Estradiol enhances the resistance of LDL to oxidation by stabilizing apoB-100 conformation

Among different proposed mechanisms to account for the protection exerted by estrogens against cardiovascular diseases, the antioxidant effect has attracted considerable attention. We confirmed that 17-beta-estradiol (E2), when added to human LDL at a 6:1 ratio to apoB-100, markedly delays the phase of massive LDL lipid peroxidation induced by Cu(2+). We also observed an increased oxidative resistance of E2-treated LDL by monitoring the early phase of oxidative degradation on the basis of increased LDL surface polarity by the generalized polarization of the lipophilic fluorescent probe 2-(dimethylamino)-6-lauroylnaphthalene (Laurdan). A scavenging of free radicals by E2 is ruled out since, consistent with its structure, its rate constant for the reduction of peroxy radicals is extremely low, i.e., 0.02% of that of vitamin E. Tryptophan fluorescence lifetime and circular dichroism measurements revealed that (i) apoB-100 undergoes a conformational modification and a progressive loss of secondary structure during lipid peroxidation; (ii) E2 increases apoB-100 secondary structure and modifies its conformation; and (iii) the apoB-100 conformational change induced by E2 makes this protein resistant to modifications brought about by lipid peroxidation. We propose that E2, by affecting apoB-100 secondary structure and conformation, modifies the interaction of this protein with the outer layer of the LDL particle thus increasing its overall oxidative resistance.     

Modification of the core lipids of low density lipoproteins produces selective alterations in the expression of apoB-100 epitopes.

The conformation of the apolipoprotein B-100 associated with low density lipoproteins (LDL) is not fixed. Rather, the conformations of several regions are subject to alteration by a variety of metabolic and therapeutic perturbations that change either the lipid compositions and/or sizes of LDL particles. However, because these perturbations simultaneously alter several structural-compositional features of the particles it has been difficult to relate structural-compositional features of LDL to apoB-100 conformations. Furthermore, in in vivo studies several days pass between samplings, thus different sets of particles are studied before and after experimental perturbation. In the present experiments more discrete perturbations of LDL were obtained in vitro by incubating LDL with very low density lipoproteins (VLDL) in the presence of partially purified human plasma lipid transfer proteins. The conformations of apoB on the LDL particles then were examined a) by probing epitope expression and b) by examining interactions between LDL and LDL-receptors in cultured human fibroblasts. During incubations with VLDL and lipid transfer proteins, the diameters of LDL particles decreased; the percentage composition of LDL triglycerides increased three-to fivefold; concomitantly, cholesteryl esters decreased. Lipid transfer protein was required for the transfer to occur and the magnitude of the increase in LDL-triglycerides depended upon the duration of incubation, the ratio of VLDL/LDL, and unknown properties specific to the various LDL preparations. The fact that the triglyceride contents of all LDL preparations were not identically affected suggests that initial packaging of the core region may affect capacity for lipid exchange.(ABSTRACT TRUNCATED AT 250 WORDS)     

The molecular mechanisms underlying the reduction of LDL apoB-100 by ezetimibe plus simvastatin

The combination of ezetimibe, an inhibitor of Niemann-Pick C1-like 1 protein (NPC1L1), and an HMG-CoA reductase inhibitor decreases cholesterol absorption and synthesis. In clinical trials, ezetimibe plus simvastatin produces greater LDL-cholesterol reductions than does monotherapy. The molecular mechanism for this enhanced efficacy has not been defined. Apolipoprotein B-100 (apoB-100) kinetics were determined in miniature pigs treated with ezetimibe (0.1 mg/kg/day), ezetimibe plus simvastatin (10 mg/kg/day), or placebo (n = 7/group). Ezetimibe decreased cholesterol absorption (-79%) and plasma phytosterols (-91%), which were not affected further by simvastatin. Ezetimibe increased plasma lathosterol (+65%), which was prevented by addition of simvastatin. The combination decreased total cholesterol (-35%) and LDL-cholesterol (-47%). VLDL apoB pool size decreased 26%, due to a 35% decrease in VLDL apoB production. LDL apoB pool size decreased 34% due to an 81% increase in the fractional catabolic rate, both of which were significantly greater than monotherapy. Combination treatment decreased hepatic microsomal cholesterol (-29%) and cholesteryl ester (-65%) and increased LDL receptor (LDLR) expression by 240%. The combination increased NPC1L1 expression in liver and intestine, consistent with increased SREBP2 expression. Ezetimibe plus simvastatin decreases VLDL and LDL apoB-100 concentrations through reduced VLDL production and upregulation of LDLR-mediated LDL clearance.     

