Oleic acid allows more apoprotein A-1 to bind with higher affinity to large emulsion particles saturated with cholesterol

Apoprotein (apo) A-1 binding to large triolein-rich emulsion particles saturated with cholesterol has been examined as a function of the oleic acid content. Six emulsion systems were formed containing 0.3-1.0% (by weight) oleic acid, 82.9-86.3% triolein, 10.6-7.2% egg yolk phosphatidylcholine, and 6.7-5.5% cholesterol. The average emulsion particle diameters calculated from these lipid compositions ranged between 84 and 116 nm. Negative stain electron microscopy of an emulsion containing 1% oleic acid showed a polydisperse population of only large spherical particles with a mean diameter of 116 +/- 54 nm. The calculated cholesterol concentrations of the particles surface and core for the six emulsions were 43.3 +/- 1.1 and 5.6 +/- 0.2 mol%, respectively, and were rather constant. Therefore, when the surface oleic acid concentrations increased from 2.6 to 10.1 mol%, the phospholipid concentration decreased from 55.1 to 45.9 mol%. In the core, oleic acid increased at the expense of triolein. In the range studied a nearly 4-fold increase in the surface oleic acid content produces a similar increase in the binding capacity (N) and reduces the dissociation constant (Kd). The changes in the Kd and N values were linearly dependent on the surface oleic acid concentration. These data show that oleic acid allows more apoA-1 to bind with higher affinity to large emulsion particles saturated with cholesterol.     

Interaction of cholesterol, phospholipid and apoprotein in high density lipoprotein recombinants.

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of 3H cholesterol/14C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27 degrees C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1, or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. 160 X 55 A. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins

The N-terminal 17% of apolipoprotein B (apoB-17) readily associates with dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV) to form large (240-A diameter) discoidal particles. Because apoB is normally secreted with triacylglycerol (TAG)-rich lipoproteins, we studied the binding of apoB-17 to triolein-rich emulsions modeling nascent TAG-rich very low density-like lipoproteins. Emulsions with the following composition (by weight) were prepared: 85--89% triolein, 1.1--1.4% cholesterol, and 10--14% phosphatidylcholines (PC) including either egg yolk (EY)-, dimyristoyl (DM)-, or dipalmitoyl (DP)-PC representing (at 25 degrees C), respectively, a fluid surface, a surface at transition, and a mainly solid surface. The respective sizes were 1,260 +/- 500, 1,070 +/- 450, and 830 +/- 300 A mean diameter. The emulsions were incubated with conditioned medium containing apoB-17, and then reisolated by ultracentrifugation. Analysis of the emulsion-bound proteins by gel electrophoresis showed that all three emulsions bound primarily apoB-17. The DPPC emulsions bound more apoB-17 than EYPC or DMPC emulsions. Immunoaffinity-purified apoB-17 exhibited saturable, high affinity binding to EYPC and DPPC emulsions. The respective K(d) values were 32 +/- 23 and 85 +/- 27 nM and capacities (N) were 10 and 58 molecules of apoB-17 per particle. When apoB-17 bound to emulsions was incubated with DMPC MLV at 26 degrees C for 18 h, it remained bound to the emulsions, indicating that once bound to these emulsions it is unable to exchange off and solubilize DMPC into discs. In contrast, apoE-3 bound to emulsions dissociated from the emulsions when incubated with DMPC MLV and formed discs.Thus, apoB-17 binds strongly and irreversibly to emulsions modeling nascent lipoproteins. It therefore may play an important role in the stabilization of nascent VLDL and chylomicrons.- Herscovitz, H., A. Derksen, M. T. Walsh, C. J. McKnight, D. L. Gantz, M. Hadzopoulou-Cladaras, V. Zannis, C. Curry, and D. M. Small. The N-terminal 17% of apoB binds tightly and irreversibly to emulsions modeling nascent very low density lipoproteins. J. Lipid Res. 2001. 42: 51;-59.     

