Asteriscus v. lapillus: comparing the chemistry of two otolith types and their ability to delineate riverine populations of common carp Cyprinus carpio.

The chemical composition of common carp Cyprinus carpio asteriscus (vaterite) and lapillus (aragonite) otoliths from the same individual and reflecting the same growth period was measured to (1) determine whether there are differences in the uptake of trace metals (Mg:Ca, Mn:Ca, Sr:Ca and Ba:Ca ) and Sr isotope ratios ((87)Sr:(86)Sr) in co-precipitating lapilli and asterisci and (2) compare the ability of multi-element and isotopic signatures from lapilli, asterisci and both otolith types combined to discriminate C. carpio populations over a large spatial scale within a river basin. Depth profile analyses at the otolith edge using laser-ablation inductively coupled plasma mass spectrometry showed that asterisci were enriched in Mg and Mn and depleted in Sr and Ba relative to lapilli, whilst (87)Sr:(86)Sr values were nearly identical in both otolith types. Significant spatial differences among capture locations were found when all trace element and Sr isotope ratio data were aggregated into a multi-element and isotopic signature, regardless of which otolith type was used or if they were used in combination. Discriminatory power was enhanced, however, when data for both otolith types were combined, suggesting that analysis of multiple otolith types may be useful for studies attempting to delineate C. carpio populations at finer spatial or temporal scales.

Direct binding of the signaling adapter protein Grb2 to the activation loop tyrosines on the nerve growth factor receptor tyrosine kinase, TrkA.

We demonstrate that the signaling adapter, Grb2, binds directly to the neurotrophin receptor tyrosine kinase, TrkA. Grb2 binding to TrkA is independent of Shc, FRS-2, phospholipase Cgamma-1, rAPS, and SH2B and is observed in in vitro binding assays, yeast two-hybrid assays, and in co-immunoprecipitation assays. Grb2 binding to TrkA is mediated by the central SH2 domain, requires a kinase-active TrkA, and is phosphotyrosine-dependent. By analyzing a series of rat TrkA mutants, we demonstrate that Grb2 binds to the carboxyl-terminal residue, Tyr(794), as well as to the activation loop tyrosines, Tyr(683) and Tyr(684). By using acidic amino acid substitutions of the activation loop tyrosines on TrkA, we can stimulate constitutive kinase activity and TrkA-Shc interactions but, importantly, abolish TrkA/Grb2 binding. Thus, in addition to providing the first evidence of direct Grb2 binding to the neurotrophin receptor, TrkA, these data provide the first direct evidence that the activation loop tyrosines of a receptor tyrosine kinase, in addition to their essential role in kinase activation, also serve a direct role in the recruitment of intracellular signaling molecules.

The signaling adapter FRS-2 competes with Shc for binding to the nerve growth factor receptor TrkA. A model for discriminating proliferation and differentiation.

We have isolated a human cDNA for the signaling adapter molecule FRS-2/suc1-associated neurotrophic factor target and shown that it is tyrosine-phosphorylated in response to nerve growth factor (NGF) stimulation. Importantly, we demonstrate that the phosphotyrosine binding domain of FRS-2 directly binds the Trk receptors at the same phosphotyrosine residue that binds the signaling adapter Shc, suggesting a model in which competitive binding between FRS-2 and Shc regulates differentiation versus proliferation. Consistent with this model, FRS-2 binds Grb-2, Crk, the SH2 domain containing tyrosine phosphatase SH-PTP-2, the cyclin-dependent kinase substrate p13(suc1), and the Src homology 3 (SH3) domain of Src, providing a functional link between TrkA, cell cycle, and multiple NGF signaling effectors. Importantly, overexpression of FRS-2 in cells expressing an NGF nonresponsive TrkA receptor mutant reconstitutes the ability of NGF to stop cell cycle progression and to stimulate neuronal differentiation.

