Outer Membrane Of Rat Liver Mitochondria Research Articles

Amidoxime Reductase System Containing Cytochrome b5 Type B (CYB5B) and MOSC2 Is of Importance for Lipid Synthesis in Adipocyte Mitochondria

Reduction of hydroxylamines and amidoximes is important for drug activation and detoxification of aromatic and heterocyclic amines. Such a reductase system was previously found to be of high activity in adipose tissue and liver, and furthermore, in vitro studies using recombinant truncated and purified enzymes suggested the participation of cytochrome b(5) reductase (CYB5R), cytochrome b(5) (CYB5), and molybdenum cofactor sulfurase C-terminal containing 1 and 2 (MOSC1 and -2). Here, we show that purified rat liver outer mitochondrial membrane contains high amidoxime reductase activity and that MOSC2 is exclusively localized to these membranes. Moreover, using the same membrane fraction, we could show direct binding of a radiolabeled benzamidoxime substrate to MOSC2. Following differentiation of murine 3T3-L1 cells into mature adipocytes, the MOSC2 levels as well as the amidoxime reductase activity were increased, indicating that the enzyme is highly regulated under lipogenic conditions. siRNA-mediated down-regulation of MOSC2 and the mitochondrial form of cytochrome b(5) type B (CYB5B) significantly inhibited the reductase activity in the differentiated adipocytes, whereas down-regulation of MOSC1, cytochrome b(5) type A (CYB5A), CYB5R1, CYB5R2, or CYB5R3 had no effect. Down-regulation of MOSC2 caused impaired lipid synthesis. These results demonstrate for the first time the direct involvement of MOSC2 and CYB5B in the amidoxime reductase activity in an intact cell system. We postulate the presence of a novel reductive enzyme system of importance for lipid synthesis that is exclusively localized to the outer mitochondrial membrane and is composed of CYB5B, MOSC2, and a third unknown component (a CYB5B reductase).

Post-translational modifications of rat liver mitochondrial outer membrane proteins identified by mass spectrometry

The identification of post-translational modifications is difficult especially for hydrophobic membrane proteins. Here we present the identification of several types of protein modifications on membrane proteins isolated from mitochondrial outer membranes. We show, in vivo, that the mature rat liver mitochondrial carnitine palmitoyltransferase-I enzyme is N-terminally acetylated, phosphorylated on two threonine residues, and nitrated on two tyrosine residues. We show that long chain acyl-CoA synthetase 1 is acetylated at both the N-terminal end and at a lysine residue and tyrosine residues are found to be phosphorylated and nitrated. For the three voltage-dependent anion channel isoforms present in the mitochondria, the N-terminal regions of the protein were determined and sites of phosphorylation were identified. These novel findings raise questions about regulatory aspects of carnitine palmitoyltransferase-I, long chain acyl-CoA synthetase and voltage dependent anion channel and further studies should advance our understanding about regulation of mitochondrial fatty acid oxidation in general and these three proteins in specific.

Expression of outer mitochondrial membrane cytochrome b 5 in Escherichia coli. Purification of the recombinant protein and studies of its interaction with electron-transfer partners

In the present work, we report expression in Escherichia coli, purification, and characterization of recombinant full-length cytochrome b(5) from outer mitochondrial membrane. Optimization of expression conditions for cytochrome b(5) from outer mitochondrial membrane allowed reaching expression level up to 10(4) nmol of the hemeprotein per liter of culture. Recombinant cytochrome b(5) from outer mitochondrial membrane was purified from cell lysate by using metal-affinity chromatography. It has physicochemical, spectral, and immunochemical properties similar to those of cytochrome b(5) from rat liver outer mitochondrial membrane. Immobilized recombinant mitochondrial cytochrome b(5) was used as affinity ligand to study its interaction with electron transfer proteins. By using this approach, it is shown that in interaction of NADPH:cytochrome P450 reductase with both forms of cytochrome b(5) an important role is played by hydrophobic interactions between proteins, although the contribution of these interactions in complex formation with NADPH:cytochrome P450 reductase is different for isoforms of cytochrome b(5).

