Structure of hemocyanin II from the horseshoe crab, Limulus polyphemus. Sequences of the overlapping peptides, ordering the CNBr fragments, and the complete amino acid sequence

The amino acid sequence of the largest fragment, CNBr Ia (203 residues) has been reported (Yokota, E., and Riggs, A. F. (1984) J. Biol. Chem. 259, 4739-4749). The amino acid sequences of the second largest fragment, CNBr Ib (142 residues), and of the 12 smaller fragments are reported in accompanying papers (Moore, M. D., Behrens, P. Q., and Riggs, A. F. (1986) J. Biol. Chem. 261, 10511-10519; Behrens, P. Q., Nakashima, H., and Riggs, A. F. (1986) J. Biol. Chem. 261, 10520-10525). The complete amino acid sequence of hemocyanin component II has been established by isolation and analysis of 13 methionine-containing peptides from either a tryptic digest or a Staphylococcus aureus strain V8 protease digest of whole carboxamidomethylated hemocyanin II. Hemocyanin II is composed of 628 residues and has a molecular weight with two copper atoms of 72,946.     

Reconstitution of the proton translocating ATPase from bovine heart mitochondria into planar phospholipid bilayer membranes

Proton translocating ATPase (F0F1) from bovine heart mitochondria was reconstituted into planar phospholipid bilayers, and its electrogenicity was directly demonstrated. The F0F1 ATPase was solubilized using 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS) as a detergent followed by sucrose density gradient centrifugation according to the method originally described by McEnery et al. for rat liver mitochondria (McEnery et al. (1986) J. Biol. Chem. 259, 4642-4651), with minor modifications. The purified ATPase was reconstituted into proteoliposomes and then reconstituted into planar phospholipid bilayers by the modified fusion method (Hirata et al. (1986) J. Biol. Chem. 261, 9839-9843). A short-circuit current of up to 0.4 pA was induced by adding ATP, and this current was suppressed by the F1 ATPase inhibitor NaN3 or by a specific mitochondrial F0 inhibitor, oligomycin. The direction of the current corresponded to the flow of positive charges from the F1 side to the F0 side. All these facts clearly demonstrate that the mitochondrial F0F1 ATPase was successfully reconstituted into planar phospholipid bilayers, and the current was generated by the ATPase.     

Molecular species composition of membrane phosphatidylcholine influences the rate of cholesterol efflux from human erythrocytes and vesicles of erythrocyte lipid

The efflux of [3H]cholesterol from prelabelled human erythrocytes having modified phosphatidylcholine compositions was measured during 24-h incubations in the presence of unlabelled acceptor liposomes composed of equimolar amounts of egg phosphatidylcholine and cholesterol. The cells were modified by replacement of part of the native phosphatidylcholine with either dipalmitoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine or dilinoleoylphosphatidylcholine catalyzed by phosphatidylcholine-specific transfer protein from bovine liver. The results indicated that the efflux of [3H]cholesterol was faster from erythrocytes in which the dipalmitoylphosphatidylcholine content was increased from 7 to 25% of the total, than from cells enriched in palmitoyloleoylphosphatidylcholine or dioleoylphosphatidylcholine. Incorporation of dilinoleoylphosphatidylcholine to a level of 13% of the total phosphatidylcholine slowed the rate of efflux of [3H]sterol. The phosphatidylcholine replacements produced no significant differences in cholesterol/phospholipid ratio before or after 24 h of incubation with the acceptor egg phosphatidylcholine-cholesterol vesicles. Using vesicles prepared from erythrocyte lipid, modified to reflect the changes in the phosphatidylcholine composition induced in the whole cells, the same influence of composition on the rate of cholesterol exchange was evident. Enhancement of the dipalmitoylphosphatidylcholine content from 7 to 25% of the total phosphatidylcholine pool increased the rate of [3H]cholesterol efflux, while the addition of the same amount of dilinoleoylphosphatidylcholine slowed it compared to controls. The magnitude of the effect was comparable in intact cells and erythrocyte lipid vesicles enriched in dipalmitoylphosphatidylcholine, while the influence of dilinoleoylphosphatidylcholine was more marked in the intact cells. These results demonstrate that changes in the molecular species composition of the phosphatidylcholine pool can influence the rate of exchange of cholesterol but not necessarily the cellular content of sterol in the human erythrocyte. The influence of this phospholipid appears to be expressed independently of the presence of membrane protein or an underlying cytoskeleton.     