Interaction of cholesterol,phospholipid and apoprotein in high density lipoprotein recombinants

To examine the effect of incorporation of cholesterol into high density lipoprotein (HDL) recombinants, multilamellar liposomes of ³H cholesterol/¹⁴C dimyristoyl phosphatidylcholine were incubated with the total apoprotein (apoHDL) and principal apoproteins (apoA-1 and apoA-2) of human plasma high density lipoprotein. Soluble recombinants were separated from unreacted liposomes by centrifugation and examined by differential scanning calorimetry and negative stain electron microscopy. At 27°C, liposomes containing up to approx. 0.1 mol cholesterol/mol dimyristoyl phosphatidylcholine (DMPC) were readily solubilized by apoHDL, apoA-1 or apoA-2. However, the incorporation of DMPC and apoprotein into lipoprotein complexes was markedly reduced when liposomes containing a higher proportion of cholesterol were used. For recombinants prepared from apoHDL, apoA-1 or apoA-2, the equilibrium cholesterol content of complexes was approx. 45% that of the unreacted liposomes. Electron microscopy showed that for all cholesterol concentrations, HDL recombinants were predominantly lipid bilayer discs, approx. $\text{160 × 55}\text{A}\text{?}$. Differential scanning calorimetry of cholesterol containing recombinants of DMPC/cholesterol/apoHDL or DMPC/cholesterol/apoA-1 showed, with increasing cholesterol content, a linear decrease in the enthalpy of the DMPC gel to liquid crystalline transition, extrapolating to zero enthalpy at 0.15 cholesterol/DMPC. The enthalpy values were markedly reduced compared to control liposomes, where the phospholipid transition extrapolated to zero enthalpy at approx. 0.45 cholesterol/DMPC. The calorimetric and solubility studies suggest that in high density lipoprotein recombinants cholesterol is excluded from 55% of DMPC molecules bound in a non-melting state by apoprotein.     

Binding affinity and reactivity of lecithin cholesterol acyltransferase with native lipoproteins.

The first step in the reaction of lecithin cholesterol acyltransferase (LCAT) with lipoproteins is the interfacial binding of the enzyme to the lipid surfaces. In this study the equilibrium dissociation constants (Kds) for the interaction of pure human plasma LCAT with LDL, HDL2, HDL3, and a reconstituted discoidal HDL (rHDL) were determined by the activity-inhibition method. In addition, enzyme kinetics were measured with each of the lipoprotein substrates. Based on phospholipid concentrations, the Kd values (0.9 x 10(-5) to 4.6 x 10(-5) M) increased in the order rHDL = HDL3 相似文献   

Binding of lipoproteins and regulation of cholesterol synthesis in cultured mouse adipose cells

Binding of human lipoproteins to cultured mouse Ob17 preadipose and adipose cells was studied, using labeled VLDL, LDL and apoprotein E-free HDL. In each case, saturation curves were obtained, yielding linear Scatchard plots. The Kd values were found to be respectively 6.4, 31 and 24 micrograms/ml for VLDL, LDL and apoprotein E-free HDL, whereas the maximal numbers of binding sites per cell were 4.2 X 10(4), 1.5 X 10(4) and 2.5 X 10(5). The binding of 125I-LDL was competitively inhibited by LDL greater than VLDL greater than total HDL; human LDL and mouse LDL were equipotent in competition assays. Methylated LDL and apoprotein E-free HDL were not competitors. In contrast, the binding of 125I-apoprotein E-free HDL was competitively inhibited by apoprotein E-free HDL greater than total HDL and the binding of 125I-HDL3 by mouse HDL. Thus, mouse adipose cells possess distinct apoprotein B, E and apoprotein E-free HDL binding sites which can recognize heterologous or homologous lipoproteins. The cell surface receptor of LDL in mouse preadipose cells shows similarities with that described for human fibroblasts, since: (1) the LDL binding initiated the process of internalization and degradation of the apoprotein B and apoprotein E-containing lipoproteins; (2) receptor-mediated uptake of cholesterol LDL led to a parallel but incomplete decrease in the [14C]acetate incorporation into cholesterol and in the activity of HMG-CoA reductase. Growing (undifferentiated) or growth-arrested cells (differentiated or not) showed no significant changes in the Kd values for lipoprotein binding. In contrast, the maximal number of binding sites correlated with the proliferative state of the cells and was independent of cell differentiation. The results are discussed with respect to cholesterol accumulation in adipose cells.     

Lecithin:cholesterol acyltransferase deficiency increases atherosclerosis in the low density lipoprotein receptor and apolipoprotein E knockout mice.