A targeted proteomic approach for the analysis of rat liver mitochondrial outer membrane proteins with extensive sequence coverage

Membrane proteins play an important role in cellular function. However, their analysis by mass spectrometry often is hindered by their hydrophobicity and/or low abundance. In this article, we present a method for the mass spectrometric analysis of membrane proteins based on the isolation of the resident membranes, isolation of the proteins by gel electrophoresis, and electroelution followed by enzymatic digestion by both trypsin and proteinase K. With this method, we have achieved 82–99% sequence coverage for the membrane proteins carnitine palmitoyltransferase-I (CPT-I), long-chain acyl-CoA synthetase (LCAS), and voltage-dependent anion channel (VDAC), isolated from rat liver mitochondrial outer membranes, including the transmembrane domains of these integral membrane proteins. This high sequence coverage allowed the identification of the isoforms of the proteins under study. This methodology provides a targeted approach for examining membrane proteins in detail.

Mitochondrial and Microsomal Ferric b5 Cytochromes Exhibit Divergent Conformational Plasticity in the Context of a Common Fold

Native-state hydrogen-deuterium exchange (HDX) monitored by NMR spectroscopy has been used to compare conformational plasticity in ferric rat liver outer mitochondrial membrane cytochrome b5 (rOM b5) and ferric bovine liver microsomal cytochrome b5 (bMc b5). Analysis of the data indicated that rOM b5 is the less conformationally flexible protein on the time scale probed by the HDX experiments. The data also suggest a likely contributor to the much higher kinetic barrier for the release of hemin from OM b5s in comparison to Mc b5s, a characteristic that may be to a large extent the source of their divergent functional properties. Specifically, the data indicate that conformational mobility within helices alpha4 and alpha5, which flank the loop harboring axial ligand His63, is considerably more restricted in rOM b5 than in bMc b5. The lower conformational flexibility of alpha4 and alpha5 in rOM b5 can reasonably be attributed to more extensive hydrophobic packing in that region of the protein, arising from two conserved side chain packing motifs in OM cytochrome b5s [Altuve, A., Wang, L., Benson, D. R., and Rivera, M. (2004) Biochem. Biophys. Res. Commun. 314, 602-609].

Modulation of redox potential in electron transfer proteins: effects of complex formation on the active site microenvironment of cytochrome b5.

The reduction potential of cytochrome b5 is modulated via the formation of a complex with polylysine at the electrode surface (Rivera et al., Biochemistry, 1998, 37, 1485). This modulation is thought to originate from the neutralization of a solvent exposed heme propionate and from dehydration of the complex interface. Although direct evidence demonstrating that neutralization of the charge on the heme propionate contributes to the modulation of the redox potential of cytochrome b5 has been obtained, evidence demonstrating that water exclusion from the complex interface plays a similar role has not been conclusive. Herein we report the preparation of the V45I/V61I double mutant of rat liver outer mitochondrial membrane (OM) cytochrome b5. This mutant has been engineered with the aim of restricting water accessibility to the exposed heme edge of cytochrome b5. The X-ray crystal structure of the V45I/V61I mutant revealed that the side chain of Ile at positions 45 and 61 restricts water accessibility to the interior of the heme cavity and protects a large section of the heme edge from the aqueous environment. Electrochemical studies performed with the V45I/V61I mutant of cytochrome b5, and with a derivative in which the heme propionates have been converted into the corresponding dimethyl ester groups, clearly demonstrate that dehydration of the heme edge contributes to the modulation of the reduction potential of cytochrome b5. In fact, these studies showed that exclusion of water from the complex interface exerts an effect (approximately 40 mV shift) that is comparable, if not larger, than the one originating from neutralization of the charge on the solvent exposed heme propionate (approximately 30 mV shift).

Phosphatidic Acid Synthesis in Mitochondria: TOPOGRAPHY OF FORMATION AND TRANSMEMBRANE MIGRATION

The topography of formation and migration of phosphatidic acid (PA) in the transverse plane of rat liver mitochondrial outer membrane (MOM) were investigated. Isolated mitochondria and microsomes, incubated with sn-glycerol 3-phosphate and an immobilized substrate palmitoyl-CoA-agarose, synthesized both lyso-PA and PA. The mitochondrial and microsomal acylation of glycerophosphate with palmitoyl-CoA-agarose was 80-100% of the values obtained in the presence of free palmitoyl-CoA. In another series of experiments, both free polymyxin B and polymyxin B-agarose stimulated mitochondrial glycerophosphate acyltransferase activity approximately 2-fold. When PA loaded mitochondria were treated with liver fatty acid binding protein, a fifth of the phospholipid left the mitochondria. The amount of exportable PA reduced with the increase in the time of incubation. In another approach, PA-loaded mitochondria were treated with phospholipase A(2). The amount of phospholipase A(2)-sensitive PA reduced when the incubation time was increased. Taken together, the results suggest that lysophosphatidic acid (LPA) and PA are synthesized on the outer surface of the MOM and that PA moves to the inner membrane presumably for cardiolipin formation.