The structure of hemocyanin II from the horseshoe crab, Limulus polyphemus. The amino acid sequences of the smaller cyanogen bromide fragments

Fourteen fragments have been isolated from hemocyanin component II of Limulus polyphemus by cleavage with CNBr. The amino acid sequences of the two largest fragments, CNBr Ia and Ib, have been determined (Yokota, E., and Riggs, A. F. (1984) J. Biol. Chem. 259, 4739-4749; Behrens, P. Q., Nakashima, H., Yokota, E., and Riggs, A. F. (1986) J. Biol. Chem. 261, 10520-10525). We have determined the amino acid sequence of the remaining 12 smaller fragments.     

Regulation of sterol transport between membranes and NPC2

Niemann-Pick disease type C (NPC) is caused by defects in either the NPC1 or NPC2 gene and is characterized by accumulation of cholesterol and glycolipids in the late endosome/lysosome compartment. NPC2 is an intralysosomal protein that binds cholesterol in vitro. Previous studies demonstrated rapid rates of cholesterol transfer from NPC2 to model membranes [Cheruku, S. R., et al. (2006) J. Biol. Chem. 281, 31594-31604]. To model the potential role of NPC2 as a lysosomal cholesterol export protein, in this study we used fluorescence spectroscopic approaches to examine cholesterol transfer from membranes to NPC2, assessing the rate, mechanism, and regulation of this transport step. In addition, we examined the effect of NPC2 on the rate and kinetic mechanism of intermembrane sterol transport, to model the movement of cholesterol from internal lysosomal membranes to the limiting lysosomal membrane. The results support the hypothesis that NPC2 plays an important role in endo/lysosomal cholesterol trafficking by markedly accelerating the rates of cholesterol transport. Rates of sterol transfer from and between membranes were increased by as much as 2 orders of magnitude by NPC2. The transfer studies indicate that the mechanism of NPC2 action involves direct interaction of the protein with membranes. Such interactions were observed directly using FTIR spectroscopy and protein tryptophan spectral shifts. Additionally, cholesterol transfer by NPC2 was found to be greatly enhanced by the unique lysosomal phospholipid lyso-bisphosphatidic acid (LBPA), suggesting an important role for LBPA in NPC2-mediated cholesterol trafficking.     

Reconstitution of mitochondrial F0.F1-ATPase with phosphatidylcholine using the nonionic detergent, octylglucoside

A reconstitution procedure has been developed for the incorporation of the mitochondrial F0.F1-ATPase into the bilayer of egg phosphatidylcholine vesicles. The nonionic detergent, octylglucoside, egg phosphatidylcholine, and the lipid-deficient, oligomycin-sensitive F0.F1-ATPase (Serrano, R., Kanner, B., and Racker, E. (1976) J. Biol. Chem. 251, 2453-2461) were combined in a 4770:320:1 detergent/phospholipid/protein molar ratio and then centrifuged on a discontinuous sucrose gradient to isolate the F0.F1-phosphatidylcholine complex. The specific activity of the reconstituted F0.F1-ATPase was as high as 14.5 mumol/min/mg protein, whereas with no added lipid the activity ranged between 1.4 and 2.2 mumol/min/mg protein. This reconstituted preparation exhibited greater than 90% oligomycin sensitivity which demonstrated the intactness of the multisubunit enzyme complex. The phosphatidylcholine/protein molar ratio of the reconstituted F0.F1 was 250:1 with less than 0.4% of the added octylglucoside remaining. Titrations with both phosphatidylcholine and octylglucoside demonstrated that the specific activity and oligomycin sensitivity were highly dependent on the concentrations of both phospholipid and detergent in the original reconstitution mixture. Analysis of the reconstituted ATPase by electron microscopy demonstrated that the catalytic portion of the enzyme complex projected from the phospholipid bilayer with an orientation similar to that observed with submitochondrial particles. The F0.F1-phosphatidylcholine complex was able to trap inulin, which suggests a vesicular structure impermeable to macromolecules. The electrophoretic mobility of the complex was identical to that for liposomes of egg phosphatidylcholine alone. The reconstitution conditions utilized give rise to an enzyme-phospholipid complex with very low ionic charge that demonstrates high oligomycin-sensitive ATPase activity.     

