidant action of steroids was possible as a result of to the modification of mitochondrial membrane through its lipid content and changing of its cholesterol: phospholipids content.     

Cross-linking studies of the cholesterol hydroxylation system from bovine adrenocortical mitochondria

Cytochrome P-450SCC and adrenodoxin were cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The sample containing 94% of cross-linked complex and 6% of free cytochrome P-450SCC was obtained after purification on cholate-Sepharose. Cytochrome P-450SCC in cross-linked complex completely preserves its high-spin form in the presence of Tween 20 or pregnenolone. Utilization of radioactively labelled adrenodoxin, chemical cleavage of cytochrome P-450SCC from cross-linked complex with o-iodosobenzoic acid and HPLC for separation of peptides allow us to conclude that the complex of cytochrome P-450SCC with adrenodoxin was cross-linked through two amino acid sequences of cytochrome P-450SCC-Leu-88-Thr-107 and Leu-368-Gly-416. The cross-linked complex of adrenodoxin reductase, adrenodoxin and cytochrome P-450SCC with an apparent molecular mass of 114 kDa was obtained with N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. The composition of cross-linked complex was determined by immunoblotting and by evaluation of radioactivity using preliminary N-ethyl[2,3-14C]maleimide-modified adrenodoxin. From this data it appears that the ternary complex may exist in solution.     

Purification and characterization of elongation factor G from bovine liver mitochondria

The mitochondrial protein synthesis translocase elongation factor Gmt (EF-Gmt) from bovine liver has been purified to greater than 90% homogeneity by a combination of conventional gravity and high performance liquid chromatography. The purification scheme results in an approximate overall 14,000-fold purification with 2% total recovery of EF-Gmt activity. Gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that the mitochondrial factor is a single polypeptide with a molecular weight of 80,000. EF-Gmt displays similar levels of activity on its homologous mitochondrial ribosomes and on Escherichia coli ribosomes. The mitochondrial translocase is sensitive to temperatures above 37 degrees C, but the factor is partially protected from heat inactivation in the presence of GTP or GDP. The activity of EF-Gmt is inhibited by treatment of the factor with N-ethylmaleimide. In contrast to all other translocases tested to date, EF-Gmt is completely resistant to the inhibiting effect of fusidic acid when tested on its homologous ribosomes. It displays weak sensitivity to this antibiotic when assayed in the presence of heterologous E. coli ribosomes.     

Orientation of ferrochelatase in bovine liver mitochondria

The orientation of ferrochelatase (protoheme ferro-lyase, EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, was examined in bovine liver mitochondria. The ability of a membrane-impermeable sulfhydryl reagent, 4,4'-dimaleimidylstilbene-2,2'-disulfonic acid, to inactivate ferrochelatase in intact or disrupted mitochondria and mitoplasts was examined. Using succinate dehydrogenase as an internal marker, it was found that ferrochelatase was inactivated only in disrupted mitochondria and mitoplasts, suggesting an internal location for the active site of the enzyme. In addition, antibodies raised against purified ferrochelatase were found to inhibit activity only in disrupted but not in intact mitoplasts. These data demonstrate that in bovine liver mitochondria ferrochelatase is located on the matrix side of the inner mitochondrial membrane. Data obtained with the membrane-impermeable amino reagent isethionyl acetimidate indicate that ferrochelatase physically spans the inner mitochondrial membrane with portions of the protein exposed on both sides of the membrane.     

Purification and properties of the soluble carnitine palmitoyltransferase from bovine liver mitochondria.

A new carnitine palmitoyltransferase (CPT) was purified to homogeneity from bovine liver mitochondria which were 96% free of peroxisomal contamination, as judged by catalase and glutamate dehydrogenase activities. The enzyme is easily removed from mitochondria, without the use of detergent. It is monomeric (Mr 63,500), unlike other preparations of CPT from mitochondria, and is most active with myristoyl-CoA and palmitoyl-CoA. The Km values are between 0.8 and 4 microM for a range of substrates from hexanoyl-CoA to stearoyl-CoA; these are much lower than values reported for other purified CPT preparations. The Km for L-carnitine is 185 microM measured with palmitoyl-CoA, and does not vary greatly with the chain length. This is also lower than the values reported for other CPT preparations, but higher than those cited for the medium-chain transferases. Kinetic and inhibitor studies were consistent with a rapid-equilibrium random-order mechanism. 2-Bromopalmitoyl-CoA, which is an inhibitor of the outer CPT, inhibited the enzyme competitively with palmitoyl-CoA as the variable substrate, when added without preincubation. If the enzyme was preincubated with 2-bromopalmitoyl-CoA and carnitine, the activity did not reappear after gel filtration of the protein. The inhibitor was bound in a 1:1 stoichiometry per subunit of enzyme.     

