Characterization of Rat Testes Mitochondrial Retinoylating System and Its Partial Purification

Retinoylation (retinoic acid acylation), a posttranslational modification of proteins occurring in a variety of eukariotic cell lines both in vivo and in vitro, was studied in rat testes mitochondria. all-trans-Retinoic acid, a highly active form of vitamin A in inducing cellular differentiation, is incorporated covalently into proteins of rat testes mitochondria. The maximum retinoylation activity of rat testes mitochondrial proteins was 21.6 pmoles mg protein(-1) 90 min(-1) at 37 degrees C. The activation energy was 44 kJ mol(-1) from 5 to 37 degrees C. The retinoylation activity had a pH optimum of 7.5. The retinoylation process was specific for the presence of ATP, ADP, and GTP (even if only 30% of the control). The half saturation constant (Km) was 0.69 microM for all-trans-retinoic acid, while the inhibition constant (Ki) was 1.5 microM for 13-cis-retinoic acid. Retinoylation was not inhibited by high concentrations of myristic acid (MA) and palmitic acid (PA), indicating that retinoylation and acylation reactions involved different rat testes mitochondrial proteins. The ATP or CoASH saturation curves of retinoylation reaction showed sigmoidal behavior with apparent half saturation constants (K0.5) of 6.5 mM ATP and 40.6 microM CoASH. On SDS-gel electrophoresis, the hydroxylapaptite/celite eluate showed various protein bands between 25 and 80 kDa. This retinoylated protein was purified 17-fold with respect to the mitochondrial extract.     

Influence of all-trans-retinoic acid on oxoglutarate carrier via retinoylation reaction

All-trans-retinoic acid (atRA), an activated metabolite of vitamin A, is incorporated covalently into proteins both invivo and invitro. AtRA reduced the transport activity of the oxoglutarate carrier (OGC) isolated from testes mitochondria to 58% of control via retinoylation reaction. Labeling of testes mitochondrial proteins with (3)HatRA demonstrated the binding of atRA to a 31.5 KDa protein. This protein was identified as OGC due to the competition for the labeling reaction with 2-oxoglutarate, the specific OGC substrate. The role of retinoylated proteins is currently being explored and here we have the first evidence that retinoic acids bind directly to OGC and inhibit its activity in rat testes mitochondria via retinoylation reaction. This study indicates the evidence of a specific interaction between atRA and OGC and establishes a novel mechanism for atRA action, which could influence the physiological biosynthesis of testosterone in situations such as retinoic acid treatment.     

Influence of all-trans-retinoic acid on oxoglutarate carrier via retinoylation reaction

All-trans-retinoic acid (atRA), an activated metabolite of vitamin A, is incorporated covalently into proteins both invivo and invitro. AtRA reduced the transport activity of the oxoglutarate carrier (OGC) isolated from testes mitochondria to 58% of control via retinoylation reaction. Labeling of testes mitochondrial proteins with ³HatRA demonstrated the binding of atRA to a 31.5 KDa protein. This protein was identified as OGC due to the competition for the labeling reaction with 2-oxoglutarate, the specific OGC substrate. The role of retinoylated proteins is currently being explored and here we have the first evidence that retinoic acids bind directly to OGC and inhibit its activity in rat testes mitochondria via retinoylation reaction. This study indicates the evidence of a specific interaction between atRA and OGC and establishes a novel mechanism for atRA action, which could influence the physiological biosynthesis of testosterone in situations such as retinoic acid treatment.     

Dietary fatty acid composition differently influences retinoylation reaction in rat testes mitochondria

All-trans-retinoic acid (atRA) is incorporated covalently into proteins of rat testes mitochondria. In this study, the effect of three diets with different fatty acid composition on the retinoylation of proteins of rat testes mitochondria has been investigated. Different groups of rats were fed on a basal diet supplemented with 15% of either coconut oil (CO), olive oil (OO) or fish oil (FO). We found that, when compared with CO, the binding of retinoic acid was decreased in FO- and OO-fed rats. Mitochondrial phospholipids composition was differently influenced by dietary treatments; minor changes were observed in fatty acid composition of phospholipids. Few differences were observed in the Arrhenius plots among the three groups of rats. Kinetic analysis revealed a decrease in the V _max value in FO- and OO- as compared with CO-fed rats. No difference among the three groups were observed in the K _M value. The retinoylation reaction was inhibited by 13-cis-RA and 9-cis-RA.     

