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PubMed1999

Cysteine 29 is the major palmitoylation site on stomatin.

The 31 kDa membrane protein stomatin was metabolically labeled with tritiated palmitic acid in the human amniotic cell line UAC and immunoprecipitated. We show that the incorporated palmitate is sensitive to hydroxylamine, indicating the binding to cysteine residues. Stomatin contains three cysteines. By expressing a myc-tagged stomatin and substituting the three cysteines by serine, individually or in combination, we demonstrate that Cys-29 is the predominant site of palmitoylation and that Cys-86 accounts for the remaining palmitate labeling. Disruption of Cys-52 alone does not show any detectable reduction of palmitic acid incorporation. Given the organization of stomatin into homo-oligomers, the presence of multiple palmitate chains is likely to increase greatly the affinity of these oligomers for the membrane and perhaps particular lipid domains within it.

Binding Sites↗

PubMed1990

Acylation of exogenous glycosylsphingosines by intact neuroblastoma (NCB-20) cells.

Acylation of exogenously added galactosylsphingosine was demonstrated in intact NCB-20 neuroblastoma cells, a cell line that normally does not synthesize galactosylceramide. Labeling of cells with [3H]palmitic acid for 6 h in the presence of 100 microM exogenous galactosylsphingosine (GalSph) resulted in a more than 3-fold increase in the incorporation of label into the ceramide monohexoside fraction relative to controls. This increase, which was almost entirely due to the incorporation of labeled nonhydroxy fatty acid into galactosylceramide, was linear over a concentration range of 1-100 microM galactosylsphingosine and for the first 5 h after the addition of galactosylsphingosine. Similarly, the addition of 100 microM glucosylsphingosine resulted in a 3-fold increase of label incorporated into glucosylceramide. Incubation of cells with 100 microM GalSph and labeled fatty acids of various chain lengths revealed that the acylation of GalSph was specific for medium chain (C16-C18) nonhydroxy fatty acids, suggesting that this was an enzyme-mediated reaction. The enzymatic nature of GalSph acylation was further demonstrated when cells were incubated for 72 h with 15 microM [3H]galactosylsphingosine labeled in the galactose moiety. [3H]Galactosylceramide containing only medium chain non-hydroxy fatty acids accumulated linearly with time reaching a maximum at 48 h and was observed to be further metabolized to ceramide dihexoside. This acylation reaction may be potentially important for the removal of glycosylsphingosines in the cell.

Acylation↗

PubMed1995

Dietary fat alters the distribution of cholesterol between vesicles and micelles in hamster bile.

The type of dietary fat strongly affects the incidence of gallstones in the hamster model of cholesterol cholelithiasis. The present study was designed to determine whether dietary fats could affect gallstone formation by altering the microstructure (vesicular/micellar ratio) of cholesterol in bile. Golden Syrian hamsters from Sasco (Omaha, NE) or Charles River (Wilmington, MA) were fed nutritionally adequate semipurified diets to which were added: (i) 4.0% butterfat without added cholesterol; (ii) 1.2% palmitic acid plus 0.3% cholesterol; or (iii) 4.0% safflower oil plus 0.3% cholesterol. Gallstone incidence and the percentage of cholesterol in vesicles and micelles were determined after two- or six-week feeding periods. Three out of ten Sasco hamsters fed the 1.2% palmitic acid diet for two weeks had cholesterol stones, while none of the eight Charles River animals had stones. In the Sasco hamsters, a significant proportion of the biliary cholesterol was found in void volume vesicles (28.8%) and small vesicles (17.1%); Charles River hamsters had negligible proportions (1.1%) of cholesterol in void volume vesicles and 15.4% in small vesicles. Cholesterol gallstones were most abundant in Sasco hamsters fed 1.2% palmitic acid for six weeks (nine out of ten animals); the mean cholesterol saturation index of the bile was 1.27. A significant proportion of the biliary cholesterol was eluted in the void volume vesicles (21.4%) and in small vesicles (15.0%). Five of the eight identically treated Charles River hamsters had cholesterol stones; the cholesterol saturation index averaged 1.36, and the biliary cholesterol was present in void volume vesicles (31.3%) and small vesicles (14.3%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1980

Thermally induced heterogeneity in microsomal membranes of fatty acid-supplemented Tetrahymena: lipid composition, fluidity and enzyme activity.