Differential scanning calorimetric study of the effect of sterol side chain length and structure on dipalmitoylphosphatidylcholine thermotropic phase behavior.

We have investigated the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers containing a series of cholesterol analogues varying in the length and structure of their alkyl side chains. We find that upon the incorporation of up to approximately 25 mol % of any of the side chain analogues, the DPPC main transition endotherm consists of superimposed sharp and broad components representing the hydrocarbon chain melting of sterol-poor and sterol-rich phospholipid domains, respectively. Moreover, the behavior of these components is dependent on sterol side chain length. Specifically, for all sterol/DPPC mixtures, the sharp component enthalpy decreases linearly to zero by 25 mol % sterol while the cooperativity is only moderately reduced from that observed in the pure phospholipid. In addition, the sharp component transition temperature decreases for all sterol/DPPC mixtures; however, the magnitude of the decrease is dependent on the sterol side chain length. With respect to the broad component, the enthalpy initially increases to a maximum around 25 mol % sterol, thereafter decreasing toward zero by 50 mol % sterol with the exception of the sterols with very short alkyl side chains. Both the transition temperature and cooperativity of the broad component clearly exhibit alkyl chain length-dependent effects, with both the transition temperature and cooperativity decreasing more dramatically for sterols with progressively shorter side chains. We ascribe the chain length-dependent effects on transition temperature and cooperativity to the hydrophobic mismatch between the sterol and the host DPPC bilayer (see McMullen, T. P. W., Lewis, R. N. A. H., and McElhaney, R. N. (1993) Biochemistry 32:516-522).(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of sterol structure on phospholipid phase behavior as detected by parinaric acid fluorescence spectroscopy

Phospholipid-sterol interactions were investigated using parinaric acid fluorescence spectroscopy. Cholesterol and cholesterol analogues which were modified in the sterol nucleus or side chain were added at 50 mol % to multilamellar vesicles of model phospholipids selected to be representative of major components in an LM cell plasma membrane. These included sphingomyelins and saturated and monounsaturated phosphatidylcholines and phosphatidylethanolamines. Based on the changes in cis-parinaric acid steady-state fluorescence polarization observed with addition of sterol, 50 mol % cholesterol abolished the phase transition of all the model phospholipids. Dihydrocholesterol and trans-22-dehydrocholesterol behaved like cholesterol in the two systems studied. 24-Methylcholesterols interacted well with all phospholipids except phosphatidylethanolamine which contained an unsaturated fatty acid. 24-Alkyl,trans-22-dehydrocholesterols abolished the phase transition in only two systems: sphingomyelins and phosphatidylcholines possessing relatively short saturated acyl chains. Since steady-state anisotropy is a function of fluorescence lifetime, rotational diffusion rates, and limiting anisotropy, we determined these parameters for two of the phospholipid systems. The results show that steady-state anisotropy values for phospholipid-sterol interactions correlate closely with limiting anisotropy and to a lesser extent with rotational relaxation time. The behavior of the sterols in the model phospholipids are used to interpret 1) fluorescence polarization measurements made with phospholipids extracted from LM cell plasma membranes, and 2) changes in membrane lipid composition which accompany growth of LM cells on various sterols.     