Metabolic behavior of hepatic VLDL and plasma LDL apoB-100 in African green monkeys

Recently, evidence has accumulated suggesting that significant amounts of plasma low density lipoproteins (LDL) may be derived by direct production. These plasma very low density lipoprotein (VLDL)-independent sources include the production and secretion of LDL-like particles directly by the liver, and/or a small pool of nascent precursor particles that are converted rapidly to LDL. The current studies were designed to test the hypothesis that hepatic VLDL represent a rapidly turning over precursor pool to plasma LDL in African green monkeys. Livers from African green monkeys were perfused with serum-free medium containing [3H]leucine or 3H-labeled amino acids for 4-6 hr. Hepatic [3H]VLDL and autologous plasma 125I-labeled LDL were injected simultaneously into recipient animals and density gradient ultracentrifugation and gel filtration were used to characterize the distribution of 3H and 125I radioactivity at selected times after injection. These studies show that 4 to 66% of the injected dose of hepatic VLDL [3H]apoB-100 was metabolized extremely rapidly into particles that resembled the recipient's plasma LDL by size and density. Based on the kinetic model developed to describe the metabolic behavior of hepatic VLDL [3H]apoB-100, the estimated maximal pool size of hepatic VLDL apoB-100 in these animals was very small (0.042 and 0.112 mg) and represented, at best, approximately 10% of the average plasma VLDL apoB-100 mass found in cholesterol-fed African green monkeys. In addition, the radiolabeled hepatic LDL appear to be metabolized similarly to plasma LDL. That is, the rapid conversion of hepatic VLDL as well as the direct production of hepatic particles within the LDL density range appear to contribute to plasma LDL. Metabolic heterogeneity was also seen within the LDL class. The more buoyant subfraction (LDL1) had a higher turnover rate than the more dense subfraction (LDL2) and hepatic VLDL-derived [3H]LDL1 had a slower final rate of plasma disappearance than the plasma-derived 125I-labeled LDL1 in most animals. The results from these studies suggest that a small pool of hepatic VLDL can be converted very rapidly to plasma LDL and may contribute significantly to the large plasma pool of LDL seen in cholesterol-fed African green monkeys. This pathway may be analogous to the pathway in some human subjects in which a portion of human plasma VLDL is converted rapidly into LDL without passing through a delipidation cascade, often referred to as direct LDL production.     

Insulin decreases the secretion of apoB-100 from hepatic HepG2 cells but does not decrease the secretion of apoB-48 from intestinal CaCo-2 cells

We compared the acute effect of insulin on the human colonic intestinal epithelial cell line CaCo-2 and the transformed human hepatic cell line HepG2. Over 24 h, 100 nM and 10 µM insulin significantly inhibited the secretion of apolipoprotein (apo) B-100 from HepG2 cells to 63 and 49% of control, respectively. Insulin had no effect on the secretion of apoB-48 from CaCo-2 cells. There was no effect of insulin on the cholesterol ester or free cholesterol concentrations in HepG2 or CaCo-2 cells. HepG2 and CaCo-2 cells bound insulin with high affinity, leading to similar stimulation of insulin receptor protein tyrosine kinase activation. Protein kinase C or mitogen-activated protein kinase activity in the presence or absence of insulin was not correlated with apoB-48 production in CaCo-2 cells. Therefore, insulin acutely decreases the secretion of apoB-100 in hepatic HepG2 cells, but does not acutely modulate the production or secretion of apoB-48 from CaCo-2 intestinal cells.     

