3-oxoacid CoA-transferase Research Articles

Identification of capacitation associated tyrosine phosphoproteins in buffalo ( Bubalus bubalis) and cattle spermatozoa

At ejaculation mammalian sperm lack fertilizing ability as they are released in a functionally immature form. The capacity to fertilize eggs is only acquired after they have been educated in the female reproductive tract and this phenomenon is termed as capacitation. Sperm capacitation includes a cascade of biochemical modifications, including cholesterol efflux, Ca 2+ influx and cAMP/PKA-dependent/independent protein tyrosine phosphorylation which is specifically considered as the biochemical marker for capacitation. The identification of tyrosine phosphoproteins shall be useful in delineating their physiological role in different events associated with sperm capacitation. The present study was conducted to identify the tyrosine phosphoproteins in the capacitated buffalo and cattle spermatozoa using 2D immunoblotting and mass spectrometry. Among several proteins identified in the buffalo capacitated sperm, serine/threonine-protein phosphatase PP1-gamma catalytic subunit, MGC157332 protein, alpha-enolase, 3-oxoacid CoA transferase 2 and actin-like protein 7A were identified as new tyrosine phosphorylation substrates in mammalian spermatozoa. Cattle sperm also contain proteins such as serine/threonine-protein phosphatase PP1-alpha catalytic subunit and membrane metallo-endopeptidase-like 1 which have not been reported as tyrosine phosphorylated in any other species. Though the presence of serine/threonine-protein phosphatase PP1-alpha catalytic subunit was demonstrated for the first time in mammalian sperm, further studies are required for its existence and possible role in different sperm functions.

Catalytic Role of the Conformational Change in Succinyl-CoA:3-Oxoacid CoA Transferase on Binding CoA,

Catalysis by succinyl-CoA:3-oxoacid CoA transferase proceeds through a thioester intermediate in which CoA is covalently linked to the enzyme. To determine the conformation of the thioester intermediate, crystals of the pig enzyme were grown in the presence of the substrate acetoacetyl-CoA. X-ray diffraction data show the enzyme in both the free form and covalently bound to CoA via Glu305. In the complex, the protein adopts a conformation in which residues 267-275, 280-287, 357-373, and 398-477 have shifted toward Glu305, closing the enzyme around the thioester. Enzymes provide catalysis by stabilizing the transition state relative to complexes with substrates or products. In this case, the conformational change allows the enzyme to interact with parts of CoA distant from the reactive thiol while the thiol is covalently linked to the enzyme. The enzyme forms stabilizing interactions with both the nucleotide and pantoic acid portions of CoA, while the interactions with the amide groups of the pantetheine portion are poor. The results shed light on how the enzyme uses the binding energy for groups remote from the active center of CoA to destabilize atoms closer to the active center, leading to acceleration of the reaction by the enzyme.

Adaptation of Myocardial Substrate Metabolism to a Ketogenic Nutrient Environment

Heart muscle is metabolically versatile, converting energy stored in fatty acids, glucose, lactate, amino acids, and ketone bodies. Here, we use mouse models in ketotic nutritional states (24 h of fasting and a very low carbohydrate ketogenic diet) to demonstrate that heart muscle engages a metabolic response that limits ketone body utilization. Pathway reconstruction from microarray data sets, gene expression analysis, protein immunoblotting, and immunohistochemical analysis of myocardial tissue from nutritionally modified mouse models reveal that ketotic states promote transcriptional suppression of the key ketolytic enzyme, succinyl-CoA:3-oxoacid CoA transferase (SCOT; encoded by Oxct1), as well as peroxisome proliferator-activated receptor alpha-dependent induction of the key ketogenic enzyme HMGCS2. Consistent with reduction of SCOT, NMR profiling demonstrates that maintenance on a ketogenic diet causes a 25% reduction of myocardial (13)C enrichment of glutamate when (13)C-labeled ketone bodies are delivered in vivo or ex vivo, indicating reduced procession of ketones through oxidative metabolism. Accordingly, unmetabolized substrate concentrations are higher within the hearts of ketogenic diet-fed mice challenged with ketones compared with those of chow-fed controls. Furthermore, reduced ketone body oxidation correlates with failure of ketone bodies to inhibit fatty acid oxidation. These results indicate that ketotic nutrient environments engage mechanisms that curtail ketolytic capacity, controlling the utilization of ketone bodies in ketotic states.

