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PubMed1984

Acylation of bovine rhodopsin by [3H]palmitic acid.

Bovine retinas or preparations of rod outer segments incorporate [3H]palmitic acid into rhodopsin. The incorporation is both time- and temperature-dependent. The major product retains the chromatographic and electrophoretic properties of rhodopsin and remains photosensitive as demonstrated by alteration of its chromatographic behavior upon exposure to light. The incorporated radioactivity resists extraction with organic solvents and is not dissociated from the protein by detergents or under the denaturing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactive free fatty acid can, however, be released by alkaline hydrolysis. Hydroxylamine treatment yields a mixture of the free fatty acid and the fatty acyl hydroxamate. These results demonstrate the formation of an ester bond between [3H]palmitic acid and rhodopsin. Cycloheximide fails to inhibit the incorporation. This finding along with the ability of rod outer segments to support the incorporation point to the acylation of rhodopsin as a late post-translational event.

Acylation↗
PubMed1975

Uptake and compartmental distribution of fatty acid by rat small intestine in vivo.

The uptake and esterification of micellar [3-H]oleate and [14-C] palmitate were uniform along the entire length of the small intestine in vivo. Fatty acids (FA) radioactivity taken up by the small intestine could be described in terms of four functionally distinct compartments analogous to those described in vitro. The KRP-extractable compartment (KEC) and albumin-extractable compartment (AEC) contained reversibly adherent unesterified FA radioactivity, while the tissue free and esterified FA compartments contained irreversibly bound radioactivity. Wheras 27% and 63% of FA uptake were reversibly bound in the KEC and AEC by the most proximal and most distal regions of the small intestine in vitro (15), less than 10% was contained in these compartments in vivo, independent of location. Linear inverse relationships were found betweeen tissue FA esterification and proportion of FA radioactivity present in the KEC,AEC, and the tissue free FA compartment in vivo. These observations allow for the possibility that FA molecules pass through these compartments prior to esterification.

Animals↗
PubMed1972

Synthesis of fatty acids from (1- 14 C)acetyl-coenzyme A in subcellular particles of rat epididymal adipose tissue.

1. Mitochondrial and microsomal fractions of rat epididymal adipose tissue incorporated [1-(14)C]acetyl-CoA equally well into various fatty acids by a chain-elongation mechanism. C(18) and C(20) fatty acids were the two major products, and comprised about 80% of the total fatty acids synthesized in both particles. 2. When incubated in air, mitochondria synthesized stearic acid, octadecenoic acid and eicosamonoenoic acid in almost equal amounts (about 20% each), whereas in microsomal fractions, the synthesis of octadecenoic acid was more than fivefold the stearic acid formation. In both fractions, major components of synthesized monoenoic fatty acids were the Delta(11:12) isomers. Hexadecenoic acid and octadecenoic acid from whole adipose tissue contained approx. 11 and 14% of the Delta(11:12) isomer respectively. 3. When mitochondria or microsomal fractions were incubated in nitrogen, there was increased synthesis of stearic acid and palmitic acid and less of C(16) and C(18) monoenoic acids; synthesis of C(20) acids remained predominantly of the monoenoic acids. Determination of the position of the double bond in the monoenoic acids supported the view that the synthesis of hexadecenoic acid and octadecenoic acid involves a desaturase activity, whereas eicosamonoenoic acid and eicosadienoic acid are formed only by elongation of endogenous fatty acids. 4. Most of the radioactivity was found in free fatty acids (63%) and the phospholipid (26%) fraction. In phospholipids, phosphatidylcholine and phosphatidylethanolamine were the two major components. 5. Most of the fatty acids synthesized, including those not normally found in particle lipids (arachidic acid, eicosamonoenoic acid and eicosadienoic acid) were distributed fairly evenly in the phospholipid and free fatty acid fractions. However, stearic acid was found predominantly in the phospholipid fraction.

Adipose Tissue↗
PubMed1985

Incorporation of 14C acetate into lipids of the ICR mouse lens.

Lenses from ICR mice 6, 12 and 35 days of age were incubated with 14C acetate in Medium 199 for 10 hours, and lenses from 35-day-old mice were also incubated for 18 hours. Acetate was incorporated into the phospholipid and free fatty acid fractions. Minimal radioactivity was incorporated into cholesterol. Phosphatidyl choline was the major labeled phospholipid. The longer incubation time resulted in a decrease in the total amount of label retained, indicating a fatiguing of the incubation system. The free fatty acids were extracted and subjected to analysis by gas chromatography. Although myristic acid (14:0) accounted for only 4% to 6% of the free fatty acids, it had the highest specific activity and the greatest percentage of incorporated radioactivity of the fatty acids for the 10-hour incubation period. The specific activity of all the fatty acids decreased dramatically after 18 hours incubation. Palmitic acid (16:0) had the greatest incorporation of label. It was suggested that exogenous acetate in the mouse lens preferentially entered a pool for energy production or storage rather than a pool for the synthesis of structural lipids as evidenced by minimal incorporation into cholesterol.