ApoB-48 and apoB-100 differentially influence the expression of type-III hyperlipoproteinemia in APOE*2 mice

Apolipoprotein E (apoE) is essential for the clearance of plasma chylomicron and VLDL remnants. The human APOE locus is polymorphic and 5-10% of APOE*2 homozygotes exhibit type-III hyperlipoproteinemia (THL), while the remaining homozygotes have less than normal plasma cholesterol. In contrast, mice expressing APOE*2 in place of the mouse Apoe (Apoe(2/2) mice) are markedly hyperlipoproteinemic, suggesting a species difference in lipid metabolism (e.g., editing of apolipoprotein B) enhances THL development. Since apoB-100 has an LDLR binding site absent in apoB-48, we hypothesized that the Apoe(2/2) THL phenotype would improve if all Apoe(2/2) VLDL contained apoB-100. To test this, we crossed Apoe(2/2) mice with mice lacking the editing enzyme for apoB (Apobec(-/-)). Consistent with an increase in remnant clearance, Apoe(2/2). Apobec(-/-) mice have a significant reduction in IDL/LDL cholesterol (IDL/LDL-C) compared with Apoe(2/2) mice. However, Apoe(2/2).Apobec(-/-) mice have twice as much VLDL triglyceride as Apoe(2/2) mice. In vitro tests show the apoB-100-containing VLDL are poorer substrates for lipoprotein lipase than apoB-48-containing VLDL. Thus, despite a lowering in IDL/LDL-C, substituting apoB-48 lipoproteins with apoB-100 lipoproteins did not improve the THL phenotype in the Apoe(2/2).Apobec(-/-) mice, because apoB-48 and apoB-100 differentially influence the catabolism of lipoproteins.     

Increased production of VLDL apoB-100 in subjects with familial hypercholesterolemia carrying the same null LDL receptor gene mutation

Early radiokinetic studies revealed that the classical metabolic defect in patients with familial hypercholesterolemia (FH) is hypocatabolism of LDL due to decreased LDL receptor activity. However, recent studies have suggested that hepatic oversecretion of apolipoprotein B-100 (apoB-100)-containing lipoproteins could also contribute to the markedly elevated plasma concentrations of LDL-cholesterol found in FH. The aim of this study was to examine the kinetics of apoB-100 labeled with a stable isotope (l-[5,5,5-D(3)] leucine) in five normolipidemic controls and in seven well-characterized FH subjects that included six FH heterozygotes and one FH homozygote carrying the same null LDL receptor gene mutation. As compared with controls, the VLDL apoB-100 production rate was increased by 50% in the FH heterozygotes and by 109% in the FH homozygote. Furthermore, FH subjects had significantly higher LDL apoB-100 pool size and lower LDL apoB-100 fractional catabolic rate than controls. These results indicate that the elevation of plasma LDL-cholesterol found in FH is attributable to both decreased clearance of LDL and increased hepatic production of apoB-100-containing lipoproteins.     

High carbohydrate fat-free diet modulates epitope expression of LDL-apoB-100 and interaction of LDL with human fibroblasts

High carbohydrate diets are known to increase the concentration of very low density lipoprotein (VLDL) and to lower the concentrations of low density lipoprotein (LDL) and high density lipoprotein (HDL) in plasma. Such diets also alter lipoprotein compositions and metabolism. The aims of the present study were to assess in detail the effects of a virtually fat-free high carbohydrate (CHO) diet (CHO greater than 85% and fat less than 1% of calories) on various aspects of LDL. Thirteen healthy normolipidemic volunteers ate a basal "American" diet and the CHO diet for 7 days each in a forward or reverse sequence. Fasting blood samples were drawn at the ends of each study period and analyzed for lipoprotein lipid and apolipoprotein concentrations. Compositions of LDL particles isolated by ultracentrifugation were characterized chemically, LDL sizes were assessed by nondenaturing gradient electrophoresis on 2-16% gels, and association and degradation of LDL with normal human skin fibroblasts were quantified in cell cultures. Immunoreactivities of apoB in LDL were tested in solid phase competitive binding radioimmunoassays using five monoclonal anti-LDL antibodies that reacted with defined epitopes of apoB-100. The study diet produced consistent decreases of LDL cholesterol and apoB concentrations by 25% and 17%, respectively. LDL compositions were altered. Mean LDL triglycerides increased 3% to 4% of total LDL mass (P less than 0.004), and LDL particle sizes decreased (P less than 0.01). In radioimmunoassays that contained monoclonal antibody B1B3, an antibody that inhibits binding of LDL to the LDL receptor, the mean ED50 value for LDL protein was reduced from 3.75 to 2.66 micrograms (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