The purpose of the present study was to test the hypothesis that lecithin:cholesterol acyltransferase (LCAT) deficiency would accelerate atherosclerosis development in low density lipoprotein (LDL) receptor (LDLr-/-) and apoE (apoE-/-) knockout mice. After 16 weeks of atherogenic diet (0.1% cholesterol, 10% calories from palm oil) consumption, LDLr-/- LCAT-/- double knockout mice, compared with LDLr-/- mice, had similar plasma concentrations of free (FC), esterified (EC), and apoB lipoprotein cholesterol, increased plasma concentrations of phospholipid and triglyceride, decreased HDL cholesterol, and 2-fold more aortic FC (142 +/- 28 versus 61 +/- 20 mg/g protein) and EC (102 +/- 27 versus 61+/- 27 mg/g). ApoE-/- LCAT-/- mice fed the atherogenic diet, compared with apoE-/- mice, had higher concentrations of plasma FC, EC, apoB lipoprotein cholesterol, and phospholipid, and significantly more aortic FC (149 +/- 62 versus 109 +/- 33 mg/g) and EC (101 +/- 23 versus 69 +/- 20 mg/g) than did the apoE-/- mice. LCAT deficiency resulted in a 12-fold increase in the ratio of saturated + monounsaturated to polyunsaturated cholesteryl esters in apoB lipoproteins in LDLr-/- mice and a 3-fold increase in the apoE-/- mice compared with their counterparts with active LCAT. We conclude that LCAT deficiency in LDLr-/- and apoE-/- mice fed an atherogenic diet resulted in increased aortic cholesterol deposition, likely due to a reduction in plasma HDL, an increased saturation of cholesteryl esters in apoB lipoproteins and, in the apoE-/- background, an increased plasma concentration of apoB lipoproteins.     

Testing the role of apoA-I,HDL, and cholesterol efflux in the atheroprotective action of low-level apoE expression

Low levels of transgenic mouse apolipoprotein E (apoE) suppress atherosclerosis in apoE knockout (apoE-/-) mice without normalizing plasma cholesterol. To test whether this is due to facilitation of cholesterol efflux from the vessel wall, we produced apoA-I-/-/apoE-/- mice with or without the transgene. Even without apoA-I and HDL, apoA-I-/-/apoE-/- mice had the same amount of aorta cholesteryl ester as apoE-/- mice. Low apoE in the apoA-I-/-/apoE-/- transgenic mice reduced aortic lesions by 70% versus their apoA-I-/-/apoE-/- siblings. To define the free cholesterol (FC) efflux capacity of lipoproteins from the various genotypes, sera were assayed on macrophages expressing ATP-binding cassette transporter A1 (ABCA1). Surprisingly, ABCA1 FC efflux was twice as high to sera from the apoA-I-/-/apoE-/- or apoE-/- mice compared with wild-type mice, and this activity correlated with serum apoA-IV. Immunodepletion of apoA-IV from apoA-I-/-/apoE-/- serum abolished ABCA1 FC efflux, indicating that apoAI-V serves as a potent acceptor for FC efflux via ABCA1. With increasing apoE expression, apoA-IV and FC acceptor capacity decreased, indicating a reciprocal relationship between plasma apoE and apoA-IV. Low plasma apoE (1-3 x 10(-8) M) suppresses atherosclerosis by as yet undefined mechanisms, not dependent on the presence of apoA-I or HDL or an increased capacity of serum acceptors for FC efflux.     

Phospholipase A2-treated human high-density lipoprotein and cholesterol movements: exchange processes and lecithin: cholesterol acyltransferase reactivity