Acetates↗
PubMed1981

In vitro conversion of saturated to monounsaturated fatty acid by Ehrlich ascites cells.

In this paper, evidence is presented on the capacity of Ehrlich ascites cells to synthesize in vitro monounsaturated fatty acids from radioactive palmitate. Localization of the double bond was determined by ozonolysis and subsequent reduction of the ozonides to aldesters followed by gas liquid chromatography. These results proved that Ehrlich ascites cells have a delta 9 desaturase that catalyzes the biosynthesis of palmitoleic acid from palmitic acid and oleic and vaccenic acid via elongation-desaturation and desaturation-elongation, respectively, using palmitic acid as substrate. Furthermore, it is shown that, as in the hepatic cells, delta 9 desaturase enzyme activity of the tumoral cells is associated with the endoplasmic reticulum. The electron transport components involved in the desaturase system, i.e., NADH-cytochrome b5 reductase and NADH-cytochrome c reductase, were also measured. The activities of these enzymes do not appear to be rate-limiting in the desaturase activity of these tumoral cells.

Animals↗
PubMed1986

'Dystrophic' lipid myopathy in two sisters.

Two sisters with progressive myopathy demonstrated microscopic and biochemical evidence of lipid storage in skeletal muscle. Their muscle biopsy specimens resembled those seen in Duchenne's muscular dystrophy and some of the biochemical features were similar, including increased muscle concentration of long-chain acyl-coenzyme A (a fatty oxidation intermediate) and decreased oxidation of radioactively labeled fatty acids by muscle homogenates in vitro. Although the site of the defect was not localized, the data suggested impairment of intramitochondrial beta-oxidation of fatty acids. These two patients may be important in understanding the pathogenesis of muscular dystrophy.

Child↗
PubMed1997

A phospholipase A2 kinetic and binding assay using phospholipid-coated hydrophobic beads.

A novel kinetic and membrane-binding assay for phospholipase A2 (PLA2) has been developed utilizing phospholipid-coated hydrophobic styrene-divinylbenzene beads (5.2 +/- 0.3 microm diameter). Phospholipids formed a stable monolayer film on styrene-divinylbenzene beads with average surface packing density of (1.3 +/- 0.2) x 10(-2) molecule/A2. Secretory PLA2 readily hydrolyzed 1-palmitoyl-2-[3H]-oleoyl-sn-glycero-3-phosphoglycerol coated on styrene-divinylbenzene beads which could be easily monitored by measuring the radioactivity of fatty acid released to solution in the presence of bovine serum albumin. For human cytosolic PLA2 with high specificity for sn-2 arachidonyl group, styrene-divinylbenzene beads coated with 1-stearoyl-2-[14C]-arachidonyl-sn-glycero-3-phosphocholine and dioleoylglycerol (7:3, mol/mol) were used as substrate. PLA2 activity was linearly proportional to the enzyme concentration in the range from 1 to 150 nM for human class II secretory PLA2 and from 1 to 20 nM for cytosolic PLA2; the specific activity was 1.6 and 1.7 micromol/min/mg, respectively. Finally, styrene-divinylbenzene beads coated with polymerized 1,2-bis[12-(lipoyloxy) dodecanoyl]-sn-glycero-3-phosphoglycerol were used to measure the membrane binding affinity of PLA2, which in conjunction with kinetic data provides important insights into how PLA2 interacts with membranes.

Animals↗
PubMed1977

p-Chlorophenoxyisobutyrate enhanced retention of homologous lipoproteins by human aortic smooth muscle cells.

