Correlations in palmitoylation and multiple phosphorylation of rat bradykinin B2 receptor in Chinese hamster ovary cells.
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Rat bradykinin B2 receptor from unstimulated Chinese hamster ovary cells transfected with the corresponding cDNA has been isolated, and subsequent mass spectrometric analysis of multiple phosphorylated species and of the palmitoylation attachment site is described. Bradykinin B2 receptor was isolated on oligo(dT)-cellulose using N-(epsilon-maleimidocaproyloxy)succinimide-Met-Lys-bradykinin coupled to a protected (dA)30-mer. This allowed a one-step isolation of the receptor on an oligo(dT)-cellulose column via variation solely of salt concentration. After enzymatic in-gel digestion, matrix-assisted laser desorption ionization and electrospray ion trap mass spectrometric analysis of the isolated rat bradykinin B2 receptor showed phosphorylation at Ser365, Ser371, Ser378, Ser380, and Thr374. Further phosphorylation at Tyr352 and Tyr161 was observed. Rat bradykinin receptor B2 receptor is also palmitoylated at Cys356. All of the phosphorylation sites except for Tyr161 cluster at the carboxyl-terminal domain of the receptor located on the cytoplasmic face of the cell membrane. Surprisingly, many of the post-translational modifications were shown by MSn mass spectroscopic analysis to be correlated pairwise, e.g. diphosphorylation at Ser365 and Ser371, at Ser378 and Ser380, and at Thr374 and Ser380 as well as mutually exclusive phosphorylation at Tyr352 and palmitoylation at Cys356. The last correlation may be involved in a receptor internalization motif. Pairwise correlations and mutual exclusion of phosphorylation and palmitoylation suggest critical roles of multiple post-translational modifications for the regulation of activity, coupling to intracelluar signaling pathways, and/or sequestration of the bradykinin receptor. (+info)
Role of the cysteine-rich domain of the t-SNARE component, SYNDET, in membrane binding and subcellular localization.
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Wild-type syndet is efficiently recruited at the plasma membrane in transfected AtT-20 cells. A deletion at the cysteine-rich domain abolishes palmitoylation, membrane binding, and plasma membrane distribution of syndet. Syndet, SNAP-25A, and SNAP-25B share four cysteine residues, of which three, Cys2, Cys4, and Cys5, are absolutely conserved in all three homologs. Mutations at any pair of cysteines within cysteines 2, 4, and 5 shift syndet from the cell surface into the cytoplasm. Thus, at least two cysteines within the conserved triplet are necessary for plasma membrane localization. Syndet C1S/C3S, with substitutions at the pair Cys1 and Cys3, distributes to the plasma membrane, a Golgi-like compartment, and the cytosol. We conclude that Cys1 and Cys3 are not absolutely necessary for membrane binding or plasma membrane localization. Our results show that the cysteine-rich domain of syndet plays a major role in its subcellular distribution. (+info)
Total plasmalogens and O-(acylalkylglycerophosphoryl) ethanolamine from labelled hexadecanol and palmitate during hypoxia and anoxia in rat heart.
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By the use of the Langendorff technique, surviving isolated rat hearts were perfused with [1-14 C] palmitate, [1-14C] hexadecanol or [1-14C,1-3H] hexadecanol under normal or anoxic conditions. After perfusion for 30min with either precursor, when oxygenated or in an hypoxic condition, or when 1mM-KCN was included in the system, the heart tissues showed no significant chemical changes in their content of total lipids, total phospholipids or total ethanolamine-containing phospholipids. Changes were observed in the ratio of alkyl-to alk-1-enyl-glycerophosphorylethanolamine in the tissue perfused with N2+CO1 plus CN-. A slight increase from 4.0+/-0.3 to 4.9+/-0.2% in alkyl derivatives and a decrease from 11.2+/-0.4 to 9.4+/-0.3% in alk-1-enyl derivatives was observed. The incorporation of the [14C] palmitate and the [14C] hexadecanol into the recovered phospholipids and plasmalogens was severely decreased in the tissues perfused with CN-: in the hypoxic state only a mild inhibition was observed compared with the oxygenated systems. Considerable 3H from [1-14C, 1-3H] hexadecanol was retained (25-35%) in the alk-1-enylether chains of plasmalogens under both the oxygenated conditions and with CN-, suggesting that the same mechanism of incorporation is operational at high or low O2 concentrations. The results are consistent with an O2-dependent, CN-sensitive step in the biosynthesis of plasmalogens in the rat heart. (+info)
The glycerol phosphate, dihydroxyacetone phosphate and monoacylglycerol pathways of glycerolipid synthesis in rat adipose-tissue homogenates.