Complete isomer-specific 1H and 13C NMR assignments of the heme resonances of rat liver outer mitochondrial membrane cytochrome b5.

Singly and doubly labeled delta-aminolevulinic acid derivatives were used to prepare rat liver outer mitochondrial membrane (OM) cytochrome b5 containing a 13C-labeled heme active site. A variety of NMR experiments, including HMBC and INADEQUATE in conjunction with the more commonly used HMQC, NOESY, and COSY, were conducted to make unambiguous assignments of protonated carbons and the quaternary pyrrole-alpha and -beta carbons in both isomeric forms of the paramagnetic active center of OM cytochrome b5. Because the long interpulse delays in the HMBC experiment have a detrimental effect on the detectability of fast relaxing paramagnetically affected resonances. INADEQUATE is proposed as the experiment of choice for assigning quaternary carbons in paramagnetic hemes with carefully chosen macrocycle labeling patterns. Furthermore, the applicability of the INADEQUATE experiment to paramagnetic heme active sites should be facilitated greatly by the availability of biosynthetic methods for producing isotopically labeled b-hemes and, more recently, isotopically labeled c-hemes.

Electrochemical measurement of second-order electron transfer rate constants for the reaction between cytochrome b5 and cytochrome c.

The second-order electron transfer reaction between cytochrome b5 and cytochrome c has been studied by cyclic voltammetry utilizing a gold electrode modified with beta-mercaptopropionate. When cyclic voltammetry is performed on a solution containing a mixture of cytochrome b5, cytochrome c and polylysine, cytochrome b5 undergoes reversible electrochemistry at the electrode surface while cytochrome c discriminates against the electrode surface. The selectivity of the modified electrode for negatively charged proteins makes it possible to selectively reduce a protein possessing a net negative charge and a relatively low reduction potential (outer mitochondrial membrane cytochrome b5, Eo = -102 mV; microsomal cytochrome b5, Eo = 3 mV) in the presence of another protein possessing a net positive charge and a relatively high reduction potential (cytochrome c, Eo = 265 mV). The electrochemical reduction of ferricytochrome b5 at the electrode surface is followed by a second-order electron transfer reaction between ferrocytochrome b5 and ferricytochrome c that yields ferricytochrome b5 and ferrocytochrome c. This fast homogeneous electron transfer reaction which is preceded by a heterogeneous electron transfer reaction results in a characteristic cyclic voltammogram containing a pre-peak to the reduction current. The second-order rate constant for the homogeneous reaction was obtained by invoking the above reaction scheme for digital simulation of a cyclic voltammogram which was subsequently fitted to the experimental data. Second-order rate constants obtained with this method are 2.9 x 10(8) and 8.9 x 10(8) for the homogeneous electron transfer reactions between rat liver outer mitochondrial membrane (OM) ferrocytochrome b5 and beef liver microsomal ferrocytochrome b5, with horse heart ferricytochrome c, respectively. These values are in good agreement with second-order rate constants obtained for the same protein systems by flash photolysis. [Meyer, T. E., Rivera, M., Walker, F. A., Mauk, M. R., Mauk, A. G., Cusanovich, M. A., & Tollin, G. (1993) Biochemistry 32, 622-627].