Human HDL3 (d 1.125-1.21 g/ml) were treated by an exogenous phospholipase A2 from Crotalus adamenteus in the presence of albumin. Phosphatidylcholine hydrolysis ranged between 30 and 90% and the reisolated particle was essentially devoid of lipolysis products. (1) An exchange of free cholesterol was recorded between radiolabelled erythrocytes at 5-10% haematocrit and HDL3 (0.6 mM total cholesterol) from 0 to 12-15 h. Isotopic equilibration was reached. Kinetic analysis of the data indicated a constant rate of free cholesterol exchange of 13.0 microM/h with a half-time of equilibration around 3 h. Very similar values of cholesterol exchange, specific radioactivities and kinetic parameters were measured when phospholipase-treated HDL replaced control HDL. (2) The lecithin: cholesterol acyltransferase reactivity of HDL3, containing different amounts of phosphatidylcholine, as achieved by various degrees of phospholipase A2 treatment, was measured using a crude preparation of lecithin: cholesterol acyltransferase (the d 1.21-1.25 g/ml plasma fraction). The rate of esterification was determined between 0 and 12 h. Following a 15-30% lipolysis, the lecithin: cholesterol acyltransferase reactivity of HDL3 was reduced about 30-40%, and then continued to decrease, though more slowly, as the phospholipid content was further lowered in the particle. (3) The addition of the lecithin: cholesterol acyltransferase preparation into an incubation medium made of labelled erythrocytes and HDL3 promoted a movement of radioactive cholesterol out of cells, above the values of exchange, and an accumulation of cholesteryl esters in HDL. This reflected a mass consumption of free cholesterol, from both the cellular and the lipoprotein compartments upon the lecithin: cholesterol acyltransferase action. As a consequence of a decreased reactivity, phospholipase-treated HDL (with 2/3 of phosphatidylcholine hydrolyzed) proved much less effective in the lecithin: cholesterol acyltransferase-induced removal of cellular cholesterol.     

Secretion of lecithin: cholesterol acyltransferase from isolated rat hepatocytes.

1. Lecithin:cholesterol acyltransferase is secreted from isolated rat heptocytes. 2. The secretion is stimulated when serum is added to the incubation medium. 3. Optimal conditions for secretion are: 5-10(6) hepatocytes per ml, 5 h incubation, pH 7.3-7.4 and 25% serum in the incubation medium. 4. Concomitantly with the secretion of lecithin:cholesterol acyltransferase there is a secretion of unesterified cholesterol and triacylglycerol. 5. Colchicine or cycloheximide in the incubation medium inhibits secretion of lecithin:cholesterol acyltransferase.     

Multiple classes of binding sites for palmitic acid on the fatty acid-binding protein molecule

Binding characteristics of fatty acid-binding protein (FABP) toward palmitic acid were studied. On the analysis of the interaction between FABP and [3H]palmitic acid over a wide range of concentrations of the fatty acid, at least three saturation plateaux were observed. By Scatchard-plot analysis, it appeared that FABP possesses three classes of binding sites for palmitic acid with different affinities [Kd1 = 1 x 10(-6) M (N = 1), Kd2 = 4 x 10(-6) M (N = 2), Kd3 = 2 x 10(-5) M (N = 10)]. Results of both sedimentation analyses and chromatofocusing of FABPs labeled with various concentrations of [3H]palmitic acid suggested that the FABP used was homogeneous. These results indicate that several classes of binding sites for palmitic acid with different affinities are present on the FABP molecule.     

Reconstitution of lipoproteins. II. Lipid-protein interaction between dimyristoyl-lecithin and dimyristoyl-lecithin:cholesterol vesicles and purified apolipoprotein C-I and C-III2 studied by isomeric spin-labelled lecithins

The reconstitution of purified apolipoprotein C-I and C-III2 with sn-3-dimyristoyl-lecithin and sn-3-dimyristoyl-lecithin:cholesterol (10:1) vesicles was studied by electron spin resonance spectroscopy using isomeric 5'-, 12'-, and 16'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithin probes. Results obtained from the temperature-induced changes of lipoprotein recombinants showed the hydrophilic nature of the lipid-protein interactions. The temperature-induced phospholipid phase transition, as measured by 5'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithin probe in recombinants containing apoprotein C-1 or apoprotein C-iii2, is very broad and has a small cooperative unit indicative of extensive lipid-protein interactions occurring at the head group region of the phospholipid bilayer. When 12"- and 16'-(N-oxyl-4",4"-dimethyloxazolidine)stearoyl spin-labelled lecithins are used as probes in the same system, similar sharper and more cooperative lipid phase changes are detected. These results indicate a surface location for both apoprotein C-I and apoprotein C-III2 with respect to the phospholipid bilayer in lipoprotein recombinants with and without cholesterol.     