The Nitrated Proteome in Heart Mitochondria of the db/db Mouse Model: Characterization of Nitrated Tyrosine Residues in SCOT

A proteomic strategy combining 2DE, Western blot, and mass spectrometry was implemented to survey the status of tyrosine nitration in mouse heart mitochondria. Compared to normal mice, nitrated proteins in the heart mitochondria of the db/db mouse model were significantly augmented due to diabetic development. A total of 18 proteins were identified as the nitration targets. Of the nitrated proteins, succinyl-CoA:3-oxoacid CoA-transferase (SCOT) is a key enzyme involved in ketolysis and has yet to be explored how its catalysis is affected by nitration. We therefore initiated a systematic investigation toward the nitrated site(s) and the corresponding changes of SCOT catalysis. To monitor modification kinetics and nitrated residue(s), recombinant SCOT was incubated with peroxynitrite followed by examination of nitration development as well as catalytic activity changes. The nitration of recombinant SCOT steadily increased in response to increasing concentrations of peroxynitrite, while its catalysis was gradually attenuated. The nitrated sites of modified SCOT were further identified by LC-ESI-MS/MS. The MS/MS spectra indicated a +45 mass unit ion shift from [M + H](+) m/z at Tyr(4) and Tyr(76). Through site-directed mutagenesis, we found that mutation of tyrosine residues at Tyr(4) or Tyr(76) did not only significantly protect SCOT from peroxynitrite modification, but it also dramatically prevented loss of enzymatic activity. Taken together, these results indicate that the two tyrosine residues of SCOT are the priority sites attacked by NO, and their nitration status is a causal factor leading to inhibition of SCOT catalysis.

Crystal structure of 4-hydroxybutyrate CoA-transferase from Clostridium aminobutyricum

4-Hydroxybutyrate CoA-transferases (4-HB-CoAT) takes part in the fermentation of 4-aminobutyrate to ammonia, acetate, and butyrate in anaerobic bacteria such as Clostridium aminobutyricum and Porphyromonas gingivalis or facultative anaerobic bacteria such as Shewanella oneidensis. Site-directed mutagenesis of the highly active enzyme has identified the catalytic glutamate residue as E238. Crystal structure of this enzyme has been determined at a resolution of 1.85 A. The 438-amino acid residue polypeptide chain folds into two topologically similar domains with an open alpha/beta-fold, which is also found in other CoAT family I and family II members. The data indicate that the members of CoAT families I and II are closely related; the latter only lacking the catalytic glutamate residue. A putative co-substrate binding site for the 4-HB-CoAT was identified, in which a 4-hydroxybutyrate molecule has been modeled. This site is also responsible for binding the acetyl group of acetyl-CoA or the succinyl group of succinyl-CoA in succinyl-CoA:3-oxoacid CoA-transferase from mammalian mitochondria. Mutations of relevant active site amino acid residues have been produced and their activities tested to corroborate the proposed structural model for substrate binding. 4-HB-CoAT from C. aminobutyricum represents the only functionally characterized 4-HB-CoAT present in the structural database.

Increased Glucose Uptake and Oxidation in Mouse Hearts Prevent High Fatty Acid Oxidation but Cause Cardiac Dysfunction in Diet-Induced Obesity

