Laurens L. M. Van Deenen

ATP-dependent translocation of amino phospholipids across the human erythrocyte membrane

Trace amounts of radiolabeled phospholipids were inserted into the outer membrane leaflet of intact human erythrocytes, using a non‐specific lipid transfer protein. Phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine were transferred from the donor lipid vesicles to the membrane of the intact red cell with equal ease, whilst sphingomyelin was transferred 6‐times less efficiently. The transbilayer mobility and equilibrium distribution of the labeled phospholipids were assessed by treatment of the intact cells with phospholipases. In fresh erythrocytes, the labeled amino phospholipids appeared to move rapidly towards the inner leaflet. The choline phospholipids, on the other hand, approached an equilibrium distribution which strongly favoured the outer leaflet. In ATP‐depleted erythrocytes, the relocation of the amino phospholipids was markedly retarded.

Effect of dimyristoyl phosphatidylcholine on intact erythrocytes. Release of spectrin-free vesicles without ATP depletion

Incubation of human erythrocytes with suspensions of sonicated dimyristoyl phosphatidylcholine resulted in dramatic morphological changes of the cells and release of membrane vesicles. The shedding of membrane vesicles was not preceded by ATP depletion and only occurred at temperatures of incubation that were above the phase transition temperature of dimyristoyl phosphatidylcholine. Membrane vesicles were separated from intact erythrocytes and exogenous dimyristoyl phosphatidylcholine by a series of centrifugation steps. The lipid composition of the membrane vesicles was similar to that of the native erythrocyte, and the predominant membrane proteins were band 3, glycophorin and acetylcholinesterase. Spectrin was not detected. Freeze-fracture electron microscopy showed vesicles (150 nm in diameter) with protein particles embedded in the lipid bilayer.

Phosphatidylinositol exchange protein effects of membrane structure on activity and evidence for a ping-pong mechanism

Abstract Phosphatidylinositol exchange protein, purified from bovine cerebral cortex, catalyzes the transfer of phosphatidylinositol and, to a lesser extent, phosphatidylcholine between rat liver microsomes and egg phosphatidylcholine liposomes. Transfer activity is sensitive to pH, temperature, and the method of liposome preparation. Variation of the phospholipid composition of the liposomes produces vesicles for which the apparent Michaelis constant decreases with increasing molar proportions of phosphatidylinositol. Interaction of exchange protein with liposomes containing radioactively labeled phosphatidylcholine allows the isolation of a phospholipid-protein complex; dissociation of this complex occurs upon subsequent interaction with unlabeled liposomes. Changes in the concentration of the two membrane species, microsomes and liposomes, yield results which are interpreted in terms of a ping-pong kinetic mechanism for the protein-catalyzed, intermembrane transfer of phospholipids.

Structural and dynamic aspects of phosphatidylcholine in the human erythrocyte membrane

Abstract A protein responsible for the phosphatidylcholine-specific transfer of phospholipid across membranes provides a useful tool for retailoring the composition of the phosphatidylcholine species in the erythrocyte membrane. Using the same protein, the transbilayer mobility of this phospholipid in normal and abnormal erythrocytes can be determined.

Asymmetric incorporation of Na+, K+-ATPase into phospholipid vesicles

Abstract Purified lamb kidney Na + , K + -ATPase, consisting solely of the M τ = 95,000 catalytic subunit and the M τ ∼- 44,000 glycoprotein, was solubilized with Triton X-100 and incorporated into unilamellar phospholipid vesicles. Freeze-fracture electron microscopy of the vesicles showed intramembranous particles of approximately 90–100 A in diameter, which are similar to those seen in the native Na + ,K + -ATPase fraction. Digestion of the reconstituted proteins with neuraminidase indicated that the glycoprotein moiety of the Na + ,K + -ATPase was asymmetrically oriented in the reconstituted vesicles, with greater than 85% of the total sialic acid directed toward the outside of the vesicles. In contrast, in the native Na + ,K + -ATPase fraction, the glycoprotein was symmetrically distributed. Purified glycoprotein was also asymmetrically incorporated into phospholipid vesicles using Triton X-100 and without detergents as described by R. I. MacDonald and R. L. MacDonald (1975 , J. Biol. Chem. 250 , 9206–9214). The glycoprotein-containing vesicles were 500–1000 A in diameter, unilamellar, and, in contrast to the vesicles containing the Na + ,K + -ATPase, did not contain the 90- to 100-A intramembranous particles. These results indicate that the intramembranous particles observed in the native Na + ,K + -ATPase and in the reconstituted Na + ,K + -ATPase are not due to the glycoprotein alone, but represent either the catalytic subunit, or the catalytic plus the glycoprotein subunit.

Selective elimination of malaria infected erythrocytes by a modified phospholipase A2 in vitro

Pig pancreatic phospholipase A2 does not act on normal erythrocytes, but the membrane penetrating capacity is enhanced by the covalent attachment of one fatty acyl chain to Lys-116 of the enzyme. Taking advantage of the impaired packing of phospholipids in the membrane of Plasmodium infected erythrocytes it was demonstrated that a lauric acid derivative of phospholipase A2 is capable of exclusively attaching the infected erythrocytes in vitro, leaving the uninfected cells undisturbed. The chemically modified phospholipase A2 appeared to cause death of the parasite in cell cultures of infected erythrocytes.