Activity-dependent interaction of the intracellular domain of rat trkA with intermediate filament proteins, the beta-6 proteasomal subunit, Ras-GRF1, and the p162 subunit of eIF3.

Many responses to nerve growth factor (NGF) are regulated through the receptor tyrosine kinase trkA. To understand more fully the functions of trkA in NGF responsive cells, we have expressed the intracellular domain of rat trkA as a fusion protein with the yeast gal4 transcription factor, and used the fusion protein to probe rat and mouse cDNA libraries by the yeast two-hybrid system. We have identified a direct interaction between the intracellular domain of trkA and two members of the intermediate filament (IF) family of proteins, the guanine-nucleotide exchange protein Ras-GRF1, the p162 subunit of eIF3, and the beta-6 proteasome subunit. The interactions are dependent on an active trkA kinase, and RasGRF1, the beta-6 proteasomal subunit, and peripherin are directly phosphorylated by trkA. The interaction with trkA is not affected by mutations at either Tyr499 or Tyr794, the two major phosphotyrosine residues essential to the activation and receptor binding of Shc, FRS-2/SNT, and phospholipase Cgamma-1, and it is highly specific in vitro for trkA, with little or no binding observed with trkB and/or trkC. The results show that trkA may play a regulatory role in a variety of cellular functions in addition to neuritogenesis, including regulated protein degradation and transcriptional activation.

A novel juxtamembrane deletion in rat TrkA blocks differentiative but not mitogenic cell signaling in response to nerve growth factor.

We have generated a novel rat TrkA receptor mutant (TrkAS3) by deletion of five conserved residues (493IMENP497) in the juxtamembrane domain. TrkAS3 receptors cannot support nerve growth factor (NGF)-induced cell cycle arrest or neuronal differentiation but retain cell survival responses as well as Ras-dependent mitogenic signaling. Cells of the nnr5 line stably expressing TrkAS3 induce NGF-dependent SHC phosphorylation and phosphatidylinositol 3-kinase, phospholipase Cgamma-1, and prolonged mitogen-activated protein kinase activation to absolute levels comparable to those in PC12 cells. Although the stoichiometry of TrkAS3-SHC binding is reduced, cells overexpressing TrkAS3 exhibit NGF-dependent SHC-Grb-2/Sos binding, essential for Ras activation, as well as NGF-dependent SNT phosphorylation to absolute levels comparable to those in PC12 cells. Collectively, these data suggest that the TrkAS3 deletion either directly affects a novel Ras-independent TrkA binding protein or that the decrease in TrkAS3-SHC association affects a Ras-independent SHC binding protein essential for cell cycle arrest and/or neurite outgrowth.

A kinase insert isoform of rat TrkA supports nerve growth factor-dependent cell survival but not neurite outgrowth.

To investigate potential differences between the family of Trk receptors that might have differential consequences on cell signaling, we generated a rat TrkA homologue of the 14-amino acid kinase insert isoform of TrkC termed TrkAKi. Signal transduction by the TrkAKi receptor has been investigated and compared with the homologous signaling defective TrkC(Ki14) receptor. Herein, we demonstrate that TrkAKi receptors show a decrease in the absolute amount of kinase activity relative to wild-type TrkA, yet retain normal patterns of receptor tyrosine phosphorylation, as determined by phosphopeptide mapping studies, unlike TrkC(Ki14). nnr5 cell clones expressing TrkAKi receptors show a decrease in nerve growth factor (NGF)-mediated SHC tyrosine phosphorylation and a loss of high-affinity TrkA-SHC interaction comparable to those expressing TrkC(Ki14). Moreover, nnr5 cells expressing TrkAKi receptors fail to demonstrate NGF-dependent tyrosine phosphorylation of the signaling molecules phospholipase Cgamma-1, MAP kinase/ERK-1, and SNT. TrkAKi receptors internalize NGF comparable to wild-type TrkA, but do not stimulate neurite outgrowth. It is interesting that, unlike TrkC(Ki14), TrkAKi receptors retain phosphatidylinositol 3-kinase activity and nnr5 cells stably expressing TrkAKi receptors retain NGF-dependent cell survival under serum-free conditions. Lastly, TrkAKi receptors fail to stimulate three immediate-early genes (NGF1A, NGF1B, and c-fos), suggesting that these gene products are not required for NGF-dependent cell survival responses.

