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The characteristics of the microsomal stearoyl CoA desaturase (EC 1.14.99.5) of vegetative Fusarium oxysporum cells grown at different temperatures were studied. The enzyme had an unusual preference for NADPH (Km = 38 micrometers) over NADH (Km = 89 micrometers) as electron donor, and a relatively high optimum pH of 8.3. Enzyme activity was highest in microsomes from cells grown at 37 degrees C and lowest in cells grown at 15 degrees C. This result correlated well with the observed changes in oleic acid content of the microsomal lipids. Both NADPH-linked reductase activities and hemoprotein content were lowest in cells grown at 37 degrees C. Spectrophotometric analysis of the microsomal hemoproteins indicated the absence of cytochrome b5 and the presence of a b-type heme with a pyridine hemochrome alpha band absorption maximum at 565 nm. Labile sulfide analysis and inhibitor studies with thenoyltrifluoroacetone suggested a role for an iron-sulfur protein in the electron transfer system associated with the desaturase. 相似文献
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William Samuel Chandrasekharam N Nagineni R Krishnan Kutty W Tony Parks Joel S Gordon Stephen M Prouty John J Hooks Barbara Wiggert 《The Journal of biological chemistry》2002,277(1):59-66
The regulation of stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the synthesis of unsaturated fatty acids, is physiologically important because the ratio of saturated to unsaturated fatty acids is thought to control cellular functions by modulating the structural integrity and fluidity of cell membranes. Transforming growth factor-beta (TGF-beta), a multifunctional cytokine, increased SCD mRNA expression in cultured human retinal pigment epithelial cells. This response was elicited by all three TGF-beta isoforms, beta1, beta2, and beta3. However, SCD mRNA expression was not increased either by other members of the TGF-beta family or by other growth factors or cytokines. TGF-beta also increased SCD mRNA expression in several other cell lines tested. The increase in SCD mRNA expression was preceded by a marked increase in Smad2 phosphorylation in TGF-beta-treated human retinal pigment epithelial cells. TGF-beta did not induce SCD mRNA expression in a Smad4-deficient cell line. However, Smad4 overexpression restored the TGF-beta effect in this cell line. Moreover, TGF-beta-induced SCD mRNA expression was effectively blocked by the overexpression of Smad7, an inhibitory Smad. Thus, a TGF-beta signal transduction pathway involving Smad proteins appears to regulate the cellular expression of the SCD gene, and this regulation may play an important role in lipid metabolism. 相似文献
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The NADH-dependent stearoyl CoA desaturase of hepatic microsomes (EC 1.14.99.5) is an enzyme system consisting of cytochrome b5 reductase (EC 1.6.2.2), cytochrome b5, and the terminal desaturase. We have developed a simple method for routine assay of the terminal enzyme based on complementation of the enzyme with chick embryo liver microsomes lacking desaturase activity. Desaturation of [1-14C]stearoyl CoA by the enzyme-microsome mixture is then assayed by thin-layer chromatography of the reaction products and determination of the amount of oleate formed. Microsomes from the livers of starved-refed rats were used as the source of the stearoyl CoA desaturase. The enzyme alone, solubilized and free from cytocrome b5 reductase and cytochrome b5, was unable to catalyze the desaturation of stearoyl CoA. However, after preincubation with chick embryo liver microsomes in the presence of 1% Triton X-100, the enzyme was active. The enzyme activity was linear with time and desaturase protein under the conditions described and depended on the concentrations of Triton X-100 present in the preincubation and the assay. The optimum concentrations of Triton X-100 were 1% for the preincubation and 0.1-0.15% in the assay. The desaturation activity was dependent on NADH and O2, and was inhibited 95% by 1 mM KCN. The use of chick embryo liver microsomes in this method eliminates the need to use purified cytochrome b5 reductase, cytochrome b5, and liposomes for routine assays and greatly reduces the complexities of timing and order of addition encountered in the existing assays. 相似文献