Human aortic smooth muscle cells (SMC) specifically bind and take up indiscriminately both the lipid and protein moieties of homologous 25I-very low density lipoproteins (VLDL) and 125I-low density lipoproteins LDL). Sixty-five to 80% of absorbed lipids are incorporated into the cell lipids, preferentially into the phospholipid fraction. Twenty to 35% of the lipid bound and the protein moiety are eliminated from the cells. Half of the eliminated protein label is recovered as TCA soluble products. Five mM of p-chlorophenoxyisobutyrate (CPIB) raise the level of intracellular radioactivity derived from the lipid moieties of VLDL and LDL by about 40% via a reduced elimination. The processing of the protein moiety and lipoprotein binding to the cell surface are not affected by 5.0 mM of CPIB. CPIB lowers the incorporation of 14C-acetate, 14C-pyruvate, and 32phosphate radioactivity into fatty acids and phospholipids of aortic SMC. Five mM of CPIB reduce the overall palmitic acid synthesis by shifting from de novo synthesis to the mechanism of chain elongation, although the further elongation to saturated C18-C24 fatty acids is also depressed. The CPIB-enhanced retention of the lipid-derived lipoprotein radio-activity is interpreted as a compensatory mechanism providing cellular fatty acids which are deficient as a result of the CPIB inhibited synthetic processes.

Aorta, Thoracic↗
PubMed1979

Use of liposomes in probing the uptake of liposomal phosphatidylcholine by rabbit lung in vitro.

The purpose of this study was to characterize the uptake of liposomal phosphatidylcholine by lung tissue and its subcellular organelles. Multilamellar liposomes were prepared from egg yolk phosphatidylcholine, dicetyl phosphate, and cholesterol (molar ratio 7 : 2 : 1). Liposomal phosphatidylcholine labeled with [1-14C]dipalmitoyl phosphatidylcholine was taken up by lung slices and incorporated into subcellular organelles including lamellar bodies, mitochondria, and microsomes. In addition, when liposomes were incubated with lamellar bodies, mitochondria, or microsomes, the transfer of liposomal phosphatidylcholine to these subcellular fractions was facilitated by the cytosolic fraction. In tissue slice experiments after 1 h of incubation, about 86% of the total radioactivity absorbed by lung slices and subcellular organelles was recovered in phosphatidylcholine. The ratio of the radioactivity of fatty acids at 1- and 2-positions of dipalmitoyl phosphatidylcholine recovered from all fractions was nearly 1 : 1. This suggests that most phosphatidylcholine molecules were taken up intact. In conclusion, this study provides a method using liposomes as a tool for probing the phosphatidylcholine transfer mechanism in lung.

Animals↗
PubMed1984

Hyperlipidemia in tumor-bearing rats.

Hyperlipidemia occurs in animals bearing tumors but the mechanism of its development is uncertain. We have measured triacylglycerol clearance and production rate in rats bearing a transplantable sarcoma. The plasma content of very-low-density lipoprotein triacylglycerol was increased in these tumor-bearing rats but our data excluded a primary clearance defect because the rate of triacylglycerol accumulation (mg/min) after Triton injection was equal to or greater than in normal control rats, except in cachectic rats with very large tumors. The fractional clearance of injected radioactive triacylglycerols was less in tumor-bearing rats than in controls, but the turnover (mg/min) was probably not decreased in the tumor-bearing rats because of their expanded plasma pool. Also inconsistent with a decreased turnover was our finding of a greater production of radioactive plasma triacylglycerols after injection of a tracer dose of radioactive free fatty acid, and unchanged production in Triton-treated rats. Therefore, in the fasted state, the hyperlipidemia of the tumor-bearing rats was associated with an unchanged or possibly an increased flux of hepatic triacylglycerols and a primary clearance defect was excluded. After fat-feeding, rats with tumors developed a higher post-prandial hyperlipidemia than control rats. Therefore, the clearance mechanism for the plasma triacylglycerols was close to saturation in the fasted state, and the added influx of exogenous triacylglycerols was removed less efficiently in the tumor-bearing rats.

Animals↗
PubMed1989

Synthesis of glutamate and glutamine in dibutyryl cyclic AMP-treated astrocytes.

The relative contributions of radioactively labeled fatty acids and glucose to synthesis of glutamate and glutamine were compared in native and dibutyryl cyclic AMP (diBcAMP)-treated primary rat astrocytes. The intracellular specific activities of glutamate and glutamine were 10-fold greater than the specific activities of aspartate or alanine. Butyrate, octanoate and palmitate were equally as effective as precursors for glutamate and glutamine while glucose was 50% as effective as the fatty acids. The specific activity of glutamate and glutamine were identical in the absence of diBcAMP. In diBcAMP treated cells the specific activity of glutamine was greater than that of glutamate when octanoate and palmitate were the labeled precursors. This suggests that cultured astrocytes preferentially utilize free fatty acids for glutamate/glutamine synthesis and that diBcAMP-treated astrocytes contain more than one glutamate compartment.

Animals↗
PubMed1980

Labelling in vivo and in vitro of molecular species of lipids from chloroplast envelopes and thylakoids.