Deletions in the extracellular domain of rat trkA lead to an altered differentiative phenotype in neurotrophin responsive cells.

We have investigated the role(s) of conserved sequences in the extracellular domain of rat trkA by generating 5' and 3' deletions and assaying changes in neurotrophin binding, tyrosine kinase activity, and neurite outgrowth. Essential sequences required for both nerve growth factor (NGF) and neurotrophin-3 (NT-3) binding were mapped to the immunoglobulin-like domains. Small deletions in the second immunoglobulin-like domain and in the juxtamembrane region abolished neurotrophin binding. Dose-response curves on cells expressing full-length trkA were identical for NGF and NT-3 (0.1 ng/ml-50 ng/ml) while cells expressing leucine rich motif (LRM) minus receptors required high concentrations of NT-3 (50 ng/ml). Scatchard analysis revealed a loss of high-affinity NT-3, but not NGF, binding to the LRM minus receptor consistent with the neurite dose-response curves. Moreover, cells expressing the LRM minus receptors failed to fasciculate and showed delayed arborization in comparison to cells expressing wild-type trkA, suggesting a possible role for the LRM's in neurotrophin-induced differentiation and in high-affinity NT-3 binding.

Stimulation of phosphatidylcholine biosynthesis in mouse MLE-12 type-II cells by conditioned medium from cortisol-treated rat fetal lung fibroblasts.

Treatment of murine adult MLE-12 type-II and fetal-rat type-II cells with fetal-rat-fibroblast-conditioned medium (FFCM) resulted in a 2-fold stimulation of [14C]choline incorporation into phosphatidylcholine. Soluble CTP:phosphocholine cytidylyltransferase (CT) activity was increased approx. 3-fold in FFCM-treated fetal-rat type-II cells but was not changed in MLE-12 cells. Neither choline kinase nor cholinephosphotransferase activities were affected by treatment of MLE-12 cells with FFCM. Long-term labelling of MLE-12 cells with [14C]choline, followed by a 14-18 h chase with FFCM, resulted in a 2.5-fold decrease in [14C]phosphocholine levels relative to controls, suggesting that CT was being activated. In contrast, oleate treatment increased CT activity in the particulate fraction in both cells. Western blots indicate that soluble CT undergoes dephosphorylation in response to FFCM, but no translocation to the particulate fraction was noted. Treatment with oleate stimulated a marked translocation. Tryptic phosphopeptide maps from FFCM-treated cells revealed only minor alterations in the phosphorylation pattern. It is concluded that FFCM and oleate activate CT through different mechanisms. The results are consistent with FFCM activating CT in MLE-12 as well as fetal type-II cells. However, the reason why this activation cannot be detected in vitro is not known.

Identification of the nuclear localization signal of rat liver CTP:phosphocholine cytidylyltransferase.