Thermally induced phase separation was observed to occur in microsomal membranes of the ciliate Tetrahymena pyriformis, using the technique of freeze-fracture electron microscopy. In the present study, we attempted to fractionate the phase-separated membranes which were produced by chilling cells by sucrose density gradient centrifugation. When Tetrahymena was grown in the presence of palmitic acid, cells rapidly incorporated the fatty acid into their phospholipids. The resulting endoplasmic reticulum containing a high level of palmitic acid was more susceptible to thermotropic phase separation. Despite the profound alterations in the fatty acid composition, the cells retained normal growth rate, appearance and cell motility. Smooth microsomes isolated from palmitic acid-supplemented Tetrahymena cells were sonicated and then fractionated into three major subfractions. Fraction-I with lower buoyant density was rich in phospholipids and saturated fatty acids, while Fraction-III with higher density was rather rich in proteins and contained more unsaturated fatty acids in the phospholipids. A significant change was also observed in the polar head composition of phospholipids in these fractions. ESR analysis demonstrated that the extracted lipids from Fraction-III were more fluid than those from Fraction-I. In addition, the motion of the spin probe in the native membranes was more restricted than in extracted lipids. These results indicate that the lipid phase separation causes "squeezing out" of the membrane proteins from the less fluid to the fluid areas. Furthermore, we examined the temperature dependence of the activities of glucose-6-phosphatase and palmitoyl CoA desaturase.

Animals↗

PubMed1988

Kinetics of long-chain (sphingoid) base biosynthesis in intact LM cells: effects of varying the extracellular concentrations of serine and fatty acid precursors of this pathway.

Serine palmitoyltransferase (EC 2.3.1.50) catalyzes the condensation of L-serine and palmitoyl-CoA to yield 3-ketosphinganine in the first unique reaction of long-chain (sphingoid) base biosynthesis. The kinetic effects of changing the extracellular concentrations of the precursors for this pathway were studied with LM cells by following the incorporation of L-[3-14C]serine into the long-chain base (i.e., sphinganine and sphingenine) backbones of complex sphingolipids. [14C]Serine was taken up by the cells and rapidly reached steady-state concentrations similar to those of the medium. From the cellular [14C]serine concentrations and specific activities, the apparent Vmax [14 pmol min-1 (10(6) cells)-1] and Km (0.23 mM) values for long-chain base synthesis were determined and found to be essentially identical with those for serine palmitoyltransferase assayed in vitro [i.e., 13 pmol min-1 (10(6) cells)-1 and 0.27 mM, respectively]. The other precursor, palmitic acid, was also taken up rapidly and increased long-chain base biosynthesis in a concentration-dependent manner. This effect was limited to palmitic acid and matched the known specificity of serine palmitoyltransferase for saturated fatty acyl-CoA's of 16 +/- 1 carbon atoms. These studies delineate the influence of extracellular precursors on the formation of the sphingolipid backbone and suggest that the kinetic properties of serine palmitoyltransferase govern this behavior of long-chain base synthesis in intact cells.

Acyltransferases↗

PubMed1986

The role of the hepatocellular redox state in the hepatic triglyceride accumulation following acute ethanol administration.

