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.     

Unsaturated fatty acids promote proliferation via ERK1/2 and Akt pathway in bovine mammary epithelial cells

GPR40 has recently been identified as a G protein-coupled cell-surface receptor for long-chain fatty acids (LCFAs). The mRNA of the bovine ortholog of GPR40 (bGPR40) was detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and in the bovine mammary gland at various stages of lactation. Oleate and linoleate caused an increase in intracellular Ca²⁺ concentrations in these cells, and significantly reduced forskolin-induced cAMP concentrations. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and Akt kinase, which regulates cell proliferation and survival, was rapidly increased by oleate. Incubation with oleate and linoleate for 24 h significantly promoted cell proliferation. Moreover, in serum-free medium, oleate significantly stimulated cell proliferation during a 7-day culture. These results suggest that bGPR40 mediates LCFA signaling in mammary epithelial cells and thereby plays an important role in cell proliferation and survival.     

发酵法生产长链二元酸研究进展

发酵法生产长链二元酸相对于化工法而言有着无可比拟的优势。本文综述了发酵法生产长链二元酸的微生物源、产酸机理、产酸条件和产物分离技术等方面的研究进展 ,并简要介绍了其工业应用前景。     

Long-chain acyl-CoA hydrolase in the brain

Summary. Long-chain acyl-CoA hydrolases are a group of enzymes that cleave acyl-CoAs into fatty acids and coenzyme A (CoA-SH). Because acyl-CoAs participate in numerous reactions encompassing lipid synthesis, energy metabolism and regulation, modulating intracellular levels of acyl-CoAs would affect cellular functions. Therefore, acyl-CoA synthetases have been intensively studied. In contrast, acyl-CoA hydrolases have been less investigated, especially in the brain despite the fact that its long-chain acyl-CoA hydrolyzing activity is much higher than that in any other organ in the body. However, recent studies have dissected the multiplicity of this class of enzymes on a genomic basis, and have allowed us to discuss their function. Here, we describe a cytosolic long-chain acyl-CoA hydrolase (referred to as BACH) that is constitutively expressed in the brain, comparing it with other acyl-CoA hydrolases found in peripheral organs that have a role in fatty acid oxidation.     

A new,simple assay for long-chain acyl-CoA dehydrogenase in cultured skin fibroblasts using stable isotopes and GC-MS

In this paper, we present a new method for measurement of long-chain acyl-CoA dehydrogenase (LCAD) activities in cultured skin fibroblasts. The method is based upon gas chromatographic/mass spectrometric determination of 3-OH-hexadecanoic acid formed during incubation of fibroblasts in a medium containing palmitoyl-CoA and crotonase, to convert the enoyl-CoA ester produced into the 3-hydroxyacyl-CoA ester. The validity of the method demonstrated by the finding of a full deficiency of LCAD in fibroblasts from three patients with an established deficiency of LCAD.     

Diversity of dioxygenases that catalyze the first step of oxidation of long-chain n-alkanes in Acinetobacter sp. M-1

Abstract Three kinds of enzymes, designated A, B and C, involved in n -alkane oxidation were found in the cytoplasm of n -alkanegrown Acinetobacter sp. M-1. All catalyzed the dioxygenation of n -alkanes to the corresponding n -alkyl hydroperoxides. Purified enzyme A consisted of four identical subunits having a molecular mass of 72 kDa. The enzyme was strongly inhibited by several iron-chelating agents, such as o -phenanthroline, 8-hydroxyquinoline and α,α'-dipyridyl, and could be distinguished from enzyme C, a Cu²⁺-requiring flavoprotein. Enzyme B was relatively unstable on purification. The three enzymes used n -alkanes, n -alkenes, and aryl compounds with longer alkyl side chains as substrates. Enzymes B and C were more active toward relatively short n -alkanes (C_12–16). Enzyme A oxidized solid n -alkanes well, the most preferable substrate being tetracosane (C₂₄). Enzyme A is responsible for about 80% of the total activity found in the soluble fraction of n -alkane-grown Acinetobacter sp. M-1, indicating that the enzyme plays a major role during growth on solid n -alkanes.     

