CTP:phosphocholine cytidylyltransferase and protein kinase C recognize different physical features of membranes: differential responses to an oxidized phosphatidylcholine

Protein kinase C (PKC) and CTP:phosphocholine cytidylyltransferase (CT) are two examples of enzymes that are regulated by reversible binding to membranes, and this binding is influenced by membrane physical properties. CT activation by oxidized phosphatidylcholines was recently demonstrated and was linked to the acyl chain disordering effect of the oxidized species (Biochemistry 38, 15606). In this paper, we compare the responses of PKC and CT to an oxidized PC, and investigate the physical properties of lipid bilayers that modulate the activity of these enzymes. We show that 1-palmitoyl, 2-(11,15 dihydroxy) eicosatrienoyl PC (diOH-PAPC) caused less of an increase in the temperature of the lamellar to hexagonal II transition (T_H) of an unsaturated PE, compared to its parent, PAPC. Using a polarity-sensitive interfacial probe, we also found evidence to suggest that this oxidized PC increases interfacial packing pressure. We found that whereas diOH-PAPC activates CT, it inhibits PKC relative to the parent PAPC. The activities of both CT and PKC are known to increase in the presence of non-lamellar forming lipids. The greater activating effect of diOH-PAPC compared with PAPC, is consistent with a stimulation of the activity of CT by negative curvature strain. However, this is not the case with PKC, for which we suggest that surface packing pressure is of prime importance.     

CTP:phosphocholine cytidylyltransferase activation by oxidized phosphatidylcholines correlates with a decrease in lipid order: a 2H NMR analysis

The enzyme CTP:phosphocholine cytidylyltransferase (CT) binds reversibly to membranes and is active only in its membrane-bound form. Membrane lipid composition influences the equilibrium between its soluble and membrane-bound forms. Whereas the enzyme is not activated by phosphatidylcholine (PC) vesicles, it is activated by PC vesicles that have been oxidized with HClO(4) [Drobnies, A. E., et al. (1998) Biochim. Biophys. Acta 1393, 90-98]. Here we explore the mechanism of activation of CT by a PC oxidized with lipoxidase. Multilamellar vesicles (MLVs) containing > or =5 mol % oxidized 1-palmitoyl-2-arachidonoylPC (PAPC) progressively activated the enzyme, which was fully activated by 25 mol % oxidized PC. The effect of oxidized PAPC on lipid order was investigated by (2)H NMR, using MLVs containing PAPC perdeuterated on the palmitoyl chain. Spectral depaking generated order parameter profiles along the sn-1 chain. The average order parameter (S(CD)) in the plateau region at 37 degrees C decreased from 0.18 to 0.15 with increasing percent of oxidized PAPC (0-25%). The change in S(CD) was even greater near the end of the palmitoyl chain. CT activation was inversely related to lipid order. The major component of the lipoxidase-oxidized PAPC was purified and characterized by mass spectrometry and NMR. This component, 1-palmitoyl-2-(11,15-dihydroxy)eicosatrienoylPC (dihydroxyPAPC), incorporated into PAPC MLVs, also stimulated CT activity and reduced the lipid order parameter. Both effects were reversed by egg sphingomyelin. We propose that CT activation by oxidized PAPC is mediated by effects on lipid packing perturbations. This is the first study to report the effects of a purified oxidized PC on the orientational order along the acyl chain and to correlate the lipid disordering of the oxidized PC with the activation of a membrane-associated regulatory enzyme.     

A mechanism underlying stimulation and inhibition of protein kinase C by lyso-PC: A role of membrane physical state

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Inhibition of LPS- and CpG DNA-induced TNF-alpha response by oxidized phospholipids

