scholarly journals Characterization and purification of neutrophil ecto-phosphatidic acid phosphohydrolase

1997 ◽  
Vol 324 (3) ◽  
pp. 941-950 ◽  
Author(s):  
Denis ENGLISH ◽  
Margaret MARTIN ◽  
Kevin A. HARVEY ◽  
Luke P. AKARD ◽  
Ruth ALLEN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phosphatidic Acid ◽  
Molecular Mass ◽  
Biological Systems ◽  
Short Chain ◽  
Intact Cells ◽  
Ability To Act ◽  
Phosphatidic Acids ◽  
Hydrolysis Of ◽  
Long Chain

Phosphatidic acid and its derivatives play potentially important roles as extracellular messengers in biological systems. An ecto-phosphatidic acid phosphohydrolase (ecto-PAPase) has been identified which effectively regulates neutrophil responses to exogenous phosphatidic acid by converting the substrate to diacylglycerol. The present study was undertaken to characterize this ecto-enzyme on intact cells and to isolate the enzyme from solubilized neutrophil extracts. In the absence of detergent, short chain phosphatidic acids were hydrolysed most effectively by neutrophil plasma membrane ecto-PAPase; both saturated and unsaturated long chain phosphatidic acids were relatively resistant to hydrolysis. Both long (C18:1) and short (C8) chain lyso-phosphatidic acids were hydrolysed at rates comparable with those observed for short chain (diC8) phosphatidic acid. Activity of the ecto-enzyme accounted for essentially all of the N-ethylmaleimide-insensitive, Mg2+-independent PAPase activity recovered from disrupted neutrophils. At 37 °C and pH 7.2, the apparent Km for dioctanoyl phosphatidic acid (diC8PA) was 1.4×10-3 M. Other phosphatidic acids and lysophosphatidic acids inhibited hydrolysis of [32P]diC8PA in a rank order that correlated with competitor solubility, lysophosphatidic acids and unsaturated phosphatidic acids being much more effective inhibitors than long chain saturated phosphatidic acids. Dioleoyl (C18:1) phosphatidic acid was an unexpectedly strong inhibitor of activity, in comparison with its ability to act as a direct substrate in the absence of detergent. Other inhibitors of neutrophil ecto-PAPase included sphingosine, dimethyl- and dihydro-sphingosine, propranolol, NaF and MgCl2. Of several leucocyte populations isolated from human blood by FACS, including T cells, B cells, NK lymphocytes and monocytes, ecto-PAPase was most prevalent on neutrophils; erythrocytes were essentially devoid of activity. A non-hydrolysable, phosphonate analogue of phosphatidic acid, phosphonate 1, efficiently solubilized catalytic activity from intact neutrophils without causing cell disruption or increasing permeability. Enzyme activity in solubilized extracts was purified in the absence of detergent by successive heparin–Sepharose, gel filtration and anion exchange chromatography. By assaying activity in renatured SDS/polyacrylamide gel slices, the molecular mass of neutrophil ecto-PAPase was estimated to be between 45 and 52 kDa, similar to the molecular mass of previously purified plasma membrane PAPases. Since a large portion of neutrophil plasma membrane PAPase is available for hydrolysis of exogenous substrates, ecto-PAPase may play an important role in regulating inflammatory cell responses to extracellular phosphatidic acid in biological systems.

scholarly journals Fluorescent, short-chain C6-NBD-sphingomyelin, but not C6-NBD-glucosylceramide, is subject to extensive degradation in the plasma membrane: implications for signal transduction related to cell differentiation

1995 ◽  
Vol 309 (3) ◽  
pp. 905-912 ◽  
Author(s):  
J W Kok ◽  
T Babia ◽  
K Klappe ◽  
D Hoekstra
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Cell Differentiation ◽  
Plasma Membranes ◽  
Cell Types ◽  
Short Chain ◽  
Synthetic Activity ◽  
Intact Cells ◽  
Ht29 Cells ◽  
Extensive Degradation