Acidic phospholipids strikingly potentiate sterol carrier protein 2 mediated intermembrane sterol transfer

A liposomal membrane model system was developed to examine the mechanism of spontaneous and protein-mediated intermembrane cholesterol transfer. Rat liver sterol carrier protein 2 (SCP2) and fatty acid binding protein (FABP, also called sterol carrier protein) both bind sterol. However, only SCP2 mediates sterol transfer. The exchange of sterol between small unilamellar vesicles (SUV) containing 35 mol % sterol was monitored with a recently developed assay [Nemecz, G., Fontaine, R. N., & Schroeder, F. (1988) Biochim. Biophys. Acta 943, 511-541], modified to continuous polarization measurement and not requiring separation of donor and acceptor membrane vesicles. As compared to spontaneous sterol exchange, 1.5 microM rat liver SCP2 enhanced the initial rate of sterol exchange between neutral zwwitterionic phosphatidylcholine SUV 2.3-fold. More important, the presence of acidic phospholipids (2.5-30 mol %) stimulated the SCP2-mediated increase in sterol transfer approximately 35-42-fold. Thus, acidic phospholipids strikingly potentiate the effect of SCP2 by 15-18 times as compared to SUV without negatively charged lipids. Rat liver FABP (up to 60 microM) was without effect on sterol transfer in either neutral zwitterionic or anionic phospholipid containing SUV. The potentiation of SCP2 action by acidic phospholipids was suppressed by high ionic strength, neomycin, and low pH. The results suggest that electrostatic interaction between SCP2 and negatively charged membranes may play an important role in the mechanism whereby SCP2 enhances intermembrane cholesterol transfer.     

Influence of apolipoproteins AI, AII, and Cs on the metabolism of membrane and lysosomal cholesterol in macrophages.

We have demonstrated previously that HDL-mediated efflux of plasma membrane cholesterol is independent of specific binding of apolipoproteins to the high density lipoprotein (HDL) receptor in either control or cholesterol-enriched cells (Karlin, J. B., Johnson, W. J., Benedict, C. R., Chacko, G. K., Phillips, M. C., and Rothblat, G. H. (1987) J. Biol. Chem. 262, 12557-12564 and Johnson, W. J., Mahlberg, F. H., Chacko, G. K., Phillips, M. C., and Rothblat, G. H. (1988) J. Biol. Chem. 263, 14099-14106). The present studies were conducted to determine if the process for removal of intracellular (lysosomal) cholesterol is similar to that of membrane cholesterol or if, in contrast, it is selectively regulated by specific apolipoproteins of HDL. For these reasons, we examined the influence of each of the major apolipoproteins of human HDL, apoAI, apoAII, and apoCs on the metabolism of membrane and lysosomal cholesterol in a macrophage foam cell model. We developed an experimental system which allows, for the first time, the simultaneous determination of lysosomal hydrolysis of cholesteryl ester and efflux and esterification of both lysosomal and membrane cholesterol. J774 and elicited mouse peritoneal macrophages were loaded with cholesteryl ester within lysosomes through phagocytosis of sonicated lipid droplets. Membrane and lysosomal pools of cholesterol were differentially radiolabeled. Discoidal complexes of egg phosphatidylcholine and purified apolipoproteins having a similar size and composition were used as cholesterol acceptors. Our results demonstrate that lysosomal hydrolysis of cholesteryl ester is independent of the presence of extracellular acceptors. Lysosomal production of cholesterol stimulates the esterification by acyl-CoA:cholesterol acyltransferase of membrane and lysosomal cholesterol. All the particles tested induce the efflux of both pools of cholesterol at a similar ratio. As efflux is stimulated, esterification by acyl-CoA:cholesterol acyltransferase is reduced. We conclude that none of these apolipoproteins selectively influences the efflux or the esterification of membrane of lysosomal cholesterol. In addition, we observe that particles containing apoAI are the most efficient acceptors, but this effect is not linked to specific binding to the HDL receptor.     