Lipid oxidation is commonly seen in the innate immune response, in which reactive oxygen intermediates are generated to kill pathogenic microorganisms. Although oxidation products of phospholipids have generally been regarded to play a role in a number of chronic inflammatory processes, several studies have shown that oxidized phospholipids inhibit the LPS-induced acute proinflammatory response in cultured macrophages and endothelial cells. We report in this study that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), but not nonoxidized PAPC, significantly inhibits the LPS-induced TNF-alpha response in intact mice. Oxidized PAPC also inhibits the 2'-deoxyribo(cytidine-phosphate-guanosine) (CpG) DNA-induced TNF-alpha response in cultured macrophages and intact mice. To elucidate the mechanisms of action, we show that oxidized PAPC, but not nonoxidized PAPC, inhibits the LPS- and CpG-induced activation of p38 MAPK and the NF-kappaB cascade. These results suggest a role for oxidized lipids as a negative regulator in controlling the magnitude of the innate immune response. Further studies on the mechanisms of action may lead to development of a new type of anti-inflammatory drug for treatment of acute inflammatory diseases such as sepsis.     

Phospholipid composition of reconstituted high density lipoproteins influences their ability to inhibit endothelial cell adhesion molecule expression

The ability of different phosphatidylcholine (PC) species to inhibit cytokine-induced expression of vascular cell adhesion molecule 1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) was investigated. PC species containing palmitoyl- in the sn-1 position and palmitoyl- (DPPC), arachidonyl- (PAPC), linoleoyl- (PLPC) or oleoyl- (POPC) in the sn-2 position were compared. These PC species were studied as components of reconstituted high density lipoproteins (rHDL) (containing apolipoprotein A-I [apoA-I] as the sole protein) or as small unilamellar vesicles (SUVs). The rHDL containing PLPC and PAPC inhibited VCAM-1 expression in activated HUVECs by 95 and 70%, respectively, at an apoA-I concentration of 16 micrometer. At this concentration of apoA-I, POPC rHDL inhibited by only 16% and DPPC rHDL did not inhibit at all. These differences could not be explained by differential binding of the rHDL to HUVECs. The same hierarchy of inhibitory activity was observed when these PC species were presented to the cells as SUVs but only when the SUVs also contained an antioxidant. It was concluded that rHDL PC is responsible for their inhibitory activity and that this varies widely with different PC species.     

Regulation of CTP: phosphocholine cytidylyltransferase activity by the physical properties of lipid membranes: an important role for stored curvature strain energy

CTP:Phosphocholine cytidylyltransferase (CT) catalyzes the key step in phosphatidylcholine (PC) synthesis. CT is activated by binding to certain lipid membranes. The membrane binding affinity of CT can vary from micromolar to millimolar K(d), depending on the lipid composition of the target membrane. Class II CT activators like diacylglycerols and unsaturated phosphatidylethanolamines (PE) favor inverted lipid phase formation. The mechanism(s) governing CT's association with class II lipid membranes and subsequent activation are relatively unknown. We measured CT activation by vesicles composed of PC and one of three unsaturated PEs, dioleoylglycerol (DOG), or cholesterol. For each lipid system, we estimated the stored curvature strain energy of the monolayer when confined to a relatively flat bilayer. CT binding and activation correlate very well with the curvature strain energy of several chemically distinct class II lipid systems, with the exception of those containing cholesterol, in which CT activation was less than the increase in curvature strain. CT activation by membranes containing DOG was reversed by inclusion of specific lysolipids, which reduce curvature strain energy. LysoPC, which has a larger positive curvature than lysoPE, produced greater inhibition of CT activation. Stored curvature strain energy is thus an important determinant of CT activation. Membrane interfacial polarity was investigated using a membrane-anchored fluorescent probe. Decreases in quenching of this interfacial probe by doxyl-PCs in class II membranes suggest the probe adopts a more superficial membrane location. This may reflect an increased surface hydrophobicity of class II lipid membranes, implying a role for surface dehydration in CT's interactions with membranes containing class II lipids. Cholesterol, a poor activator of CT, did not affect the positioning of the polarity-sensitive probe, suggesting that one reason for its ineffectiveness is an inability to enhance surface hydrophobicity.     

