Fusion of small unilamellar liposomes with phospholipid planar bilayer membranes and large single-bilayer vesicles

Small unilamellar phosphatidylserine/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca²⁺ concentration was increased to a threshold of 3–5 mM in 100 mM NaCl, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 μm diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca²⁺-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca²⁺ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca²⁺ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca²⁺ concentration.     

Characterization of two fluorescent tryptophans in recombinant human granulocyte-colony stimulating factor: Comparison of native sequence protein and tryptophan-deficient mutants

In order to probe the role of the individual tryptophans of granulocyte-colony stimulating factor (G-CSF) inpH and guanidine HCl-induced fluorescence changes, site-directed mutagenesis was used to generate mutants replacing Trp¹¹⁸, Trp⁵⁸, or both with phenylalanine. Neither Trp to Phe mutation affected the folding or activity of the recombinant G-CSF, and the material expressed in yeast behaved identically to that expressed inEscherichia coli. All of the G-CSF species responded topH and guanidine HCl in qualitatively the same manner. Trp⁵⁸ has a fluorescence maximum at 350 nm and is quenched to a greater extent by the addition of guanidine HCl, indicating that it is fully solvent-exposed. Trp¹¹⁸ has a fluorescence maximum at 344 nm, and is less solvent-accessible than Trp⁵⁸. The analog in which both tryptophans have been replaced with phenylalanine shows only tyrosine fluorescence, with a peak at 304 nm which decreases with increasingpH. The intensity of the tyrosine fluorescence in this analog is much greater than that of the native sequence protein or single tryptophan mutants, indicating that energy transfer is taking place from tyrosine to tryptophan in these molecules. Below neutralpH the tyrosine fluorescence is much greater in the [Phe⁵⁸]G-CSF than in the [Phe¹¹⁸]G-CSF, indicating that Trp⁵⁸ might be a more efficient recipient of energy transfer from the tyrosine(s).     

Membrane interaction of neuropeptide Y detected by EPR and NMR spectroscopy

Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammals. It belongs to the best-conserved peptides in nature, i.e., the amino acid sequences of even evolutionary widely separated species are very similar to each other. Using porcine NPY, which differs from human NPY only at position 17 (a leucine residue exchanged for a methionine), labeled with a TOAC spin probe at the 2nd, 32nd, or 34th positions of the peptide backbone, the membrane binding and penetration of NPY was determined using EPR and NMR spectroscopy. The vesicular membranes were composed of phosphatidylcholine and phosphatidylserine at varying mixing ratios. From the analysis of the EPR line shapes, the spectral contributions of free, dimerized, and membrane bound NPY could be separated. This analysis was further supported by quenching experiments, which selected the contributions of the bound NPY fraction. The results of this study give rise to a model where the α-helical part of NPY (amino acids 13-36) penetrates the membrane interface. The unstructured N-terminal part (amino acids 1-12) extends into the aqueous phase with occasional contacts with the lipid headgroup region. Besides the mixing ratio of zwitterionic and negatively charged phospholipid species, the electrostatic peptide membrane interactions are influenced by the pH value, which determines the net charge of the peptide resulting in a modified membrane binding affinity. The results of these variations indicate that NPY binding to phospholipid membranes depends strongly on the electrostatic interactions. An estimation of the transfer energy of the peptide from aqueous solution to the membrane interface ΔG supports the preferential interaction of NPY with negatively charged membranes.     

Orientation of the tryptophanyl side chain in [(d)TrpA1] and [TrpA1]insulin

Insulin analogues withl- andd-tryptophan instead of glycine in A1 permit an estimate of the proximity relationship between the indole residue of tryptophan and B19-tyrosine by evaluation of singlet-singlet resonance energy transfer. A significantly higher transfer efficiency is observed with [(d)Trp^A1]insulin than with the [Trp^A1]analogue. On the basis of this result it is possible to deduce the arrangement of the side chains and the α-amino groups in position A1 of [(d)Trp^A1] and [Trp^A1]insulin.     