Lipid composition of mitochondria from bovine heart, liver, and kidney

Highly purified preparations of mitochondria from bovine heart, liver, and kidney were isolated and characterized by electron microscopy, oxidative phosphorylation ability, cytochrome c reductase activity, and cytochrome content. Components of lipid extracts of the preparations were determined by thin-layer chromatography, diethylaminoethyl-cellulose column chromatography, and spectrophotometric procedures. The major phospholipids were identified by their chromatographic behavior, IR spectrometry, and paper chromatography of their hydrolysis products. The lipid content of the mitochondria paralleled that of the components of the electron transfer chain, heart mitochondria being richest and liver mitochondria poorest in lipid. Heart mitochondria contain equal concentrations of coenzyme Q and cholesterol (1%); the highest cholesterol content (4.7%) was found in mitochondria from kidney. The phospholipids of mitochondria from the three organs were qualitatively and quantitatively very similar. The major polar lipid components (cardiolipin, choline glycerophosphatides, and ethanolamine glycerophosphatides) were present in a molar ratio of 1:4:4. It is suggested that mitochondria from different sources contain characteristic lipids, mainly phospholipids, of which cardiolipin is particularly diagnostic of the source of the mitochondria.     

Comparison of 3-hydroxybutyrate dehydrogenase from bovine heart and rat liver mitochondria

3-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with an absolute requirement of phosphatidylcholine for enzymatic activity. Purification of the enzyme to homogeneity from bovine heart mitochondria was described more than a decade ago [H. G. Bock and S. Fleischer (1975) J. Biol. Chem. 250, 5774-5781]. We have modified the purification procedure so that it is faster, the yield has been improved, and the specific activity is greater by approximately 50%. The updated procedure has also been applied to isolate the enzyme from rat liver mitochondria. Characteristics of the enzyme from bovine heart and rat liver mitochondria have been compared and found to be similar with respect to: (1) purification characteristics; (2) amino acid composition; (3) pH optimum for enzymatic activity; (4) kinetic characteristics; (5) molecular weight as determined by sedimentation equilibrium in guanidine hydrochloride; (6) peptide maps; (7) immunological cross-reactivity. These studies show that 3-hydroxybutyrate dehydrogenase from bovine heart and rat liver mitochondria, though similar, are not identical.     

Microcalorimetric study of mitochondria isolated from fish liver tissue

We determined the thermogenesis curves of mitochondria isolated from fish liver tissue by using an LKB 2277 Bioactivity Monitor. After isolation from the fish liver, mitochondria still have activity and can live for a long time by using the stored nutrients. We calculated the recovery rate constants of mitochondria. We found that the thermogenesis curves of mitochondria are similar to those obtained from prokaryotic cells, but not similar to those obtained from eukaryotic cells. We determined the metabolic thermogenesis curves of mitochondria isolated from two kinds of carp liver tissue, scattered-scaled mirror carp and harvest carp. There are some important similarities and some important differences between these thermogenesis curves.     

Purification and characterization of branched chain alpha-ketoacid dehydrogenase from bovine liver mitochondria.

Branched chain alpha-ketoacid dehydrogenase (EC 1.2.4.3(4)) was solubilized and purified from bovine liver mitochondria for the first time. Decarboxylation of alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, and alpha-ketoisocaproate was catalyzed by this multienzyme complex and this activity was co-purified for each substrate. Three enzymatic functions were contained in the complex including decarboxylation of the above ketoacids, transacylation of their simple acid derivatives, and reduction of NAD+ as an overall reaction. Product stoichiometry of these three reactions was 1 CO2:1 acyl-CoA:1 NADH. Activity depended upon the addition of thiamin pyrophosphate, CoASH, and NAD+ which were dissociable cofactors. Physically, two active forms of the enzyme complex were found: a 275,000-dalton unit and a 2 x 10(6)-dalton component. Both showed a characteristic flavin spectra and catalyzed all functions of the complex, implying that 10 small units aggregated into the larger unit. The soluble complex as visualized by electron microscopy had a diameter ranging from 12 to 24 nm corresponding to a molecular weight of 2 x 10(6). The size of the native membrane-bound component remains to be determined.     

Acetyl-CoA acetyltransferase from bovine liver mitochondria. Molecular properties of multiple forms.