Shift of myocardial substrate preference has been observed in many chronic diseases such as diabetes and heart failure. This study was undertaken to elucidate the mechanisms underlying the chronic substrate switch in adult hearts and to determine the functional consequences of the switch. Transgenic mice with cardiac-specific overexpression of the insulin-independent glucose transporter GLUT1 (TG) were used to increase intracellular glucose in cardiac myocytes. A high-fat diet was used to increase the fatty acid supply to the heart. High-fat diet induced a 40% increase in fatty acid oxidation in wild-type hearts, whereas glucose oxidation was decreased to 30% of the control. In contrast, glucose oxidation was >2-fold higher in TG hearts, and the high-fat diet failed to upregulate fatty acid oxidation in these hearts. Glucose induced changes in the expression of multiple metabolic genes, including peroxisome proliferator-activated receptor-alpha (decreased by 51%), 3-oxoacid CoA transferase (decreased by 67%), and acetyl-CoA carboxylase (increased by 4-fold), resulting in a remodeling of the metabolic network to favor a shift of substrate preference toward glucose. Although TG mice on a normal diet maintained normal cardiac energetics and function, the inability to upregulate myocardial fatty acid oxidation in TG mice fed a high-fat diet resulted in increased oxidative stress in the heart, activation of p38 mitogen-activated protein kinase, and contractile dysfunction. We have demonstrated that chronic increases in myocardial glucose uptake and oxidation reduce the metabolic flexibility and render the heart susceptible to contractile dysfunction.

Genetic Obesity Affects Neural Ketone Body Utilization in the Rat Brain

Obesity causes various physiological disorders between the central nervous system and peripheral tissues. Ketone bodies have a neuro-protective role and are strongly affected by obesity-related metabolic disorders. To clarify the effects of obesity on ketone body utilization in brain, we examined the mRNA localization of acetoacetyl-CoA synthetase (AACS), which activates ketone bodies for the synthesis of fatty acid and cholesterol, in various brain regions of Zucker fatty rats by in situ hybridization. The AACS mRNA level was increased in the paraventricular thalamic nucleus (PVT) but not affected in the cerebrum and hippocampus in Zucker fatty rats. In contrast, the AACS mRNA level was reduced in the arcuate hypothalamic nucleus (Arc) and ventromedial hypothalamic nucleus (VMH) in the hypothalamus. Succinyl-CoA:3-oxoacid CoA-transferase (SCOT) mRNA level was decreased only in the PVT but not affected in the Arc and VMH. These data raise the possibility that AACS is regulated by the leptin signaling pathway in the hypothalamus but not in the PVT. As AACS was expressed in neural-like cells, ketone bodies are assumed to be utilized for the synthesis of lipidic substances and to cause metabolic disorders in the nervous system.

Ketone Body Utilization is Regulated by Male-specific Factors in Rat Subcutaneous Adipocytes

Acetoacetyl-CoA synthetase (AACS) is the enzyme responsible for the utilization of ketone bodies for the synthesis of cholesterol and fatty acids and the gene is highly expressed only in the male subcutaneous white adipose tissue (WAT). This study is to elucidate how AACS is involved in sex-dependent metabolic differences in adipose tissues. To answer this question, we examined the effect of castration on AACS mRNA expression in rat WAT. Castration did not affect any changes in the level of serum ketone bodies, but it significantly decreased AACS gene expression in subcutaneous WAT. In contrast, expression of the other ketone body utilizing enzyme, succinyl-CoA: 3-oxoacid CoA-transferase (SCOT), did not change. Moreover, the AACS gene was highly expressed in larger-sized adipocytes obtained from rat subcutaneous WAT, and was up-regulated by testosterone in differentiated adipocytes. Our results suggest that AACS plays male-specific metabolic roles in the regulation of ketone body-utilization for lipogenesis and that sex-specific ketone body-utilization is restricted in subcutaneous WAT.

Influence of ketone bodies on oxidative stress parameters in brain of developing rats in vitro