Amino acids 149 and 294 of human lecithin:cholesterol acyltransferase affect fatty acyl specificity

We identified two regions of human LCAT (hLCAT) that when mutated separately to the corresponding rat sequence (E149A and Y292H/W294F) and transiently expressed in COS-1 cells increased phospholipase A2 (PLA2) activity by 5.5- and 2.8-fold, respectively, and increased cholesteryl ester (CE) formation by 2.9- and 1.4-fold, respectively, relative to hLCAT using substrate particles containing 1-16:0,2-20:4-sn-glycero-3-phosphocholine (PAPC). In contrast, both activities with 1-16:0,2-18:1-sn-glycero-3-phosphocholine (POPC) substrate were similar among the three LCAT proteins. The triple mutant (E149A/Y292H/W294F) had increased PLA2 activity with PAPC similar to that observed with the E149A mutation alone; however, unlike E149A, the triple mutant demonstrated a 50% decrease in activity with POPC for both PLA2 activity and CE formation, suggesting an interaction between the two regions of LCAT. Additional mutagenesis studies demonstrated that W294F, but not Y292H, increased PLA2 activity by 3-fold with PAPC without affecting activity with POPC. The E149A/W294F double mutation mimicked the LCAT activity phenotype of the triple mutant (more activity with PAPC, less with POPC). In conclusion, separate mutation of two amino acids in hLCAT to the corresponding rat sequence increases activity with PAPC, whereas the combined mutations increase PAPC and decrease POPC activity, suggesting that these amino acids participate in the LCAT PC binding site and affect fatty acyl specificity.     

Oxidized phospatidylcholine but not native phosphatidylcholine inhibits inducible nitric oxide synthase in RAW 264.7 macrophages

This study was designed to compare the effects of oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC) and native PAPC on the inducible nitric oxide synthase (iNOS) in the macrophage cell line RAW 264.7. Macrophages stimulated by bacterial lipopolysaccharide (1 microg/ml) were incubated with increasing amounts of native or oxidized PAPC (oxPAPC, 10-20 microg/ml). Cells incubated with oxPAPC showed a dose-dependent inhibition of inducible nitric oxide synthesis, as well as reduced iNOS protein expression and mRNA levels. Additionally, chromatin immunoprecipitation assay revealed that oxPAPC reduced the interaction of the active NF-kappaB subunit p65 with the iNOS promoter region when compared to native PAPC.     

Xenopus paraxial protocadherin inhibits Wnt/β-catenin signalling via casein kinase 2β

Xenopus paraxial protocadherin (PAPC) regulates cadherin-mediated cell adhesion and promotes the planar cell polarity (PCP) pathway. Here we report that PAPC functions in the Xenopus gastrula as an inhibitor of the Wnt/β-catenin pathway. The intracellular domain of PAPC interacts with casein kinase 2 beta (CK2β), which is part of the CK2 holoenzyme. The CK2α/β complex stimulates Wnt/β-catenin signalling, and the physical interaction of CK2β with PAPC antagonizes this activity. By this mechanism, PAPC restricts the expression of Wnt target genes during gastrulation. These experiments identify a novel function of protocadherins as regulators of the Wnt pathway.     

Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3

Treatment of human artery wall cells with apolipoprotein A-I (apoA-I), but not apoA-II, with an apoA-I peptide mimetic, or with high density lipoprotein (HDL), or paraoxonase, rendered the cells unable to oxidize low density lipoprotein (LDL). Human aortic wall cells were found to contain 12-lipoxygenase (12-LO) protein. Transfection of the cells with antisense to 12-LO (but not sense) eliminated the 12-LO protein and prevented LDL-induced monocyte chemotactic activity. Addition of 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE] and 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] dramatically enhanced the nonenzymatic oxidation of both 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and cholesteryl linoleate. On a molar basis 13(S)-HPODE and 15(S)-HPETE were approximately two orders of magnitude greater in potency than hydrogen peroxide in causing the formation of biologically active oxidized phospholipids (m/z 594, 610, and 828) from PAPC. Purified paraoxonase inhibited the biologic activity of these oxidized phospholipids. HDL from 10 of 10 normolipidemic patients with coronary artery disease, who were neither diabetic nor receiving hypolipidemic medications, failed to inhibit LDL oxidation by artery wall cells and failed to inhibit the biologic activity of oxidized PAPC, whereas HDL from 10 of 10 age- and sex-matched control subjects did.We conclude that a) mildly oxidized LDL is formed in three steps, one of which involves 12-LO and each of which can be inhibited by normal HDL, and b) HDL from at least some coronary artery disease patients with normal blood lipid levels is defective both in its ability to prevent LDL oxidation by artery wall cells and in its ability to inhibit the biologic activity of oxidized PAPC.     