Sphingosine inhibits the activity of rat liver CTP:phosphocholine cytidylyltransferase

We report CTP:phosphocholine cytidylyltransferase (CT) as another target enzyme of sphingosine actions in addition to the well-characterized protein kinase C. Effects of sphingosine and lysophingolipids were studied on the activity of purified cytidylyltransferase prepared by the method of Weinhold et al. (Weinhold, P. A., Rounsifer, M.E., and Feldman, D.A. (1986) J. Biol. Chem. 261, 5104-5110). The sphingolipids were tested as components of egg phosphatidylcholine (PC) vesicles, 25 mol% sphingosine inhibited the CT activity by about 50%. The inhibition of CT by sphingosine and lysosphingolipids was reversible. Sphingosine was found to be a reversible inhibitor of CT with respect to the activating lipids such as phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and fatty acid:phosphatidylcholine vesicles. Egg PC vesicles containing sphingosine, psychosine (galactosylsphingosine), glucopsychosine (glucosylsphingosine), and lysosphingomyelin (sphingosylphosphorylcholine) suppressed the activation by PC/oleic acid vesicles, whereas the parent sphingolipids did not. Egg PC vesicles containing oleylamine and hexadecyltrimethylamine inhibited CT activity, whereas egg PC-octylamine vesicles did not alter the enzyme activity. This indicates the importance of an amino group and long alkyl chain. LysoPC, a known detergent, did not inhibit the enzyme activity under the same assay conditions in which sphingosine inhibited. These results are the first report of a lipid inhibitor of purified CT.     

Vasopressin stimulates the phosphorylation of an 83,000M r protein in the superior cervical ganglion

1. 32P-Labeled proteins from the superior cervical ganglion of the rat were separated by two-dimensional gel electrophoresis and visualized by autoradiography. 2. The most heavily labeled phosphoprotein in the ganglion had a relative molecular weight of 83,000 and a pI of 4.5. Phosphorylation of this protein was increased by phorbol 12,13-dibutyrate, an activator of the Ca2+/phospholipid-dependent protein kinase, protein kinase C. This protein appears to be similar or identical to a specific protein kinase C substrate that has been described in other tissues (Blackshear, P. J., et al., J. Biol. Chem. 261:1459-1469, 1986). 3. Phosphorylation of this protein was also increased by treatment of the ganglion with phospholipase C (Bacillus cereus) but was not increased by 8-bromo-cyclic AMP or by nicotinic agonists. Vasopressin increased the hydrolysis of inositol-containing phospholipids in the ganglion and also increased the labeling of the 83,000 Mr protein. Thus, vasopressin appears to activate protein kinase C in the ganglion. 4. Muscarine, which also increased phospholipid metabolism in the ganglion, did not increase the phosphorylation of the 83,000 Mr protein. Muscarine and vasopressin stimulate phospholipid metabolism in different structures within the ganglion (Horwitz, J., et al., J. Pharmacol. Exp. Ther. 237:312-317, 1986). Muscarine may increase phospholipid metabolism in structures that do not contain significant amounts of the 83,000 Mr protein.     

Crystallographic and biochemical studies of the (inactive) Lys-49 phospholipase A2 from the venom of Agkistridon piscivorus piscivorus.

Chemical, genetic, and structural studies have defined a critical role for Asp-49 in the calcium-mediated activation of extracellular phospholipases A2 (PLA2). In 1984, a new class of PLA2 was isolated in which this invariant aspartate was replaced with a lysine (Maragnore, J.M., Merutka, G., Cho, W., Welches, W., Kezdy, F.J., and Heinrikson, R.L. (1984) J. Biol. Chem. 259, 13839-13843; Maragnore, J.M., and Heinrikson, R.L. (1986) J. Biol. Chem. 261, 4797-4804). The enzymatic activity of Lys-49 PLA2s has been questioned based on biochemical, mutational, and structural studies (van den Bergh, C.J., Slotboom, A.J., Verheij, H.M., and de Haas, G.H. (1988) Eur. J. Biochem. 176, 353-357). In this paper, we describe the structures of two crystal forms of the Lys-49 PLA2 isolated from the venom of Agkistridon piscivorus piscivorus. The refined models, along with complementary biochemical analysis, clarify the structural basis for the enzymatic inactivity of Lys-49 proteins.     