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DesA3 (Rv3229c) from Mycobacterium tuberculosis is a membrane-bound stearoyl coenzyme A Delta(9) desaturase that reacts with the oxidoreductase Rv3230c to produce oleic acid. This work provides evidence for a mechanism used by mycobacteria to regulate this essential enzyme activity. DesA3 expressed as a fusion with either a C-terminal His(6) or c-myc tag had consistently higher activity and stability than native DesA3 having the native C-terminal sequence of LAA, which apparently serves as a binding determinant for a mycobacterial protease/degradation system directed at DesA3. Fusion of only the last 12 residues of native DesA3 to the C terminus of green fluorescent protein (GFP) was sufficient to make GFP unstable. Furthermore, the comparable C-terminal sequence from the Mycobacterium smegmatis DesA3 homolog Msmeg_1886 also conferred instability to the GFP fusion. Systematic examination revealed that residues with charged side chains, large nonpolar side chains, or no side chain at the last two positions were most important for stabilizing the construct, while lesser effects were observed at the third-from-last position. Using these rules, a combinational substitution of the last three residues of DesA3 showed that either DKD or LEA gave the best enhancement of stability for the modified GFP in M. smegmatis. Moreover, upon mutagenesis of LAA at the C terminus in native DesA3 to either of these tripeptides, the modified enzyme had enhanced catalytic activity and stability. Since many proteases are conserved within bacterial families, it is reasonable that M. tuberculosis will use a similar C-terminal degradation system to posttranslationally regulate the activity of DesA3 and other proteins. Application of these rules to the M. tuberculosis genome revealed that approximately 10% the proteins encoded by essential genes may be susceptible to C-terminal proteolysis. Among these, an annotation is known for less than half, underscoring a general lack of understanding of proteins that have only temporal existence in a cell. 相似文献
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Hepatic poly(A+) RNA from rats induced for stearyl-CoA desaturase was used for primer-extension of cDNA coding for stearyl-CoA desaturase. Previously, Northern blot analysis showed that translatable desaturase mRNA is 4,900 nucleotides in length (Thiede, M. A., and Strittmatter, P. (1985) J. Biol. Chem. 260, 14459-14463). Six overlapping cDNAs, ranging from 850 to 1450 bases, were used to compile the 4,689-nucleotide sequence. The cDNA includes a 1,074-base open reading frame coding for 358 amino acids, corresponding to a molecular mass of 41,400 daltons. Positive identification of this open reading frame was accomplished by matching the amino acid sequence of both amino-terminal and cyanogen bromide peptides of the purified enzyme with regions of the sequence deduced from the cDNA. Amino acid composition data from the cDNA compares well with that from the desaturase. The protein contains 62% hydrophobic amino acids. An interesting feature of this mRNA is the 3,500-base 3' noncoding region, which has been localized on a single 3' exon by Southern blot analysis. 相似文献
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Regulation of hepatic acetyl coenzyme A carboxylase by phosphorylation and dephosphorylation 总被引:9,自引:0,他引:9
Acetyl CoA carboxylase, in a partially purified preparation, was inactivated by ATP in a time- and temperature-dependent reaction. Adenosine 3′,5′-monophosphate did not affect the inactivation. Further purification separated the carboxylase from a protein fraction which could greatly enhance the inactivation of the enzyme.Inactivation of the enzyme with [γ-32P]ATP resulted in the incorporation of 32P which copurified with the enzyme. No label was incorporated when [U-14C]ATP was used. When carboxylase inactivated by exposure to [γ-32P]ATP was precipitated with antibody, isotope incorporation into the precipitate paralleled enzyme inactivation. The phosphate was bound to serine and threonine residues by an ester linkage.Sodium fluoride completely inhibited the activation of partially purified enzyme by magnesium ions. Activation by magnesium, accompanied by the release of protein-bound 32P, was antagonistic to inactivation of the enzyme by ATP.The data presented in this communication are consistent with a mechanism for controlling acetyl CoA carboxylase activity by interconversion between phosphorylated and dephosphorylated forms. Phosphorylation of the enzyme by a portein kinase decreases enzyme activity, whereas dephosphorylation by a protein phosphatase reactivates the enzyme. 相似文献
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beta-Amyloid-specific upregulation of stearoyl coenzyme A desaturase-1 in macrophages 总被引:3,自引:0,他引:3
