Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.4.1.2 (acetyl-CoA carboxylase)
2,876 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Biotin uptake, utilization, and efflux were studied in normal and biotin-deficient cultured rat hepatocytes. Biotin-deficient cells accumulate about 16-fold more biotin than do normal cells when incubated with a physiological concentration of biotin for 24 h. This difference is due to the greater amount of protein-bound biotin relative to free biotin in biotin-deficient hepatocytes, and is attributable to the presence of more apocarboxylases in deficient cells. The rate of biotin uptake and the rate of activation of the carboxylases, acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, and beta-methylcrotonyl-CoA carboxylase, are proportional to the concentration of exogenous biotin. Increases in carboxylase activities are proportional to the concentration of biotin only at exogenous biotin concentrations of less than 410 nM. Concentrations of 410 nM or more biotin increase carboxylase activities to normal or near normal. Biocytin inhibits biotin uptake at very high concentrations, whereas desthiobiotin and lipoic acid have no effect. Biocytin in the medium results in carboxylase activation either intracellularly or extracellularly by conversion to biotin by biotinidase. Investigation of the efflux of biotin from normal and biotin-deficient cells preincubated with the vitamin showed greater retention of biotin by biotin-deficient cells than by normal cells over 24 h. Retention of free biotin is similar in biotin-deficient and normal cells. The greater amount of biotin retained by biotin-deficient cells is accounted for by the greater amount of bound biotin in these cells. These results suggest that the free and bound biotin pools are independently regulated. The ready loss of free biotin from these cells has implications for the treatment of inherited, biotin-responsive carboxylase deficiencies.
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PMID:Biotin uptake, utilization, and efflux in normal and biotin-deficient rat hepatocytes. 179 12

Peroxidase-conjugated avidin was used to detect biotin-containing carboxylases in rat liver. By a transblot method, avidin-peroxidase interacted with liver proteins with estimated molecular masses of 120 and 74 kDa. The proteins were identified as pyruvate carboxylase (120 kDa, 6.4 pI) and methylcrotonyl-CoA carboxylase (74 kDa, 7.2 pI) by two-dimensional gel electrophoresis and transblot method. An additional band with estimated molecular mass of 220 kDa was detected in the cytosol fraction of rat liver, compatible with acetyl-CoA carboxylase. Rat liver proteins were prepared and treated with avidin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblot with avidin-peroxidase. A 190-kDa band was found with a parallel decrease in the 120-kDa band determined by Coomassie blue staining; however, these proteins did not stain by the transblot avidin-peroxidase method. When the transblot of parallel proteins was incubated with biotin and subsequently with avidin-peroxidase, two additional bands, namely 190 and 145 kDa, were detected while the 74-kDa band disappeared correlated with decreased staining of the 120-kDa band. The present procedure is a simple, rapid, and inexpensive method for detecting biotin-containing proteins in various tissues and organs and in determining the occurrence of nonspecific staining with the avidin-biotin complex method of immunoblot.
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PMID:Transblot identification of biotin-containing proteins in rat liver. 274 54

Incubation of cultured cells with [3H]biotin leads to the labelling of acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase and methylcrotonyl-CoA carboxylase. The biotin-containing subunits of the last two enzymes from rat cell lines are not separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, but adequate separation is achieved with the enzymes from human cells. Since incorporated biotin is only released upon complete protein breakdown, the loss of radioactivity from gel slices coinciding with fluorograph bands was used to quantify degradation rates for each protein. In HE(39)L diploid human fibroblasts, the degradation rate constants are 0.55, 0.40, 0.31 and 0.19 day-1 for acetyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase respectively. A similar series of rate constants is found for AG2804 transformed fibroblasts. The degradation rate constants are decreased by 31-67% in the presence of 50 micrograms of leupeptin/ml plus 5 mM-NH4Cl. Although the largest percentage effect was noted with the most stable enzyme, propionyl-CoA carboxylase, the absolute change in rate constant produced by the lysosomotropic inhibitors was similar for the three mitochondrial carboxylases, but greater for the cytosolic enzyme acetyl-CoA carboxylase. The heterogeneity in degradation rate constants for the mitochondrial carboxylases indicates that only part of their catabolism can occur via the autophagy-mediated unit destruction of mitochondria. Calculations showed that the autophagy-linked process had degradation rate constants of 0.084 and 0.102 day-1 respectively in HE(39)L and AG2804 cells. It accounted for two-thirds of the catabolic rate of propionyl-CoA carboxylase and a lesser proportion for the other enzymes.
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PMID:Distribution and degradation of biotin-containing carboxylases in human cell lines. 286 10