A comparison of the packing behavior of egg phosphatidylcholine with cholesterol and biogenically related sterols in Langmuir monolayer films

Cholesterol and selected derivatives were studied as mixed Langmuir monolayers with egg phosphatidylcholine (PC). As an extension of our earlier work, which employed binary sterol/PC mixtures, here we examined ternary mixed monolayers containing cholesterol along with an alternate sterol and PC in different molar ratios, using pressure-area isotherms. The ternary systems behaved similarly to the binary sterol/PC systems reported previously, with similar condensation noted for the sterol/PC films. To better understand how variations in sterol structure affect sterol packing in such membrane monolayers, binary mixtures containing cholestenone, cholestanol, and lanosterol with PC were also studied. Cholestanol behaved similarly to cholesterol when incorporated with PC, while cholestenone and lanosterol did not cause as much film condensation. The observed differences in molecular packing, and attributed sterol structural differences, are considered within the context of sterol/phospholipid mixtures in biological membranes.     

Cholesterol crystallization in model biles: effects of bile salt and phospholipid species composition

Cholesterol in human bile is solubilized in micelles by (relatively hydrophobic) bile salts and phosphatidylcholine (unsaturated acyl chains at sn-2 position). Hydrophilic tauroursodeoxycholate, dipalmitoyl phosphatidylcholine, and sphingomyelin all decrease cholesterol crystal-containing zones in the equilibrium ternary phase diagram (van Erpecum, K. J., and M. C. Carey. 1997. Biochim. Biophys. Acta. 1345: 269-282) and thus could be valuable in gallstone prevention. We have now compared crystallization in cholesterol-supersaturated model systems (3.6 g/dl, 37 degrees C) composed of various bile salts as well as egg yolk phosphatidylcholine (unsaturated acyl chains at sn-2 position), dipalmitoyl phosphatidylcholine, or sphingomyelin throughout the phase diagram. At low phospholipid contents [left two-phase (micelle plus crystal-containing) zone], tauroursodeoxycholate, dipalmitoyl phosphatidylcholine, and sphingomyelin all enhanced crystallization. At pathophysiologically relevant intermediate phospholipid contents [central three-phase (micelle plus vesicle plus crystal-containing) zone], tauroursodeoxycholate inhibited, but dipalmitoyl phosphatidylcholine and sphingomyelin enhanced, crystallization. Also, during 10 days of incubation, there was a strong decrease in vesicular cholesterol contents and vesicular cholesterol-to-phospholipid ratios (approximately 1 on day 10), coinciding with a strong increase in crystal mass. At high phospholipid contents [right two-phase (micelle plus vesicle-containing) zone], vesicles were always unsaturated and crystallization did not occur. Strategies aiming to increase amounts of hydrophilic bile salts may be preferable to increasing saturated phospholipids in bile, because the latter may enhance crystallization.     

Protein-kinase-C-catalyzed phosphorylation of the microtubule-binding domain of microtubule-associated protein 2 inhibits its ability to induce tubulin polymerization

It has previously been demonstrated that microtubule-associated protein 2 (MAP2) is a good substrate for the purified protein kinase C [Tsuyama, S., Bramblett, G. T., Huang, K.-P. & Flavin, M. (1986) J. Biol. Chem. 261, 4110-4116; Akiyama, T., Nishida, E., Ishida, J., Saji, N., Ogawara, H., Hoshi, M., Miyata, Y. & Sakai, H. (1986) J. Biol. Chem. 261, 15648-15651]. We have shown here that phosphorylation of MAP2, catalyzed by protein kinase C, reduces the ability to induce tubulin polymerization. MAP2 is divided into two domains by digestion with alpha-chymotrypsin; the microtubule-binding and the non-binding (projection) domains. The limited chymotryptic digestion following phosphorylation of MAP2 by protein kinase C has shown that both the domains of MAP2 were phosphorylated by protein kinase C, 50-60% of the incorporated phosphates being detected in the microtubule-binding domain. Polypeptide fragments, containing the microtubule-binding domain of MAP2, were purified by DEAE-cellulose column chromatography after chymotryptic digestion of MAP2. The purified microtubule-binding fragments were competent to polymerize tubulin, and served as good substrates for protein kinase C. The phosphorylation of the microtubule-binding fragments by protein kinase C reduced their ability to induce tubulin polymerization. These results suggest that the ability of MAP2 to induce tubulin polymerization is inhibited by phosphorylation of the microtubule-binding domain catalyzed by protein kinase C.     