Biosynthesis of Long-Chain Alcohols by Developing and Regenerating Rat Sciatic Nerve

Abstract: Cell-free preparations of rat sciatic nerve were found to catalyze the reduction of fatty acid to alcohol in the presence of NADPH as reducing cofactor. The reductase was membrane-bound and associated primarily with the microsomal fraction. When fatty acid was the substrate, ATP, coenzyme A (CoA), and Mg²⁺ were required, indicating the formation of acyl CoA prior to reduction. When acyl CoA was used as substrate, the presence of albumin was required to inhibit acyl CoA hydro-lase activity. Fatty acid reductase activity was highest with palmitic and stearic acids, and somewhat lower with lauric and myristic acids. It was inhibited by sulfhydryl reagents, indicating the participation of thiol groups in the reduction. Only traces of long-chain aldehyde could be detected or trapped as semicarbazone. Fatty acid reductase activity in rat sciatic nerve was highest between the second and tenth days after birth and decreased substantially thereafter. Microsomal preparations of sciatic nerve from 10-day-old rats exhibited about four times higher fatty acid reductase activity than brain or spinal cord microsomes from the same animals. Wallerian degeneration and regeneration of adult rat sciatic nerve resulted in enhanced fatty acid reductase activity, which reached a maximum at about 12 days after crush injury.     

Biological decomposition of dichloromethane from a chemical process effluent

The application of specialized microorganisms to treat dichloromethane (DM) containing process effluents was studied. An aerobic fluidized bed reactor with a working volume of 801 filled with sand particles as carriers for the bacteria was used. Oxygen was introduced into the recycle stream by an injector device. DM was monitored semi-continuously. A processor controlled the feed volume according to the DM effluent concentration. Mineralization rates of 12 kg DM/m_bioreactor ³ · d were reached within about three weeks using synthetic wastewater containing 2000 mg/l DM as single carbon compound. DM from process water of a pharmaceutical plant was reduced from about 2000 mg/l in the feed to below 1 mg/l in the effluent at volumetric loading rates of 3 to 4 kg DM/m_bioreactor ³ · d. Degradation of wastewater components like acetone and isopropanol were favoured, thus making the process less attractive for waste streams containing high amounts of DOC other than of DM. DM concentrations of up to 1000 mg/l were tolerated by the immobilized microorganisms and did not influence their DM degradation capacity. The ability to mineralize DM was lost when no DM was fed to the reactor for 10 days.     

Evidence for two fatty alcohol oxidases in the biosurfactant-producing yeast Candida (Torulopsis) bombicola

Fatty alcohol oxidase activities in Candida (Torulopsis) bombicola ATCC 22214, which produces large amounts of glycolipids consisting of omega- and (omega-1)-hydroxyfatty acids and a sugar moiety, occurred only in the microsomal fraction whether the cells had grown on n-alkanes, carbohydrates or a mixture of the two. High activities occurred in glucose-grown cells. Aliphatic alcohols from octanol to hexadecanol were oxidized with two maxima in activities for decanol and for tetradecanol. Differences in their pH optima and in temperature stability suggest two separate enzymes are present. Long chain diols, but not omega-hydroxyfatty acids, were also oxidized.     

Alcohol oxidase of Aspergillus flavipes grown on hexadecanol

Abstract: The activity of alcohol oxidase in Aspergillus flavipes was induced by growth on hexadecanol, though highest activities were obtained using a mixture of hexadecanol and olive oil. The enzyme showed a wide range of substrate specificity towards aliphatic primary alcohols from C₈ to C₁₈. The preferred substrate was decanol. The enzyme had an optimum pH of 9.5. It also used cis -unsaturated alcohols better than the trans -isomers. ω-Hydroxy fatty acids and α,ω-diols were not attacked.