Anti-inflammatory apoA-I-mimetic peptides bind oxidized lipids with much higher affinity than human apoA-I

4F is an anti-inflammatory, apolipoprotein A-I (apoA-I)-mimetic peptide that is active in vivo at nanomolar concentrations in the presence of a large molar excess of apoA-I. Physiologic concentrations ( approximately 35 microM) of human apoA-I did not inhibit the production of LDL-induced monocyte chemotactic activity by human aortic endothelial cell cultures, but adding nanomolar concentrations of 4F in the presence of approximately 35 microM apoA-I significantly reduced this inflammatory response. We analyzed lipid binding by surface plasmon resonance. The anti-inflammatory 4F peptide bound oxidized lipids with much higher affinity than did apoA-I. Initially, we examined the binding of PAPC (1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine) and observed that its oxidized products bound 4F with an affinity that was approximately 4-6 orders of magnitude higher than that of apoA-I. This high binding affinity was confirmed in studies with defined lipids and phospholipids. 3F-2 and 3F(14) are also amphipathic alpha-helical octadecapeptides, but 3F-2 inhibits atherosclerosis in mice and 3F(14) does not. Like 4F, 3F-2 also bound oxidized phospholipids with very high affinity, whereas 3F(14) resembled apoA-I. The extraordinary ability of 4F to bind pro-inflammatory oxidized lipids probably accounts for its remarkable anti-inflammatory properties.     

Role of sn-1-saturated,sn-2-polyunsaturated phospholipids in control of membrane receptor conformational equilibrium: effects of cholesterol and acyl chain unsaturation on the metarhodopsin I in equilibrium with metarhodopsin II equilibrium.

The effect of phospholipid bilayer acyl chain packing free volume on the equilibrium concentration of the form of photolyzed rhodopsin which initiates visual signal transduction, metarhodopsin II (meta II), is examined in reconstituted systems formed from the saturated phospholipid dimyristoylphosphatidylcholine (DMPC) and in the polyunsaturated phospholipid sn-1-palmitoyl-sn-2-arachidonoylphosphatidylcholine (PAPC) with and without 30 mol% cholesterol. The extent of meta II formation is determined from both flash photolysis measurements and rapidly acquired absorbance spectra. Equilibrium and dynamic properties of the lipid bilayer are characterized by the dynamic fluorescence properties of 1,6-diphenyl-1,3,5-hexatriene (DPH). DPH orientational properties are characterized by fv, a parameter which reflects the volume available for probe reorientation in the bilayer, relative to that available in an unhindered, isotropic environment [Straume, M., & Litman, B. J. (1987) Biochemistry 26, 5121-5126]. The metarhodopsin I in equilibrium with meta II equilibrium constant, Keq has a linear relationship with fv for rhodopsin in PAPC vesicles with and without cholesterol as well as for rhodopsin in DMPC vesicles, and these two correlation lines have different slopes. The correlations between Keq and fv in PAPC and DMPC systems are compared with a similar correlation in the native rod outer segment disk membrane and one reported previously in an egg phosphatidylcholine (egg PC) system [Mitchell, D. C., Straume, M., Miller, J. L., & Litman, B. J. (1990) Biochemistry 29, 9143-9149].(ABSTRACT TRUNCATED AT 250 WORDS)     

Packing characteristics of highly unsaturated bilayer lipids: Raman spectroscopic studies of multilamellar phosphatidylcholine dispersions