The contribution of helical potential to the in vitro receptor binding activity of a neuropeptide Y N-terminal deletion fragment

In its dimeric form neuropeptide Y (NPY) folds into a compact structure in which the antiparallel oriented proline and α-helices apparently associate to form a primitive hydrophobic core. To investigate the contribution of helical stability to the receptor binding activity of NPY and its N-terminal deletion fragments, we synthesized and studied the solution conformational properties and in vitro activities of NPY, N^α-acetyl-NPY_2–36, NPY_15–36, N^α-propinonly-NPY_15–36, and N^α-succinyl-NPY_15–36 is significantly less helical than both NPY and N^α-acetyl-NPY_2–36, and this decreased helical potential is attributed of the absence of the intramolecular stabilizing interaction afforded by the proline helix in the latter analogues. However, in accord with the helix dipole model, the helical potential of NPY_15–36 is significantly increased by N-terminal succinlyation, whereas propionylation has no effect. In addition to an increase in helical potential, N^α-succinyl-NPY_15–36 is 2.5 and 4.6 times more active than NPY_15–36 and N^α-propionly-NPY_15–36, respectively and is equipotent with N^α-acteyl-NPY_2–36 in displacing 1mM[³H]-NPY from specific binding sites in rat brain membranes. The demonstration of positive correlation between % α-helix content and in vitro binding activity suggests that the helical potential of N-terminal NPY deletion fragments contributes to their in vitro activity in the rat brain, and that a second role of the proline helix might be to stabilize the receptor-active conformation of the NPY α-helix. © 1993 John Wiley & Sons, Inc.     

[3H]-BIBP3226 and [3H]-NPY Binding to Intact SK-N-MC Cells and CHO Cells Expressing the Human Y1 Receptor

Abstract

We have studied the binding of [³H]-NPY and the newly developed non-peptide Y₁ receptor antagonist [³H]-BIBP3226 to intact SK-N-MC cells and CHO-K1 cells transfected with the human NPY Y₁ receptor gene i.e. CHO-Y1 cells. Whereas the association and dissociation of the specific [³H]-NPY binding was slow, the binding kinetics of [³H]-BIBP3226 binding was very rapid. Saturation binding of both radioligands reveal the presence of an apparently homogeneous population of high affinity binding sites in both cell lines. The corresponding equilibrium dissociation constants are similar for the two cell lines and are close to those obtained from previous competition binding experiments. The specific binding of both radioligands was completely and with high affinity displaced by BIBP3226 and its inactive (S)-enantiomer BIBP3435 was much less potent. Whilst the NPY Y₁ agonists NPY, PYY and [Leu³¹-Pro³⁴]-NPY completely and potently displaced [³H]-NPY binding, they could only displace 70 to 80 % of the [³H]-BIBP3226 binding sites in CHO-Y1 and SK-N-MC cells. A possible explanation can be that only part of the receptors are G-protein coupled. In agreement pertussis toxin was found to reduce high affinity [³H]-NPY binding sites in CHO-Y1 cells whereas [³H]-BIBP3226 binding parameters remained unchanged.     

Substitution of D-Trp32 in NPY Destabilizes the Binding Transition State to the Y1 Receptor Site in SK-N-MC Cell Membranes

The retention rate of the spin label 3-isothiocyanto methyl-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl spin label (proxyl) attached to the porcine N-acetyl-NPY peptide and the porcine N-acetyl-D-Trp³²-NPY peptide at Lys⁴ was investigated using SK-N-MC neuroblastoma cell membranes containing the Y1 receptor. The release rate of the spin labeled peptides was monitored by electron spin resonance and the K_D was determined by a direct radiolabeled NPY displacement binding assay. The analyses show that for the porcine [Ac-Tyr¹N⁴-proxyl]-NPY, the K_D was 8 × 10^–10 M and k_off was 2.7 × 10^–4 sec^–1 yielding a value for k_on of 3.3 × 10⁵ sec^–1 M^–1. The [Ac-Tyr¹, N⁴-proxyl,-D-Trp³²]-NPY antagonist ligand had a value of K_D equal to 1.35 × 10^–7 M and k_off was 1.7 × 10^–4 sec^–1 leading to a value for k_on of 1.2 × 10³ sec^–1 M^–1. The difference in the k_on rates of two orders of magnitude is interpreted as demonstrating the N-acetyl-N⁴ proxyl-D-Trp³²-NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence.     