Lipid mixtures from chloroplast envelope and thylakoid membrane were isolated after different labelling times in vivo and in vitro and separated into major components. The isolated compounds were subjected to analyses such as separation of molecular species, determination of radioactivity in fatty acids and water-soluble hydrolysis products and radio gas-liquid chromatography of fatty acid mixtures. In the case of monogalactosyl and digalactosyl diacylglycerol these procedures were also applied to several individual molecular species. To investigate the extent of de novo synthesis these species were also used for methylation studies and their fatty acids subjected to alpha-oxidation. In envelope membranes diacylglycerols and monogalactosyl diacylglycerols may each be separated into several distinct and non-mixing pools. Molecules made de novo with oligoene fatty acids are very efficient substrates for galactosylation in vivo. The time-dependent changes in patterns of galactolipid molecular species may indicate a desaturation of acyl chains operating in close contact to intact lipids. After isolation, envelopes incorporated UDP-[14C]galactose into completely different patterns of galactolipids and molecular species pointing to changed properties of this membrane system or to a loss of regulatory factors.

Chloroplasts↗
PubMed1988

Regulation of the metabolism of lipid emulsion model lipoproteins by a saturated acyl chain at the 2-position of triacylglycerol.

Lipid emulsion particles were prepared by sonicating four different lipid mixtures (triacylglycerol (TAG), 70%; phospholipid, 25%; cholesteryl oleate (CO), 3%; and free cholesterol, 2%), then purified by density gradient ultracentrifugation. For three test mixtures, the TAG contained 50, 75, or 100% 1,3-dioleyl-2-stearylglycerol (OSO) with the remainder being triolein (OOO); 100% triolein in the lipid mixture was used as the control. After intravenous injection of the lipid particles into unanesthetized rats, removal of radioactive TAG fatty acid and CO from plasma was measured for 30 min, then liver and spleen uptakes were measured. When emulsions contained 75% or 100% OSO as TAG, the plasma removal rates of CO were, respectively, 60% or 30% of the rate when the TAG was 100% triolein; smaller recoveries of CO were found in the liver. The clearances of TAG fatty acid did not differ significantly and the recoveries of TAG fatty acid in the organs were not affected by the type of emulsion injected. Remnant particles were derived from donor rats in which uptake was blocked by exclusion of liver and other viscera from the circulation before injection of 100% OOO and 100% OSO emulsions. When injected into recipient intact rats, the removal of remnants from plasma was slower for remnants derived 15 min after injection of 100% OSO emulsions than from 100% OOO emulsions, showing that the slower removal of emulsion CO was due to slower remnant uptake from the plasma with OSO emulsions.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMedDate not supplied

[Metabolic effects of ethanol and lipid metabolism in the rat liver].

Effect of ethanol intoxication of lipid metabolism in rat liver tissue was studied after daily intraperitoneal administration of 2g ethanol per kg of body mass within a week. Administration of ethanol led to an increase in content of total lipids, cholesterol and triacylglycerides in liver tissue as well as to elevation of relative content of lysophosphatidyl choline and cardiolipin and to a decrease of phosphatidyl choline. Under conditions of the intoxication incorporation of I-14C-acetate into triacylglycerides, phosphatidyl choline and phosphatidyl ethanolamine was decreased mainly as a result of a decrease in specific radioactivity of fatty acids of these lipids.

Alcoholic Intoxication↗
PubMed1982

Specific, saturable binding and uptake of rat chylomicron remnants by rat skin fibroblasts.

To investigate the possible contribution of chylomicron remnants to the accumulation of cholesterol in non-hepatic tissues, rat chylomicron remnants were incubated with rat skin fibroblasts. The binding of remnants was saturable and specific. Native, undegraded chylomicrons were almost as effective as unlabeled remnants in displacing the uptake of labeled remnants. Rat low density and high density lipoproteins were relatively ineffective in displacing the uptake of labeled remnants. Accumulation of radioactive unesterified fatty acids occurred in proportion to the uptake of labeled remnants, indicating probable internalization and degradation of the particles after binding. The incorporation of added [14C]acetate into cell non-saponifiable lipids was not significantly suppressed by added remnants, indicating an apparent lack of feedback regulation of cholesterol biosynthesis after remnant uptake. Our results show that the physiological mechanism underlying uptake of remnants by hepatic parenchymal cells might not be accounted for by tissue or cellular specificity, but may perhaps arise because of the lower capillary permeability of extra-hepatic sites compared with the hepatic sinusoid.

Animals↗