Pro-oxidant and antioxidant properties have been found for acetoacetate (AcAc) and beta-hydroxybutyrate (BHB) in peripheral tissues. In the present study we investigated the role of AcAc and BHB at concentrations found in diabetic patients during ketoacidotic crises and in individuals affected by succinyl CoA: 3-oxoacid CoA transferase and acetoacetyl-CoA thiolase deficiencies, disorders clinically characterized by neurological symptoms, on a large number of oxidative stress parameters in fresh cerebral cortex of developing rats. Lipid peroxidation (chemiluminescence and thiobarbituric acid-reactive substances levels), protein oxidative damage (carbonyl formation and sulfhydryl oxidation), 2',7'-dichlorofluorescin diacetate oxidation and the non-enzymatic (total antioxidant reactivity and glutathione levels) and enzymatic (glutathione peroxidase, superoxide dismutase and catalase activities) antioxidant defenses were not changed by doses of BHB and AcAc as high as 25 mM in cortical supernatants under basal conditions. Furthermore, BHB did not affect the increased thiobarbituric acid-reactive substances levels provoked by 3-hydroxy-3-methylglutaric and 3-methylglutaconic acids and by a hydroxyl-induced generation system. Finally, BHB and AcAc were not able to oxidize sulfhydryl groups from a commercial GSH solution. Therefore, under basal conditions or under situations with high production of free radicals, AcAc and BHB were not able to reduce or increase the oxidative stress parameters in the brain. Taken together, our present results do not support the hypothesis that BHB and AcAc act as potent direct or indirect pro-oxidants or antioxidants in the CNS.

Clozapine causes oxidation of proteins involved in energy metabolism: a possible mechanism for antipsychotic-induced metabolic alterations

Although atypical antipsychotics are widely known to induce alterations in lipid and glucose metabolism, the mechanisms by which these alterations occur remain unknown. Several recent studies have shown that atypical antipsychotics induce oxidative stress and oxidative cell injury by increasing levels of lipid and protein oxidation. In this study, a novel proteomic approach was used to identify specific proteins oxidized after clozapine treatment. Differentiated neuroblastoma SKNSH cells were treated with 0, 5 or 20 mum clozapine for 24 h and protein extracts were labelled with 6-iodoacetamide fluorescein (6-IAF). The lack of incorporation of 6-IAF to cysteine residues is an indicator of protein oxidation. Labelled proteins were exposed to 2D electrophoresis, and differential protein labelling was assessed. Increased oxidation after clozapine treatment was observed in 10 protein spots (p<0.05), although only four of them remained significant after correcting for analysis with two drug concentrations. Five proteins, corresponding to nine of the spots, were identified by HPLC-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) as mitochondrial ribosomal protein S22, mitochondrial malate dehydrogenase, calumenin, pyruvate kinase and 3-oxoacid CoA transferase. The latter four proteins play important roles in energy metabolism. These results suggest that oxidative stress may be a mechanism by which antipsychotics increase the risk for metabolic syndrome and diabetes.

Tamoxifen Induces Oxidative Stress and Mitochondrial Apoptosis via Stimulating Mitochondrial Nitric Oxide Synthase

Tamoxifen is an anticancer drug that induces oxidative stress and apoptosis via mitochondria-dependent and nitric oxide (NO)-dependent pathways. The present report shows that tamoxifen increases intramitochondrial ionized Ca(2+) concentration and stimulates mitochondrial NO synthase (mtNOS) activity in the mitochondria from rat liver and human breast cancer MCF-7 cells. By stimulating mtNOS, tamoxifen hampers mitochondrial respiration, releases cytochrome c, elevates mitochondrial lipid peroxidation, increases protein tyrosine nitration of certain mitochondrial proteins, decreases the catalytic activity of succinyl-CoA:3-oxoacid CoA-transferase, and induces aggregation of mitochondria. The present report suggests a critical role for mtNOS in apoptosis induced by tamoxifen.

Protein profile of human hepatocarcinoma cell line SMMC-7721: Identification and functional analysis

To investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy. Total proteins from human hepatocarcinoma cell line SMMC-7721 were separated by two-dimensional electrophoresis (2DE). The silver-stained gel was analyzed by 2DE software Image Master 2D Elite. Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin, endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein I, peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed. Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

Transposon-mediated gene trapping in zebrafish

The Tol2 transposon system can create chromosomal insertions in the zebrafish germ lineage very efficiently. We constructed a Tol2-based gene trap vector, T2KSAG, which contains a splice accepter, the GFP gene and the polyA signal. In the pilot screen for gene trapping using T2KSAG, we identified 38 fish lines expressing GFP in specific organs and tissues. In the SAGp53A line, GFP is expressed in the forebrain and midbrain, and the insertion of the gene trap construct captured a transcript of the kab gene encoding a zebrafish homolog of the human KARP (Ku86 autoantigen related protein)-binding protein (KAB). In the SAGm18B line, GFP is expressed in the central nervous system, and the insertion captured a transcript of a gene for succinyl CoA:3-oxoacid CoA-transferase (SCOT). Here, we describe how we performed the gene trap screen and characterized the gene trap insertions and will discuss the outcome of the pilot screen.