Polarized distribution of Na, K-ATPase α-subunit isoforms in electrocyte membranes

We have previously demonstrated that Na⁺, K⁺-ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the α subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of α isoforms (α1 and α2) in the electrocyte. Dose-response curves showed that non-innervated membranes present a Na⁺, K⁺-ATPase activity 2.6-fold more sensitive to ouabain (I₅₀=1.0±0.1 μM) than the activity of innervated membranes (I₅₀=2.6±0.2 μM). As depicted in [³H]ouabain binding experiments, when the [³H]ouabain-enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na⁺, K⁺-ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of α1 and α2 isoforms, restricted to the innervated and non-innervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na⁺, K⁺-ATPase α-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell.     

Lipid composition and fluidity in the jejunal brush-border membrane of spontaneously hypertensive rats. Effects on activities of membrane-bound proteins

The lipid composition and fluidity of jejunal brush-border membrane vesicles (BBMV) have been studied in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats. The activities of both Na⁺-dependent D-glucose cotransport and Na⁺-H⁺ antiport have also been determined. A significant increase in the level of free cholesterol was observed in jejunal BBMV from SHR compared to WKY rats. Since phospholipid values did not change in either group of animals, a significant enhancement in the free cholesterol/phospholipid ratio was observed in SHR. A decrease in the levels of phosphatidylethanolamine together with an increase in the values of phosphatidylserine was observed in hypertensive rats. Although the content of phosphatidylcholine (PC) and sphingomyelin (SM) was not singificantly altered in SHR, the ratio PC/SM significantly increased in these animals when compared to WKY rats. The major fatty acids present in bursh-border membranes prepared from SHR and WKY rats were palmitic (160), stearic (180), oleic (181, n-9) and linoleic (182, n-6), and the fatty acid composition was not modified by the hypertension. A decreased fluorescence polarization, i.e., increased membrane fluidity, was observed in SHR, which was not correlated to the increased ratio of cholesterol/phospholipid found in the brush-border membrane isolated from these animals. These structural changes found in SHR were associated to an enhancement in both Na⁺-dependent D-glucose transport and Na⁺-H⁺ antiport activity in the jejunal BBMV of SHR.Abbreviations BBMV brush-border membrane vesicles - DPH 1,6-diphenyl-1,3,5-hexatriene - FC free cholesterol - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SM sphingomyelin - SHR spontaneously hypertensive rat - p steady-state fluoroscence polarization - r_s steady-state fluorescence anisotropy - WKY Wistar Kyoto     

Effect of phosphatidylinositol replacement by diacylglycerol on various physical properties of artificial membranes with respect to the role of phosphatidylinositol response

In an attempt to gain insight into the physiological role of phosphatidylinositol turnover enhanced by extracellular stimuli, the physical properties of artificial membranes (egg yolk phosphatidylcholine/bovine brain phosphatidylserine) containing phosphatidylinositol or diacylglycerol were studied by ESR using spin probes and freeze-fracture electron microscopy. Diacylglycerol lost both the ability to form lipid bilayer structures and its susceptibility to calcium ions. Yeast phosphatidylinositol included in dipalmitoylphosphatidylcholine liposomes lowered the phase transition temperature of dipalmitoylphosphatidylcholine and expanded the temperature range of phase transition. However, diacylglycerol at the same concentration did not undergo the effects caused by phosphatidylinositol but the phase transition temperature was slightly raised. Phase separation of phosphatidylserine induced by calcium ions was enhanced when the phosphatidylinositol was replaced by diacylglycerol in phosphatidylcholine/ phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) mixtures. The mobility of phosphatidylcholine spin probe was decreased in phosphatidylcholine/ phosphatidylserine/diacylglycerol (3:5:2, by molar ratio) liposomes compared with phosphatidylcholine/phosphatidylserine/phosphatidylinositol (3:5:2, by molar ratio) liposomes. An additional component from protonated stearic acid spin probes was observed in phosphatidylcholine/phosphatidylinositol (8:2, by molar ratio) liposomes at 40°C, whereas the component was not seen in phosphatidylcholine/diacylglycerol (8:2, by molar ratio) liposomes. This may indicate the alteration of surface charge induced by the replacement of phosphatidylinositol by diacylglycerol. Indeed, in the presence of 1 mM Ca²⁺, the additional component was removed by an electrostatic interaction between Ca²⁺ and phosphatidylinositol molecules in phosphatidylcholine/phosphatidylinositol liposomes at 40°C. These results support the hypothesis that the enhanced turnover of phosphatidylinositol may play a triggering role for various cellular responses to exogenous stimuli by altering membrane physical states.     