Activation of lecithin: cholesterol acyltransferase by human apolipoprotein A-IV

Human plasma apoproteins (apo) A-I and A-IV both activate the enzyme lecithin:cholesterol acyltransferase (EC 2.3.1.43). Lecithin:cholesterol acyltransferase activity was measured by the conversion of [4-14C] cholesterol to [4-14C]cholesteryl ester using artificial phospholipid/cholesterol/[4-14C]cholesterol/apoprotein substrates. The substrate was prepared by the addition of apoprotein to a sonicated aqueous dispersion of phospholipid/cholesterol/[4-14C]cholesterol. The activation of lecithin:cholesterol acyltransferase by apo-A-I and -A-IV differed, depending upon the nature of the hydrocarbon chains of the sn-L-alpha-phosphatidylcholine acyl donor. Apo-A-I was a more potent activator than apo-A-IV with egg yolk lecithin, L-alpha-dioleoylphosphatidylcholine, and L-alpha-phosphatidylcholine substituted with one saturated and one unsaturated fatty acid regardless of the substitution position. When L-alpha-phosphatidylcholine esterified with two saturated fatty acids was used as acyl donor, apo-A-IV was more active than apo-A-I in stimulating the lecithin:cholesterol acyltransferase reaction. Complexes of phosphatidylcholines substituted with two saturated fatty acids served as substrate for lecithin:cholesterol acyltransferase even in the absence of any activator protein. Essentially the same results were obtained when substrate complexes (phospholipid-cholesterol-[4-14C]cholesterol-apoprotein) were prepared by a detergent dialysis procedure. Apo-A-IV-L-alpha-dimyristoylphosphatidylcholine complexes thus prepared were shown to be homogeneous particles by column chromatography and density gradient ultracentrifugation. It is concluded that apo-A-IV is able to facilitate the lecithin:cholesterol acyltransferase reaction in vitro.     

Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein

The low density lipoprotein receptor-related protein (LRP) from rat liver membranes binds apoprotein E (apoE)-enriched rabbit beta-migrating very low density lipoproteins (beta-VLDL) in a ligand blotting assay on nitrocellulose membranes. Binding was markedly activated when the beta-VLDL was preincubated with recombinant human apoE-3, native human apoE-3 or E-4, or native rabbit apoE. Human apoE-2, which binds poorly (1-2% of apo E-3 binding) to low density lipoprotein receptors, was approximately 40% as effective as apoE-3 or apoE-4 in binding to LRP. Stimulation of apoE-dependent binding to LRP was blocked by the inclusion of a mixture of human apoC proteins, but not apoA-I or A-II, in the preincubation reaction. High concentrations of apoE did not overcome the apoC inhibition. The effects of apoE and apoC on the ligand blotting assay were paralleled by similar effects in the ability of beta-VLDL to stimulate cholesteryl ester synthesis in mutant human fibroblasts that lack low density lipoprotein receptors. These properties of LRP are consistent with the known effects of apoE and apoC on uptake of chylomicron and very low density lipoprotein remnants in the liver and raise the possibility that LRP functions as a receptor for apoE-enriched forms of these lipoproteins in intact animals.     

Effect of N-benzoyl-D-phenylalanine and metformin on insulin receptors in neonatal streptozotocin-induced diabetic rats: studies on insulin binding to erythrocytes

In the present study, we focused on the insulin-receptor binding in circulating erythrocytes of N-benzoyl-D-phenylalanine (NBDP) and metformin in neonatal streptozotocin (nSTZ)-induced male Wistar rats. We measured blood levels of glucose and plasma insulin and the binding of insulin to cell-membrane ER receptors in NBDP and metformin-treated diabetic rats. The mean specific binding of insulin to ER was significantly lower in diabetic control rats (DC) (53.0 +/- 3.1%) than in NBDP (62.0 +/- 3.1%), metformin (66.0 +/- 3.3%) and NBDP and metformin combination-treated (72.0 +/- 4.2%) diabetic rats, resulting in a significant decrease in plasma insulin. Scatchard plot analysis demonstrated that the decrease in insulin binding was accounted for by a lower number of insulin receptor sites per cell in DC rats when compared with NBDP and metformin-treated rats. High-affinity (Kd1), low-affinity (Kd2), and kinetic analysis revealed an increase in the average receptor affinity in ER from NBDP and metformin-treated diabetic rats having NBDP 2.0 +/- 0.10 x 10(-10) M(-1) (Kd1); 12.0 +/- 0.85 x 10(-8) M(-1) (Kd2), Metformin 2.1 +/- 0.15 x 10(-10) M(-1) (Kd1); 15.0 +/- 0.80 x 10(-8) M(-1) (Kd2), NBDP and metformin 2.7 +/- 0.10 x 10(-10) M(-1) (Kd1); 20.0 +/- 1.2 x 10(-8) M(-1) (Kd2) compared with 0.9 +/- 0.06 x 10(-10) M(-1) (Kd1); 6.0 +/- 0.30 x 10(-8) M(-1) (Kd2) in DC rats. The results suggest an acute alteration in the number of insulin receptors on ER membranes in nSTZ induced diabetic control rats. Treatment with NBDP along with metformin significantly improved specific insulin binding, with receptor number and affinity binding reaching almost normal non-diabetic levels. The data presented here show that NBDP along with metformin increase total ER membrane insulin binding sites with a concomitant significant increase in plasma insulin.     