Extracts of 3T3-L1 cells prepared after labelling the monolayer cultures with [3H]biotin contained numerous protein bands that were detected by fluorography of dried SDS/polyacrylamide electrophoresis gels. All labelled proteins in the extracts could be removed by avidin affinity chromatography. The biotin-containing subunits of acetyl-CoA carboxylase, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase, with molecular masses of approx. 220, 120, 75 and 72 kDa respectively, were detected together with minor bands at 100, 85 and 37 kDa that did not appear to be partial degradation products. Additional labelled bands increased in amount during incubation of cell extracts or did not occur in extracts prepared with trichloroacetic acid, 9.5 M-urea or proteolytic inhibitors, and were tentatively classified as partial degradation products. The unknown bands were not removed by incubation of cell monolayers for 24 h, a treatment that gave degradation rate constants of 0.47 day-1 for acetyl-CoA carboxylase and 0.28 day-1 for pyruvate carboxylase. Upon two-dimensional electrophoresis, pyruvate carboxylase, methylcrotonyl-CoA carboxylase and propionyl-CoA carboxylase had isoelectric points of 6.4, 7.2 and 6.4 respectively. Several additional discrete spots with isoelectric points below 6.2 were also present. All the unknown biotin-containing proteins banded with intact mitochondria during density-gradient centrifugation. We conclude that several unknown biotin-containing proteins are present in the mitochondria of 3T3-L1 cells, whereas others are partial breakdown products of mitochondrial proteolysis.
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PMID:Multiple biotin-containing proteins in 3T3-L1 cells. 380 Aug 73

Biotin carboxylases in mammalian cells are regulatory enzymes in lipogenesis and gluconeogenesis. In this study, endogenous biotin in skeletal and cardiac muscle was detected using avidin conjugated with alkaline phosphatase and applied in high concentrations to muscle sections. The avidin binding was subsequently visualized by histochemical demonstration of the alkaline phosphatase activity. All cardiac muscle cells showed high affinity for avidin with only the nuclei and the intercalated discs remaining unstained. In skeletal muscle a diffuse reaction could be detected in the sarcoplasm of the muscle fibres. A granular reaction was noted in the same fibres that showed activity for succinic dehydrogenase. The specificity of the coloured reaction product in the muscle sections was investigated and is suggested to be caused by avidin binding to biotin moieties in mitochondria and the cytosol. Mitochondrial and cytosolic preparations of skeletal muscle were electrophoresed in sodium dodecyl sulphate gels. After blotting and incubation with conjugated avidin, two bands with molecular weights of 75 kDa and 130 kDa respectively were evident in the mitochondrial preparation. It is suggested that the 75-kDa band represents comigration of the biotin-containing subunits of propionyl-CoA carboxylase and methylcrotonyl-CoA carboxylase. The 130-kDa band may represent the biotin-containing pyruvate carboxylase. In the cytosolic preparation a 270-kDa band was stained in blots that had been incubated with conjugated avidin; this band is suggested to represent acetyl-CoA carboxylase. A 190-kDa cytosolic band might be a cleavage product of acetyl-CoA carboxylase. We propose that using alkaline phosphatase-conjugated avidin it is possible to detect the mitochondrial and cytosolic biotin-dependent carboxylases in striated muscle.
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PMID:Biotin carboxylases in mitochondria and the cytosol from skeletal and cardiac muscle as detected by avidin binding. 816 85