The involvement of the plasma membrane in the metabolism of the sphingolipids sphingomyelin (SM) and glucosylceramide (GlcCer) was studied, employing fluorescent short-chain analogues of these lipids, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]hexanoylsphingosylphosphorylcholine (C6-NBD-SM), C6-NBD-GlcCer and their common biosynthetic precursor C6-NBD-ceramide (C6-NBD-Cer). Although these fluorescent short-chain analogues are metabolically active, some caution is to be taken in view of potential changes in biophysical/biochemical properties of the lipid compared with its natural counterpart. However, these short-chain analogues offer the advantage of studying the lipid metabolic enzymes in their natural environment, since detergent solubilization is not necessary for measuring their activity. These studies were carried out with several cell types, including two phenotypes (differing in state of differentiation) of HT29 cells. Degradation and biosynthesis of C6-NBD-SM and C6-NBD-GlcCer were determined in intact cells, in their isolated plasma membranes, and in plasma membranes isolated from rat liver tissue. C6-NBD-SM was found to be subject to extensive degradation in the plasma membrane, due to neutral sphingomyelinase (N-SMase) activity. The extent of C6-NBD-SM hydrolysis showed a general cell-type dependence and turned out to be dependent on the state of cell differentiation, as revealed for HT29 cells. In undifferentiated HT29 cells N-SMase activity was at least threefold higher than in its differentiated counterpart. In contrast, in all cell types studied, very little if any biosynthesis of C6-NBD-SM from the precursor C6-NBD-Cer occurred. Moreover, in the case of C6-NBD-GlcCer, neither hydrolytic nor synthetic activity was found to be associated with the plasma membrane. These results are discussed in the context of the involvement of the sphingolipids SM and GlcCer in signal transduction pathways in the plasma membrane.

scholarly journals The effect of intracellular pH on long-chain fatty acid uptake in 3T3-L1 adipocytes: evidence that uptake involves the passive diffusion of protonated long-chain fatty acids across the plasma membrane

1996 ◽  
Vol 313 (2) ◽  
pp. 487-494 ◽  
Author(s):  
Bernardo L. TRIGATTI ◽  
Gerhard E. GERBER
Keyword(s):  
Fatty Acids ◽  
Plasma Membrane ◽  
Fatty Acid ◽  
Chain Fatty Acid ◽  
Long Chain Fatty Acids ◽  
Cytoplasmic Ph ◽  
Long Chain Fatty Acid ◽  
Intact Cells ◽  
Fatty Acid Binding ◽  
Long Chain

To understand the mechanism of long-chain fatty acid permeation of the plasma membrane in mammalian cells, the effects of changes in the cytoplasmic pH on the internalization of physiologically relevant, submicromolar concentrations of uncomplexed long-chain fatty acids were investigated in 3T3-L1 adipocytes. The acidification of the cytoplasm upon NH4Cl prepulsing of intact cells was accompanied by a rapid reduction of cellular long-chain fatty acid uptake (measured as the total accumulation of [9,10-3H]oleate). This was followed by a slow recovery to normal levels of uptake as the cytoplasmic pH recovered. Conventional filtration assays do not distinguish between fatty acid movement across the plasma membrane and intracellular steps, such as binding to cytoplasmic fatty acid-binding proteins or metabolism. While the in vitro binding of a photoreactive fatty acid, 11-m-diazirinophenoxy[11-3H]undecanoate, to a cytoplasmic fatty acid-binding protein was insensitive to changes in pH from pH 7.5 to 5.5, the in vitro conversion of oleate into oleoyl-CoA by cellular acyl-CoA synthetase decreased dramatically. Therefore, the labelling of the 15 kDa cytoplasmic fatty acid-binding protein in intact cells by the photoreactive fatty acid was used as a more direct measure of the permeation of the probe across the plasma membrane. Acidification of the cytoplasm resulted in an immediate reduction in the labelling of this protein in intact adipocytes. Its photolabelling recovered, however, upon the recovery of the cytoplasmic pH to normal levels. This was due to effects of the cytoplasmic pH on the permeation of the photoreactive fatty acid across the plasma membrane rather than its binding to the 15 kDa protein or metabolism in vivo. This is the first demonstration that the movement of physiologically relevant, submicromolar concentrations of uncomplexed long-chain fatty acids across the plasma membrane of intact cells is coupled to the cytoplasmic pH and suggests that it occurs by the diffusion of the protonated long-chain fatty acid through the lipid bilayer.

scholarly journals Higher-plant medium- and short-chain acyl-CoA oxidases: identification, purification and characterization of two novel enzymes of eukaryotic peroxisomal β-oxidation

1996 ◽  
Vol 320 (2) ◽  
pp. 607-614 ◽  
Author(s):  
Mark A. HOOKS ◽  
Kornelia BODE ◽  
Ivan COUÉE
Keyword(s):  
Molecular Mass ◽  
Subcellular Location ◽  
Maximal Activity ◽  
Fatty Acyl ◽  
Short Chain ◽  
Higher Plant ◽  
Medium Chain ◽  
Chain Acyl ◽  
Acyl Coa Oxidase ◽  
Long Chain