CTP:phosphocholine cytidylyltransferase (CT) is a major regulatory enzyme in phosphatidylcholine synthesis in mammalian cells. CT is found in both soluble and particulate forms, both of which are nuclear. We report here the identification of a 21-residue sequence at the amino terminus of CT, 8KVNSRKRRKEVPGPNGATEED28, which was sufficient to direct beta-galactosidase into the cell nucleus. Further deletions from either end of this sequence greatly reduced the nuclear localization of beta-galactosidase. Deletions of amino acids within the nuclear localization signal or of the entire signal disrupted CT nuclear localization, but CT was not completely excluded from the nucleus. Clones of stable transfectants of the nuclear localization signal-deficient CT expressed in Chinese hamster ovary (CHO) 58 cells, which is temperature-sensitive for growth and CT activity, were isolated and characterized. The deletion mutants were active under the same conditions as the wild-type enzyme. Despite the difference in subcellular location from wild-type CT, the nuclear localization mutants were fully able to complement the CT-deficient cell line CHO 58 for both growth and choline incorporation into phosphatidylcholine at the nonpermissive temperature. The mobility of the mutant enzymes on SDS gels was altered relative to the mobility of wild-type CT; however, the extent of phosphorylation of the mutant enzymes was decreased only slightly. Thus, the distribution of CT in both cytoplasm and nucleus, rather than exclusively nucleus, has little effect on the ability of CT to function in growing CHO cells.

Effect of probenecid on the formation and elimination kinetics of the sulphate and glucuronide conjugates of diflunisal.

The effect of probenecid on the pharmacokinetics of diflunisal and its glucuronide and sulphate conjugates was studied in 8 healthy volunteers. Diflunisal 250 mg b.d. was administered p.o. for 15 days and its steady state pharmacokinetics was evaluated on Day 16 after the last dose (control phase). Probenecid 500 mg b.d. was co-administered throughout the entire study period in the treatment phase of the study. The steady state plasma concentration of diflunisal was significantly higher during the probenecid treatment phase as compared to the control phase (104.0 vs. 63.1 micrograms.ml-1). This was the result of a significant decrease in the plasma clearance of diflunisal from 5.8 (control) to 3.4 ml.min-1 (probenecid co-administration). The metabolite formation clearances of both glucuronides were significantly decreased by probenecid, -45% and -54% for the phenolic and acyl glucuronide, respectively. The metabolite formation clearance of the sulphate conjugate was not affected by probenecid coadministration. Steady state plasma concentrations of the sulphate and glucuronide conjugates of diflunisal were 2.5- to 3.1-fold higher during probenecid co-administration, due to a significant reduction in the renal clearance of the three diflunisal conjugates. Probenecid also reduced the plasma protein binding of diflunisal, but only to a minor extent; the unbound plasma fraction of diflunisal at steady state averaged between 5 and 30% higher during probenecid co-administration.

Identification of phosphorylation sites in rat liver CTP: phosphocholine cytidylyltransferase.

CTP:phosphocholine cytidylyltransferase (CT) is an important regulatory enzyme in phosphatidylcholine biosynthesis. The enzyme exists as a soluble, inactive form that is highly phosphorylated; activation of the enzyme is accompanied by dephosphorylation and translocation to the membrane. We have used a recombinant baculovirus clone to obtain CT labeled in vivo with 32PO4. The tryptic phosphopeptide pattern of the baculovirus-expressed CT was the same as for CT expressed in mammalian cells, indicating that insect cells modify the same phosphorylation sites as do mammalian cells. 32PO4-labeled, baculovirus-expressed CT was digested with trypsin and the peptides purified by reversed-phase high performance liquid chromatography. Phosphoamino acid analysis of the complete protein as well as individual peptides revealed that only serine residues were phosphorylated. Sequence analysis of purified radioactive peptides revealed that phosphorylation of CT was confined to the carboxyl-terminal region and that all or nearly all Ser residues from Ser315 to the carboxyl terminus were labeled. Ser315, Ser319, Ser329, Ser323, Ser331, Ser343, and Ser347 all reside in potential sites for proline-directed kinases. Two other phosphorylated serine residues, Ser315 and Ser333, are found within protein kinase C consensus phosphorylation sites. Ser321, Ser322, Ser333, Ser345, Ser346, Ser350, Ser352, and Ser362 were also found to be phosphorylated. Serine362 resides within a putative casein kinase II phosphorylation site, and there are five potential sites for phosphorylation by glycogen synthase kinase 3. Identification of these sites will allow investigations that focus on the establishment of the physiological function of phosphorylation at each site.