Mature dry pea seeds contain three major biotinylated proteins. Two of these of subunit molecular mass about 75 kDa and 200 kDa are associated with 3-methylcrotonyl-CoA carboxylase (EC 6.4.1.4) and acetyl-CoA carboxylase activities (EC 6.4.1.2) respectively. The third does not exhibit any of the biotin-dependent carboxylase activities found in higher organisms and represents the major part of the total protein-bound biotin in the seeds. This novel protein has been purified from a whole pea seed extract. Because in SDS/polyacrylamide gels the protein migrates with an apparent molecular mass of about 65 kDa, it is referred to as SBP65, for 65 kDa seed biotinylated protein. The molecular mass of native SBP65 is greater than 400 kDa, suggesting that the native protein assumes a polymeric structure, resulting from the association of six to eight identical subunits. The results of CNBr cleavage experiments suggest that biotin is covalently bound to the protein. The stoichiometry is 1 mol of biotin per 1 mol of 65 kDa polypeptide. The temporal and spatial pattern of expression of SBP65 is described. SBP65 is specifically expressed in the seeds, being absent from leaf, root, stem, pod and flower tissues of pea plants. The level of SBP65 increases dramatically during seed development. The protein is not detectable in very young seeds. Its accumulation pattern parallels that for storage proteins, being maximally expressed in the mature dry seeds. SBP65 disappears at a very high rate during seed germination. The level of free biotin has also been evaluated for various organs of pea plants. In all proliferating tissues examined (young developing seeds, leaf, root, stem, pod and flower tissues), free biotin is in excess of protein-bound biotin. Only in the mature dry seeds is protein-bound biotin (i.e. that bound to SBP65) in excess of free biotin. These temporal expression patterns, and the strict organ specificity for expression of SBP65, are discussed with regard to the possibility that in plants, as in mammals, biotin plays a specialized role in cell growth and differentiation.
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PMID:Developmental patterns of free and protein-bound biotin during maturation and germination of seeds of Pisum sativum: characterization of a novel seed-specific biotinylated protein. 816 32

Peroxidase-conjugated avidin was used to detect the endogenous avidin-binding proteins in rat tissues. By a transblot method, avidin-peroxidase interacted with proteins of mitochondrial fractions of rat liver with estimated molecular weights of 120,000 and 74,000. The proteins were identified as pyruvate carboxylase (120 kDa, pI 6.4) and methylcrotonyl-CoA carboxylase (74 kDa, pI 7.2) by two-dimensional gel electrophoresis and transblot method. The estimated molecular weight of an additional band (220,000) detected in the cytosolic fraction of rat liver was consistent with acetyl-CoA carboxylase. Intense staining also occurred with kidney, heart, ovary and adipose tissue, moderate with large and small intestine, cerebrum and cerebellum, and very faint with testis, lung and spleen. The sections of rat liver embedded in LR white were incubated with avidin-colloidal gold conjugate and examined under an electron microscope. The glutaraldehyde-perfused rat liver blocks were also incubated with streptavidin-ferritin conjugate and the ultrathin sections were cut and examined. The majority of gold and ferritin particles were found in the mitochondria of liver cells. No other cellular compartment was labeled except the cytosol which accounted for approximately 20% of the total labeling of the hepatocytes. The present procedure is a simple, rapid and inexpensive method for detecting the intracellular localization of endogenous avidin-binding proteins in the cells.
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PMID:Detection of endogenous avidin-binding proteins in rat liver cells by transblot and electron microscopy. 825 80