The bridging function of hepatic lipase clears plasma cholesterol in LDL receptor-deficient "apoB-48-only" and "apoB-100-only" mice

Hepatic lipase clears plasma cholesterol by lipolytic and nonlipolytic processing of lipoproteins. We hypothesized that the nonlipolytic processing (known as the bridging function) clears cholesterol by removing apoB-48- and apoB-100-containing lipoproteins by whole particle uptake. To test our hypotheses, we expressed catalytically inactive human HL (ciHL) in LDL receptor deficient "apoB-48-only" and "apoB-100-only" mice. Expression of ciHL in "apoB-48-only" mice reduced cholesterol by reducing LDL-C (by 54%, 46 +/- 6 vs. 19 +/- 8 mg/dl, P < 0.001). ApoB-48 was similarly reduced (by 60%). The similar reductions in LDL-C and apoB-48 indicate cholesterol removal by whole particle uptake. Expression of ciHL in "apoB-100-only" mice reduced cholesterol by reducing IDL-C (by 37%, 61 +/- 19 vs. 38 +/- 12 mg/dl, P < 0.003). Apo-B100 was also reduced (by 27%). The contribution of nutritional influences was examined with a high-fat diet challenge in the "apoB-100-only" background. On the high fat diet, ciHL reduced IDL-C (by 30%, 355 +/- 72 vs. 257 +/- 64 mg/dl, P < 0.04) but did not reduce apoB-100. The reduction in IDL-C in excess of apoB-100 suggests removal either by selective cholesteryl ester uptake, or by selective removal of larger, cholesteryl ester-enriched particles. Our results demonstrate that the bridging function removes apoB-48- and apoB-100-containing lipoproteins by whole particle uptake and other mechanisms.     

Loss of apoB-100 secondary structure and conformation in hydroperoxide rich, electronegative LDL(-).

A subpopulation of low-density lipoproteins (LDL) is present in human plasma that contains lipid hydroperoxides and is more negatively charged (LDL(-)) than normal native LDL. By circular dichroism and tryptophan lifetime measurements we found that apoB-100 secondary structure is markedly decreased and its conformation is severely altered in LDL(-). The low tryptophan fluorescence intensity confirms the oxidative degradation of the lipoprotein, and the very long lifetime value of one of its decay components indicates a low polarity environment for the remaining unbleached residues. Either a peculiar folding or, most likely, a sinking of the apoB-100 into the lipid core can account for the observed long lifetime component. Oxidation in vitro produces a similar unfolding of the apolipoprotein but the lifetime of tryptophan fluorescence is shifted to lower values, indicating that the denatured apoprotein remains at the hydrophilic surface of the lipoprotein particle. A disordering and an increased polarity of the LDL(-) surface lipids was demonstrated by measuring the generalized polarization of 2-dimethylamino-6-lauroylnaphthalene (Laurdan). The looser monolayer packing apparently favors the new conformation of apoB-100 and its sinking into a more hydrophobic environment, possibly accounting for it reduced receptor binding properties.     

Acidity and lipolysis by group V secreted phospholipase A(2) strongly increase the binding of apoB-100-containing lipoproteins to human aortic proteoglycans

Local acidic areas characterize diffuse intimal thickening (DIT) and advanced atherosclerotic lesions. The role of acidity in the modification and extra- and intracellular accumulation of triglyceride-rich VLDL and IDL particles has not been studied before. Here, we examined the effects of acidic pH on the activity of recombinant human group V secreted phospholipase A(2) (sPLA(2)-V) toward small VLDL (sVLDL), IDL, and LDL, on the binding of these apoB-100-containing lipoproteins to human aortic proteoglycans, and on their uptake by human monocyte-derived macrophages. At acidic pH, the ability of sPLA(2)-V to lipolyze the apoB-100-containing lipoproteins was moderately, but significantly, increased while binding of the lipoproteins to proteoglycans increased >60-fold and sPLA(2)-V-modification further doubled the binding. Moreover, acidic pH more than doubled macrophage uptake of soluble complexes of sPLA(2)-V-LDL with aortic proteoglycans. Proteoglycan-affinity chromatography at pH 7.5 and 5.5 revealed that sVLDL, IDL, and LDL consisted of populations with different proteoglycan-binding affinities, and, surprisingly, the sVLDL fractions with the highest proteoglycan-affinity contained only low amounts of apolipoproteins E and C-III. Our results suggest that in atherosclerotic lesions with acidic extracellular pH, sPLA(2)-V is able to lipolyze sVLDL, IDL, and LDL, and increase their binding to proteoglycans. This is likely to provoke extracellular accumulation of lipids derived from these atherogenic lipoprotein particles and to increase the progression of the atherosclerotic lesions.     