Ca2+-induced phase separation in phosphatidylserine,phosphatidylethanolamine and phosphatidylcholine mixed membranes

Ca²⁺-induced phase separation in phosphatidylserine/phosphatidylethanolamine and phosphatidylserine/phosphatidylethanolamine/phosphatidylcholine model membranes was studied using spin-labeled phosphatidylethanolamine and phosphatidylcholine and compared with that in phosphatidylserine/phosphatidylcholine model membranes studied previously. The phosphatidyl-ethanolamine-containing membranes behaved in qualitatively the same way as did phosphatidylserine/phosphatidylcholine model membranes. There were some quantitative differences between them. The degree of phase separation was higher in the phosphatidylethanolamine-containing membranes. For example, the degree of phase separation in phosphatidylserine/phosphatidylethanolamine membranes containing various mole fractions of phosphatidylserine was 94–100% at 23°C and 84–88% at 40°C, while the corresponding value for phosphatidylserine/phosphatidylcholine membranes was 74–85% at 23°C and 61–79% at 40°C. Ca²⁺ concentration required for the phase separation was lower for phosphatidylserine/phosphatidylethanolamine than that for phosphatidylserine/phosphatidylcholine membranes; concentration to cause a half-maximal phase separation was $\text{1.4 · 10}{}^{\mathrm{?7}}$ M for phosphatidylserine-phosphatidylethanolamine and $\text{1.2 · 10}{}^{\mathrm{?6}}$ M for phosphatidylserine/phosphatidylcholine membranes. The phase diagram of phosphatidylserine/phosphatidylethanolamine membranes in the presence of Ca²⁺ was also qualitatively the same as that of phosphatidylserine/phosphatidylcholine except for the different phase transition temperatures of phosphatidylethanolamine (17°C) and phosphatidylcholine (?15°C). These differences were explained in terms of a greater tendency for phosphatidylethanolamine, compared to phosphatidylcholine, to form its own fluid phase separated from the Ca²⁺-chelated solid-phase phosphatidylserine domain.     

Conformational and functional studies of gomesin analogues by CD, EPR and fluorescence spectroscopies

The aim of this work was to examine the bioactivity and the conformational behavior of some gomesin (Gm) analogues in different environments that mimic the biological membrane/water interface. Thus, manual peptide synthesis was performed by the solid-phase method, antimicrobial activity was evaluated by a liquid growth inhibition assay, and conformational studies were performed making use of several spectroscopic techniques: CD, fluorescence and EPR. [TOAC¹]-Gm; [TOAC¹, Ser^2,6,11,15]-Gm; [Trp⁷]-Gm; [Ser^2,6,11,15, Trp⁷]-Gm; [Trp⁹]-Gm; and [Ser^2,6,11,15, Trp⁹]-Gm were synthesized and tested. The results indicated that incorporation of TOAC or Trp caused no significant reduction of antimicrobial activity; the cyclic analogues presented a β-hairpin conformation similar to that of Gm. All analogues interacted with negatively charged SDS both above and below the detergent's critical micellar concentration (cmc). In contrast, while Gm and [TOAC¹]-Gm required higher LPC concentrations to bind to micelles of this zwitterionic detergent, the cyclic Trp derivatives and the linear derivatives did not seem to interact with this membrane-mimetic system. These data corroborate previous results that suggest that electrostatic interactions with the lipid bilayer of microorganisms play an important role in the mechanism of action of gomesin. Moreover, the results show that hydrophobic interactions also contribute to membrane binding of this antimicrobial peptide.     