Formation of retinoylated proteins from retinoyl-CoA in rat tissues

Retinoylation (acylation of proteins by retinoic acid) is considered as one mechanism of retinoic acid (RA) action occurring in cells in vitro and in vivo. Previously, our studies showed that in rat tissues the formation of retinoyl-CoA from RA, the first step of retinoylation, required ATP, CoA and MgCl(2). In the current study, we examined whether the transfer of retinoyl-CoA into proteins, the second step of retinoylation, occurs in rat tissues. [(3)H]-Labeled-retinoyl-CoA bound covalently to proteins in rat liver, kidney, testis, and brain. The levels of incorporation of retinoyl-CoA into proteins were higher in vitamin A-deficient rats than in normal ones. The formation of retinoylated proteins depended on the incubation time, and the concentrations of retinoyl-CoA and homogenate. The reaction was suppressed by fatty acyl-CoAs and palmitic acid, but not by arachidonic acid. The Vmax and Km values for retinoyl-CoA in the formation of retinoylated proteins using a crude liver extract were estimated to be 2,597.3 pmol/min/mg protein and 9.5 x 10(-5) M, respectively. Retinoylated proteins formed from retinoyl-CoA, including a 17 kDa protein exhibiting high radioactivity, disappeared in the presence of 2-mercaptoethanol, indicating that RA was linked to the proteins through a thioester bond. These results demonstrate that retinoylation in rat tissues occurs via retinoyl-CoA formed from RA. This process may play a significant physiological role in cells.     

Differential binding to soluble nuclear receptors and effects on cell viability of retinol and retinoic acid in cultured retinoblastoma cells

Human retinoblastoma cells in culture contain soluble receptors for both ³H-retinol and ³H-retinoic acid which are separate and distinct as assessed by specificity, enzyme susceptibility and binding affinity. Total cellular binding of retinol correlates well with its rapid effect on cell mortality. A limited number of soluble nuclear receptor sites for ³H-retinoic acid but not ³H-retinol are observed after incubation of the ³H-retinoid with intact cells indicating a possible preferential effect of retinoic acid at the gene level.     

Retinoic acid-promoted expansion of total cellular ATP pools in 3T3 cells can mediate its stimulatory and growth inhibitory effects

Exposure of Swiss 3T3 cells to micromolar quantities of β-all-trans-retinoic acid (RA) results in either inhibition of growth or stimulation of cellular responsiveness to mitogens, depending on the length of treatment. Inhibition of growth is produced by treatment of the cells with RA for at least 48 hours. The total cellular pools of adenosine 5′-triphosphate (ATP) are markedly increased after 48-hour RA treatment and dose dependence studies show a correlation between the expanded ATP pools and the inhibitory effects. The expansion of total cellular ATP pools by retinoic acid occurs throughout the cell cycle and parallels the cell cycle-dependent fluctuations in total cellular ATP pools of untreated cells. Studies of [³H]thymidine incorporation and labeling indices in intact cells and [³H]dTTP incorporation and labeling indices in isolated nuclei of RA-treated and control cultures suggest that cellular acid-soluble nucleotide pools mediate the inhibition of DNA replication in the 48-hour-RA-treated cells. The stimulatory activity of RA for mitogenic responsiveness is demonstrated by treatment of G₀/G₁-arrested 3T3 cells with micromolar levels of RA for a maximum of 18 hours resulting in the potentiation of phorbol myristate acetate (PMA)-stimulated transition into S phase of the cell cycle. Marked increases in total cellular ATP and UTP pools are produced by 18-hour treatment of G₀/G₁-arrested cells with RA, before their exposure to PMA.     

Retinoylation of the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases in HL60 cells.

