Specific and sensitive assay for alkaline and neutral ceramidases involving C12-NBD-ceramide.
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A fluorescent analogue of ceramide, C12-NBD-ceramide, was found to be hydrolyzed much faster than 14C-labeled ceramide by alkaline ceramidase from Pseudomonas aeruginosa and neutral ceramidase from mouse liver, while this substrate was relatively resistant to acid ceramidase from plasma of the horseshoe crab. The radioactive substrate was used more preferentially by the acid ceramidase. It should be noted that C6-NBD-ceramide, which is usually used for ceramidase assays, was hardly hydrolyzed by any of the enzymes examined, compared to C12-NBD-ceramide. For the alkaline and neutral enzymes, the Vmax and k (Vmax/Km) with C12-NBD-ceramide were much higher than those with 14C-ceramide. In contrast, for the acid enzyme these parameters with C12-NBD-ceramide were less than half those with the radioisotope-labeled substrate. It is noteworthy that the labeling of ceramide with NBD did not itself reduce the Km of the alkaline enzyme, but did that of the neutral enzyme. It was also found that C12-NBD-ceramide was preferentially hydrolyzed by the alkaline and neutral enzymes, but not the acid one, in several mammalian cell lines. This study clearly shows that the attachment of NBD, but not dansyl, increases the susceptibility of ceramide to alkaline and neutral enzyme, and decreases that to acid enzymes. Thus the use of this substrate provides a specific and sensitive assay for alkaline and neutral ceramidases. (+info)
Maturation of the axonal plasma membrane requires upregulation of sphingomyelin synthesis and formation of protein-lipid complexes.
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Neuronal maturation is a gradual process; first axons and dendrites are established as distinct morphological entities; next the different intracellular organization of these processes occurs; and finally the specialized plasma membrane domains of these two compartments are formed. Only when this has been accomplished does proper neuronal function take place. In this work we present evidence that the correct distribution of a class of axonal membrane proteins requires a mechanism which involves formation of protein-lipid (sphingomyelin/cholesterol) detergent-insoluble complexes (DIGs). Using biochemistry and immunofluorescence microscopy we now show that in developing neurons the randomly distributed Thy-1 does not interact with lipids into DIGs (in fully developed neurons the formation of such complexes is essential for the correct axonal targeting of this protein). Using lipid mass spectrometry and thin layer chromatography we show that the DIG lipid missing in the developing neurons is sphingomyelin, but not cholesterol or glucosylceramide. Finally, by increasing the intracellular levels of sphingomyelin in the young neurons the formation of Thy-1/DIGs was induced and, consistent with a role in sorting, proper axonal distribution was facilitated. These results emphasize the role of sphingomyelin in axonal, and therefore, neuronal maturation. (+info)
Saturable stimulation of fatty acid transport through model cytoplasm by soluble binding protein.
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To better define the role of soluble binding proteins in the cytoplasmic transport of amphipathic molecules, we measured the diffusional mobility of a fluorescent long-chain fatty acid, 12-N-methyl-(7-nitrobenz-2-oxa-1,3-diazol)aminostearate (NBD-stearate), through model cytoplasm as a function of soluble binding protein concentration. Diffusional mobilities were correlated with the partition of the fatty acid between membrane and protein binding sites. Cytoplasm was modeled as a dense suspension of liposomes, and albumin was used as a model binding protein. Albumin saturably increased NBD-stearate mobility through the membrane suspension approximately eightfold. Fatty acid mobility in the absence of albumin was identical to the mobility of the membrane vesicles (1.99 +/- 0.33 x 10(-8) cm(2)/s), whereas the mobility at saturating concentrations was identical to the mobility of albumin (1.65 +/- 0.12 x 10(-7) cm(2)/s). The protein concentration producing half-maximal stimulation of NBD-stearate diffusion (42.8 +/- 0.3 microM) was unexpectedly greater than that required to solubilize half of the NBD-stearate (17.9 +/- 3.0 microM). These results support a proposed mechanism for cytoplasmic transport of small amphipathic molecules in which aqueous diffusion of the protein-bound form of the molecule largely determines the transport rate. However, slow interchange of fatty acid between the binding protein and membranes also appears to influence the transport rate in this model system. (+info)
Fluorescent phosphoinositide derivatives reveal specific binding of gelsolin and other actin regulatory proteins to mixed lipid bilayers.