Physical and chemical characteristics of apolipoprotein A-I-lipid complexes produced by Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene

Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene linked to the human metallothionein gene promoter region secrete large quantities of apolipoprotein A-I (7.1 +/- 0.4% total secreted protein) in the presence of zinc. Approx. 16% of the secreted apolipoprotein A-I is complexed with lipid and can be isolated ultracentrifugally at d less than or equal to 1.21 g/ml. The latter complexes are composed of discs and vesicles as judged by electron microscopy and can be further separated by column chromatography into three fractions: fraction I, mostly vesicles (60-260 nm) and large discs (18-20 nm diameter); fraction II, discs 14.2 +/- 2.6 nm diameter; and fraction III, nonresolvable by electron microscopy. The latter fraction is extremely lipid-poor (94% protein, 6% phospholipid); in contrast, the protein, phospholipid and unesterified cholesterol content for the other fractions are 43, 33 and 24%, respectively, for fraction I and 53, 33 and 14%, respectively, for fraction II. Fraction II particles contain three and four apolipoprotein A-Is per particle as determined by protein crosslinking while large structures in fraction I contain primarily six to seven apolipoprotein A-Is per particle. Following incubation with purified lecithin: cholesterol acyltransferase, discoidal particles were transformed into apparent spherical particles 12.9 +/- 3.4 nm diameter; this transformation coincided with 19-21% conversion of unesterified cholesterol to esterified cholesterol. The apolipoprotein A-I-lipid complexes isolated from Chinese hamster ovary cell media are similar to nascent HDL found in plasma of lecithin:cholesterol acyltransferase-deficient patients and those secreted by the human hepatoma line, Hep G2. The ability of the Chinese hamster ovary cell nascent HDL-like particles to undergo transformation in the presence of purified lecithin:cholesterol acyltransferase indicates that they are functional particles.     

Effect of pH and different substrates on the electrokinetic properties of (Na+, K+)-ATPase vesicles

Some biophysical properties of a (Na+, K+)-ATPase preparation from guinea-pig kidney have been analysed. The recently developed technique of laser Doppler spectroscopy was applied to measure particle mobility under electrophoretic conditions. The following results were obtained: 1. magnesium ions at pH 7.3 decrease the mobility of the ATPase containing vesicles by binding to negatively charged surface groups. At pH 3.3 the competitive binding of protons causes a shift of the mobility vs. [Mg2+] curve to higher values of [Mg2+], 2. binding of ATP at pH 7.3 (Kd = 0.9 X 10(-4) M for (mM 1 NaCl, 0.2 KCl, 0.1 MgCl2, 0.1 Tris) was measured as an increase in particle mobility depending also on [Mg2+]. At pH 3.3 also unspecific ATP-binding occurred, 3. ITP and GTP had the same Kd value as ATP; ADP a slightly lower one (Kd = 1.2 X 10(-4) M). Tris-H3PO4 (Kd = 2.6 X 10(-4) M) was also able to increase particle mobility, but only at higher concentrations and not to the same extent as ATP; AMP induced only very small changes, 4. from the mobility-pH curve an isoelectric point of 4.1 is derived (buffer: 1 mM NaCl, 0.2 mM KCl, 0.1 mM MgCl2, 0.1 mM Tris). In the presence of 0.9 mM ATP the isoelectric point is shifted to 3.2. As the electrophoretic mobility is directly proportional to the net charge of the vesicles, the results may be interpreted as changes in surface charge density, originating from both a conformational change of the ATPase polypeptide and a decrease in vesicle size.     