Regulation of CTP:phosphocholine cytidylyltransferase in HeLa cells. Effect of oleate on phosphorylation and intracellular localization.

The mechanism by which oleate induced the translocation of CTP:phosphocholine cytidylyltransferase from the soluble to particulate fraction was studied in HeLa cells. Addition of 0.5 mM oleate caused a 10-fold stimulation of [methyl-3H]choline incorporation into phosphatidylcholine. The stimulation of phosphatidylcholine synthesis was correlated with a decrease in phosphocholine and an increase in CDP-choline, indicating that cytidylyltransferase is regulatory under these conditions. The stimulation of cytidylyltransferase activity upon oleate treatment was concomitant with the translocation of this enzyme from the soluble to particulate fraction. Immunoblot analysis revealed the soluble form as multiple slowly migrating bands, and the particulate form as a faster migrating, single band. The slowly migrating form could be converted to the fast migrating form by phosphatase treatment, suggesting that the mobility difference was due to phosphorylation. In vivo 32P labeling showed that the soluble cytidylyltransferase was highly phosphorylated and the particulate form was much less phosphorylated. The kinetics of the translocation showed that the conversion of the majority of cytidylyltransferase from the soluble to the particulate form occurred within about 15 min following addition of oleate and that the translocation showed a good correlation with dephosphorylation. In contrast, the relocation of cytidylyltransferase from the particulate to the soluble fraction after the removal of oleate occurred within 1 min, and extensive phosphorylation was not required for cytidylyltransferase to leave the membrane. The relocated, soluble cytidylyltransferase was phosphorylated at a much slower rate than that at which the enzyme left the membrane. Immunolocalization showed that the cytidylyltransferase translocated to the nuclear envelope instead of the endoplasmic reticulum. These results indicate that activation of cytidylyltransferase by dephosphorylation and translocation to the nuclear envelope may be a general mechanism occurring in many cell types. Moreover, in HeLa cells, multiple mechanism exist for regulating cytidylyltransferase activity.

Baculovirus-mediated expression of rat liver CTP:phosphocholine cytidylyltransferase.

We describe herein the expression and purification of milligram quantities of rat liver CTP:phosphocholine cytidylyltransferase in recombinant baculovirus-infected insect cells. The enzyme was purified by incorporating modifications to a previously published procedure (P. A. Weinhold and D. A. Feldman, 1992, in "Methods in Enzymology" (E. A. Dennis and D. E. Vance, Eds.), Vol. 209, pp. 248-258, Academic Press, San Diego, CA). Like cytidylyltransferase purified from rat liver, the purified recombinant cytidylyltransferase has the same molecular weight (42 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and reacts to antibodies generated against the N and C termini of rat liver cytidylyltransferase. In addition, like the purified rat liver enzyme, the recombinant cytidylyltransferase is activated by lipids. We also expressed rat liver cytidylyltransferase as a fusion protein with glutathione-S-transferase and with a thrombin cleavage site between the two enzymes for rapid isolation of cytidylyltransferase. Thrombin cleavage was, however, incomplete and prolonged exposure to thrombin resulted in the degradation of cytidylyltransferase itself.

Both phenolic and acyl glucuronidation pathways of diflunisal are impaired in liver cirrhosis.

The pharmacokinetics of diflunisal, a salicylate derivative that undergoes phenolic and acyl glucuronidation as well as sulphate conjugation, has been studied after a single oral dose (250 mg) in patients with cirrhosis (n = 5) and in healthy controls (n = 5). The plasma clearance of total (bound + unbound) diflunisal was 10.2 ml.min-1 in the control subjects and it was not affected by cirrhosis (10.9 ml.min-1). The plasma protein binding of diflunisal was significantly reduced in cirrhosis; the percentage of unbound diflunisal in plasma was 0.089 in the controls and 0.147 in the patients with cirrhosis. Plasma clearance of unbound diflunisal was significantly impaired in cirrhosis: 11.5 l.min-1 in control subjects vs 7.41.min-1 in cirrhotics. In cirrhotic patients, the unbound partial clearances to the phenolic and acyl glucuronides were both significantly reduced, by approximately 38%. The unbound partial clearance to the sulphate conjugate was not significantly affected by cirrhosis. The results show that both the phenolic and acyl glucuronidation pathways of diflunisal are equally susceptible to the effects of liver cirrhosis.