The thermotropic properties and acyl chain packing characteristics of multilamellar dispersions of highly unsaturated lipids were examined by Raman spectroscopy. Bilayer assemblies were composed of POPC (1-palmitoyl-2-oleoylphosphatidylcholine), PAPC (1-palmitoyl-2-arachidonylphosphatidylcholine), and PDPC (1-palmitoyl-2-docosahexaenoylphosphatidylcholine), lipid systems possessing saturated sn-1 chains and unsaturated sn-2 chains with one, four, and six double bonds, respectively. Raman spectra were recorded in the acyl chain 2800-3100-cm-1 carbon-hydrogen (C-H) stretching and 1100-1200-cm-1 carbon-carbon (C-C) stretching mode regions, spectral intervals reflecting both the inter- and intrachain order/disorder properties of the various lipid dispersions. In order to obtain C-H stretching mode spectra relevant solely to the sn-1 chains of PAPC and PDPC, liquid-phase spectra of arachidonic and docosahexaenoic acid, respectively, were subtracted from the observed phospholipid spectra. The unsaturated sn-2 chains of PAPC and PDPC undergo minimal conformational reorganizations as the bilayers pass from the gel to liquid-crystalline phases. Phase transition temperatures, Tm, derived from statistically fitting the temperature-dependent Raman spectral data are approximately -2.5, -22.5, and -3 degrees C for POPC, PAPC, and PDPC, respectively. As the degree of unsaturation increases from POPC to PAPC and PDPC, the cooperativity of the phase transition, as measured by its breadth, decreases. Estimates of the transition widths from the temperature profiles are approximately 15 degrees C for PAPC and 20 degrees C for PDPC. The behavior of various Raman spectral parameters for the lipid gel phase reflects the formation of lateral microdomains, or clusters, whose packing properties maximize the van der Waals interactions between sn-1 chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the binding preference of human groups IIA and X phospholipases A2 for membranes with anionic phospholipids

Mammals contain 9-10 secreted phospholipases A(2) (sPLA(2)s) that display widely different affinities for membranes, depending on the phospholipid composition. The much higher enzymatic activity of human group X sPLA(2) (hGX) compared with human group IIA sPLA(2) (hGIIA) on phosphatidylcholine (PC)-rich vesicles is due in large part to the higher affinity of the former enzyme for such vesicles; this result also holds when vesicles contain cholesterol and sphingomyelin. The inclusion of anionic phosphatidylserine in PC vesicles dramatically enhances interfacial binding and catalysis of hGIIA but not of hGX. This is the result of the large number of lysine and arginine residues scattered over the entire surface of hGIIA, which cause the enzyme to form a supramolecular aggregate with multiple vesicles. Thus, high affinity binding of hGIIA to anionic vesicles is a complex process and cannot be attributed to a few basic residues on its interfacial binding surface, as is also evident from mutagenesis studies. The main reason hGIIA binds poorly to PC-rich vesicles is that it lacks a tryptophan residue on its interfacial binding surface, a residue that contributes to the high affinity binding of hGX to PC-rich vesicles. Results show that the lag in the onset of hydrolysis of PC vesicles by hGIIA is due in part to the poor affinity of this enzyme for these vesicles. Binding affinity of hGIIA, hGX, and their mutants to PC-rich vesicles is well correlated to the ability of these enzymes to act on the PC-rich outer plasma membrane of mammalian cells.     

Dissociation of protein kinase C activation and sn-1,2-diacylglycerol formation. Comparison of phosphatidylinositol- and phosphatidylcholine-derived diglycerides in alpha-thrombin-stimulated fibroblasts

Diacylglycerols (DAGs) derived from phosphatidylcholine (PC) hydrolysis have been shown to activate protein kinase C (PKC) in vitro, but it is not known whether this event occurs in response to DAGs generated via agonist-induced PC hydrolysis in intact cells. In this report we have addressed this question directly, using alpha-thrombin stimulation of IIC9 fibroblasts. PKC activation in intact cells was assessed in two ways, by measuring: 1) PKC membrane association as determined by kinase activity and Western blot analysis and 2) the phosphorylation of an endogenous PKC substrate, an 80-kDa protein. Treatment with 500 ng/ml alpha-thrombin has been shown to stimulate both phosphoinositide and PC hydrolysis, whereas treatment with 100 pg/ml alpha-thrombin stimulates only PC breakdown. Using these two conditions, we show that DAG produced from phosphoinositide, but not PC hydrolysis, is associated with the activation of PKC.     