Retinoylation (retinoic acid acylation) is a post-translational modification of proteins occurring in a variety of eukaryotic cell lines. There are at least 20 retinoylated proteins in the human myeloid leukemia cell line HL60 (N. Takahashi and T.R. Breitman (1990) J. Biol. Chem. 265, 19, 158-19, 162). Here we found that some retinoylated proteins may be cAMP-binding proteins. Five proteins, covalently labeled by 8-azido-[32P]cAMP which specifically reacts with the regulatory subunits of cAMP-dependent protein kinase, comigrated on two-dimensional polyacrylamide gel electrophoresis with retinoylated proteins of Mr 37,000 (p37RA), 47,000 (p47RA), and 51,000 (p51RA) labeled by [3H]retinoic acid treatment of intact cells. Furthermore, p47RA coeluted on Mono Q anion exchange chromatography with the type I cAMP-dependent protein kinase holoenzyme and p51RA coeluted on Mono Q anion exchange chromatography with the type II cAMP-dependent protein kinase holoenzyme. An antiserum specific to RI, the cAMP-binding regulatory subunit of type I cAMP-dependent protein kinase, immunoprecipitated p47RA. An antiserum specific to RII, the cAMP-binding regulatory subunit of type II cAMP-dependent protein kinase, immunoprecipitated p51RA. These results indicate that both the RI and the RII regulatory subunits of cAMP-dependent protein kinase are retinoylated. Thus, an early event in RA-induced differentiation of HL60 cells may be the retinoylation of subpopulations of both RI and RII.     

Covalent binding of perfluorinated fatty acids to proteins in the plasma, liver and testes of rats.

Perfluorinated fatty acids alter hepatic lipid metabolism and are potent peroxisome proliferators in rodents. Two such perfluorinated acids, perfluorodecanoic acid (PFDA) and perfluorooctanoic acid (PFOA), were examined to determine if they covalently bind cellular proteins. PFDA and PFOA were found to covalently bind proteins when administered to rats in vivo. The liver, plasma and testes of male rats treated with [1-14C]PFDA or PFOA (9.4 mumol/kg) contained detectable levels of covalently bound 14C (0.1-0.5% of the tissue 14C content). Characterization of PFDA covalent binding to albumin in vitro showed that cysteine significantly decreased binding with no effect of methionine, suggesting protein sulfhydryl groups are involved. In cytosolic and microsomal incubation there was no effect of the addition of CoA, ATP or NADPH on the magnitude of the covalent binding of PFDA. Therefore PFDA need not be metabolically activated to form covalent adducts. Despite demonstration of covalent binding of PFDA and PFOA to proteins both in vivo and in vitro, the role of this macromolecular binding in perfluorinated fatty acid toxicity is not known.     

Affinity purification of retinoic acid-binding proteins using immobilized 4-(2-hydroxyethoxy)retinoic acid

An affinity chromatographic matrix that purifies cellular retinoic acid-binding protein to near homogeneity from rat testes cytosol has been developed. The three-step procedure includes an acid precipitation, a batch treatment with CM Bio-Gel, and affinity chromatography on 4-(2-hydroxyethoxy)retinoic acid coupled to epoxy-activated Sepharose 6B. The binding protein was purified approximately 8500-fold based on total soluble testicular protein and with a recovery in excess of 80%. In addition, further enhancement of the purity of the protein can be attained by size-exclusion HPLC to increase purification to 21,000-fold. The recovered protein has an apparent M(r) 14,300 as determined by size-exclusion HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein is isolated in the apo-form and retains its ability to bind retinoic acid as evidenced by the binding of [3H]retinoic acid. An apparent retinoic acid-binding protein of M(r) 18,000 has also been isolated from rat testes nuclei by the affinity chromatography step. The affinity phase has been used for 6 months without any detectable loss in its ability to purify cellular retinoic acid-binding protein.     