Possible involvement of neuropeptide Y Y1 receptors in antidepressant like effect of agmatine in rats

Agmatine and neuropeptide Y (NPY) are widely distributed in central nervous system and critically involved in modulation of depressive behavior in experimental animals. However their mutual interaction, if any, in regulation of depression remain largely unexplored. In the present study we explored the possible interaction between agmatine and neuropeptide Y in regulation of depression like behavior in forced swim test. We found that acute intracerebroventricular (i.c.v.) administration of agmatine (20–40 μg/rat), NPY (5 and 10 μg/rat) and NPY Y1 receptor agonist, [Leu³¹, Pro³⁴]-NPY (0.4 and 0.8 ng/rat) dose dependently decreased immobility time in forced swim test indicating their antidepressant like effects. In combination studies, the antidepressant like effect of agmatine (10 μg/rat) was significantly potentiated by NPY (1 and 5 μg/rat, icv) or [Leu³¹, Pro³⁴]-NPY (0.2 and 0.4 ng/rat, icv) pretreatment. Conversely, pretreatment of animals with NPY Y1 receptor antagonist, BIBP3226 (0.1 ng/rat, i.c.v.) completely blocked the antidepressant like effect of agmatine (20–40 μg/rat) and its synergistic effect with NPY (1 μg/rat, icv) or [Leu³¹, Pro³⁴]-NPY (0.2 ng/rat, icv). The results of the present study showed that, agmatine exerts antidepressant like effects via NPYergic system possibly mediated by the NPY Y1 receptor subtypes and suggest that interaction between agmatine and neuropeptide Y may be relevant to generate the therapeutic strategies for the treatment of depression.     

Comparative dynamics and location of chain spin-labelled sphingomyelin and phosphatidylcholine in dimyristoyl phosphatidylcholine membranes studied by EPR spectroscopy

The dynamics and environment of sphingomyelin spin-labelled at different positions in the N-acyl chain have been studied in dimyristoyl phosphatidylcholine bilayer membranes by using electron spin resonance spectroscopy. Comparison was made with phosphatidylcholine spin-labelled on the sn-2 acyl chain in the same host membrane. Spin-labelled sphingomyelin was found to mix well with the host phosphatidylcholine lipids in both gel and fluid phase membranes. At 1 mol%, mutual spin-spin interactions are no greater than for spin-labelled phosphatidylcholine. In the fluid membrane phase, the effective chain order parameters and polarity-sensitive isotropic hyperfine coupling constants of spin-labelled sphingomyelin display a similar dependence on the position of labelling to those of spin-labelled phosphatidylcholine. The values of both parameters are, however, generally larger for sphingomyelin than for phosphatidylcholine at equivalent positions of acyl chain labelling. This difference is attributed to the different chain linkage of sphingo- and glycero-lipids, combined with an offset of approximately one C-atom in transbilayer register between the respective N-acyl and O-acyl chains. In the gel phase, differences in chain configuration between sphingomyelin and phosphatidylcholine are indicated by differences in spin label spectral anisotropy between the two lipids, which appears to reverse towards the terminal methyl chain end.     

Novel Cell Line Selectively Expressing Neuropeptide Y‐Y2 Receptors

Abstract

Neuropeptide Y (NPY) recognition by the human neuroblastoma cell lines SiMa, Kelly, SH‐SY5Y, CHP‐234, and MHH‐NB‐11 was analyzed in radioactive binding assays using tritiated NPY. For the cell lines CHP‐234 and MHH‐NB‐11 binding of [³H]propionyl‐NPY was observed with K_d‐values of 0.64 ± 0.07 nM and 0.53 ± 0.12 nM, respectively, determined by saturation analysis with non‐linear regression. The receptor subtype was determined by competition analysis using the subtype selective NPY analogues [Leu³¹, Pro³⁴]‐NPY (NPY‐Y₁, NPY‐Y₅), [Ahx^5‐24]‐NPY (NPY‐Y₂), [Ala³¹, Aib³²]‐NPY (NPY‐Y₅), NPY [3‐36] (NPY‐Y₂, NPY‐Y₅), and NPY [13‐36] (NPY‐Y₂). Both cell lines, CHP‐234 and MHH‐NB‐11, the latter one being characterized for NPY receptors for the first time, showed exclusive expression of NPY‐Y₂ receptors. In both cell lines binding of NPY induced signal transduction, which was monitored as reduction of forskolin‐induced cAMP production in an ELISA.     

