Delta3,5,7,Delta2,4,6-trienoyl-CoA isomerase, a novel enzyme that functions in the beta-oxidation of polyunsaturated fatty acids with conjugated double bonds.
(17/3819)
The mitochondrial metabolism of unsaturated fatty acids with conjugated double bonds at odd-numbered positions, e.g. 9-cis, 11-trans-octadecadienoic acid, was investigated. These fatty acids are substrates of beta-oxidation in isolated rat liver mitochondria and hence are expected to yield 5,7-dienoyl-CoA intermediates. 5, 7-Decadienoyl-CoA was used to study the degradation of these intermediates. After introduction of a 2-trans-double bond by acyl-CoA dehydrogenase or acyl-CoA oxidase, the resultant 2,5, 7-decatrienoyl-CoA can either continue its pass through the beta-oxidation cycle or be converted by Delta3,Delta2-enoyl-CoA isomerase to 3,5,7-decatrienoyl-CoA. The latter compound was isomerized by a novel enzyme, named Delta3,5,7,Delta2,4, 6-trienoyl-CoA isomerase, to 2,4,6-decatrienoyl-CoA, which is a substrate of 2,4-dienoyl-CoA reductase (Wang, H.-Y. and Schulz, H. (1989) Biochem. J. 264, 47-52) and hence can be completely degraded via beta-oxidation. Delta3,5,7,Delta2,4,6-Trienoyl-CoA isomerase was purified from pig heart to apparent homogeneity and found to be a component enzyme of Delta3,5,Delta2,4-dienoyl-CoA isomerase. Although the direct beta-oxidation of 2,5,7-decatrienoyl-CoA seems to be the major pathway, the degradation via 2,4,6-trienoyl-CoA makes a significant contribution to the total beta-oxidation of this intermediate. (+info)
Genetic engineering of the unsaturation of fatty acids in membrane lipids alters the tolerance of Synechocystis to salt stress.
(18/3819)
The role of unsaturated fatty acids in membrane lipids in the tolerance of the photosynthetic machinery to salt stress was studied by comparing the desA-/desD- mutant of Synechocystis sp. PCC 6803, which contained monounsaturated fatty acids, with the wild-type strain, which contained a full complement of polyunsaturated fatty acids. In darkness, the loss of oxygen-evolving photosystem II activity in the presence of 0.5 M NaCl or 0.5 M LiCl was much more rapid in desA-/desD- cells than in wild-type cells. Oxygen-evolving activity that had been lost during incubation with 0.5 M NaCl in darkness returned when cells were transferred to conditions that allowed photosynthesis or respiration. Recovery was much greater in wild-type than in desA-/desD- cells, and it was prevented by lincomycin. Thus, the unsaturation of fatty acids is important in the tolerance of the photosynthetic machinery to salt stress. It appears also that the activity and synthesis of the Na+/H+ antiporter system might be suppressed under high-salt conditions and that this effect can be reversed, in part, by the unsaturation of fatty acids in membrane lipids. (+info)
Isolation and characterization of the cis-trans-unsaturated fatty acid isomerase of Pseudomonas oleovorans GPo12.
(19/3819)
Pseudomonas oleovorans contains an isomerase which catalyzes the cis-trans conversion of the abundant unsaturated membrane fatty acids 9-cis-hexadecenoic acid (palmitoleic acid) and 11-cis-octadecenoic acid (vaccenic acid). We purified the isomerase from the periplasmic fraction of Pseudomonas oleovorans. The molecular mass of the enzyme was estimated to be 80 kDa under denaturing conditions and 70 kDa under native conditions, suggesting a monomeric structure of the active enzyme. N-terminal sequencing showed that the isomerase derives from a precursor with a signal sequence which is cleaved from the primary translation product in accord with the periplasmic localization of the enzyme. The purified isomerase acted only on free unsaturated fatty acids and not on esterified fatty acids. In contrast to the in vivo cis-trans conversion of lipids, this in vitro isomerization of free fatty acids did not require the addition of organic solvents. Pure phospholipids, even in the presence of organic solvents, could not serve as substrate for the isomerase. However, when crude membranes from Pseudomonas or Escherichia coli cells were used as phospholipid sources, a cis-trans isomerization was detectable which occurred only in the presence of organic solvents. These results indicate that isolated membranes from Pseudomonas or E. coli cells must contain factors which, activated by the addition of organic solvents, enable and control the cis-trans conversion of unsaturated acyl chains of membrane phospholipids by the periplasmic isomerase. (+info)
Role of group II secretory phospholipase A2 in atherosclerosis: 2. Potential involvement of biologically active oxidized phospholipids.