Detection of eight antibodies in cancer patients’ sera against proteins derived from the adenocarcinoma A549 cell line using proteomics-based analysis

To screen cancer for specific autoantibodies, we applied the approach established by Brichory et al., who reported annexins I and II as specific antigens. Solubilized proteins from a cancer cell line (A549) were separated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), followed by Western blotting (WB) analysis, in which the sera of individual patients were tested for primary antibodies. We found 11 positive spots on PVDF membrane using a WB/enhanced chemiluminescence detection Kit, and identified eight proteins, such as alpha-enolase, inosine-5'-monophosphate dehydrogenase, aldehyde dehydrogenase, 3-phosphoglycerate dehydrogenase, 3-oxoacid CoA transferase, chaperonin, peroxiredoxin 6 and triosephosphate isomerase, that reacted with these antibodies in patients' sera using MALDI-TOF/TOF. All eight antibodies were not detected in the sera derived from lung tuberculosis and healthy controls.

Crystallographic Trapping of the Glutamyl-CoA Thioester Intermediate of Family I CoA Transferases

Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 A resolution, respectively. YdiF is organized into tetramers, with each monomer having an open alpha/beta structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent gamma-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.

Different localization in rat brain of the novel cytosolic ketone body-utilizing enzyme, acetoacetyl-CoA synthetase, as compared to succinyl-CoA:3-oxoacid CoA-transferase

In lipogenic tissue cytosol, ketone bodies are known to be activated by acetoacetyl-CoA synthetase (AACS) and incorporated into cholesterol and fatty acids. In order to investigate the physiological role of AACS in the brain, we examined the localization of AACS mRNA in rat brain by in situ hybridization using a labeled probe. High labeling was observed in the midbrain, pons/medulla, cerebral cortex, hippocampus and cerebellum, and the localization profile of AACS mRNA was different from that of succinyl-CoA:3-oxoacid CoA-transferase (SCOT), a mitochondrial ketone body-activating enzyme. In addition, the expression of AACS mRNA in the cerebellum was restricted primarily to glial cells, while in the cerebral cortex, it was restricted to neuronal cells. Streptozotocin treatment caused remarkable decreases in AACS mRNA levels in all regions where expression was observed, but changes in SCOT mRNA levels were not observed. These results suggest that the physiological role of AACS is different from that of SCOT and varies depending upon its localization in the brain.

Effect of enteral nutrition of monoacetoacetin on bacterial translocation in burned rats

Bacterial translocation (BT) occurs under stress, and enteral nutrition is said to be effective in counteracting this effect. The usefulness of i.v. nutrition of monoacetoacetine (MA) under stress has been reported previously, and we studied the effect of enteral nutrition of MA as it is related to BT. Rats were given 13.6% MA or 15% glucose (Glu) via gastrostomy, and after 4 days a 30% full-thickness burn was made. Before and after administration of the burn, we measured serum cytokines; amounts of bacteria in the mesenteric lymph node (MLN), liver, and spleen (estimation of BT); mucosal thickness of the terminal ileum; and body weight changes. To confirm the effectiveness of MA in the small intestine, we estimated succinyl-CoA:3-oxoacid CoA-transferase (SCOT) expression in the terminal ileum by immunohistochemical staining and Western blot analysis. At 6 hours after burn, all cytokines were lower, and BT in the MLN was inhibited significantly in the MA group. Ileal mucosal thickness was not significantly different, but mucosa was more edematous in the Glu group. At 3 days after burn injury, BT was significantly inhibited in the MLN and liver, and the ileal mucosa was significantly thicker in the MA group. Body weight loss after burn injury was significantly smaller in the MA group. SCOT expression was the strongest at 6 hours after burn injury by Western blot analysis but not by immunohistochemical staining. Enteral nutrition of MA may be useful for the inhibition of intestinal mucosal atrophy and the prevention of multiple organ dysfunction syndrome caused by the inhibition of BT and subsequent overproduction of cytokines.