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Fluorescent derivatives of phosphatidyl inositol (PtdIns)-(4,5)-P2 were synthesized and used to test the effects of the PtdIns-(4, 5)-P2-regulated proteins gelsolin, tau, cofilin, and profilin on labeled PtdIns-(4,5)-P2 that was either in micellar form or mixed with phosphatidylcholine (PtdCho) in bilayer vesicles. Gelsolin increased the fluorescence of 7-nitrobenz-2-oxa-1,3-diazole (NBD)- or pyrene-labeled PtdIns-(4,5)-P2 and NBD-PtdIns-(3,4,5)-P3. Cofilin and profilin produced no detectable change at equimolar ratios to PtdIns-(4,5)-P2, while tau decreased NBD-PtdIns-(4,5)-P2 fluorescence. Fluorescence enhancement by gelsolin of NBD-PtdIns-(4, 5)-P2 in mixed lipid vesicles depended on the mole fraction of PtdIns-(4,5)-P2 in the bilayer. Specific enhancement of 3% NBD-PtdIns-(4,5)-P2 : 97% PtdCho was much lower than that of 10% PtdIns-(4,5)-P2 : 90% PtdCho, but the enhancement of 3% NBD-PtdIns-(4,5)-P2 could be increased by addition of 7% unlabeled PtdIns-(4,5)-P2. The gelsolin-dependent increase in NBD-PtdIns-(4, 5)-P2 fluorescence was reversed by addition of Ca2+ or G-actin. Significant, but weaker, fluorescence enhancement was observed with the gelsolin N-terminal domain (residues 1-160) and a peptide comprised of gelsolin residues 150-169. Fluorescence energy transfer from gelsolin to pyrene-PtdIns-(4,5)-P2 was much stronger with intact gelsolin than the N-terminal region of gelsolin containing the PtdIns-(4,5)-P2 binding sites, suggesting that PtdIns-(4,5)-P2 may bind near a site formed by the juxtaposition of the N- and C-terminal domains of gelsolin. (+info)
Rapid transbilayer movement of fluorescent phospholipid analogues in the plasma membrane of endocytosis-deficient yeast cells does not require the Drs2 protein.
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Evidence is presented that endocytosis-deficient Saccharomyces cerevisiae end4 yeast cells rapidly internalize the fluorescent phospholipid analogues 1-palmitoyl-2-{6-[7-nitro-2,1, 3-benzoxadiazol-4-yl(NBD)amino] caproyl}phosphatidylcholine (P-C6-NBD-PtdCho) and P-C6-NBD-phosphatidylserine (P-C6-NBD-PtdSer). Both analogues redistributed between the exoplasmic and cytoplasmic leaflet with a half-time of < 15 min at 0 degrees C. The plateau of internalized analogues was about 70%. Transbilayer movement is probably protein-mediated, as the flip-flop of both analogues was very slow in liposomes composed of plasma-membrane lipids. Rapid analogue internalization was not abolished on depletion of intracellular ATP by about 90%. For P-C6-NBD-PtdCho only was a moderate decrease in the plateau of internalized analogues of about 20% observed, while that of P-C6-NBD-PtdSer was not affected. The Drs2 protein plays only a minor role, if any, in the rapid transbilayer movement of analogues in S. cerevisiae end4 cells. In S. cerevisiae end4 Deltadrs2 cells harbouring both an end4 allele and a drs2 null allele, about 60% and 50% of P-C6-NBD-PtdCho and P-C6-NBD-PtdSer, respectively, became internalized within 15 min at 0 degrees C. The preferential orientation of P-C6-NBD-PtdSer to the cytoplasmic leaflet is in qualitative agreement with the sequestering of endogenous phosphatidylserine to the cytoplasmic leaflet, as assessed by binding of annexin V. Virtually no binding of annexin V to spheroplasts of the parent wild-type strain or the mutant strains was observed. Likewise, no difference in the exposure of endogenous aminophospholipids to the exoplasmic leaflet between these strains was found by labelling with trinitrobenzenesulfonic acid. Thus, lipid asymmetry, at least of aminophospholipids, was preserved in S. cerevisiae end4 cells independently of the presence of the Drs2 protein. (+info)
Cytoplasmic transport of fatty acids in rat enterocytes: role of binding to fatty acid-binding protein.
