| 1. |
- Florén, Claes-Henrik, et al.
(författare)
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Binding, interiorization and degradation of cholesteryl ester labelled chylomicron remnant particles by rat hepatocyte monolayers
- 1977
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Ingår i: The Biochemical journal. - : Portland Press Ltd.. - 0264-6021. ; 168:3, s. 483-494
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Tidskriftsartikel (refereegranskat)abstract
- 1. The cholesteryl ester of isolated chylomicron-remnant particles was efficiently degraded by hepatocyte monolayers. The degradation was sensitive to metabolic inhibitors. 2. With increasing amounts of remnant cholesteryl ester the rate of uptake approached saturation and conformed to a linear double-reciprocal plot. The V(max.) was determined as 80ng of cholesteryl ester/h per mg of protein and the apparent K(m) as 1.4mug of cholesteryl ester per mg of protein. The time course for the uptake and hydrolysis suggested that binding of particles to the cell surface preceded the degradation. 3. Cholesteryl esters of native chylomicrons were degraded to a much smaller extent and their presence had only a small inhibitory effect on the degradation of chylomicron remnants. Intestinal very-low-density lipoproteins were degraded somewhat faster than chylomicrons, and caused more inhibition of remnant degradation. Rat high-density lipoproteins inhibited the hydrolysis of remnant cholesteryl ester by up to 50%, but had less influence on the amount of cholesteryl ester that was bound to the cells. Serum decreased both the uptake and hydrolysis, whereas d=1.21 infranatant had no effect. 4. The cholesteryl ester hydrolysis after the uptake by the cells was inhibited by chloroquine and by colchicine. Only 28-36% of the unhydrolysed cholesteryl ester could be released from these cells by trypsin treatment, indicating that the major portion was truly intracellular. The particles that could be released from the cell surface by trypsin and those remaining in the medium had the same triacylglycerol/cholesteryl ester ratio as the added remnant particles. Significant amounts of denser particles were thus not formed during contact with the cell surface. 5. The presence of heparin, as well as preincubation of the cells with heparin, increased the uptake of chylomicron remnants. This effect was most marked in the presence of serum. A much smaller proportion of the other serum lipoproteins was taken up, and this proportion was not increased by heparin.
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| 2. |
- Wu, Jun, et al.
(författare)
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Functional studies of human intestinal alkaline sphingomyelinase by deglycosylation and mutagenesis
- 2005
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Ingår i: Biochemical Journal. - 0264-6021. ; 386, s. 153-160
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Tidskriftsartikel (refereegranskat)abstract
- Intestinal alk-SMase (alkaline sphingomyelinase) is an ectoenzyme related to the NPP (nucleotide phosphodiesterase) family. It has five potential N-glycosylation sites and predicated transmembrane domains at both the N- and C-termini. The amino acid residues forming the two metal-binding sites in NPP are conserved, and those of the active core are modified. We examined the functional changes of the enzyme induced by deglycosylation and mutagenesis. Treating alk-SMase cDNA-transfected COS-7 cells with tunicamycin rendered the expressed enzyme completely inactive. Mutations of the five potential N-glycosylation sites individually and in combination showed that these sites were all glycosylated and deficient glycosylation decreased the enzyme activity. Immunogold labelling showed that the wild-type enzyme was mainly located in the plasma membrane, whereas the C-terminal domain-truncated enzyme was released into the medium. Deglycosylation blocked the release of the enzyme that accumulated in endosome-like structures. The enzyme activity was also decreased by mutations of the residues forming the putative metalbinding sites and the active core. Substitution of the active core sequence with that of NPP or mutation of T75 in the core abolished the enzyme activity against sphingomyel in but failed to render the enzyme NPP active. Our results indicate that alk-SMase activity is severely affected by defective N-glycosylation and structural alterations of the putative metal-binding sites and the predicted active core.
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| 3. |
- Wu, Jun, et al.
(författare)
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Intestinal alkaline sphingomyelinase hydrolyses and inactivates platelet-activating factor by a phospholipase C activity.
- 2006
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Ingår i: The Biochemical journal. - 1470-8728. ; 394, s. 299-308
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Tidskriftsartikel (refereegranskat)abstract
- Alkaline sphingomyelinase (alk-SMase) is a new member of the NPP (nucleotide pyrophosphatase/phosphodiesterase) family that hydrolyses SM (sphingomyelin) to generate ceramide in the intestinal tract. The enzyme may protect the intestinal mucosa from inflammation and tumorigenesis. PAF (platelet-activating factor) is a pro-inflammatory phospholipid involved in pathogenesis of inflammatory bowel diseases. We examined whether alk-SMase can hydrolyse and inactivate PAF. [3H]Octadecyl-labelled PAF was incubated with purified rat intestinal alk-SMase or recombinant human alk-SMase expressed in COS-7 cells. The hydrolytic products were assayed with TLC and MS. We found that alkSMase cleaved the phosphocholine head group from PAF and generated 1-O-alkyl-2-acetyl-sn-glycerol. Differing from the activity against SM, the activity against PAF was optimal at pH 7.5, inhibited by EDTA and stimulated by 0.1-0.25 mM Zn2+. The activity was abolished by site mutation of the predicted metal-binding sites that are conserved in all NPP members. Similar to the activity against SM, the activity against PAF was dependent on bile salt, particularly taurocholate and taurochenodeoxycholate. The V(max) for PAF hydrolysis was 374 mumol x h(-1) x (mg of protein)(-1). The hydrolysis of PAF and SM could be inhibited by the presence of SM and PAF respectively, the inhibition of PAF hydrolysis by SM being stronger. The PAF-induced MAPK (mitogen-activated protein kinase) activation and IL-8 (interleukin 8) release in HT-29 cells, and chemotaxis in leucocytes were abolished by alk-SMase treatment. In conclusion, alk-SMase hydrolyses and inactivates PAF by a phospholipase C activity. The finding reveals a novel function, by which alk-SMase may counteract the development of intestinal inflammation and colon cancer.
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