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Secretory nonpancreatic phospholipase A2 (group II sPLA2) is induced in inflammation and present in atherosclerotic lesions. In an accompanying publication we demonstrate that transgenic mice expressing group II sPLA2 developed severe atherosclerosis. The current study was undertaken to determine whether 1 mechanism by which group II sPLA2 might contribute to the progression of inflammation and atherosclerosis is by increasing the formation of biologically active oxidized phospholipids. In vivo measurements of bioactive lipids were performed, and in vitro studies tested the hypothesis that sPLA2 can increase the accumulation of bioactive phospholipids. We have shown previously that 3 oxidized phospholipids derived from the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) stimulated endothelial cells to bind monocytes, a process that is known to be an important step in atherogenesis. We now show that these 3 biologically active phospholipids are significantly increased in livers of sPLA2 transgenic mice fed a high-fat diet as compared with nontransgenic littermates. We present in vitro evidence for several mechanisms by which these phospholipids may be increased in sPLA2 transgenics. These studies demonstrated that polyunsaturated free fatty acids, which are liberated by sPLA2, increased the formation of bioactive phospholipids in LDL, resulting in increased ability to stimulate monocyte-endothelial interactions. Moreover, sPLA2-treated LDL was oxidized by cocultures of human aortic endothelial cells and smooth muscle cells more efficiently than untreated LDL. Analysis by electrospray ionization-mass spectrometry revealed that the bioactive phospholipids, compared with unoxidized PAPC, were less susceptible to hydrolysis by human recombinant group II sPLA2. In addition, HDL from the transgenic mice and human HDL treated with recombinant sPLA2 in vitro failed, in the coculture system, to protect against the formation of biologically active phospholipids in LDL. This lack of protection may in part relate to the decreased levels of paraoxonase seen in the HDL isolated from the transgenic animals. Taken together, these studies show that levels of biologically active oxidized phospholipids are increased in sPLA2 transgenic mice; they also suggest that this increase may be mediated by effects of sPLA2 on both LDL and HDL. (+info)
Signal transduction in spontaneous myogenic tone in isolated arterioles from rat skeletal muscle.
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OBJECTIVE: The mechanism of spontaneous myogenic tone was investigated in isolated arteriolar segments. METHODS: Arterioles were isolated from rat cremaster muscle. Segments were endothelium-denuded and mounted in a pressure myograph at 75 mmHg. Under this condition, segments spontaneously constricted from a passive diameter of 167 +/- 3 to 82 +/- 4 microns (n = 41). The effects of several inhibitors were tested on the maintenance of myogenic tone. RESULTS: Gadolinium (10(-6)-10(-4) M), a putative inhibitor of stretch-activated cation channels, was ineffective. The phospholipase C (PLC) inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC) induced a dose-dependent inhibition of tone. NCDC inhibited phenylephrine- (10(-6) M), but not potassium buffer-induced (100 mM) constriction. The protein kinase C (PKC) inhibitors staurosporine, chelerythrine and calphostin C inhibited myogenic tone in a concentration-dependent manner. At an intermediate concentration, calphostin C selectively inhibited phenylephrine-induced constriction. However, all PKC inhibitors abolished responses to phenylephrine and potassium buffer at higher concentrations. The cytochrome P450 inhibitor 17-ODYA (0.3-3 x 10(-6) M) did not inhibit myogenic tone. CONCLUSIONS: No evidence was found for a role of gadolinium-sensitive, stretch-activated cation channels or cytochrome P450 metabolites. On the other hand, both PLC and PKC contribute to the maintenance of myogenic tone. (+info)
Pentobarbital-sensitive EDHF comediates ACh-induced arteriolar dilation in the hamster microcirculation.