beta-Amyloid peptide (A beta), a major component of senile plaques, the formation of which is characteristic of Alzheimer's disease (AD), is believed to induce inflammation of the brain mediated by microglia, leading to neuronal cell loss. In this study, we performed an oligonucleotide microarray analysis to investigate the molecular events underlying the A beta-induced activation of macrophages and its specific suppression by the A beta-specific-macrophage-activation inhibitor, RS-1178. Of the approximately 36,000 genes and expressed sequence tags analyzed, eight genes were specifically and significantly upregulated by a treatment with interferon gamma (IFN gamma) and A beta compared to a treatment with IFN gamma alone (p<0.002). We found that the gene for a well-characterized lipogenetic enzyme, stearoyl coenzyme A desaturase-1 (SCD-1), was specifically upregulated by A beta treatment and was suppressed to basal levels by RS-1178. Although the underlying mechanisms remain unknown, our results suggest the presence of a link between AD and SCD-1. 相似文献
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Regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase. Developmental pattern 总被引:6,自引:0,他引:6
D J McNamara F W Quackenbush V W Rodwell 《The Journal of biological chemistry》1972,247(18):5805-5810
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Coenzyme A (CoA) and acyl carrier protein are two cofactors in fatty acid metabolism, and both possess a 4'-phosphopantetheine moiety that is metabolically derived from the vitamin pantothenate. We studied the regulation of the metabolic pathway that gives rise to these two cofactors in an Escherichia coli beta-alanine auxotroph, strain SJ16. Identification and quantitation of the intracellular and extracellular beta-alanine-derived metabolites from cells grown on increasing beta-alanine concentrations were performed. The intracellular content of acyl carrier protein was relatively insensitive to beta-alanine input, whereas the CoA content increased as a function of external beta-alanine concentration, reaching a maximum at 8 microM beta-alanine. Further increase in the beta-alanine concentration led to the excretion of pantothenate into the medium. Comparing the amount of pantothenate found outside the cell to the level of intracellular metabolites demonstrates that E. coli is capable of producing 15-fold more pantoic acid than is required to maintain the intracellular CoA content. Therefore, the supply of pantoic acid is not a limiting factor in CoA biosynthesis. Wild-type cells also excreted pantothenate into the medium, showing that the beta-alanine supply is also not rate limiting in CoA biogenesis. Taken together, the results point to pantothenate kinase as the primary enzymatic step that regulates the CoA content of E. coli. 相似文献
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R E Dugan G C Ness M R Lakshmanan C M Nepokroeff J W Porter 《Archives of biochemistry and biophysics》1974,161(2):499-504
We have previously established that insulin causes a marked and rapid stimulation of hepatic β-hydroxy-β-methylglutaryl coenzyme A reductase activity in normal and diabetic rats [Biochem. Biophys. Res. Commun.50, 504 (1973)], whereas l-triiodothyronine stimulates the reductase activity to supranormal levels in hypophysectomized rats two days after administration [Proc. Nat. Acad. Sci. (1974) In press]. In the present investigation it is demonstrated that the stimulation of the reductase activity in hypophysectomized-diabetic rats requires the mediation of both insulin and l-triiodothyronine. Neither hormone alone is effective. The rapid stimulation of reductase activity by insulin and the delayed stimulation elicited by l-triiodothyronine are both inhibited by either glucagon or hydrocortisone. Thus, an interplay of hormones regulates reductase activity and consequently cholesterol biosynthesis. 相似文献
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Rimoldi OJ Finarelli GS Brenner RR 《Biochemical and biophysical research communications》2001,283(2):323-326
It has been recognized that rat liver microsomal Delta6 desaturation activity is defective in experimental diabetes, a fact that may be reverted by means of insulin treatment. In the present study, we used streptozotocin-induced diabetic rats in order to determine the regulatory role of insulin on the expression of hepatic Delta6 desaturase gene. The abundance of hepatic Delta6 desaturase mRNA in the diabetic rats is sevenfold lower than in the control. Insulin administration to diabetic rats induces Delta6 desaturase mRNA eightfold within 24 h. The effect of insulin on the Delta6 desaturase mRNA was inhibited 70% with dibutyryl-cAMP and theophylline administration and 90% by cycloheximide administration. Therefore, our data demonstrate that the activity of hepatic Delta6 desaturase in response to insulin is, at least in part, regulated by pretranslational events that require the synthesis of an unknown protein(s). 相似文献