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1. Fat-free homogenates from the epididymal fat-pads of rats were used to measure the rate of palmitate esterification with different substrates. The effectiveness of the acyl acceptors decreased in the order glycerol phosphate, dihydroxyacetone phosphate, 2-octadecenyl-glycerol and 2-hexadecylglycerol. 2. Glycerol phosphate and dihydroxyacetone phosphate inhibited their rates of esterification in a mutually competitive manner. 3. The esterification of glycerol phosphate was also inhibited in a partially competitive manner by 2-octadecenylglycerol and to a lesser extent by 2-hexadecylglycerol. However, glycerol phosphate did not inhibit the esterification of 2-octadecenylglycerol. 4. The esterification of dihydroxyacetone phosphate and 2-hexadecylglycerol was more sensitive to inhibition by clofenapate than was that of glycerol phosphate. Norfenfluramine was more effective in inhibiting the esterification of 2-hexadecylglycerol than that of glycerol phosphate or dihydroxyacetone phosphate. 5 It is concluded that rat adipose tissue can synthesize glycerolipids by three independent routes. (+info)
Hepatic glucose cycling does not contribute to the development of hyperglycemia in Zucker diabetic fatty rats.
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Hepatic glucose cycling, whereby glucose is taken up by the liver, partially metabolized, then recycled to glucose, makes a substantial contribution to the development of hyperglycemia in IDDM. This stimulation of glucose cycling appears to be associated with elevated rates of fatty acid oxidation. Whether hepatic glucose cycling also contributes to the development of hyperglycemia in NIDDM is unclear. Using a model of NIDDM, the Zucker diabetic fatty (ZDF) rat, we determined whether glucose cycling was enhanced. Hepatocytes from ZDF rats exhibited higher rates of glucose phosphorylation and glycolysis, but there was no increase in the rate of cycling between glucose and glucose-6-phosphate or between glycolytically derived pyruvate and glucose. Despite the increased rates of glycolysis, the production of CO2 in liver cells from ZDF rats was no different from rates measured in cells from control animals. Instead, there was a large increase in the accumulation of lactate and pyruvate in the ZDF liver cells. The addition of 2-bromopalmitate, an inhibitor of fatty acid oxidation that inhibited glucose cycling in hepatocytes from IDDM rats, had no effect on glucose cycling in cells from ZDF rats. We therefore conclude that, unlike in IDDM, hepatic glucose cycling does not contribute to the development of hyperglycemia in the NIDDM Zucker rat. (+info)
The tammar wallaby, Macropus eugenii, has a ruminant-like digestive system which may make a significant concentration of amino acids and fatty acids available to the blastocyst via uterine fluids. Fluorescent and radioisotope analyses were performed to determine the rate of glutamine and palmitate use by blastocysts recovered on day 0, 3, 4, 5 and 10 after reactivation induced by removal of pouch young (RPY). Between day 0 and 4 glutamine uptake increased from 15.6 +/- 6.6 to 36.1 +/- 2.7 pmol per embryo h-1 (P < 0.01) and ammonium production increased from 8.2 +/- 4.3 to 26.6 +/- 3.0 pmol per embryo h-1 (P < 0.01). Glutamine oxidation did not increase until day 10 after RPY (P < 0.01), but the percentage of glutamine oxidized increased from 4.5 +/- 3.1% during diapause to 31.2 +/- 12.6% (P < 0.01) by day 5 after RPY and increased further to 51.0 +/- 15.8% (P < 0.01) by day 10 after RPY. Palmitate oxidation also increased from 0.3 +/- 0.1 by day 0 blastocysts to 3.8 +/- 1.7 pmol per embryo h-1 (P < 0.01) by day 4 blastocysts. This increase provides a greater potential for ATP production, possibly to supply increased demand due to the coincident resumption of mitoses. The ATP:ADP ratio within blastocysts had reduced by the time of the first measurement at day 3 (0.5 +/- 0.2 pmol per embryo h-1; P < 0.01) compared with day 0 blastocysts (1.4 +/- 0.3 pmol per embryo h-1). It is likely that metabolism of amino acids and fatty acids contributes to the energy supply during reactivation of tammar wallaby blastocysts after embryonic diapause. (+info)
The mechanism of inhibition of beta-oxidation by aspirin metabolites in skin fibroblasts from Reye's syndrome patients and controls.