Trans fatty acids alter the lipid composition and size of apoB-100-containing lipoproteins secreted by HepG2 cells

This study was conducted to determine the secretion rate and composition of lipoproteins secreted by HepG2 cells as influenced by the type of fatty acid present in the incubation medium. Cells were preincubated for 24 h with palmitic, oleic, elaidic, linoleic or conjugated linoleic acid (CLA), and the lipoproteins secreted during a subsequent incubation period of 24 h were collected for analysis. The secretion rate of apolipoprotein B-100 (apoB) was significantly greater in HepG2 cells preincubated with elaidic acid compared with those preincubated with palmitic or oleic acid; apoB secretion was greater in cells preincubated with CLA compared with those preincubated with linoleic acid. The lipid composition of secreted lipoproteins was also influenced by fatty acid treatment, resulting in significantly smaller lipoprotein particles secreted by cells preincubated with elaidic acid and CLA compared with those secreted by cells treated with oleic acid and linoleic acid, respectively. Our results are relevant to human metabolism for the following reasons: (1) the size of plasma low-density lipoproteins (LDLs) is determined, at least in part, by the composition of apoB-containing lipoproteins secreted by the liver; (2) small plasma LDL particles are associated with an increased risk of coronary heart disease; and (3) specific dietary fatty acids can affect the composition and size of plasma LDLs, thereby imparting a relative atherogenicity to plasma LDLs independent of LDL cholesterol concentration. The present study therefore suggests that elaidic acid and CLA promote the hepatic secretion of small apoB-containing lipoproteins, which could lead to an increased production of small plasma LDL particles.     

Effects on apoB-100 secretion and bile acid synthesis by redirecting cholesterol efflux from HepG2 cells

This study determined the effects of apoA-I, HDL3, or hydroxy-beta-cyclodextrin on apoB-100 secretion and bile acid synthesis by HepG2 cells. The principal observations were that: 1) ApoB-100 secretion into the medium was significantly less after the addition of any of the three agents. 2) Triglyceride mass was not significantly changed from control in the medium but was significantly, although modestly, reduced in the cells. 3) Neither free cholesterol (FC) nor cholesteryl ester (CE) mass in the medium was changed; by contrast, CE mass was reduced within the cells although FC was not. 4) Although the total mass of cholesterol in the medium was unaffected, the proportion associated with apoB-100 was reduced, whereas the proportion associated with the non-apoB-100 fraction was increased. 5) There was also an unanticipated, but substantial, increase in bile acid synthesis induced by apoA-I, HDL3, or hydroxy-beta-cyclodextrin, which was time and concentration dependent, and which was associated with marked increases in cholesterol 7 alpha-hydroxylase activity. There were no significant changes in ACAT activity and only modest increases in HMG-CoA reductase activity. These findings support previous clinical observations that an elevated apoB-100 can accompany a low HDL cholesterol in normotriglyceridemic subjects. They also point to physiologically important, although still only partially understood, metabolic relationships amongst hepatic apoB-100 secretion, cholesterol efflux, and bile acid synthesis.     