Fatty acyl coenzyme A-sensitive adenine nucleotide transport in a reconstituted liposome system

The adenine nucleotide translocase was purified from bovine heart mitochondria and incorporated into membranes of phospholipid liposomes. The rate of transport of the adenine nucleotides was competitively inhibited by oleoyl coenzyme A with an approximate Ki of 1.0 microM. Significant inhibition was limited to those fatty acyl coenzyme A esters which are carnitine dependent for their oxidation in isolated mitochondria. Octanoyl coenzyme A was almost completely inactive as was palmitic acid and palmitoyl carnitine. By comparing the inhibitory characteristics of carboxyatractylate and bongkrekic acid with those of oleoyl-CoA, it was determined that the fatty acyl-CoA esters could produce inhibition whether the carrier was inserted into the liposome in either the conventional (65%) or reverse (30%) orientation. The results demonstrate that the interaction of long chain fatty acyl-CoA esters with the ADP/ATP carrier in a purified reconstituted system mimics their effects with isolated mitochondria and inverted submitochondrial particles. In general, these findings are consistent with the role of acyl-CoA esters acting as natural ligands and biological effectors of the translocator.     

Enzymes and binding proteins affecting retinoic acid concentrations

Free retinoids suffer promiscuous metabolism in vitro. Diverse enzymes are expressed in several subcellular fractions that are capable of converting free retinol (retinol not sequestered with specific binding proteins) into retinal or retinoic acid. If this were to occur in vivo, regulating the temporal-spatial concentrations of functionally-active retinoids, such as RA (retinoic acid), would be enigmatic. In vivo, however, retinoids occur bound to high-affinity, high-specificity binding proteins, including cellular retinol-binding protein, type I (CRBP) and cellular retinoic acid-binding protein, type I (CRABP). These binding proteins, members of the superfamily of lipid binding proteins, are expressed in concentrations that exceed those of their ligands. Considerable data favor a model pathway of RA biosynthesis and metabolism consisting of enzymes that recognize CRBP (apo and holo) and holo-CRABP as substrates and/or affecters of activity. This would restrict retinoid access to enzymes that recognize the appropriate binding protein, imparting specificity to RA homeostasis; preventing, e.g. opportunistic RA synthesis by alcohol dehydrogenases with broad substrate tolerances. An NADP-dependent microsomal retinol dehydrogenase (RDH) catalyzes the first reaction in this pathway. RDH recognizes CRBP as substrate by the dual criteria of enzyme kinetics and chemical crosslinking. A cDNA of RDH has been cloned, expressed and characterized as a short-chain alchol dehydrogenase. Retinal generated in microsomes from holo-CRBP by RDH supports cytosolic RA synthesis by an NAD-dependent retinal dehydrogenase (RalDH). RalDH has been purified, characterized with respect to substrate specificity, and its cDNA has been cloned. CRABP is also important to modulating the steady-state concentrations of RA, through sequestering RA and facilitating its metabolism, because the complex CRABP/RA acts as a low K_m substrate.     

Homozygous deletion of the CRABPI gene in AB1 embryonic stem cells results in increased CRABPII gene expression and decreased intracellular retinoic acid concentration

The cellular retinoic acid (RA) binding proteins I and II (CRABPI and CRABPII), intracellular proteins which bind retinoic acid with high affinity, are involved in the actions of RA, though their exact roles are not fully understood. We have generated several genetically engineered AB1 cell lines in which both alleles of the CRABPI gene have been deleted by homologous recombination. We have used these CRABPI knockout cell lines to examine the consequences of functional loss of CRABPI on RA-induced gene expression and RA metabolism in the murine embryonic stem cell line, AB1, which undergoes differentiation in response to RA. Complete lack of CRABPI results in decreased intracellular [3H]RA concentrations under conditions in which external concentrations of [3H]RA are low (1-10nM) and in an altered distribution of [3H] polar metabolites of [3H]RA in the cell and in the medium. Fewer [3H] polar metabolites are retained within the CRABPI(-/-) cells compared to the wild-type cells. These data suggest that CRABPI functions to regulate the intracellular concentrations of retinoic acid and to maintain high levels of oxidized retinoic acid metabolites such as 4-oxoretinoic acid within cells.     