Medium- and short-chain acyl-CoA oxidases were identified in and subsequently purified from dark-grown maize plantlets. The oxidase showing preference for medium-chain fatty acyl-CoAs (C10–C14) was purified to homogeneity. The oxidase showing preference for short-chain fatty acyl-CoAs (C4–C8) was purified over 150-fold. Various catalytic properties confirmed these enzymes to be true acyl-CoA oxidases. They produced trans-2-enoyl-CoA and H2O2 from the saturated acyl-CoA, as verified by various independent assay techniques. They also exhibited FAD-dependent activity; i.e. removal of loosely bound FAD by gel filtration markedly reduced activity, which could be restored upon re-addition of FAD. They showed apparent Km values between 2 and 10 µM for the acyl-CoA substrate giving maximal activity, no activity with the corresponding free fatty acid, high pH optima (8.3–8.6) and a peroxisomal subcellular location. The medium-chain acyl-CoA oxidase was determined to be a monomeric protein with a molecular mass of 62 kDa. The short-chain acyl-CoA oxidase was shown to have a native molecular mass of 60 kDa, but exhibited a labile multimeric structure, as indicated by the elution of multiple peaks of activity during several chromatographic steps, and ultimately by the purification of a subunit of molecular mass 15 kDa. The medium- and short-chain acyl-CoA oxidases were demonstrated to be distinct from the maize equivalent of the cucumber glyoxysomal long-chain acyl-CoA oxidase previously purified and characterized [Kirsch, Loffler and Kindl (1986) J. Biol. Chem. 261, 8570–8575]. The maize long-chain acyl-CoA oxidase was partially purified to permit determination of its substrate specificity; it showed activity with a broad range of acyl-CoAs of chain length greater than C8, and maximal activity with C16. The implications of the existence of multiple acyl-CoA oxidases in the regulation of plant peroxisomal β-oxidation are discussed.

scholarly journals Phosphatidylinositol and phosphatidic acid transport between the ER and plasma membrane during PLC activation requires the Nir2 protein

2016 ◽  
Vol 44 (1) ◽  
pp. 197-201 ◽  
Author(s):  
Yeun Ju Kim ◽  
Maria Luisa Guzman-Hernandez ◽  
Eva Wisniewski ◽  
Nicolas Echeverria ◽  
Tamas Balla
Keyword(s):  
Plasma Membrane ◽  
Phosphatidic Acid ◽  
Lipid Transfer ◽  
Acid Transport ◽  
Contact Zones ◽  
Quiescent Cells ◽  
Associated Proteins ◽  
Phosphatidylinositol 4 ◽  
Hydrolysis Of ◽  
Lateral Sclerosis

Phospholipase C (PLC)-mediated hydrolysis of the limited pool of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] requires replenishment from a larger pool of phosphatidylinositol (PtdIns) via sequential phosphorylation by PtdIns 4-kinases and phosphatidylinositol 4-phosphate (PtdIns4P) 5-kinases. Since PtdIns is synthesized in the endoplasmic reticulum (ER) and PtdIns(4,5)P2 is generated in the PM, it has been postulated that PtdIns transfer proteins (PITPs) provide the means for this lipid transfer function. Recent studies identified the large PITP protein, Nir2 as important for PtdIns transfer from the ER to the PM. It was also found that Nir2 was required for the transfer of phosphatidic acid (PtdOH) from the PM to the ER. In Nir2-depleted cells, activation of PLC leads to PtdOH accumulation in the PM and PtdIns synthesis becomes severely impaired. In quiescent cells, Nir2 is localized to the ER via interaction of its FFAT domain with ER-bound VAMP-associated proteins VAP-A and–B. After PLC activation, Nir2 also binds to the PM via interaction of its C-terminal domains with diacylglycerol (DAG) and PtdOH. Through these interactions, Nir2 functions in ER–PM contact zones. Mutations in VAP-B that have been identified in familial forms of amyotrophic lateral sclerosis (ALS or Lou-Gehrig's disease) cause aggregation of the VAP-B protein, which then impairs its binding to several proteins, including Nir2. These findings have shed new lights on the importance of non-vesicular lipid transfer of PtdIns and PtdOH in ER–PM contact zones with a possible link to a devastating human disease.

Polyphosphate Content and Fine Structure of Acidocalcisomes ofPlasmodium falciparum

2004 ◽  
Vol 10 (5) ◽  
pp. 563-567 ◽  
Author(s):  
Felix A. Ruiz ◽  
Shuhong Luo ◽  
Silvia N.J. Moreno ◽  
Roberto Docampo
Keyword(s):  
Plasmodium Falciparum ◽  
Fine Structure ◽  
Trophozoite Stage ◽  
Intact Cells ◽  
X Ray ◽  
Apicomplexan Parasites ◽  
Ofplasmodium Falciparum ◽  
Irregular Form ◽  
Hydrolysis Of ◽  
Long Chain