LM cell growth and membrane lipid adaptation to sterol structure

Using a sterol auxotroph of the LM cell mouse fibroblast, we demonstrate that relatively few cholesterol analogues can substitute for cholesterol as a growth factor. The auxotroph grows normally on desmosterol and trans-22-dehydrocholesterol and at reduced rates on dihydrocholesterol, campesterol, and 22,23-dihydrobrassicasterol. It does not grow with beta-sitosterol, stigmasterol, ergosterol, or cis-22-dehydrocholesterol when the sterol is present as sole supplement but does grow at normal rates when the analogue is supplied with suboptimal amounts of cholesterol. Two contrasting types of membrane lipid changes are observed in cells grown on cholesterol analogues. In cells grown with dihydrocholesterol, a marked increase in desaturation and elongation of fatty acids is noted. Conversely, when cells are grown with cis-22-dehydrocholesterol, desaturation and elongation of fatty acids are severely curtailed. Cells grown on alkyl sterols respond like cells grown on cis-22-dehydrocholesterol but in a less pronounced fashion. The effects of sterol substitution in mammalian cells versus in lower eukaryotes are compared, and an explanation for the secondary changes in fatty acid composition in terms of phospholipid phase behavior is suggested.     

The involvement of the lipid phase transition in the plasma-induced dissolution of multilamellar phosphatidylcholine vesicles

Unsonicated liposomes prepared from dimyristoyl phosphatidylcholine were nearly completely dissolved during a 3 h incubation with rat plasma at or close to the phase-transition temperature of 24°C. At 37 or 15°C virtually no liposomal disintegration was observed even after 24 h of incubation. The liposomal solubilization, which was monitored by turbidity measurements or by determination of phospholipid sedimentability, was accompanied by the formation of a phospholipid-protein complex similar or identical to the one we previously reported to be formed from sonicated liposomes of egg phosphatidylcholine (Scherphof, G., Roerdink, F., Waite, M. and Parks, J. (1978) Biochim. Biophys. Acta 542, 296–307). Unsonicated multilamellar liposomes made of egg phosphatidylcholine were completely resistant to the dissolving potency of plasma when incubated at 37°C. Liposomes from equimolar mixtures of dimyristoyl and dipalmitoyl phosphatidylcholine were only degraded by plasma in the temperature range between 30 and 35°C at which temperature this cocrystallizing phospholipid mixture undergoes a phase transition. However, even at these temperatures the rate of dissolution of this mixture was significantly lower than of dimyristoyl phosphatidylcholine at 24°C. In the dissolving process of this mixture a slight preference for the lower-melting component was observed.The ability of cholesterol to completely abolish the susceptibility of dimyristoyl phosphatidylcholine liposomes to plasma at a 1:2 molar ratio of cholesterol to phospholipid substantiates the essential role of the phase transition in the process of liposome solubilization.When liposomes of the monotectic mixtures dimyristoyl and distearoyl phosphatidylcholine or dilauroyl and distearoyl phosphatidylcholine were incubated with plasma at temperatures in between those at which the constituent lipids undergo a phase change in the mixture, the liposomes were slowly disolved. Under those conditions a selective removal of the lipids in the liquid-crystalline phase was observed.It is concluded that for the plasma-induced dissolution of unsonicated liposomes, which is most probably achieved by interaction with (apo)lipoproteins, the presence of phase boundaries is required in much the same way as was first reported for phospholipases by Op den Kamp, J.A.F., de Gier, J. and Van Deenen, L.L.M. (1974) Biochim. Biophys. Acta 345, 253–256).