The role of the increased hepatocellular redox-state [( NADH]/[NAD+] ratio) as a mechanism underlying hepatic triglyceride deposition after acute ethanol dosing has been investigated in the rat. Following a single dose of ethanol (2 g/kg i.p.) in fasted rats, increases were observed at 1.5 hr in the hepatic [lactate]/[pyruvate] (133%), [3-hydroxybutyrate]/[acetoacetate] (69%) ratios, and the liver triglyceride concentration (129%). At the same time point, ethanol increased radioactivity incorporated into hepatic total lipid and triglyceride, after an injection of [U-14C] palmitic acid, by 76% and 158% respectively. Treatment of animals with Naloxone hydrochloride (2 mg/kg i.p.) at 1.0 hr and 2.5 hr after ethanol abolished these ethanol-mediated redox-state changes, without inhibiting ethanol oxidation or affecting hepatic acetaldehyde levels. This, however, did not prevent completely the triglyceride accumulation in the liver or reverse the enhanced uptake of radio-labelled palmitate caused by ethanol. Administration of sorbitol (3.5 g/kg i.p.) caused 109%, 57% and 200% increases in the hepatic [lactate]/[pyruvate], [3-hydroxybutyrate]/[acetoacetate] ratios and glycerol-3-phosphate concentrations respectively. However, the hepatic triglyceride concentration and the incorporation of [U-14C] palmitic acid into hepatic lipids were not influenced by this treatment. In vitro studies in which rat liver slices were incubated with [1-14C] palmitic acid also indicated that the altered [NADH]/[NAD+] ratio was not responsible for the decreased rate of fatty acid oxidation seen after ethanol administration or after the addition of ethanol to the incubation medium. In conclusion, these experiments indicate that increases in the hepatic [NADH]/[NAD+] ratio resulting from ethanol oxidation may not be directly implicated in the altered hepatic fatty acid utilisation and triglyceride deposition observed after acute ethanol administration in rats.

Acetaldehyde↗

PubMed1985

Sustained regional abnormalities in cardiac metabolism after transient ischemia in the chronic dog model.

Positron emission tomography allows noninvasive assessment of myocardial blood flow and metabolism, and may aid in defining the extent and severity of an ischemic injury. This hypothesis was tested by studying, in chronically instrumented dogs, regional blood flow and metabolism during and after a 3 hour balloon occlusion of the left anterior descending coronary artery. The metabolic findings after ischemia were compared with the recovery of regional function over a 4 week period. N-13 ammonia was used as a blood flow tracer, and C-11 palmitic acid and F-18 deoxyglucose as tracers of fatty acid and glucose metabolism, respectively. Regional myocardial function was monitored with ultrasonic crystals implanted subendocardially. Regional function improved most between 24 hours and 1 week after reperfusion, but was still attenuated at 4 weeks. The slow functional recovery was paralleled by sustained metabolic abnormalities, reflected by segmentally delayed clearance of C-11 activity from myocardium and increased uptake of F-18 deoxyglucose. Absence of blood flow and C-11 palmitic acid uptake at 24 hours of reperfusion correlated with extensive necrosis as evidenced by histologic examination. Conversely, uptake of C-11 palmitic acid with delayed C-11 clearance and increased F-18 deoxyglucose accumulation identified reversibly injured tissue that subsequently recovered functionally and revealed little necrosis. Thus, recovery of metabolism after 3 hours of ischemia is slow in canine myocardium and paralleled by slow recovery of function. Metabolic indexes by positron tomography early after reperfusion can identify necrotic and reversibly injured tissue. Positron tomography may therefore aid in defining the extent and prognosis of an ischemic injury in patients undergoing reperfusion during evolving myocardial infarction.

Animals↗

PubMed1965

Incorporation of 14C-labelled fatty acids into the mammary-gland lipids of the lactating rabbit.

1. [1-(14)C]Stearic acid, [9-(14)C]stearic acid, [9-(14)C]palmitic acid and [9-(14)C]decanoic acid were fed to lactating rabbits, and the fatty acids of the mammary gland were separated and partially degraded. 2. Most of the (14)C recovered was located in the acids fed. Stearic acid was least efficiently absorbed; decanoic acid was the most extensively metabolized. 3. Resynthesis after degradation to C(2) units led to uniform alternate labelling in the C(2)-C(10) acids, whereas C(12)-C(18) acids had excess of (14)C at the carboxyl end. 4. Acids formed by beta-oxidation down to C(12), but not below, were also present in the mammary-gland lipids. Desaturation of the administered acids was a very minor reaction.

Animals↗

PubMed1992

Evidence for multiple species of vaccinia virus-encoded palmitylated proteins.