Evidence for an in vivo modification of mitochondrial proteins by coenzyme A.

Following denaturation of mitochondrial proteins by sodium dodecyl sulfate, a [1-14C]pantothenic acid-derived radioactivity proved to be acid precipitable in the outer membrane, the intermembrane space, the inner membrane and in the matrix of rat liver mitochondria, where it had the highest specific radioactivity of 541 +/- 29 cpm/100 micrograms protein. This tightly and/or covalently bound protein radioactivity could be released by incubation in the presence of dithioerythreitol; it was identified as [14C]coenzyme A by its HPLC retention time, its absorption spectrum and its radioactivity. This acid-stable and thiol-labile coenzyme A-binding apparently refers to specific protein binding sites. With the purified, homogeneous mitochondrial matrix enzymes acetyl-CoA acetyltransferase (acetoacetyl-CoA thiolase) (EC 2.3.1.9, acetyl-CoA:acetyl-CoA C-acetyltransferase) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16) coenzyme A was found exclusively, e.g., in the modified, partially-active forms A1 und A2 of acetyl-CoA acetyltransferase and not in the unmodified fully-active enzyme. Thus it is evident that this coenzyme A modification is transient. We suggest that coenzyme A-modification is a signal involved in the assembly or the degradation process of distinct mitochondrial matrix proteins.     

The 18-kDa mouse epididymal protein (MEP 10) binds retinoic acid.

Mouse epididymal protein (MEP) 10 has recently been characterized in our laboratory. Amino acid sequence analysis of the N-terminal of MEP 10 revealed an 86% similarity in sequence with rat proteins B and C, characterized by Brooks and Higgins [J Reprod Fertil 1980; 59:363-375]. Proteins B and C, have been recently shown to possess retinoic acid-binding ability [Newcomer ME, Ong DE. J Biol Chem 1990; 265:12876-12879; Ong DE, Chytil F. Arch Biochem Biophys 1988; 267:474-478]. Therefore, it was of interest to determine whether MEP 10 possessed the same ability to bind retinoic acid. Mouse caudal fluid was trace-labeled with 3H-retinoic acid and applied to a DEAE ion-exchange column. Analysis of the fractions for both the presence of radioactivity by scintillation counting and MEP 10 by ELISA revealed that the peak of radioactivity corresponded to the peak of MEP 10 immunoreactivity. These peak fractions were pooled and used for subsequent binding analysis and SDS-PAGE and Western blot analysis. SDS-PAGE and Western blot analysis revealed that the peak fractions were enriched for a protein of 18 kDa and that this protein was MEP 10. Competitive binding assays revealed that all-trans-retinoic acid was effective in inhibiting binding of labeled retinoic acid, but that the 13-cis isomer of retinoic acid was only moderately effective in inhibiting binding of the labeled ligand. All-trans-retinol was ineffective in the binding inhibition assay. Similar ligand specificity has also been described for the rat proteins B and C by Ong and Chytil [Arch Biochem Biophys' 1988; 267:474-478].(ABSTRACT TRUNCATED AT 250 WORDS)     

Overexpression of the cellular retinoic acid binding protein-I (CRABP- I) results in a reduction in differentiation-specific gene expression in F9 teratocarcinoma cells

Treatment of F9 teratocarcinoma stem cells with retinoic acid (RA) causes their irreversible differentiation into extraembryonic endoderm. To elucidate the role of the cellular retinoic acid binding protein-I (CRABP-I) in this differentiation process, we have generated several different stably transfected F9 stem cell lines expressing either elevated or reduced levels of functional CRABP-I protein. Stably transfected lines expressing elevated levels of CRABP-I exhibit an 80-90% reduction in the RA induced expression of retinoic acid receptor (RAR) beta, laminin B1, and collagen type IV (alpha 1) mRNAs at low exogenous RA concentrations, but this reduction is eliminated at higher RA concentrations. Thus, greater expression of CRABP-I reduces the potency of RA in this differentiation system. Moreover, transfection of a CRABP-I expression vector into F9 cells resulted in five- and threefold decreases in the activation of the laminin B1 RARE (retinoic acid response element) and the RAR beta RARE, respectively, as measured from RARE/CAT expression vectors in transient transfection assays. These results support the idea that CRABP-I sequesters RA within the cell and thereby prevents RA from acting to regulate differentiation specific gene expression. Our data suggest a mechanism whereby the level of CRABP-I can regulate responsiveness to RA during development.     