In the past, lipoic acid has been administered to patients and test animals as therapy for diabetic neuropathy and various intoxications. Lipoic acid and the vitamin biotin have structural similarities. We sought to determine whether the chronic administration of lipoic acid affects the activities of biotin-dependent carboxylases. For 28 d, rats received daily intraperitoneal injections of one of the following: 1) a small dose of lipoic acid [4.3 micromol/( kg.d)]; 2) a large dose of lipoic acid [15.6 micromol/(kg.d)]; or 3) a large dose of lipoic acid plus biotin [15.6 and 2.0 micromol/(kg.d), respectively]. Another group received n-hexanoic acid [14.5 micromol/(kg.d)], which has structural similarities to lipoic acid and biotin and thus served as a control for the specificity of lipoic acid. A fifth group received phosphatidylcholine in saline injections and served as the vehicle control. The rat livers were assayed for the activities of acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, and beta-methylcrotonyl-CoA carboxylase. Urine was analyzed for lipoic acid; serum was analyzed for indicators of liver damage and metabolic aberrations. The mean activities of pyruvate carboxylase and beta-methylcrotonyl-CoA carboxylase were 28-36% lower in the lipoic acid-treated rats compared with vehicle controls (P < 0.05). Rats treated with lipoic acid plus biotin had normal carboxylase activities. Carboxylase activities in livers of n-hexanoic acid-treated rats were normal despite some evidence of liver injury. Propionyl-CoA carboxylase and acetyl-CoA carboxylase were not significantly affected by administration of lipoic acid. This study provides evidence consistent with the hypothesis that chronic administration of lipoic acid lowers the activities of pyruvate carboxylase and beta-methylcrotonyl-CoA carboxylase in vivo by competing with biotin.
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PMID:Lipoic acid reduces the activities of biotin-dependent carboxylases in rat liver. 927 59

Biotin, a water-soluble vitamin, is used as cofactor of enzymes involved in carboxylation reactions. In humans, there are five biotin-dependent carboxylases: propionyl-CoA carboxylase; methylcrotonyl-CoA carboxylase; pyruvate carboxylase, and two forms of acetyl-CoA carboxylase. These enzymes catalyze key reactions in gluconeogenesis, fatty acid metabolism, and amino acid catabolism; thus, biotin plays an essential role in maintaining metabolic homeostasis. In recent years, biotin has been associated with several diseases in humans. Some are related to enzyme deficiencies involved in biotin metabolism. However, not all biotin-responsive disorders can be explained based on the classical role of the vitamin in cell metabolism. Several groups have suggested that biotin may be involved in regulating transcription or protein expression of different proteins. Biotinylation of histones and triggering of transduction signaling cascades have been suggested as underlying mechanisms behind these non-classical biotin-deficiency manifestation in humans.
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PMID:Biotin in metabolism and its relationship to human disease. 1245 13

The biotin carboxylase family is comprised of a group of enzymes that utilize a covalently bound prosthetic group, biotin, as a cofactor. These enzymes, which include acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase, methylcrotonyl-CoA carboxylase, geranoyl-CoA carboxylase, oxaloacetate decarboxylase, methylmalonyl-CoA decarboxylase, transcarboxylase and urea amidolyase, are found in diverse biosynthetic pathways in both pro-karyotes and eukaryotes. The reactions catalyzed by most members of this group of enzymes share two common features: (1) carboxylation of biotin, apparently via the formation of a carboxyphosphate intermediate, followed by (2) transcarboxylation of CO(2) from biotin to specific acceptor molecules to yield different products. Structural determinations by NMR and X-ray crystallography, complemented by mutagenesis studies, have identified some motifs that are structurally or catalytically important. Analysis of the amino acid sequences of a number of biotin carboxylases not only shows remarkable similarities within certain domains but also that there appears to have been domain rearrangements between groups of carboxylases. Acyl-coenzyme A derivatives, which bind either as substrates or as allosteric regulators of the biotin carboxylases, do not appear to share any of the CoA binding motifs that have been identified in other CoA-SH/acyl-CoA binding proteins. Further comparisons of biotin-dependent carboxylases with other groups of enzymes in the protein data bank reveal that this family of biotin enzymes has strong similarities in specific domains to a number of ATP-utilizing enzymes and to the lipoyl-containing enzymes. These structural homologies are so extensive as to be highly suggestive of evolutionary relationships between biotin carboxylases and these other enzymes.
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PMID:The biotin enzyme family: conserved structural motifs and domain rearrangements. 1276 20


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