Biosynthetic Preparation of Isotopically Labeled Heme

An efficient method for the preparation of isotopically enriched heme has been developed. This method utilizes a commercially available bacterial host and plasmid, into which a synthetic gene encoding for rat liver outer mitochondrial membrane cytochrome b 5 a heme-binding protein, has been inserted. The method described in this report utilizes the efficient synthesis of the cytochrome b 5 polypeptide together with the enhanced biosynthesis of heme brought about by addition of the first committed precursor in heme biosynthesis, δ-aminolevulinic acid. Apocytochrome b 5 sequesters heme as the macrocycle is being synthesized in order to form holocytochrome b 5, thus avoiding toxic concentrations of free macrocycle in the cell. Relatively high concentrations of free heme in the cell have been shown to stimulate excretion of heme precursors such as coproporphyrinogen and uroporphyrinogen (W. F. Harris III, R. S. Burkhalter, W. Lin and R. Timkovich, (1993) Bioorg. Chem. 21, 209-220), therefore causing isotopic dilution of the labeled material. The heme obtained using this methodology was determined to be >85% enriched. Because the heme in cytochrome b 5 is not covalently attached to the polypeptide, it can be extracted and used in other applications. Use of glutamate, a precursor of δ-aminolevulinate biosynthesis in Escherichia coli, did not result in high levels of isotopic incorporation into heme, thus pointing out to the importance of using a labeled precursor that is committed to heme biosynthesis in order to obtain high levels of isotopic labeling.

Substrate specificity of rat liver mitochondrial carnitine palmitoyl transferase I: evidence against α-oxidation of phytanic acid in rat liver mitochondria

The two branched chain fatty acids pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) and phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) were converted to co-enzyme A thioesters by rat liver mitochondrial outer membranes. However, these branched chain fatty acids could not be converted to pristanoyl and phytanoyl carnitines, respectively, by mitochondrial outer membranes. As expected, the unbranched long chain fatty acids, stearic acid and palmitic acid, were rapidly converted to stearoyl and palmitoyl carnitines, respectively, by mitochondrial outer membranes. These observations indicate that the branched chain fatty acids could not be transported into mitochondria. The data presented strongly suggest that in rat liver, α-oxidation of phytanic acid occurs in organelles other than mitochondria.

Mature carnitine palmitoyltransferase I retains the N-terminus of the nascent protein in rat liver

Carnitine palmitoyltransferase I was isolated from octylglucoside extracts of rat liver mitochondrial outer membranes. This native enzyme was digested proteolytically with V8 protease. Five major peptides were obtained all of which were found in the amino acid sequence predicted from the full-length cDNA sequence of the protein. One peptide was found to correspond to the extreme N-terminus of the deduced amino acid sequence. Therefore, the mature protein retains the N-terminus of the nascent protein after import into the mitochondrial membrane. Knowledge of the identity of the N-terminus of the mature protein allows a reappraisal of the role of the two main, N-terminal hydrophobic domains of the protein and of the possible topology of the protein within the membrane.

One-step synthesis of radioactive acyl-CoA and acylcarnitines using rat liver mitochondrial outer membrane as enzyme source.

Rat liver mitochondrial outer membrane enriched preparations have proven to be a convenient enzyme source for synthesizing coenzyme A (CoA) and carnitine esters of radioactive fatty acids. These membranes are simple to isolate and they retain acyl-CoA ligase and carnitine palmitoyltransferase activities well upon storage. Enzyme purification is not required. A novel aspect of the present procedure is that the same enzymatic incubation step allows both the acyl-CoA and the acylcarnitine esters to be obtained simultaneously when carnitine is present, but produces acyl-CoA ester only when carnitine is not included. Under the conditions described, the conversion of [1-14C]octanoic acid to the respective esters was about 95%; the corresponding figure for [1-14C]palmitic acids was over 70%. The procedure seems suitable for synthesizing the labeled CoA and carnitine esters from a variety of radioactive fatty acids.

Development and characterization of a polyclonal antibody against rat liver mitochondrial overt carnitine palmitoyltransferase (CPT I). Distinction of CPT I from CPT II and of isoforms of CPT I in different tissues