Inhibition of cholesterol efflux by 7-ketocholesterol: comparison between cells, plasma membrane vesicles, and liposomes as cholesterol donors.

Cholesterol removal from lipid-loaded macrophages is an important, potentially antiatherogenic process, and we have previously shown that an oxysterol, 7-ketocholesterol (7K), can impair efflux to lipid-free apoprotein A-1 (apoA-1). This publication investigates whether incorporation of 7K into membranes could account for this impairment of cholesterol efflux. Cholesterol efflux was studied from lipoprotein-loaded THP-1 cells, from plasma membrane vesicles obtained from these cells, and from artificial, protein-free liposomes. Impairment of cholesterol efflux by 7K was observed for all cholesterol donor systems whether measured as decline in cholesterol removal rates or as the percentage mass of total cellular cholesterol exported. 7-Ketocholesterol itself was not removed by apoA-1 from any of the cholesterol donor systems. Increasing membrane cholesterol content increased the rate of cholesterol removal by apoA-1 (as seen with plasma membrane vesicles), the quantity of cholesterol removed at equilibrium (liposomes), or both (whole cells). Although the minimum inhibitory 7K concentrations varied between the cholesterol donor systems, 7K inhibited cholesterol efflux in all systems. It was concluded that 7K induces alteration in membranes which decreased the efficiency of cholesterol efflux and the quantity of removed cholesterol induced by apoA-1. As cell membrane proteins are not essential for cholesterol efflux in these systems, the impairment of such by 7K suggests that its effect on membrane lipid composition and its structure are key regulatory elements in this efflux process.     

13C-NMR spectroscopy of human serum high density lipoprotein enriched with labelled phospholipids.

Native human serum high density lipoprotein (HDL) (d = 1.063--1.21g x cm-3) was enriched with phosphatidylcholines labelled with 13C in the polar head group ([N-13CH3]choline) and in the fatty acyl chains ([26-13C]cholesterol) and its linoleic acid ester using the previously described exchange method (Stoffel et al. 1978). The properties of the HDL particles with the exchanged lipid classes were the same as those of the native particles (Mr, CD, fluorescence, lipid and apoprotein stoichiometry, electrophoretic mobility). The T1-times were very similar to those obtained previously with recombined apolipoprotein-[13C]lipid complexes and further support our proposals concerning lipid and apoprotein interactions in the HDL particle.     

Structural and chemical properties of a flavodoxin from Anabaena PCC 7119

Structural and chemical properties of a flavodoxin from Anabaena PCC 7119 are described. The first 36 residues of the amino-terminal amino acid sequence have been determined and show extensive homology with flavodoxins isolated from other sources. Anabaena flavodoxin exhibits a net negative change (-3) in the helix-1 segment as found with other cyanobacterial flavodoxins Synechococcus 6301 (Anacystis nidulans) and Nostoc MAC, but in contrast to the net positive charge found in this region in the case of flavodoxins isolated from nitrogen-fixing bacteria (Azotobacter and Klebsiella). The FMN cofactor can be reversibly resolved from the apoprotein by trichloroacetic acid treatment. Apoflavodoxin, thus prepared, binds FMN with a Kd value of 0.1 nM and binds riboflavin with a decreased affinity (Kd = 5 microM) at pH 7.2. The apoprotein is stable in dilute solutions at pH values around 7 but readily denatures at pH 8 as judged from loss in flavin-binding ability and by ultraviolet circular dichroism spectroscopy. Oxidation-reduction potential studies at pH values of 7 and 8 show OX/SQ couples of -195 mV and -255 mV, respectively, and show SQ/HQ couples of -390 mV and -418 mV, respectively. From these data, the binding constant for the FMN semiquinone is calculated to be approx. 5-fold tighter and the binding of the FMN hydroquinone is approx. 10(5)-fold weaker than that of the oxidized FMN to the apoprotein. Anabaena flavodoxin functions as an effective mediator of electron transfer from ferredoxin-NADP(+)-reductase to cytochrome c with a turnover number [4.5-5) x 10(3) min-1); a values similar to that determined for Anabaena ferredoxin. The flavodoxin binds tightly to the reductase with Kd values of 6.4 and 8.5 microM at pH values of 7.0 and 8.0, respectively.