Cellular retinoid-binding proteins in reginerating rat liver: demonstration of a novel cellular retinoid-binding protein

Changes in the levels of liver cellular aetinol- and retinoic acid-binding proteins were studied after partial (about 70%) hepatectomy for 14 days in the rat. It was found that a novel binding protein designated F-type appears transiently in liver cytosol 3 days after the operation. The appearance of this protein coincides with the peak level of the alpha 1-fetoproteain. In contrast, cellular retinoic acid-binding protein was detected only the first day after hepatectomy, whereas no significant change was observed in the level of the cellular retinol-binding protein during the entire observation period. [3H]Retinol or [3H]retinoic acid complexed with serum retinol-binding protein injected intravenously into vitamin A-deficient rats 1 day after the hepatectomy was recovered 5 min or 20 min later bound specifically to cellular retinol- or retinoic acid-binding protein, respectively. The results presented here strongly suggest that each of the three cellular retinoid-binding proteins plays a distinct role in cell proliferation and differentiation.     

Modulation of the human preadipocyte mitochondrial activity by beta-carotene

Increased ROS generation by the overload by metabolic substrates mitochondria paralleled by decrease of antioxidant activity are typical events found in metabolic syndrome and diabetes type 2. Metabolites of beta-carotene (BC) such as retinoic acid (RA), as well as low concentration of reactive oxygen species (ROS) modify the mitochondrial bioenergetic function. The aim of the study was to investigate the effect of beta-carotene on mitochondrial activity in human preadipocytes. BC used in concentrations, 10 or 30 μM, decreased mitochondrial membrane potential, inhibited mitochondrial respiration and decreased cellular ATP content. We conclude, that BC, the known antioxidant may decrease oxidative phosphorylation capacity of mitochondria.     

Synthesis of ribonucleic acid by isolated rat liver mitochondria

Rat liver mitochondria isolated in sucrose-N-tris(hydroxymethyl)methyl-2-aminoethane-sulphonic acid (TES) incorporated [(3)H]UTP into RNA for 1h. Incorporation was inhibited 50% by 1mug of actinomycin D/ml, 1mug of acriflavine/ml and 0.5mug of ethidium bromide/ml but was insensitive to rifampicin, rifamycin SV, streptovarcin and deoxyribonuclease. After the first 10min of incubation, the synthesis was insensitive to ribonuclease. RNA synthesis by mitochondria isolated in sucrose-EDTA was insensitive to actinomycin D and sensitive to ribonuclease during the first 10min of the incubation but thereafter the sensitivities were the same as for mitochondria isolated in sucrose-TES. In a hypo-osmotic medium the relative extent of incorporation of the four ribonucleoside triphosphates into RNA was CTP>UTP=ATP>GTP. In an iso-osmotic medium the incorporation of CTP and GTP decreased. All four nucleotides were incorporated into RNA in a DNA-dependent process, as indicated by the inhibition by actinomycin D. In addition, CTP and ATP were incorporated into the CCA end of mitochondrial tRNA. ATP was also incorporated into an unidentified acid-insoluble compound, which hydrolysed in alkali to a product that was not ATP, ADP or 5'- or 2(3')-AMP. Atractyloside inhibited the incorporation of ATP into RNA with 50% inhibition at 2-3nmol/mg of protein. The [(3)H]UTP-labelled RNA had peaks of 16S and 13S characteristic of mitochondrial rRNA. In addition a peak at 20-21S was observed as well as heterogeneous RNA sedimenting throughout the gradient. The synthesis of all these species was inhibited by actinomycin D, indicating that rat liver mitochondrial DNA codes for mitochondrial rRNA as well as other as yet unidentified species.