A.M.H.P. Van Den Besselaar

Phosphatidylcholine mobility in liver microsomal membranes.

Abstract Purified phosphatidylcholine exchange protein from bovine liver was used to exchange rat liver microsomal phosphatidylcholine for egg phosphatidylcholine. It was found that at 25 and 37°C rat liver microsomal phosphatidylcholine was completely and rapidly available for replacement by egg phosphatidylcholine. In contrast, phosphatidylcholine in vesicles prepared from total microsomal lipids could only be exchanged for about 60%. At 8 and 0°C complex exchange kinetics were observed for phosphatidylcholine in rat liver microsomes. The exchange process had neither effect on the permeability of the microsomal membrane to mannose 6-phosphate, nor on the permeability of the phosphatidylcholine vesicles to neodymium (III) cations. Purified phospholipase A 2 from Naja naja could hydrolyze some 55–60% of microsomal phosphatidylcholine at 0°C, but 70–80% at 37°C. Microsomal phosphatidylcholine, remaining after phospholipase treatment at 37°C, could be exchanged for egg phosphatidylcholine at 37°C, but at a slower rate than with intact microsomes. Microsomal phosphatidylcholine remaining after phospholipase treatment at 0 and 37°C had a lower content of arachidonic acid than the original phosphatidylcholine. These results are discussed with respect to the localization and transmembrane movement of phosphatidylcholine in liver microsomes.

Evidence for isotropic motion of phospholipids in liver microsomal membranes. A 31P NMR study

1. The motional properties of phospholipids in bovine and rat liver microsomes and aqueous dispersions of the extracted lipids have been investigated employing 31 P NMR techniques. 2. The 31P NMR spectra obtained from the microsomes indicate that a considerable portion of the constituent phospholipids experience isotropic motion on the NMR timescale (10(-5) s). This is in strong contrast to the spectra obtained from aqueous dispersions of the extracted lipids, which display the characteristic lineshape associated with liquid crystalline phospholipids in (large) bilayer structures, which experience restricted anisotropic motion. 3. Evidence is presented which strongly suggests that the isotropic motion of microsomal phospholipids does not arise from tumbling of the microsomal vesicles or from lateral diffusion of phospholipids around these vesicles. 4. These results are discussed in terms of possible transitory formation of intramembrane non-bilayer lipid configurations, with which the bulk (bilayer) phospholipids are in rapid exchange.

Outside-inside distribution and translocation of lysophosphatidylcholine in phosphatidylcholine vesicles as determined by 13C-NMR using (N-13CH3)-enriched lipids

Abstract 1. 1. The outside-inside distribution of palmitoyl lysophosphatidylcholine and dioleoyl phosphatidylcholine in mixed sonicated vesicles is measured with (N- 13 CH 3 )-labelled lipids using 13 C NMR and Dy 3+ as an impermeable shift reagent. 2. 2. Palmitoyl lysophosphatidylcholine is preferentially localised in the outside layer of the vesicle membrane. Incorporation of cholesterol in the vesicle diminishes the extent of lysophosphatidylcholine asymmetry. 3. 3. Palmitoyl lysophosphatidylcholine added to dioleoyl phosphatidylcholine vesicles is incorporated in the outer monolayer of the vesicle. Even after 40 h less than 2% of the lysophosphatidylcholine could be detected in the inner monolayer. Since in the cosonicated vesicles 17% of the lysophosphatidylcholine is present in the inner monolayer it can be concluded that the transmembrane movement of lysophosphatidylcholine across the lipid bilayer of these vesicles is an extremely slow process.

Transbilayer distribution and movement of lysophosphatidylcholine in liposomal membranes

Single bilayer vesicles were prepared by sonication of 5 mol% 1-palmitoyl lysophosphatidylcholine and 95 mol% egg phosphatidylcholine. Incubation with lysophospholipase results in a fast hydrolysis of 80–90% of lysophosphatidylcholine. The remaining lysophosphatidylcholine is only very slowly hydrolysed. There results are interpreted as lysophosphatidylcholine being asymmetrically distributed over the two halves of the bilayer. The slow phase of lysophosphatidylcholine hydrolysis sets an upper limit to the rate of transbilayer movement of lysophosphatidylcholine. The half time of this process at 37° C is estimated to be about 100 h. Incorporation of cholesterol in the vesicles reduces the distributional asymmetry of lysophosphatidylcholine to the extent of an outside-inside ratio of 60 : 40. [14C]Lysophosphatidylcholine introduced into the outer monolayer of such vesicles by intervesicular transfer of lysophosphatidylcholine remains virtually completely available for hydrolysis by lysophospholipases, corroborating the interpretation that transbilayer movement of lysophosphatidylcholine in these vesicles is an extremely slow process. In handshaken liposomes consisting of 5 mol% 1-palmitoyl lysophosphatidylcholine and 95 mol% egg phosphatidylcholine 15–20% of lysophosphatidylcholine is readily available for exogenous lysophospholipase. This pool may represent lysophosphatidylcholine in the outer monolayer of the liposomes.

Transverse distribution and movement of lysophosphatidylcholine in sarcoplasmic reticulum membranes as determined by 13C NMR and lysophospholipase.