The interaction of phospholipase A2 with phospholipid analogues and inhibitors

A series of structurally modified phospholipids have been used to delineate the structural features involved in the interaction between cobra venom (Naja naja naja) phospholipase A2 and its substrate. Special emphasis has been placed on sn-2 amide analogues of the phospholipids. These studies have led to a very potent, reversible phospholipase A2 inhibitor. A six-step synthesis of this compound, 1-palmitylthio-2-palmitoylamino-1,2-dideoxy-sn-glycero-3- phosphorylethanolamine (thioether amide-PE), was developed. Other analogues studied included 1-palmitylthio-2-palmitoylamino-1,2-dideox-sn- glycero-3-phosphorylcholine, 1-palmityl-2-palmitoylamino-2- deoxy-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-palmitoylamino-2-deoxy-sn-glycero-3- phosphorylcholine, 1-palmitylthio- 2([(tetradecyloxy)carbonyl]amino)-1,2-dideoxy-sn-glycero-3- phosphorylcholine, 1-palmitoyl- 2([(octadecylylamino)carbonyl]amino)-2-deoxy-sn-glycero-3- phosphorylcholine, and sphingomyelin. Inhibition studies used the well defined Triton X-100 mixed micelle system and the spectroscopic thio assay. The phospholipid analogues showed varying degrees of inhibition. The best inhibitor was the thioether amide-PE which had an IC50 of 0.45 microM. In contrast, sphingomyelin, a natural phospholipid that resembles the amide analogues, did not inhibit but rather activated phosphatidylcholine hydrolysis. This systematic study of phospholipase A2 inhibition led to the following conclusions about phospholipid-phospholipase A2 interactions: (i) sn-2 amide analogues bind tighter than natural phospholipids, presumably because the amide forms a hydrogen bond with the water molecule in the enzyme active site, stabilizing its binding. (ii) Inhibitor analogues containing the ethanolamine polar head group appear to be more potent inhibitors than those containing the choline group. This difference in potency may be due solely to the fact that the cobra venom phospholipase A2 is activated by choline-containing phospholipids. Thus, choline-containing non-hydrolyzable analogues both inhibit and activate this enzyme. Both of these effects must be taken into account when studying phosphatidylcholine inhibitors of the cobra venom enzyme. (iii) The potency of inhibition of these analogues is significantly enhanced by increasing the hydrophobicity of the sn-1 functional group.(ABSTRACT TRUNCATED AT 400 WORDS)     

Disruption of the maxi-K-caveolin-1 interaction alters current expression in human myometrial cells

Background  

One determinant of the total K+ myometrial smooth muscle cell (MSMC) current is the large conductance, calcium- and voltage-activated potassium channel (maxi-K channel). This channel provides a repolarizing current in response to excitatory stimuli, most notably in response to increases in the levels of intracellular Ca2+, and blocking the channel by pharmacological means induces the depolarization of MSMCs and also enhances contraction strength. In MSMCs, maxi-K channels can reside in the caveolae, where they associate with the scaffolding protein caveolin-1 (cav-1). The aim of this study was to investigate the consequences of this interaction - more specifically, how disruption of the association between the maxi-K channel and cav-1 may influence the current expression and excitability of myometrial cells - with the aim of better understanding the mechanisms that underlie the regulation of normal and aberrant uterine function.     

Ubiquitination regulates the assembly of VLDL in HepG2 cells and is the committing step of the apoB-100 ERAD pathway

Apolipoprotein B-100 (apoB-100) is degraded by endoplasmic reticulum-associated degradation (ERAD) when lipid availability limits assembly of VLDLs. The ubiquitin ligase gp78 and the AAA-ATPase p97 have been implicated in the proteasomal degradation of apoB-100. To study the relationship between ERAD and VLDL assembly, we used small interfering RNA (siRNA) to reduce gp78 expression in HepG2 cells. Reduction of gp78 decreased apoB-100 ubiquitination and cytosolic apoB-ubiquitin conjugates. Radiolabeling studies revealed that gp78 knockdown increased secretion of newly synthesized apoB-100 and, unexpectedly, enhanced VLDL assembly, as the shift in apoB-100 density in gp78-reduced cells was accompanied by increased triacylglycerol (TG) secretion. To explore the mechanisms by which gp78 reduction might enhance VLDL assembly, we compared the effects of gp78 knockdown with those of U0126, a mitogen-activated protein kinase/ERK kinase1/2 inhibitor that enhances apoB-100 secretion in HepG2 cells. U0126 treatment increased secretion of both apoB100 and TG and decreased the ubiquitination and cellular accumu-lation of apoB-100. Furthermore, p97 knockdown caused apoB-100 to accumulate in the cell, but if gp78 was concomitantly reduced or assembly was enhanced by U0126 treatment, cellular apoB-100 returned toward baseline. This indicates that ubiquitination commits apoB-100 to p97-mediated retrotranslocation during ERAD. Thus, decreasing ubiquitination of apoB-100 enhances VLDL assembly, whereas improving apoB-100 lipidation decreases its ubiquitination, suggesting that ubiquitination has a regulatory role in VLDL assembly.     