GLTP-fold interaction with planar phosphatidylcholine surfaces is synergistically stimulated by phosphatidic acid and phosphatidylethanolamine

Among amphitropic proteins, human glycolipid transfer protein (GLTP) forms a structurally-unique fold that translocates on/off membranes to specifically transfer glycolipids. Phosphatidylcholine (PC) bilayers with curvature-induced packing stress stimulate much faster glycolipid intervesicular transfer than nonstressed PC bilayers raising questions about planar cytosol-facing biomembranes being viable sites for GLTP interaction. Herein, GLTP-mediated desorption kinetics of fluorescent glycolipid (tetramethyl-boron dipyrromethene (BODIPY)-label) from lipid monolayers are assessed using a novel microfluidics-based surface balance that monitors lipid lateral packing while simultaneously acquiring surface fluorescence data. At biomembrane-like packing (30–35 mN/m), GLTP uptake of BODIPY-glycolipid from POPC monolayers was nearly nonexistent but could be induced by reducing surface pressure to mirror packing in curvature-stressed bilayers. In contrast, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) matrices supported robust BODIPY-glycolipid uptake by GLTP at both high and low surface pressures. Unexpectedly, negatively-charged cytosol-facing lipids, i.e., phosphatidic acid and phosphatidylserine, also supported BODIPY-glycolipid uptake by GLTP at high surface pressure. Remarkably, including both 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (5 mol%) and POPE (15 mol%) in POPC synergistically activated GLTP at high surface pressure. Our study shows that matrix lipid headgroup composition, rather than molecular packing per se, is a key regulator of GLTP-fold function while demonstrating the novel capabilities of the microfluidics-based film balance for investigating protein-membrane interfacial interactions.     

Structural Identification and Systematic Comparison of Phorbol Ester,Dioleoylglycerol, Alcohol and Sevoflurane Binding Sites in PKCδ C1A Domain

Protein kinase C (PKC) is a family of signal transducing enzymes that have been implicated in anesthetic preconditioning signaling cascade. Evidences are emerging that certain exogenous neuromodulators such as n-alkanols and general anesthetics can stimulate PKC activity by binding to regulatory C1A domain of the enzyme. However, the accurate binding sites in C1A domain as well as the molecular mechanism underlying binding-stimulated PKC activation still remain unelucidated. Here, we report a systematic investigation of the intermolecular interaction of human PKCδ C1A domain with its natural activator phorbol ester (PE) and co-activator dioleoylglycerol (DOG) as well as exogenous stimulators butanol, octanol and sevoflurane. The domain is computationally identified to potentially have three spatially vicinal ligand-binding pockets 1, 2 and 3, in which the pockets 1 and 2 have previously been determined as the binding sites of PE and DOG, respectively. Systematic cross-binding analysis reveals that long-chain octanol and DOG are well compatible with the flat, nonpolar pocket 2, where the nonspecific hydrophobic contacts and van der Waals packing are primarily responsible for the binding, while the general anesthetic sevoflurane prefer to interact with the rugged, polar pocket 3 through specific hydrogen bonds and electrostatic forces. Short-chain butanol appears to bind effectively none of the three pockets. In addition, the pocket 1 consists of two angled arms 1 and 2 that are also involved in pockets 2 and 3, respectively. Dynamics characterization imparts that binding of long-chain octanol and DOG to pocket 2 or binding of sevoflurane to pocket 3 can induce a conformational displacement in arm 1 or 2, thus further opening the included angle and enlarging pocket 1, which can improve the pocket 1-PE affinity via an allosteric mechanism, consequently stimulating the PE-induced PKCδ activation.     

