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Intramembranous arrangement of phospholipids and dolichol in microsomal and model membranes
Stockholm University, Faculty of Science.
1985 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The transfer of phospholipids from the endoplasmic reticulum to the inner mitochondrial membrane was investigated by pulse labeling in vivo. With [3H]gIycerol microsomal phosphatidylethanoiamine and phosphatidylcholine were rapidly labeled during the first 30 min; while maximum incorporation into the inner mitochondrial membrane occured only after about 5 hours. It appears that the in vivo transfer of these phospholipids between the two membrane comparments is a relatively slow process.

Microsomal membranes from rat liver were treated with the cross-linking reagent l,5-difluoro-2,4-dinitrobenzene (DFDNB). Experimental work showed that at a probe concentration of 0.75 mM all free phosphatidylethanoiamine (PE) and phosphatidylse- rine (PS) were found as dinitrophenyl derivatives: 29% of PE was in monomeric form, 9% dimeric, 2% interacted with PS, and 65% cross-linked to protein. The cross-linking pattern of PE was clearly different from that pattern which is present in the inner mitochondrial and erythrocyte membranes. In vivo labeling of PE with [3H]glycerol and [3H]ethanolamine followed by phospholipase A2 or DFDNB treatment of isolated microsomes established a heterogeneous labeling pattern during the first 2 hours in the different PE pools. The experiments indicate that the biosynthesis of PE takes place in a compartment that is more accessible to surface probes and that the labeled molecules are transferred in a time-dependent process to a second compartment where the lipid is not available for phospholipase A, action but is available for cross-linking to protein.

Rough microsomes from rat liver of both control and methylcholanthrene treated animals were subfractionated on a discontinuous sucrose gradient into three fractions according to their sedimentation velocity. The slowly sedimenting vesicles were enriched in electron transport enzymes, while those in the pellet showed higher phosphatase and ATPase activities. The individual phospholipids exhibited an identical distribution pattern in the three subfractions and no change occurred after induction with methylcholanthrene treatment. Nearest neighbour analysis of phosphatidylethanoiamine with dinitrodifluoroenzene revealed a similar pattern in the enzymatically different subfraction. When membrane and enzyme synthesis was induced, cross-linking to proteins were substantially decreased. The experiments indicate that the phospholipids are distributed in a homogeneous fashion in the lateral plane of the rough microsomal membrane.

Rat liver microsomes were incubated with the monofunctional aminoreagent fluo- rescamine. Although the probe easily penetrates the membrane, two pools of PE could be detected. The first pool rapidly reacts with the probe, comprising 80% of the total, while the second pool exhibits a very slow interaction, the two pools showed differences in fatty acid composition as well as in the positions they were attached. In vivo labeling with ethanolamine, glycerol, palmitic and stearic acid resulted in a higher specific activity in the first pool after 1 h; equilibration with the second pool took about 3 h. No equlibration between the pools could be detected under in vitro conditions. Injection of liposomes consisting of labeled synthetic phosphatidylethanolamines into the portal vein was followed by uptake by the hepatocytes and incorporation of the lipids into the microsomal membranes. Depending on the fatty acid composition of the injected lipid, either of the two pools became selected. It is suggested that the fatty acid composition of a given phospholipid molecule exerts a signal function directing the lipid to its final intramembraneous location.

The studies on the influence of isoprenes on phospholipid fluidity and polymorphism in model membranes revealed that dolichol, dolichol esters and dolichyl phosphate have a chain length dependent bilayer destablizing effect on unsaturated PE’s and mixtures of PE/PC, thus promoting the hexagonal II phase. An increased fatty acyl chain fluidity could be detected both in PE and PC membranes after incorporation of dolichyl phosphate, whereas dolichol only exerts this effect on PE membranes. Dolichol esters did not change their membrane fluidity, probably because they are only soluble to a certain extent in the model membranes. Dolichyl phosphate has its polar group oriented in the headgroup region perpendicular to the membrane surface, which is probably not the case with the other isoprenes.

Place, publisher, year, edition, pages
Stockholm: Stockholm University, 1985. , p. 50
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:su:diva-174601Libris ID: 7608671ISBN: 91-7146-638-X (print)OAI: oai:DiVA.org:su-174601DiVA, id: diva2:1358751
Public defence
1985-05-03, Seminarierummet 3, Arrheniuslaboratoriet, Frescati, Stockholm, 10:00
Note

Härtill 5 uppsatser

Available from: 2019-10-08 Created: 2019-10-08 Last updated: 2019-12-10Bibliographically approved

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