When cells were infected with vaccinia virus in the presence of [3H]palmitic acid, radiolabel was incorporated into six viral proteins with apparent molecular weights of 92, 41, 37, 26, 17, and 14 kDa, all of which are expressed at late times during the infection cycle. The [3H]palmitate-labeled fatty acid moieties from the modified proteins were isolated, converted to p-nitrophenacyl derivatives, and subjected to reverse phase HPLC analysis which confirmed the identity of the fatty acid group as palmitic acid. Furthermore, the radiolabeled palmitate-protein bonds were sensitive to treatment with neutral hydroxylamine, suggesting that association of the fatty acid moieties with these proteins occurs via a thioester linkage. Previous studies by other investigators have identified the 37-kDa protein as the major antigen present in the outer membrane of extracellular enveloped virions, and demonstrated that the protein is modified by palmitic acid but is not glycosylated (G. Hiller and K. Weber J. Virol. (1985) 55, 651-659). Growth of vaccinia virus in the presence of tunicamycin indicated that the 41- and 26-kDa palmitylated proteins were also subject to modification by glycosylation, whereas like the 37-kDa protein, the 92-, 17-, and 14-kDa species did not appear to be glycosylated. Subcellular fractionation studies provided evidence that all of the viral palmitylated proteins were membrane-associated. Extraction of purified vaccinia virus with NP-40 and DTT demonstrated that the palmitylated proteins were associated with one of the viral membranes rather than the core of the virion. Viewing these results together with the previous reports of myristylated VV proteins (Franke et al. J. Virol. (1989) 63, 4285-4291), suggests that acylation of VV proteins represents a major modification pathway utilized by VV proteins during the assembly of progeny virions.

Chromatography, High Pressure Liquid↗

PubMed1989

Synthesis and initial processing of gastric apomucin.

The initiation of the processing of apomucin was investigated using mucus glycoprotein synthesizing polysomes from rat gastric epithelial cells. The polysomes were isolated from cells labeled with [3H]palmitic acid and [14C]N-acetylgalactosamine, purified on Helix pomatia-Sepharose affinity column, dissociated to release peptidyl-tRNA, and chromatographed on DEAE-HPLC column to separate peptidyl-tRNA complexes from the free and ribosomal RNA and proteins. The analysis of the HPLC purified peptidyl-tRNA revealed that complexes were labeled with [3H]palmitic acid and [14C]N-acetylgalactosamine. Digestion of the peptidyl-tRNA with RNase released 3H and 14C labeled peptides, while alkaline degradation destroyed the complex and rendered the [3H]palmitic acid extractable with hexane. The treatment of the 3H and 14C labeled peptidyl-tRNA complexes with alpha-N-acetylgalactosaminidase led to the release of radiolabeled N-acetylgalactosamine, whereas alkaline borohydride reduction produced N-acetylgalactosaminitol. The fatty acid residues have been detected in peptidyl-tRNA containing 2,000Da peptides, whereas N-acetylgalactosamine was discernible on 5,000Da peptides.

Acetylgalactosamine↗

PubMed2002

How helminth lipid-binding proteins offload their ligands to membranes: differential mechanisms of fatty acid transfer by the ABA-1 polyprotein allergen and Ov-FAR-1 proteins of nematodes and Sj-FABPc of schistosomes.