Identification of a hydroxy metabolite of diflunisal in rat and human urine.

1. A new metabolite of diflunisal, a hydroxy derivative, has been identified in rat and human urine following administration of diflunisal. 2. This hydroxy metabolite of diflunisal is excreted in urine of both species as a polar conjugate, most likely a sulphate. 3. Attempts to isolate the polar conjugate in pure form were unsuccessful due to its rapid hydrolysis in the presence of acid, and organic solvents such as diethyl ether. Its breakdown product, however, was more stable and was isolated and purified by semi-preparative h.p.l.c. Unequivocal identification as 3-hydroxy-diflunisal (i.e. hydroxylation in position 3 of the salicylic acid ring) was accomplished by means of FAB-mass spectrometry and n.m.r. spectroscopy. 4. The contribution of this oxidative metabolic pathway to the overall elimination scheme of diflunisal is more important in rat than in man. Gunn rats excrete more of the hydroxy diflunisal conjugate in urine (20-30% of a 50 mg/kg i.v. dose of diflunisal) than Wistar rats. In healthy humans, hydroxylation of diflunisal contributes only to a small extent to the overall biotransformation of diflunisal.

The effect of multiple dosage on the kinetics of glucuronidation and sulphation of diflunisal in man.

1. The single (250 and 500 mg) and multiple dose (250 and 500 mg twice daily for 15 days) pharmacokinetics of diflunisal were compared in young volunteers. 2. The plasma clearance of diflunisal was lowered significantly after multiple dose administration (5.2 +/- 1.2 and 4.2 +/- 0.7 ml min-1 for the 250 and 500 mg twice daily regimens, respectively) as compared with single dose administration 11.4 +/- 3.1 and 9.9 +/- 2.0 ml min-1 for the 250 and 500 mg single doses, respectively). 3. The partial metabolic clearances of diflunisal by acyl and phenolic glucuronide formation were lowered significantly (greater than 50%) after multiple dose administration. 4. The urinary recovery of diflunisal sulphate increased as a function of dose: 6.1 +/- 2.8 and 9.1 +/- 3.5% following the 250 and 500 mg single dose, respectively, and 10.9 +/- 3.1 and 15.9 +/- 3.6% following the 250 and 500 mg twice daily regimens. The partial metabolic clearance of diflunisal by sulphate conjugation was unchanged following multiple dose administration. 5. The plasma protein binding of diflunisal was concentration-dependent. Analysis of unbound plasma clearances of diflunisal showed that its total plasma clearance following 500 mg twice daily was affected by both saturable glucuronidation and concentration-dependent plasma binding.

Sex-difference and the effects of smoking and oral contraceptive steroids on the kinetics of diflunisal.

The single dose pharmacokinetics of diflunisal were studied in 4 groups of 6 young volunteers: control men, control women, women taking low estrogen oral contraceptive steroids (OCS), and women smokers (10-20 cigarettes day). The plasma clearance of diflunisal was significantly higher in men (0.169 ml.min-1.kg-1) and in women on OCS (0.165 ml.min-1.kg-1) as compared to control women (0.108 ml.min-1.kg-1). Partial metabolic clearances of diflunisal by the three conjugative pathways (phenolic and acyl glucuronide formation, sulphate conjugation) were all increased in men and women OCS users as compared to control women. Statistically significant increases, however, were only observed for the partial metabolic clearance of diflunisal by phenolic glucuronidation between men and women (2.91 vs. 1.85 ml.min-1 respectively), and for the partial clearance by acyl glucuronidation between OCS users and control women (4.81 vs. 3.01 ml.min-1 respectively). Smoking resulted in a moderate increase (35%) in plasma diflunisal clearance. However, a significant reduction in total urinary recovery of diflunisal and its glucuronide and sulphate conjugates was found in smokers (70.5% in smokers as compared to 84.2-87.2% in the 3 other study groups). Consequently, smoking may have induced hydroxylation, a minor oxidative metabolic pathway of diflunisal recently discovered in man.