Hg2+ and Cd2+ interact differently with biomimetic erythrocyte membranes

In order to characterize the potentially deleterious effects of toxic Hg²⁺ and Cd²⁺ on lipid membranes, we have studied their binding to liposomes whose composition mimicked erythrocyte membranes. Fluorescence spectroscopy utilizing the concentration dependent quenching of Phen Green™ SK by Hg²⁺ and Cd²⁺ was found to be a sensitive tool to probe these interactions at metal concentrations ≤1 μM. We have systematically developed a metal binding affinity assay to screen for the interactions of Hg²⁺ or Cd²⁺ with certain lipid classes. A biomimetic liposome system was developed that contained four major lipid classes of erythrocyte membranes (zwitterionic lipids: phosphatidylcholine and phosphatidylethanolamine; negatively charged: phosphatidylserine and neutral: cholesterol). In contrast to Hg²⁺, which preferentially bound to the negatively charged phosphatidylserine compared to the zwitterionic components, Cd²⁺ bound stronger to the two zwitterionic lipids. Thus, the observed distinct differences in the binding affinity of Hg²⁺ and Cd²⁺ for certain lipid classes together with their known effects on membrane properties represent an important first step toward a better understanding the role of these interactions in the chronic toxicity of these metals.     

Conformational behavior of [Trp4, Met5]-enkephalin: comparison of fluorescence parameters at different Ph.

The conformational behavior of the biologically active [Trp⁴,Met⁵]-enkephalin was elucidated by evaluation of intramolecular energy transfer between Tyr¹ and Trp⁴. Identical transfer efficiencies and tyrosine fluorescence quantum yields were observed in aqueous solution at pH 1.5 and 5.5 and the use of these parameters in Förster's equation resulted in the same average Tyr-Trp separation (9.3 Å) under these two conditions. The invariability of these sensitive parameters indicates the existence of very similar types of a folded conformation in the cationic and zwitterionic form of the analog at low concentrations.     

Maintenance of epithelial surface membrane lipid polarity: A role for differing phospholipid translocation rates

Summary Large differences in lipid composition of apical and basolateral membranes from epithelial cells exist. To determine the responsible mechanism(s), rat renal cortical brush border and basolateral membrane phospholipids were labeled using³²P and either [³H]-glycerol or [2-³H] acetate for incorporation and degradation studies, respectively. Brush border and basolateral membrane fractions were isolated simultaneously from the same cortical homogenate. Different phospholipid classes were degraded at variable rates with phosphatidylcholine having the fastest decay rate. Decay rates for individual phospholipid classes were, however, similar in both brush border and basolateral membrane fractions. In phospholipid incorporation studies again, large variations existed between individual phospholipid classes with phosphatidylcholine and phosphatidylinositol showing the most rapid rates of incorporation. Sphingomyelin and phosphatidylserine showed extremely slow incorporation rates and did not enter into the isotopic decay phase for 48 hr. In contrast to degradation studies, however, the same phospholipid class labeled the two surface membrane domains at highly variable rates. The difference in these rates, with the exception of phosphatidylinositol, were identical to the differences in phospholipid compositions between the two membranes. For example, phosphatidylcholine was incorporated into the basolateral membrane 2.5 × faster than into the brush border membrane and its relative composition was 2.5 × greater in the basolateral membrane. The opposite was true for sphingomyelin. These results indicate incorporation and not degradation rates of individual phospholipids play a major role in regulating the differing phospholipid composition of brush border and basolateral membranes.     

Phosphatidylinositol-4,5-Bisphosphate Enhances Anionic Lipid Demixing by the C2 Domain of PKCα

The C2 domain of PKCα (C2α) induces fluorescence self-quenching of NBD-PS in the presence of Ca²⁺, which is interpreted as the demixing of phosphatidylserine from a mixture of this phospholipid with phosphatidylcholine. Self-quenching of NBD-PS was considerably increased when phosphatidylinositol-4,5-bisphosphate (PIP₂) was present in the membrane. When PIP₂ was the labeled phospholipid, in the form of TopFluor-PIP₂, fluorescence self-quenching induced by the C2 domain was also observed, but this was dependent on the presence of phosphatidylserine. An independent indication of the phospholipid demixing effect given by the C2α domain was obtained by using ²H-NMR, since a shift of the transition temperature of deuterated phosphatidylcholine was observed as a consequence of the addition of the C2α domain, but only in the presence of PIP₂. The demixing induced by the C2α domain may have a physiological significance since it means that the binding of PKCα to membranes is accompanied by the formation of domains enriched in activating lipids, like phosphatidylserine and PIP₂. The formation of these domains may enhance the activation of the enzyme when it binds to membranes containing phosphatidylserine and PIP₂.