Three different classes of small lipid-binding protein (LBP) are found in helminth parasites. Although of similar size, the ABA-1A1 (also designated As-NPA-A1) and Ov-FAR-1 (formerly known as Ov20) proteins of nematodes are mainly alpha-helical and have no known structural counterparts in mammals, whereas Sj-FABPc of schistosomes is predicted to form a beta-barrel structure similar to the mammalian family of intracellular fatty acid binding proteins. The parasites that produce these proteins are unable to synthesize their own complex lipids and, instead, rely entirely upon their hosts for supply. As a first step in elucidating whether these helminth proteins are involved in the acquisition of host lipid, the process by which these LBPs deliver their ligands to acceptor membranes was examined, by comparing the rates and mechanisms of ligand transfer from the proteins to artificial phospholipid vesicles using a fluorescence resonance energy transfer assay. All three proteins bound the fluorescent fatty acid 2-(9-anthroyloxy)palmitic acid (2AP) similarly, but there were clear differences in the rates and mechanisms of fatty acid transfer. Sj-FABPc displayed a collisional mechanism; 2AP transfer rates increased with acceptor membrane concentration, were modulated by acceptor membrane charge, and were not diminished in the presence of increasing salt concentrations. In contrast, transfer of ligand from Ov-FAR-1 and ABA-1A1 involved an aqueous diffusion step; transfer rates from these proteins were not modulated by acceptor membrane concentration or charge but did decrease with the ionic strength of the buffer. Despite these differences, all of the proteins interacted directly with membranes, as determined using a cytochrome c competition assay, although Sj-FABPc interacted to a greater extent than did Ov-FAR-1 or rABA-1A1. Together, these results suggest that Sj-FABPc is most likely to be involved in the intracellular targeted transport and metabolism of fatty acids, whereas Ov-FAR-1 and ABA-1A1 may behave in a manner analogous to that of extracellular LBPs such as serum albumin and plasma retinol binding protein.

Allergens↗

PubMed1983

Effect of retinoic acid on the acylation of phospholipids in granulocytes in vitro.

The influence of retinoic acid on the incorporation of [1-14C]palmitic acid and [1-14C]arachidonic acid into phospholipids was examined in guinea pig peritoneal granulocytes. All-trans-retinoic acid inhibited the incorporation of both fatty acids into phosphatidic acid and phosphatidylinositol. However, it stimulated the incorporation of both fatty acids into phosphatidylcholine but not other phospholipids. All-trans-retinoic acid was more effective than 13-cis-retinoic acid. The influence of all-trans-retinoic acid on the acylation of phospholipids was concentration-dependent with significant effect occurring at 2.1 microM. The loss of labeled fatty acids from prelabeled phospholipids and the transport of labeled fatty acids into granulocytes were not responsive to the presence of retinoic acid in the incubation media. These results suggest that retinoic acid may affect the activities of acyltransferases involved in the synthesis of phosphatidic acid, phosphatidylinositol and phosphatidylcholine.

Acylation↗

PubMed1982

Effects of fatty acid composition on the sensitivity of membrane functions to ethanol in Escherichia coli.

The effects of membrane composition on the sensitivity of membrane functions to ethanol in Escherichia coli have been investigated. The addition of ethanol (0.67M) in vitro did not cause appreciable inhibition of NADH oxidase, D-lactate oxidase or ATPase but caused an 11% to 30% inhibition of succinic dehydrogenase, glutamate uptake, proline uptake and 1ac permease. Although the sensitivities of some of these membrane functions to ethanol varied with membrane composition, none correlated with the changes in sensitivity to killing by ethanol. In contrast, leucine transport was resistant to ethanol (0.67M) in control cells and in cells enriched in vaccenic acid, but was inhibited by 25% in cells grown in palmitic acid. The release of nucleotides was examined as a comparative measure of cellular permeability. Ethanol increased nucleotide leakage. Leakage was reduced in cells grown in vaccenic acid and enhanced in cells enriched in palmitic acid. The addition of MgSO4 (10mM) reduced nucleotide leakage and enhanced survival. Based upon these results, metabolite leakage was proposed as the primary event associated with bacterial inactivation in buffered solutions of ethanol. The increase in acyl chain length is proposed as the beneficial aspect of vaccenic acid incorporation rather than the increase in membrane unsaturation.

Amino Acids↗

PubMed2006

Palmitic acid-induced activation of human T-lymphocytes and aortic endothelial cells with production of insulin receptors, reactive oxygen species, cytokines, and lipid peroxidation.