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The effects of aspirin metabolites on beta-oxidation were studied in skin fibroblasts from eight typical Reye's syndrome (RS) patients and controls. RS patients' cells did not differ from controls in rates of palmitate oxidation or in the three component activities of the mitochondrial trifunctional enzyme (MTE), indicating no inherited beta-oxidation defect. Aspirin metabolites salicylate, hydroxyhippurate and gentisate, but not aspirin, directly inhibited palmitate oxidation in control and RS cells. RS cells were significantly more sensitive to inhibition than controls at 0.5 to 5 mM salicylate. Inhibition was concentration-dependent and reversible. Inhibition did not occur in fibroblasts lacking activity of the long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) activity of MTE. Salicylate was therefore inhibiting beta-oxidation at this step. Hydroxyhippurate and salicylate reversibly inhibited HAD activities in extracts of control and RS cells. Studies with pure short-chain HAD and LCHAD (MTE) showed hydroxyhippurate and salicylate were competitive inhibitors of the former but mixed (not competitive) inhibitors of the latter. Both compounds inhibited the combined, three-step, MTE reaction measured in the physiological direction. We conclude that (1) salicylate and hydroxyhippurate decrease beta-oxidation in intact cells by reversible inhibition of LCHAD activity of the MTE, and (2) beta-oxidation in RS cells is inherently more sensitive to inhibition by low concentrations of salicylate than controls. (+info)
Development and initial evaluation of a novel method for assessing tissue-specific plasma free fatty acid utilization in vivo using (R)-2-bromopalmitate tracer.
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We describe a method for assessing tissue-specific plasma free fatty acid (FFA) utilization in vivo using a non-beta-oxidizable FFA analog, [9,10-3H]-(R)-2-bromopalmitate (3H-R-BrP). Ideally 3H-R-BrP would be transported in plasma, taken up by tissues and activated by the enzyme acyl-CoA synthetase (ACS) like native FFA, but then 3H-labeled metabolites would be trapped. In vitro we found that 2-bromopalmitate and palmitate compete equivalently for the same ligand binding sites on albumin and intestinal fatty acid binding protein, and activation by ACS was stereoselective for the R-isomer. In vivo, oxidative and non-oxidative FFA metabolism was assessed in anesthetized Wistar rats by infusing, over 4 min, a mixture of 3H-R-BrP and [U-14C] palmitate (14C-palmitate). Indices of total FFA utilization (R*f) and incorporation into storage products (Rfs') were defined, based on tissue concentrations of 3H and 14C, respectively, 16 min after the start of tracer infusion. R*f, but not Rfs', was substantially increased in contracting (sciatic nerve stimulated) hindlimb muscles compared with contralateral non-contracting muscles. The contraction-induced increases in R*f were completely prevented by blockade of beta-oxidation with etomoxir. These results verify that 3H-R-BrP traces local total FFA utilization, including oxidative and non-oxidative metabolism. Separate estimates of the rates of loss of 3H activity indicated effective 3H metabolite retention in most tissues over a 16-min period, but appeared less effective in liver and heart. In conclusion, simultaneous use of 3H-R-BrP and [14C]palmitate tracers provides a new useful tool for in vivo studies of tissue-specific FFA transport, utilization and metabolic fate, especially in skeletal muscle and adipose tissue. (+info)