Role of the N-terminus in the interaction of pancreatic phospholipase A2 with aggregated substrates. Properties and crystal structure of transaminated phospholipase A2

A free N-terminal alpha-NH3+ group is absolutely required for full catalytic activity of phospholipase A2 on aggregated substrates. To elucidate how this alpha-NH3+ group triggers catalytic activity, we specifically transaminated this group in various pancreatic phospholipases A2. Porcine, porcine iso-, equine, human, ovine, and bovine phospholipases A2 all loose catalytic activity on micellar substrates due to the inability of the transaminated proteins to bind to neutral micellar substrate analogues, as was found for the zymogens. Loss of activity is pseudo first order, the rate constants being different for the enzymes studied. The transaminated phospholipases A2 have an intact active site, as catalytic activities on monomeric substrates are comparable to those of the respective zymogens. The X-ray structure of transaminated bovine phospholipase A2 at 2.1-A resolution shows that the N-terminal region and the sequence 63-72 in this protein are more flexible than in the native enzyme. Also, in this respect, the transaminated enzyme very much resembles the zymogen structure. In good agreement with this, it was found by photochemically induced dynamic nuclear polarization 1H NMR that aromatic resonances of Trp-3 and Tyr-69 are affected by transamination. In addition, fluorescence spectroscopy of the unique Trp-3 in transaminated bovine phospholipase A2 revealed a red shift of the emission maximum indicative of a more polar environment of Trp-3 in the transaminated phospholipase A2 as compared to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the interaction of phospholipase A(2) with phosphatidylcholine-phosphatidylglycerol mixed lipids

The first requirement in the hydrolysis of phospholipid bilayers by phospholipase A(2) is the interaction of the enzyme with the bilayer surface. The catalytic ability of phospholipase A(2) has been shown to be extremely sensitive to the topology of the bilayer to which it binds and hydrolyzes. Phospholipid bilayer properties and composition such as unsaturation, charge, and the presence of reaction products are known regulators of the catalytic activity of phospholipase A(2) toward the phospholipids and influences the binding of enzyme to the membrane. We show in this paper that the effect of increased anionic lipid results in enhanced binding that can be described quantitatively in terms of a simple phenomenological model. However, the interaction with anionic lipid does not singularly dominate the thermodynamics of binding, nor can the lag phase observed in the time course of hydrolysis of large unilamellar vesicles simply be the result of limited interaction between the enzyme and the bilayer. Furthermore, we show that phospholipase A(2) from Akgistrodon piscivorus piscivorus can exist in at least two bilayer-bound states and that the absence of a fluorescence change upon mixing the enzyme with lipid bilayers does not necessarily indicate the absence of an interaction.     

Binding of thyroxine to human plasma low density lipoprotein through specific interaction with apolipoprotein B (apoB-100)

Human plasma low density lipoprotein (LDL), which binds 0.2% of plasma T4, was shown to interact with the hormone through its protein moiety, apolipoprotein B-100. LDL and LDL2, the major subfraction of LDL, were found to have 3 equivalent binding sites for T4 with Ka = 2.5 x 10(6) M-1. Photoaffinity labeling of LDL with inner ring-labeled [125I]T4, followed by SDS-PAGE or agarose-SDS-PAGE of the labeled products, revealed that apoB-100 and its proteolytic cleavage products, apoB-74 and apoB-26, bound [125I]T4. In the presence of 1 or 10 microM T4, labeling was decreased in 7 separate experiments by 40-53% or 65-86%, respectively, consistent with a Ka of approximately 10(6) M-1. Binding of T4 to apoB-100 associated with VLDL was also demonstrated by photoaffinity labeling. The observed thyroid hormone binding property of lipid-complexed apoB-100 and the knowledge that receptors for the apolipoprotein exist in various tissues suggest a possible physiological role in thyroid hormone transport.