The [3H]tetradecylglycidyl-CoA (TDG-CoA)-binding protein (Mr approx. 88,000) of purified outer membranes from rat liver mitochondria was identified by SDS/PAGE. The region in which it migrated was shown to contain another protein which stained strongly with periodic acid-Schiff reagent and could be removed from membrane extracts by incubation with Sepharose-concanavalin A. Amounts of TDG-CoA-binding protein were prepared from lectin-treated extracts using preparative SDS/PAGE and used to raise a polyclonal antibody in a sheep. The IgG fraction purified from this anti-serum reacted strongly with a protein of Mr approximately 88,000 on Western blots, and much more weakly with two other proteins of Mr approximately 76,000 and Mr approximately 53,000 in extracts of rat liver mitochondrial outer membranes. The crude IgG fraction and immunopurified IgG both removed carnitine palmitoyltransferase (CPT) I activity from very pure outer membrane extracts, suggesting that the TDG-CoA-binding protein against which the antiserum was raised also expresses CPT I activity. This was confirmed by the demonstration of a strong positive correlation between CPT I activity and the amount of immunoreactive protein of Mr approximately 88,000 in mitochondria prepared from rats in different physiological states. By contrast, the antibody did not react with CPT II either in mitochondria or in purified form. Similarly, an anti-(CPT II) antibody did not cross-react with CPT I on Western blots, proving conclusively that CPT I and CPT II are immunologically distinct proteins, as well as being of different functional molecular sizes [Zammit, Corstophine & Kelliher (1988) Biochem. J. 250, 415-420]. Immunoblots of mitochondrial proteins obtained from different tissues indicated that, of the rat tissues tested, only kidney cortex mitochondria contain the same isoform of CPT I as that in liver. Heart, skeletal muscle and brown adipose tissue mitochondria contain a slightly smaller isoform which was only weakly reactive with anti-(rat liver CPT I) antibody, indicating that these tissues contain a molecularly quite distinct isoenzyme. This would explain the previous observations that CPT I in these tissues has markedly different kinetic characteristics from the isoenzyme present in liver mitochondria.

One-step facile synthesis of radioactive acyl-coA and acylcarnitines using rat liver outer mitochondrial membrane as the enzyme source

Conference Article| April 01 1991 One-step facile synthesis of radioactive acyl-coA and acylcarnitines using rat liver outer mitochondrial membrane as the enzyme source A.K.M.J. BHUIYAN; A.K.M.J. BHUIYAN 1Laboratory of Intermediary Metabolism, Clin. Res. Inst. of Montreal, Montreal, Quebec H2W 1R7, Canada Search for other works by this author on: This Site PubMed Google Scholar S.V. PANDE S.V. PANDE 1Laboratory of Intermediary Metabolism, Clin. Res. Inst. of Montreal, Montreal, Quebec H2W 1R7, Canada Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1991) 19 (2): 174S. https://doi.org/10.1042/bst019174s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation A.K.M.J. BHUIYAN, S.V. PANDE; One-step facile synthesis of radioactive acyl-coA and acylcarnitines using rat liver outer mitochondrial membrane as the enzyme source. Biochem Soc Trans 1 April 1991; 19 (2): 174S. doi: https://doi.org/10.1042/bst019174s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1991 Biochemical Society1991 Article PDF first page preview Close Modal You do not currently have access to this content.

Carnitine palmitoyltransferase (CPT 2) from liver mitochondrial inner membrane becomes inhibitable by malonyl-CoA if reconstituted with outer membrane malonyl-CoA binding protein

A soluble extract was obtained on treatment of rat liver mitochondrial outer membranes with cholate which bound ( 14C]malonyl-CoA but was essentially free of carnitine palmitoyltransferase (CPT) activity. Extraction of mitochondrial inner membranes with cholate readily solubilized a CPT activity which was insensitive to malonyl-CoA. Combination of these two extracts caused the CPT derived from inner membranes to become inhibitable by malonyl-CoA.

Soluble and particulate forms of rat catechol-O-methyltransferase distinguished by gel electrophoresis and immune fixation.

Catechol-O-methyltransferase (COMT) was visualized in homogenates and subcellular fractions of rat tissues, including liver and brain, by gel electrophoresis, electrophoretic transfer of proteins to nitrocellulose (Western blotting), and immune fixation with antiserum to highly purified soluble rat liver COMT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of all tissue homogenates examined revealed three major immune-specific proteins with apparent molecular weights 23,000, 26,000, and 66,000 (23K, 26K and 66K). Centrifugation of homogenates at 100,000 X g for 60 min resulted in the enrichment of the 26K species protein in the pellet whereas the 23K and 66K proteins were the predominant forms in the supernatant. The 66K protein appeared in variable amounts depending on the tissue being examined and the length of transfer of protein and is assumed to be an "aggregate" of the smaller form(s). The 26K protein was essentially the only immunoreactive species seen in a purified preparation of rat liver outer mitochondrial membrane. Isoelectric focusing (IEF) under denaturing conditions and two-dimensional gel electrophoresis of brain and liver fractions showed that the 23K protein was resolved into three bands of pI 5.1, 5.2, and 5.3, whereas the 26K protein had a pI of 6.2. Analysis of COMT activity in slices from nondenaturing IEF gels indicated that the pI 5.1-5.3 species are biologically active; the pI 6.2 species could not be detected under these conditions. COMT activity was demonstrated, however, in outer mitochondrial membranes from rat liver, which contain predominantly the 26K, pI 6.2 immunoreactive species. The major form of COMT in all rat tissues examined is "soluble" with an apparent Mr of 23K and a pI of 5.2. The nature of the modifications giving rise to pI 5.1 and 5.3 forms of this enzyme are not clear, nor is the relationship between the 23K and 26K forms. Further studies are needed to elucidate the relationship of immunoreactive forms of COMT to each other, their intracellular location, and their functional significance.