Although acidocalcisomes have been well characterized morphologically in other apicomplexan parasites, no such characterization has been done in Plasmodium spp. Here, we report thatPlasmodium falciparummerozoites possess electron-dense organelles rich in phosphorus and calcium, as detected by X-ray microanalysis of intact cells, which are similar to the acidocalcisomes of other apicomplexans, but of more irregular form. In agreement with these results malaria parasites possess large amounts of short- and long-chain polyphosphate (polyP), which are associated with acidocalcisomes in other organisms. PolyP levels were highest in the trophozoite stage of the parasite. Treatment of isolated trophozoites with chloroquine resulted in a significant hydrolysis of polyP. Taken together, these results provide evidence that acidocalcisomes fromPlasmodium falciparumdo not differ significantly from acidocalcisomes of other apicomplexan parasites.

scholarly journals Partial purification of a diacylglycerol lipase from bovine aorta

1994 ◽  
Vol 298 (1) ◽  
pp. 213-219 ◽  
Author(s):  
M W Lee ◽  
D L Severson
Keyword(s):  
Lipase Activity ◽  
Specific Activity ◽  
Competitive Inhibitor ◽  
Short Chain ◽  
Partial Purification ◽  
Diacylglycerol Lipase ◽  
Bivalent Cations ◽  
Triton X ◽  
Hydrolysis Of ◽  
Long Chain

A diacylglycerol (DG) lipase has been purified from a soluble subcellular fraction of bovine aorta by (NH4)2SO4 precipitation in the presence of 5.0% (w/v) Triton X-100, followed by chromatography on DEAE-Sephacel, heparin-Sepharose and octyl-Sepharose in the presence of either CHAPS or Triton X-100 detergents. Under basal conditions, the hydrolysis of a short-chain [3H]dioctanoylglycerol ([3H]diC8) substrate was much greater than that of a long-chain 1-[1-14C]palmitoyl-2-oleoyl-sn-glycerol (1-[14C]POG) substrate. Lipase activity measured with 1-[14C]POG was markedly enhanced by Triton X-100. In the presence of 0.1% Triton X-100, specific enzyme activities in the octyl-Sepharose fraction determined with 1-[14C]POG or 1-stearoyl-2-[1-14C]-arachidonoyl-sn-glycerol as substrates were the same as that measured with [3H]diC8. MgCl2 (5mM) or CaCl2 (2 mM) also selectively stimulated lipase activity (up to 10-13-fold) measured with the long-chain (1-[14C]POG) substrate only. The increase in relative specific activity in the octyl-Sepharose fraction was 60-fold and 155-fold, based on hydrolysis of [3H]diC8 and 1-[14C]POG (+ Triton X-100), respectively. Unlabelled diC8 was a competitive inhibitor of 1-[14C]POG hydrolysis, suggesting that a single lipase hydrolyses both the short-chain and long-chain DG substrates; selective stimulatory effects of non-ionic detergents and bivalent cations on the hydrolysis of 1-[14C]POG may be due to effects on the physical properties of the substrate preparation. Monoacylglycerol lipase, DG kinase and cholesterol esterase activities could not be detected in the partially purified lipase preparation.

scholarly journals Plasma-membrane location of phosphatidylinositol hydrolysis in rabbit neutrophils stimulated with formylmethionyl-leucylphenylalanine

1982 ◽  
Vol 208 (3) ◽  
pp. 801-808 ◽  
Author(s):  
J P Bennett ◽  
S Cockcroft ◽  
A H Caswell ◽  
B D Gomperts
Keyword(s):  
Plasma Membrane ◽  
High Speed ◽  
Plasma Membranes ◽  
Diazonium Salt ◽  
Gradient Centrifugation ◽  
Particulate Fraction ◽  
Intact Cells ◽  
Hydrolysis Of ◽  
High Speed Supernatant ◽  
Sucrose Step Gradient

Rabbit peritoneal neutrophils, disrupted by sonication, were separated into three subcellular fractions by sucrose-step-gradient centrifugation and these were analysed with respect to biochemical markers. They comprised a high-speed supernatant containing the cytosol, a light particulate fraction enriched in Golgi and plasma membranes and a heavy particulate fraction enriched in granules and nuclei. The light particulate fraction was further separated into its components, which were identified as Golgi membranes (galactosyltransferase activity) and plasma membranes ((radioactivity derived from labelling intact cells with [125I]di-iodosulphanilic acid diazonium salt and [3H]formylmethionyl-leucylphenylalanine ([3H]fMet-Leu-Phe) binding)). In cells prelabelled with [3H]glycerol, the hydrolysis of phosphatidylinositol due to cell stimulation with fMet-Leu-Phe (10 nM) was shown to occur in the light particulate fraction. The [32P]Pi-labelling of phosphatidate, which is an early consequence of phosphatidylinositol hydrolysis, also occurred in this fraction. Analytical sucrose-gradient centrifugation of the light particulate fraction showed that the stimulated increment in [32P]phosphatidate (and thus by implication the initial phosphatidylinositol breakdown) was localized in the plasma membrane.

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