Association of protein kinase C with phospholipid monolayers: two-stage irreversible binding

The association of protein kinase C (PKC) with phospholipid (PL) monolayers spread at the air-water interface was examined. PKC-PL binding induced surface pressure changes that were dependent on the amount of PKC, the phospholipid composition of the monolayers, the presence of Ca2+, and the initial surface pressure of the monolayer (pi 0). Examination of surface pressure increases induced by PKC as a function of phospholipid surface pressure, pi 0, revealed that PKC-phosphatidylserine (PS) association had a critical pressure of 43 dyn/cm. Above this surface pressure, PKC cannot cause further surface pressure changes. This high critical pressure indicated that PKC should be able to penetrate many biological membranes which appear to have surface pressures of about 30 dyn/cm. PKC-induced surface pressure changes were Ca2+ dependent only for PL monolayers spread at a pi 0 greater than 26 dyn/cm. PKC alone (in the absence of PL) formed a film at the air-water interface with a surface pressure of about 26 dyn/cm. Calcium-dependent binding was studied at the higher surface pressures which effectively excluded PKC from the air-water interface. Subphase depletion measurements suggested that association of PKC with PS monolayers consisted of two stages: a rapid Ca2+-dependent interaction followed by a slower process that resulted in irreversible binding of PKC to the monolayer. The second stage appeared to involve penetration of PKC into the hydrocarbon region of the phospholipid. The commonly used in vitro substrates for PKC, histone and protamine sulfate, also associated with and penetrated PS monolayers with critical pressures of 50 and 60 dyn/cm, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C (PKC) level is increased in PC12 cells overexpressing transfected liver-type phosphofructokinase

Summary— PC12 cells which overexpress transfected liver-type phosphofructokinase (PFKL) have previously been described as a model system for PFKL overexpression in Down's syndrome and have been shown to perform glycolysis at enhanced rates. Here we report that levels of protein kinase C (PKC) in PC 12-PFKL cells were almost doubled, as estimated from in vitro activity and phorbol ester binding experiments and from an increase found in PKC-alpha mRNA levels. Most of the added PKC was found to be associated with the cellular membrane while the cytoplasmic levels of PKC were barely increased. The steady-state levels of 1,2-sn-diacylglycerol in PC12-PFKL cells were found to be unaltered, suggesting that enhanced glycolysis in these cells did not influence PKC by altering the amounts of this compound. PFKL is one of several genes known to be overexpressed in Down's syndrome. Upregulation of PKC due to PFKL overexpression could result in widespread disturbances of gene expression and play a part in causing some of the many symptoms of the disease.     

Studies of the structure and organization of cationic lipid bilayer membranes: calorimetric, spectroscopic, and x-ray diffraction studies of linear saturated P-O-ethyl phosphatidylcholines

Differential scanning calorimetry, x-ray diffraction, and infrared and (31)P-nuclear magnetic resonance ((31)P-NMR) spectroscopy were used to examine the thermotropic phase behavior and organization of cationic model membranes composed of the P-O-ethyl esters of a homologous series of n-saturated 1,2-diacyl phosphatidylcholines (Et-PCs). Differential scanning calorimetry studies indicate that on heating, these lipids exhibit single highly energetic and cooperative endothermic transitions whose temperatures and enthalpies are higher than those of the corresponding phosphatidylcholines (PCs). Upon cooling, these Et-PCs exhibit two exothermic transitions at temperatures slightly below the single endotherm observed upon heating. These cooling exotherms have both been assigned to transitions between the liquid-crystalline and gel phases of these lipids by x-ray diffraction. The x-ray diffraction data also show that unlike the parent PCs, the chain-melting phase transition of these Et-PCs involves a direct transformation of a chain-interdigitated gel phase to the lamellar liquid-crystalline phase for the homologous series of n > or = 14. Our (31)P-NMR spectroscopic studies indicate that the rates of phosphate headgroup reorientation in both gel and liquid-crystalline phases of these lipids are comparable to those of the corresponding PC bilayers. However, the shape of the (31)P-NMR spectra observed in the interdigitated gel phase indicates that phosphate headgroup reorientation is subject to constraints that are not encountered in the non-interdigitated gel phases of parent PCs. The infrared spectroscopic data indicate that the Et-PCs adopt a very compact form of hydrocarbon chain packing in the interdigitated gel phase and that the polar/apolar interfacial regions of these bilayers are less hydrated than those of corresponding PC bilayers in both the gel and liquid-crystalline phases. Our results indicate that esterification of PC phosphate headgroups results in many alterations of bilayer physical properties aside from the endowment of a positively charged surface. This fact should be considered in assessing the interactions of these compounds with naturally occurring lipids and with other biological materials.