Diabetic conditions are associated with hyperglycemia and hyperlipidemia, but the role of saturated fatty acids (SFA) vs. unsaturated fatty acids (UFA) in activation of T-lymphocytes and human aortic endothelial cells (HAEC) is not known. We investigated in vitro effects of various concentrations of SFA (palmitate) and UFA (oleic, linoleic, linolenic, and arachidonic) acids in activation of these cells. These cells in presence of palmitate, but not UFA, exhibited time, and concentration-dependent emergence of insulin receptors, GLUT 4 expression, generation of ROS, cytokines, lipid peroxidation, and IRS-1. We conclude that both T-lymphocytes and HAEC share common characteristics in exhibiting activation of these cells to palmitate, but not to UFA, by developing insulin receptors and becoming insulin responsive tissues, a hitherto unknown response to palmitate. We hypothesize that these events may serve as protective defense mechanisms against acute effects of glucotoxicity and lipotoxicity in these cells.

Aorta↗

PubMed1980

Metabolic properties and ultrastructure of alveolar type II cells isolated with elastase.

We used porcine pancreatic elastase to isolate type II cells from the lungs of rats; the yield and purity of the type II cells was better than that obtained by methods using trypsin. In 102 experiments we obtained 82 +/- 23 . 10(6) cells/rat, 68 +/- 11% (mean +/- S.D.) of which type II cells. This preparation of cells, when centrifuged over a discontinuous density gradient, yielded 25 +/- 10 . 10(6) cells/rat, 80 +/- 13% of which were type II cells (n = 102). The cells, after density gradient centrifugation, could be futher purified by centrifugal elutriation (94 +/- 3% type II cells, n = 22) or adherence in primary culture (94 +/- 2% type II cells, n = 34). Type II cells isolated with elastase are similar morphologically and biochemically to type II cells isolated from rats with trypsin. The preparations of cells appeared healthy by several different criteria: ultrastructure, exclusion of vital dye, lack of stimulation of oxygen consumption by exogenous sodium succinate, and linear rates of oxidation of [1-14C]palmitic acid and of incorporation of [1-14C]acetate into fatty acids. Type II cells consumed 75 +/- 20 nmol O2/10(6) cell per h, oxidized [1-14C]palmitic acid at a rate of 0.4 nmol/10(6) cells per h, and incorporated [1-14C]acetate into fatty acids at a rate of 7.5 nmol/10(6) cells per h.

Acetates↗

PubMed1999

Altered transmembrane ionic flux in hepatocytes isolated from cirrhotic rats.

BACKGROUND/AIMS: Although cirrhosis is known to be associated with many hepatocyte abnormalities, there is no well-established model to study the cellular drug uptake process independent of hemodynamic effects. The purpose of the present study was to test the following hypothesis: hepatocytes isolated from cirrhotic animals may be used as a model to study the cellular abnormalities associated with cirrhosis. Our hypothesis was tested by comparing the membrane potential (PD) of hepatocytes in anesthetized healthy and cirrhotic animals, and the PD and [3H]palmitic acid clearance rate of hepatocytes isolated from healthy and cirrhotic animals. METHODS: Mild to moderate cirrhosis was induced in female Sprague-Dawley rats by CCl4 administration. PD was recorded in anesthetized animals using intracellular microelectrodes. Hepatocytes from those livers were subsequently isolated by collagenase perfusion for further determinations of PD and [3H]palmitic acid uptake. RESULTS: The mean (+/-SEM) hepatocyte PD from intact rat livers was 38+/-1 mV (control) and -32+/-1 mV (cirrhosis; n=6/group, p<0.01). The PD (mean+/-SEM) in isolated hepatocytes was -21+/-1 mV (control) and -15+/-1 mV (cirrhosis, n=13/group, p<0.01). The clearance rate of [3H]palmitic acid was lower in hepatocytes isolated from cirrhotic animals (26%) than in those isolated from healthy control animals (p<0.01). CONCLUSION: The results of this study indicate that hepatocytes isolated from cirrhotic animals may be used to study the cellular abnormalities associated with cirrhosis.

Animals↗

PubMed1978

Interaction of mycobacterial polymethylpolysaccharides with paranaric acid and palmitoyl-coenzyme A: structural specificity and monomeric dissociation constants.