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It is unclear to what extent the endothelium-derived hyperpolarizing factor (EDHF) contributes to the control of microcirculatory blood flow in vivo. We analyzed, by intravital microscopy in hamster muscles, the potential role of EDHF along the vascular tree under stimulated (ACh) or basal conditions. Experiments were performed in conscious as well as anesthetized (pentobarbital, urethan) animals. Additionally, cellular effects of the potential EDHF were studied in isolated small arteries. In pentobarbital-anesthetized animals, treatment with Nomega-nitro-L-arginine (L-NNA; 30 micromol/l) and indomethacin (3 micromol/l) reduced the dilation in response to 10 micromol/l ACh from 60 +/- 6 to 20 +/- 4%. This nitric oxide/prostaglandin-independent dilation (NPID), which was of a similar magnitude in large and small arterioles, was abolished by potassium depolarization or charybdotoxin (ChTX, 1 micromol/l) but not by glibenclamide. In conscious animals, NPID amounted to 33 +/- 3%. The inhibitor of the P-450 monooxygenase 17-octadecynoic acid (ODYA) reduced NPID further to 9 +/- 4%. ChTX abolished the NPID and also reduced basal diameters (by -11 +/- 3%). The induction of anesthesia with pentobarbital reduced NPID (to 12 +/- 6%), whereas urethan anesthesia was without effect. Pentobarbital also reduced the ACh-induced hyperpolarization of vascular smooth muscle in isolated arteries, whereas ChTX abolished it. This study suggests that a considerable part of the ACh dilation in the microcirculation is mediated by EDHF, which also contributes to the control of basal tone in conscious animals. The direct inhibitory effect of pentobarbital and ODYA supports the idea that "microcirculatory" EDHF is a product of the cytochrome P-450 pathway. The role of EDHF might be underestimated in pentobarbital-anesthetized animals. (+info)
cis/trans isomerase of unsaturated fatty acids of Pseudomonas putida P8: evidence for a heme protein of the cytochrome c type.
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From a pool of 600 temperature-sensitive transposon mutants of Pseudomonas putida P8, 1 strain was isolated that carries a mini-Tn5 insertion within the cytochrome c operon. As a result, genes involved in the attachment of heme to cytochrome c-type proteins are turned off. Accordingly, cytochrome c could not be detected spectrophotometrically. The mutant also exhibited a remarkable reduction of cis-trans isomerization capability for unsaturated fatty acids. Consistent with the genetic and physiological data is the detection of a cytochrome c-type heme-binding motif close to the N terminus of the predicted polypeptide of the cis/trans isomerase (cti) gene (CVACH; conserved amino acids in italics). The functional significance of this motif was proven by site-directed mutagenesis. A possible mechanism of heme-catalyzed cis-trans isomerization of unsaturated fatty acids is discussed. (+info)
Metabolic imbalance and sporulation in an isocitrate dehydrogenase mutant of Bacillus subtilis.
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A Bacillus subtilis mutant with a deletion in the citC gene, encoding isocitrate dehydrogenase, the third enzyme of the tricarboxylic acid branch of the Krebs cycle, exhibited reduced growth yield in broth medium and had greatly reduced ability to sporulate compared to the wild type due to a block at stage I, i.e., a failure to form the polar division septum. In early stationary phase, mutant cells accumulated intracellular and extracellular concentrations of citrate and isocitrate that were at least 15-fold higher than in wild-type cells. The growth and sporulation defects of the mutant could be partially bypassed by deletion of the major citrate synthase gene (citZ), by raising the pH of the medium, or by supplementation of the medium with certain divalent cations, suggesting that abnormal accumulation of citrate affects survival of stationary-phase cells and sporulation by lowering extracellular pH and chelating metal ions. While these genetic and environmental alterations were not sufficient to allow the majority of the mutant cell population to pass the stage I block (lack of asymmetric septum formation), introduction of the sof-1 mutant form of the Spo0A transcription factor, when coupled with a reduction in citrate synthesis, restored sporulation gene expression and spore formation nearly to wild-type levels. Thus, the primary factor inhibiting sporulation in a citC mutant is abnormally high accumulation of citrate, but relief of this metabolic defect is not by itself sufficient to restore competence for sporulation. (+info)