Degradation of proteins in rat liver mitochondrial outer membrane transplanted into different cell types. Evidence for alternative processing.

The degradation of proteins in reductively [3H]methylated mitochondrial outer membrane (MOM) transplanted into cells by a poly(ethylene glycol)-mediated process has been studied. The average rate of degradation (t1/2 24-28 h) of MOM proteins transplanted into HTC cells was not the same as for endogenous MOM proteins (t1/2 56 h), mitoplast proteins (t1/2 120 h), plasma membrane proteins (t1/2 approx. 90 h) or cytosol proteins (t1/2 75 h). The degradation of transplanted MOM proteins was inhibited to the same extent (30-45%) as that of endogenous mitochondrial and plasma membrane proteins by leupeptin and NH4Cl. No inhibition of HTC cell cytosol protein degradation by NH4Cl was observed. NH4Cl differentially inhibited the degradation of endogenous MOM and mitoplast protein subunits as shown after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Proteins in MOM transplanted into tissue culture cells were degraded either with t1/2 24-28 h (MRC-5, B82 and A549 cells) or with t1/2 55-70 h (CHO-K1 and 3T3-L1 cells) similar to that of proteins in MOM transplanted into rat hepatocytes [Evans & Mayer (1983) Biochem. J. 216, 151-161]. The data suggest that membrane protein destruction is but the end part of a fundamental intracellular membrane recognition process.

Selective effects on catalysis by the multiple forms of monoamine oxidase produced by interactions of acidic phospholipids with mitochondrial membranes.

The effect of acidic phospholipids on the A and B multiple forms of membrane-bound mitochondrial monoamine oxidase has been investigated by incubating liposomes with isolated rat liver mitochondrial outer membrane preparations at lipid:protein ratios of 0.01 to 1. A strong inhibition of monoamine oxidase B was observed with phosphatidylserine and a moderate activation of monoamine oxidase A with phosphatidylinositol, while cardiolipin had no significant effect on either form. The specificity of phosphatidylserine inhibition for monoamine oxidase B was also confirmed in mitochondrial outer membrane isolated from tissues containing exclusively the A or B form of the enzyme (human placenta and bovine liver). Levels of incorporation were comparable for all the phospholipids and tissues studied and could not account for the different effects observed. Inhibition of monoamine oxidase B was found to be similar in an intact mitochondria preparation to that observed in the isolated outer membrane. A recent report of activation of both monoamine oxidase forms in delipidated whole mitochondria by the acidic phospholipids was re-examined and found to involve release of monoamine oxidase from the mitochondria. The details of the effects of phosphatidylserine and phosphatidylinositol on membrane-bound monoamine oxidase are consistent with the concept of the multiple forms as two distinct peptides, and suggest a second possible mode of in vivo regulation of substrate specificity.

In vitro synthesis of monoamine oxidase of rat liver outer mitochondrial membrane

Monoamine oxidase, an intrinsic protein of outer mitochondrial membrane, was purified from bovine liver and rabbit antibody against the enzyme was prepared. The antibody could react with the monoamine oxidase of rat liver mitochondria. When rat liver RNA was translated in vitro using rabbit reticulocyte lysate and monoamine oxidase peptide in the translation products was immunoprecipitated by the antibody, the peptide was detected in the products programmed by the messenger RNA's from total and free polysomes but not that from bound polysomes. The enzyme synthesized in vitro had the same apparent molecular size as the mature protein in outer mitochondrial membrane.