1. 1. The transverse distribution of 1-palmitoyl-sn-glycero-3-phospho-N-[Me-13C]-choline in vitro incorporated in sarcoplasmic reticulum has been measured by means of 13C NMR and DyCl3 as an impermeable shift reagent. 2. 2. Lysophosphatidylcholine added to the membranes equilibrates within 30 min at 20°C between outer and inner membrane leaflet so that 42% is located in the inner leaflet. 3. 3. Lysophosphatidylcholine diffuses back from the inner leaflet to the outer upon lysophospholipase action on the outer lysophosphatidylcholine pool.

Studies on lysophospholipases: VI. The action of two purified lysophospholipases from beef liver on membrane-bound lysophosphatidylcholine

The action of two lysophospholipases purified from beef liver on lysophosphatidylcholine in microsomal membranes has been studied. Enzyme I, which has been shown to be localized in the soluble fraction of the beef liver cell, has a higher specific activity on microsomal lysophosphatidylcholine than Enzyme II, which originates from the microsomal cell fraction. This trend is also observed with phosphatidylcholine liposomes and single bilayer vesicles in which lysophosphatidylcholine has been incorporated. At low mol fractions of lysophosphatidylcholine in liposomes, the maximum enzymatic rate is proportional to this mol fraction. Similar results are obtained with mixed micelles of lysophosphatidylcholine and Triton X-100. The results are explained in terms of a model in which the two-dimensional substrate density in the membrane surface controls the rate of enzyme action.

Availability of lysophosphatidylcholine in single bilayer vesicles for hydrolysis by lysophospholipase

Single bilayer vesicles were prepared from total rat liver microsomal lipids to which 5 mol% lysophosphatidylcholine had been added. The availability of lysophosphatidylcholine for enzymatic hydrolysis by lysophospholipase (EC 3.1.1.5) was found to be higher in vesicles prepared by the cholate dispersion technique when compared with sonicated vesicles. Sepharose 4 B chromatography showed that the vesicles prepared by the cholate technique were smaller than those prepared by sonication. This is in contrast to previous observations for egg phosphatidylcholine vesicles. Total rat liver microsomal extracts were found to contain proteolipid, which could be removed by ether precipitation. Cholate vesicles prepared from proteolipid-free extracts were still smaller than sonicated vesicles from this extract. Experiments with [14C] dextran entrapped in the vesicles indicate that there is no loss of the permeability barrier of the vesicles for high molecular weight solutes during vesicle treatment with lysophospholipase. The high availability of lysophosphatidylcholine in cholate vesicles of total rat liver microsomal lipids is discussed in terms of a highly asymmetric distribution of lysophosphatidylcholine over the inner and outer monolayer of the bilayer.

Studies on lysophospholipases: VIII. Immunochemical differences between two lysophospholipases from beef liver

Abstract 1. 1. Two distinct lysophospholipases have previously been obtained in homogeneous form from beef liver. In this paper, we demonstrate that ageing of a beef liver homogenate does not result in a change in the ratio of the two enzymatic activities, indicating that no interconversion of the lysophospholipases took place. 2. 2. Possible partial structural relationships between the two enzymes were explored by immunochemical techniques. Rabbit antisera raised against each individual lysophospholipase showed no cross-reactivity with the other enzyme. This was concluded from immuno double-diffusion experiments and from the results of immunoprecipitation of enzymatic activities in solution. 3. 3. Lysophospholipase and esterase activity in the purified preparation of lysophospholipase II from beef liver were concomitantly precipitated by antiserum against lysophospholipase II. This is further proof that both enzymatic activities reside in a single polypeptide chain, in agreement with previous results of isoelectric focusing experiments.

Transbilayer Localization and Movement of Lysophosphatidylcholine and Phosphatidylcholine in Model — and Biomembranes

It has long been recognized that biological membranes represent vectorial structures with a topological asymmetry of their protein constituents, notably transport factors, receptors and glycoproteins. Only more recently has an asymmetric disposition of the phospholipids in the transverse plane of the membrane been appreciated. Classical in this respect are studies on the phospholipid distribution in the erythrocyte membrane. The use of specific phospholipases has led to the conclusion that sphingomyelin, and the majority of phosphatidylcholine are located in the outer half of the bilayer while phosphatidylserine and most of the phosphatidylethanolamine are situated in the inner monolayer (1). The use of impermeable reagents to detect the amino-phospholipids phosphatidylethanolamine and phosphatidylserine, gave results consistent with this distribution (2). It is obvious that the asymmetric arrangement of phospholipids in biomembranes, if a general phenomenon, can have important implications for the availability of phospholipid classes or -species for the functioning of membrane proteins in general and of phospholipid metabolizing enzymes in particular. For example, can phospholipases A located in a given monolayer only release fatty acids from phospholipids in that monolayer or are the phospholipids in that monolayer in dynamic equilibrium with those in the other monolayer through a rapid transbilayer movement? Is further metabolism of the reaction products of such a phospholipase activity, i.e. lysophospholipids and free fatty acids, restricted to the monolayer in which they are formed or can they move to the other monolayer as well?