Distribution of arachidonic acid in choline- and ethanolamine-containing phosphoglycerides in subfractionated human neutrophils.

Human neutrophils were fractionated on Percoll gradients and the various subcellular fractions were analyzed for phospholipid and fatty acid composition. The results showed that plasma membranes and azurophilic granules were enriched with ethanolamine-(PE) relative to choline-(PC) containing phosphoglycerides. A remarkable degree of uniformity existed throughout the gradient with respect to the subclass composition of the subcellular PC and PE components. In each fraction 50-60% of the PC was diacyl, 40-45% was 1-O-alkyl-2-acyl (ether linked), and 2-5% was 1-O-alk-1'-enyl-2-acyl (plasmalogenic). For PE, 20-25% was diacyl, 7-12% ether linked, and 64-76% plasmalogenic. When neutrophils were incubated for 15 min with [1-14C]arachidonic acid and subfractionated most of the PC-associated label was intracellularly localized. A similar result was observed in PE, however, when the cells were allowed to stand for 2 h in fatty acid-free buffer following the 15 min of labeling and then subfractionated there was a sizable migration of [14C]arachidonate into plasma membrane PE. In all cases the diacyl subclass was labeled most heavily after 15 min but after an additional 2 h of incubation in fatty acid-free buffer there was a direct transfer of label to the ether- and plasmalogenic-linked PC and PE subclasses. It was also found that arachidonoyl-coenzyme A 1-acyl-lysophosphatide acyltransferase activity was inherent in all three major membrane types but was enriched in the endoplasmic reticulum/secondary granule fraction. Arachidonate consistently accounted for roughly 5% of the PC and 17% of the PE fatty chain composition in each subcellular fraction. These findings demonstrate that, despite the uniform arachidonate and PC and PE subclass composition within the various neutrophil subcellular fractions, the bulk of the PC- and PE-associated arachidonate is localized in intracellular membranes.

Studies on the incorporation and transacylation of various fatty acids in choline and ethanolamine-containing phosphoacylglycerol subclasses in human neutrophils.

The incorporation of eight 14C-labeled fatty acids into human neutrophil phospholipids was investigated and the results were expressed as percent of the total phospholipid associated 14C-labeled substrate incorporated after an initial 15 min labeling and a subsequent 2 h reincubation in fatty acid-free buffer. In all cases, the PC fraction accounted for more than 40% of the total phospholipid radioactivity. The inositol-containing phosphoacylglycerols were also labeled well by all the fatty acids except 22:6(n - 3) and 16:0; however, a greater percentage of [14C]22:6(n - 3) was found in PE than that of any other labeled fatty acid substrate. In all cases, most of the radioactivity in PC after 15 min was in the diacyl subclass. After 2 h, there was a shift of [14C]20:4(n - 6), [14C]20:5(n - 3), [14C]22:6(n - 3) and [14C]18:4(n - 4) into the ether-linked subclass. No such shift was observed for [14C]16:0 or [14C]18:2(n - 6) and, although there was an increase in the percent radioactive 20:3(n - 6) and 20:3(n - 9) in ether-linked PC after 2 h, the total radioactivity in this fraction remained low by comparison. A similar shift in label occurred in the plasmalogenic-linked PE subspecies in cells labeled with [14C]20:4(n - 6), [14C]20:5(n - 3) and [14C]22:6(n - 3).