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The intracellular movement of fatty acids is thought to be facilitated through codiffusion with fatty acid-binding protein (FABP). This facilitation may occur by decreasing binding to immobile membranes, leading to faster cytoplasmic diffusion. The aims of this study were to measure the intracellular transport of 12-N-methyl-(7-nitrobenzo-2-oxa-1,3-diazol)aminostearate (NBD-stearate) in villus rat enterocytes and to determine 1) the mechanism of its cytoplasmic transport and 2) if its transport rate correlated with the known variation of FABP binding capacity along the length of the small intestine. Two-dimensional laser photobleaching was used to measure the movement of a fluorescent fatty acid NBD-stearate in enterocytes isolated from different segments of rat intestine. The fraction of NBD-stearate found in the cytostol of enterocytes was determined by differential centrifugation. Cytoplasmic transport of NBD-stearate occurred solely by diffusion and not by convection. Diffusion was homogeneous (nondirectional), consistent with isotropic diffusion. The diffusion rate varied with location along the intestine, correlating with the local FABP concentration and measured cytosolic binding. We conclude that cytoplasmic proteins like FABP promote the intracellular transport of fatty acids by enhancing their diffusive flux. We suggest that facilitation is not specific for a particular cell type but occurs in a variety of cells that transport fatty acids and may contain different types of FABP. (+info)
Brownian ratchets: molecular separations in lipid bilayers supported on patterned arrays.
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Brownian ratchets use a time-varying asymmetric potential that can be applied to separate diffusing particles or molecules. A new type of Brownian ratchet, a geometrical Brownian ratchet, has been realized. Charged, fluorescently labeled phospholipids in a two-dimensional fluid bilayer were driven in one direction by an electric field through a two-dimensional periodic array of asymmetric barriers to lateral diffusion fabricated from titanium oxide on silica. Diffusion spreads the phospholipid molecules in the orthogonal direction, and the asymmetric barriers rectify the Brownian motion, causing a directional transport of molecules. The geometrical ratchet can be used as a continuous molecular sieve to separate mixtures of membrane-associated molecules that differ in electrophoretic mobility and diffusion coefficient. (+info)
Location of the catalytic nucleophile of phospholipase D of Streptomyces antibioticus in the C-terminal half domain.
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Phospholipase D (PLD) of Streptomyces antibioticus was labelled with fluorescent-labelled substrate, 1-hexanoyl-2-{6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)-amino]hexanoyl}-sn-glycero-3-phosphocholine, when it was incubated with the substrate and the reaction followed by SDS/PAGE. Mutant enzymes lacking the catalytic activity were not labelled under the same conditions, indicating that labelling of the PLD occurred as the result of its catalytic action. This confirmed that the labelled protein was the phosphatidyl PLD intermediate. PLDs contain two copies of the highly conserved catalytic HxKxxxxD (HKD) motif. Therefore, two protein fragments were separately prepared with recombinant strains of Escherichia coli. One of the fragments was the N-terminal half of the intact PLD containing one HKD motif, and the other was the C-terminal half with the other motif. An active enzyme was reconstructed from these two fragments, and therefore designated fragmentary PLD (fPLD). When fPLD was subjected to the labelling experiment, only the C-terminal half was labelled. Therefore, it was concluded that the catalytic nucleophile that bound directly to the phosphatidyl group of the substrate was located on the C-terminal half of PLD, and that the N-terminal half did not contain such a nucleophile. (+info)