The long-chain polyenoic fatty acids alpha- and beta-paranaric acid form complexes with the 6-O-methylglucose polysaccharide from Mycobacterium smegmatis as demonstrated by an enhanced fluorescence emission of the paranaric acid. This interaction is eliminated by digestion of the methylglucose polysaccharide with alpha-amylase and glucoamylase, which removes four hexose units from the nonreducing end of the chain. Titration of the methylglucose polysaccharide with either paranaric acid isomer suggests formation of a 1:1 complex with a dissociation constant (K(d)) of 0.4 muM. The fluorescence emission of this complex is quenched by palmitoyl-CoA, which indicates that the paranaric acid can be displaced by the acoyl-CoA, a conclusion confirmed by gel filtration. The presumed polysaccharide/palmitoyl-CoA complex has a K(d) of about 0.1 muM. Acoyl-CoA derivatives with shorter fatty acid chains and free palmitic acid complete less effectively, indicating that they form weaker complexes with the polysaccharide. The methylmannose polysaccharides with 12 or 13 sugar units also complex paranaric acid strongly (K(d) approximately 0.4 muM), whereas the isomer with 11 sugar units complexes weakly.The methylglucose polysaccharide has been coupled to L-tryptophan methyl ester. The fluorescence emission spectrum of the attached tryptophan group is shifted to a shorter wavelength relative to N-acetyl-L-tryptophan methyl ester, and this effect is enhanced in the corresponding derivative made with the amylase-digested polysaccharide. The circular dichroism spectrum of the polysaccharide-tryptophan derivative shows three bands with negative ellipticity, in the 270-300 nm region, not observed in the amylase-digested derivative. These results imply that the tryptophan is in a more structured environment in the former than in the latter derivative. alpha-Paranaric acid binds to the polysaccharide-tryptophan conjugate and shows an enhanced fluorescence emission with partial quenching of the tryptophan fluorescence emission, suggestive of Förster energy transfer from tryptophan to paranaric acid.

Acyl Coenzyme A↗

PubMed1990

Composition and synthesis of glycolipids in megakaryocytes and platelets: differences in synthesis in megakaryocytes at different stages of maturation.

The composition and synthesis of megakaryocyte and platelet glycolipids were compared since these lipids are thought to be important for biologic activities such as adhesion and maturation. Highly purified guinea pig megakaryocytes at different stages of maturation and platelets were studied. Glycolipids and gangliosides were extracted, separated by thin-layer chromatography, and the carbohydrate content was analyzed by gas-liquid chromatography (GLC). Synthesis of ceramides and glycolipids was determined by the incubation of megakaryocytes with [14C]acetate, [3H]palmitic acid, and [3H]galactose. A major neutral glycolipid present in guinea pig megakaryocytes and platelets was identified as asialoGM2 by selective enzymatic hydrolysis with beta-N-acetylhexosaminidase, alpha-galactosidase and endo-beta-galactosidase, and carbohydrate analysis by GLC. Trace amounts of asialoGM1 were detected immunologically. The cells also contained glucosyl ceramide and lactosyl ceramide. Several ganglosides were detected of which one was identified as GM1 by its reaction with the beta-subunit of cholera toxin and by the identification of an asialoGM1 core with anti-asialoGM1 antibody after desialylation. The synthesis of ceramides from palmitic acid and acetate was 5 and 10 times greater, respectively, in megakaryocytes than in platelets. Ceramide and glycolipid synthesis from palmitic acid occurred primarily in immature megakaryocytes while synthesis from acetate occurred primarily in more mature megakaryocytes. The glycosylation of ceramides from galactose was 42 times greater in megakaryocytes than in platelets. Thus, ceramides and glycolipids are primarily synthesized in megakaryocytes, but platelets retain the capacity to synthesize significant amounts of free ceramides. The glycosylation of free ceramides occurs almost exclusively in megakaryocytes and only in trace amounts in platelets. These data indicate that megakaryocytes determine the composition of glycolipids in platelets and that there is considerable compartmentalization of glycolipid synthesis and membrane assembly at various stages of megakaryocytes development.

Acetates↗