Bovine liver mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA:acetyl-CoA C-acetyltransferase, EC 2.3.1.9) has been obtained in three forms designated transferase I, A and B on the basis of their elution positions from chromatography on phosphocellulose. All forms have been shown to have a molecular weight of about 152 000, each being composed of four similar subunits. Amino acid analysis of transferase A and B, the two major forms, revealed a close relationship between both forms with almost identical amino acid composition and arginine as N-terminal residue. The three transferases differ with respect to their redox state and their multiplicity of forms with isoelectric points of 6.9, 7.5 and 8.8, into which the transferases I and A were spontaneously transformed upon isoelectric focusing or rechromatography on phosphocellulose. Transferase B represents a stable enzyme form with an isoelectric point of 8.8. Although the redox state of transferase B can be adjusted to that of transferase A still a difference in charge and in the multiplicity of forms exists, thus indicating different protein states.     

Determination of the sequence of the aralkyl acyl-CoA:amino acid N-acyltransferase from bovine liver mitochondria

The aralkyl acyl-CoA:amino-acid N-acyl-transferase was previously purified to homogeneity from bovine liver mitochondria. The N-terminal amino-acid sequence and sequences obtained by cyanogen bromide cleavage of the enzyme were used to design oligonucleotide probes that were used to screen a bovine liver cDNA library. Several clones were isolated and sequenced, and the sequence is given. The cDNA contains 126 bases of 5′-untranslated region and 188 bp of 3′ untranslated region. The cDNA codes for an enzyme containing 295 amino-acid residues. The sequence gives a molecular weight for the enzyme of 39,229, which is larger than previously estimated. The amino-acid composition of the enzyme, based on this sequence, is in agreement with the previously obtained amino-acid analysis on the purified kidney enzyme. © 1997 John Wiley & Sons, Inc.     

Purification and biochemical characterization of hepatic ferredoxin (hepatoredoxin) from bovine liver mitochondria

Hepatic ferredoxin (hepatoredoxin) was purified from bovine liver mitochondria. The monomeric molecular mass of the hepatoredoxin was larger than that of adrenocortical ferredoxin (adrenodoxin) from bovine adrenocortical mitochondria at 14 kDa. We studied the amino acid residues and NH2-terminal sequence of this protein. The hepatoredoxin was organ-specific protein. The optical absorption spectrum of oxidized hepatoredoxin had two peaks, at 414 and 455 nm in the visible region. Hepatoredoxin formed an immunoprecipitin line against anti-adrenodoxin immunoglobulin in Ouchterlony double diffusion, and an immunochemical staining band in Western blotting.     

Interaction of salicylate and ibuprofen with the carboxylic acid: CoA ligases from bovine liver mitochondria

Neither salicylate nor ibuprofen was a substrate or inhibitor of the long-chain fatty acid: CoA ligase. In contrast, all three xenobiotic-metabolizing medium-chain fatty acid:CoA ligases (XL-I, XL-II, and XL-III) had activity toward salicylate. The K_m value for salicylate was similar for all three forms (2 to 3 μM), but XL-II and XL-III had higher activity at V_max. For ibuprofen, only XL-III catalyzed its activation, and it had a K_m for ibuprofen of 36 μM. Studies of salicylate inhibition of XL-I, XL-II, and XL-III revealed that it inhibited the benzoate activity of all three forms with K₁ values of ca. 2 μM, which is in agreement with the K_m values obtained with salicylate as substrate. Kinetic analysis revealed that salicylate conjugation by all three forms is characterized by substrate inhibition when salicylate exceeds ca. 20 μM. Substrate inhibition was more extensive with XL-I and XL-III. Previous work on the ligases employed assay concentrations of salicylate in the range of 0.1 to 1.0 mM, which are clearly inhibitory, particularly toward XL-I and XL-III. Thus, activity was not properly measured in previous studies, which accounts for the fact that salicylate conjugation was only found with one form, which is most likely XL-II since it has the highest V_max activity and shows the least amount of substrate inhibition. Studies with ibuprofen indicated that it inhibited XL-I, XL-II, and XL-III, with K₁ values being in the range of 75–125 μM. The short-chain ligase was inhibited by both salicylate and ibuprofen with K₁ values of 93 and 84 μM, respectively. It was concluded that pharmacological doses of salicylate, but not ibuprofen, will affect the metabolism of medium-chain fatty acids and carboxylic acid xenobiotics and that the previously described mitochondrial ibuprofen:CoA ligase activity is attributable to XL-III. © 1996 John Wiley & Sons, Inc.