Studies on Acholeplasma laidlawii grown on branched-chain fatty acids

Abstract Acholeplasma laidlawii B was grown on the branched-chain fatty acids, 14-methylpentadecanoic acid and 14-methylhexadecanoic acid, and the straight-chain palmitic acid. The incorporation of the branched-chain fatty acids was very effective; more than 90% of the fatty acids of the lipids of this organism consisted of the branched-chain constituents. A somewhat smaller amount (81%) was found in the cells grown with palmitic acid. Differential scanning calorimetry of the isolated membranes showed that distinct lipid phase transitions occurred in between 15 and 31 °C for the 14-methylpentadecanoic acid, 11 and 29 °C for the 14-methylhexadecanoic acid, and 14 and 36 °C for the palmitic acid-enriched membranes. Freeze-fracture electron microscopy showed that the lipid phase transitions were accompanied by particle aggregation only in the case of palmitic acid-enriched membranes. When the branched-chain acid-enriched membranes were quenched from temperatures below the onset of the lipid phase transition, a random distribution of particles on both fracture faces of the membrane was observed. The membranes were incubated with pig pancreatic phospholipase A 2 at various temperatures. Below the onset of the lipid phase transition phosphatidylglycerol was not accessible for this enzyme in palmitate-enriched membranes. However, a fast hydrolysis of 60–75% of the phosphatidylglycerol could be measured in the branched-chain acid-enriched membranes at temperatures below the onset of the lipid phase transition. The residual phosphatidylglycerol could be hydrolyzed at a slower, temperature-dependent rate. The observations show that lipids containing branched-chain acids undergo a cooperative lipid phase transition which does not result in a tight packing of the lipids of the bilayer below the phase transition.

On the interaction between intrinsic proteins and phosphatidylglycerol in the membrane of Acholeplasma laidlawii.

About 30% of the phosphatidylglycerol in oleic acid-enriched Acholeplasma laidlawii membranes are not hydrolyzed at temperatures below 10 °C by phospholipase A2 from porcine pancreas. Removal of 53% of the membrane proteins by proteolysis did not reduce the size of this inaccessible phosphatidylglycerol pool. However, modification of the membrane proteins with 2,4,6-trinitrobenzenesulfonic acid or glutaraldehyde did make an additional 70% of this protected pool of phosphatidylglycerol accessible to phospholipase A2. Complete hydrolysis of phosphatidylglycerol at low incubation temperatures was achieved only after heat treatment of the membranes which resulted in an extensive aggregation of intrinsic membrane proteins as visualized by freeze-etch electron microscopy. Phospholipase A2 from bee venom was more effective in hydrolyzing phosphatidylglycerol at low temperature than the pancreatic enzyme. These results show that the inaccessibility of phosphatidylglycerol is not due to resealing of isolated membranes, the presence of a crystalline phase in the membrane lipids, or a shielding effect of surface proteins. The protection against hydrolysis may be due to an interaction of phosphatidylglycerol with intrinsic membrane proteins which is stabilized at low temperatures. Increasing the temperature favors the exchange of protein-bound phosphatidylglycerol with other membrane lipids resulting in complete hydrolysis.

Cooperative Protection by Vitamins E and C of Human Erythrocyte Membranes against Peroxidation

As a membrane-soluble radical scavenger, vitamin E (tocopherol) appears to be of vital importance in the protection of biological membranes against peroxidation. It was proposed by Tappel’ that vitamin C in the water phase could regenerate membrane vitamin E from vitamin E radical, thus maintaining an active vitamin E pool. This kind of interaction between vitamins E and C and the synergistic protection of membranes resulting therefrom have been observed in solution, in model membranes, and recently in a microsomal system.’ We have performed studies on the effect of vitamins E and C, alone as well as in combination, on lipid peroxidation in human erythrocyte white ghost membranes. Cumene hydroperoxide plus hemin-Fe3+ were used as oxidants, resulting in the formation of cumene (per)oxyl radicals in the membranes that initiate lipid peroxidation. The fluorescent polyunsaturated fatty acid, parinaric acid (PnA, 9,11,13,15-octadecatetraenoic acid), was used as a sensitive membrane probe for per~xidation.”~ Peroxidation of the conjugated double bond system of PnA is accompanied by a loss in its fluorescent properties, which can be detected in a direct, continuous, and sensitive way. Analysis of the PnA fluorescence decrease curves in peroxidation experiments yields information on the peroxidation process of PnA. Vitamin E incorporated in the erythrocyte membranes exhibited a clearly protective effect on the peroxidation on PnA, introducing a concentration-dependent lag phase. It can be assumed that cumene (per )oxyl radicals entering the membrane primarily react with vitamin E molecules, thus preventing peroxidation of PnA. The effect of vitamin C (ascorbate) on peroxidation is known to depend on its concentration as well as on the oxidant system. In our experiments, vitamin C appeared to have a “double dualistic” effect on PnA peroxidation (FIG. 1). The overall effect of vitamin C on peroxidation, as determined 5 or 10 min after addition of oxidants, can be proor antioxidant, depending on the initial vitamin C concentration (FIG. lB, open circles). In addition, the effect apparently changes from antioxidant (lag phase, FIG. 1A) to prooxidant within the time course of an experiment at low initial vitamin C concentrations. In the presence of both vitamins (FIG. 1, closed circles), the overall effect is always protective and exceeds the sum of the individual effects.

Transbilayer Organization and Mobility of Phospholipids in Normal and Pathologic Erythrocytes

Regarding the marked asymmetric distribution of phospholipids in the erythrocyte membrane, which is characterized by the two choline containing phospholipids, sphingomyelin (SM) and phosphatidyl- choline (PC), being mainly located in the outer monolayer, whereas most of the phosphatidylethanolamine (PE) and all of the phosphatidylserine (PS) is found in the cytoplasmic leaflet (Zwaal, et.al. 1975, Op den Kamp, 1984), some important questions may be raised. These concern: (i) its constitution during biogenesis of the cell, (ii) its maintenance during the life span of the erythrocyte, and last but not least, (iii) its physiologic significance.