Differential expression of succinyl CoA transferase (SCOT) genes in somatic and germline cells of the mouse testis

Succinyl CoA:3-oxo acid CoA transferase (SCOT/OXCT; EC 2.8.3.5) is a key mitochondrial enzyme in the metabolism of ketone bodies in various organs (but not in the liver). We identified a cDNA clone of the testicular germ cell-specific succinyl CoA transferase isozyme (SCOT-t). We then isolated a mouse orthologue of the SCOT/OXCT cDNA (SCOT-s) and determined the expression of the two types of SCOT in the testis. The mRNAs of scot-s and scot-t were expressed exclusively in testicular somatic cells (i.e. Leydig and Sertoli cells) and germ cells, respectively. SCOT enzymatic activities were assayed in Leydig cell (SCOT-s) and sperm (SCOT-t) fractions. The SCOT activity in sperm was 2.5-fold higher than that in Leydig cells. We conclude that germ cells and somatic cells differentially express the SCOT enzymes and that the SCOT activity of sperm caused exclusively by SCOT-t should play an important role in sperm activity.

Cloning and characterization of a human orthologue of testis-specific succinyl CoA: 3-oxo acid CoA transferase (Scot-t) cDNA.

Succinyl CoA: 3-oxo acid CoA transferase (scot; EC 2.8.3.5) is a key enzyme for metabolism of ketone bodies. Previously, we have cloned a testis-specific succinyl CoA: 3-oxo acid CoA transferase (scot-t) cDNA from a subtracted cDNA library of mouse testis and demonstrated its expression to be specific to late haploid male germ cells. In this study, the human orthologue of mouse scot-t was cloned and characterized. The entire coding region of the mRNA and the deduced amino acid sequence of human Scot-t (h-Scot-t) showed 75.4 and 75.8% identity with mouse scot-t respectively. The mRNA was exclusively expressed in the testis, and the protein was localized to the midpiece of ejaculated spermatozoa where mitochondria exist. We showed that the h-Scot-t gene was intronless by using a polymerase chain reaction technique and that a non-functional pseudogene, 98% similar to h-Scot-t, was also located on the human genome (1p33-34.3). Furthermore, the genomic structure of the actual h-Scot-t transcription unit was found to be located in an intron (1p34.1-35.3) of the bone morphogenetic protein 8 (BMP8) gene. In conclusion, we have demonstrated that h-Scot-t is a single intronless gene specifically expressed in the testis.

Diabetes-associated nitration of tyrosine and inactivation of succinyl-CoA:3-oxoacid CoA-transferase.

High levels of reactive species of nitrogen and oxygen in diabetes may cause modifications of proteins. Recently, an increase in protein tyrosine nitration was found in several diabetic tissues. To understand whether protein tyrosine nitration is the cause or the result of the associated diabetic complications, it is essential to identify specific proteins vulnerable to nitration with in vivo models of diabetes. In the present study, we have demonstrated that succinyl-CoA:3-oxoacid CoA-transferase (SCOT; EC 2.8.3.5) is susceptible to tyrosine nitration in hearts from streptozotocin-treated rats. After 4 and 8 wk of streptozotocin administration and diabetes progression, SCOT from rat hearts had a 24% and 39% decrease in catalytic activity, respectively. The decrease in SCOT catalytic activity is accompanied by an accumulation of nitrotyrosine in SCOT protein. SCOT is a mitochondrial matrix protein responsible for ketone body utilization. Ketone bodies provide an alternative source of energy during periods of glucose deficiency. Because diabetes results in profound derangements in myocardial substrate utilization, we suggest that SCOT tyrosine nitration is a contributing factor to this impairment in the diabetic heart.