Regional distribution, ontogeny, purification, and characterization of the Ca2+-independent phospholipase A2 from rat brain.

We purified an 80-kDa Ca2+-independent phospholipase A2 (iPLA2) from rat brain using octyl-Sepharose, ATP-agarose, and calmodulin-agarose column chromatography steps. This procedure gave a 30,000-fold purification and yielded 4 microg of a near-homogeneous iPLA2 with a specific activity of 4.3 micromol/min/mg. Peptide sequences of the rat brain iPLA2 display considerable homology to sequences of the iPLA2 from P388D1 macrophages, Chinese hamster ovary cells, and human B lymphocytes. Under optimal conditions, the iPLA2 revealed the following substrate preference toward the fatty acid chain in the sn-2 position of phosphatidylcholine: linoleoyl > palmitoyl > oleoyl > arachidonoyl. The rat brain iPLA2 also showed a head group preference for choline > or = ethanolamine >> inositol. The iPLA2 is inactivated when exposed to pure phospholipid vesicles. The only exception is vesicles composed of phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate. Studies on the regional distribution and ontogeny of various phospholipase A2 (PLA2) types in rat brain indicate that the iPLA2 is the dominant PLA2 activity in the cytosolic fraction, whereas the group IIA secreted PLA2 is the dominant activity in the particulate fraction. The activities of these two enzymes change during postnatal development.

Group-specific assays that distinguish between the four major types of mammalian phospholipase A2.

Phospholipase A2 (PLA2) constitutes a diverse superfamily of enzymes which catalyze the deacylation of phospholipids. At least four types of PLA2 are potentially involved in arachidonic acid release in cells and tissues. Since all of them catalyze the same enzymatic reaction, it is difficult to distinguish between them in mixtures of enzymes normally present in biological samples. Utilizing specific properties of each PLA2, we have designed distinct assay procedures which selectively and sensitively detect each type: Group VI Ca2+-independent PLA2, Group IV cytosolic Ca2+-dependent PLA2, Groups V and IIA secreted PLA2s. Each specific assay procedure is selective for a particular PLA2 type by at least fourfold and as high as four orders of magnitude relative to the other three enzymes. All assays can detect PLA2 activity with as low as subnanogram quantities of enzyme. Importantly, these assays are able to differentiate and quantitate the biochemically and structurally related enzymes, Group IIA and V sPLA2s in crude biological samples. Employing this system, we have found that iPLA2 is the dominant PLA2 in rat brain, and cPLA2 is the most abundant PLA2 in P388D1 macrophages and human amnionic WISH cells.

Mechanism of the apparent cooperativity in the interaction of protein kinase C with phosphatidylserine.

Protein kinase C displays high apparent cooperativity in its activation by phosphatidylserine. This contribution uses a novel approach to address the physical basis for this apparent cooperativity. We examine the binding of protein kinase C betaII to large unilamellar vesicles as a function of increasing mole fraction phosphatidylserine and as a function of increasing total lipid concentrations. Binding data are subjected to an analysis, described in the Appendix, that allows calculation of the fractional saturation of phosphatidylserine binding sites with this ligand. This analysis reveals that (1) protein kinase C betaII binds approximately eight phosphatidylserine molecules and (2) the binding of each lipid is not cooperative. Rather, the apparent cooperativity observed in protein kinase C's interaction with multiple phosphatidylserine molecules arises from effects specific to the interaction of a multivalent macromolecule with multiple membrane-associated ligands. Nor does diacylglycerol, which has been previously shown to dramatically increase protein kinase C's affinity for phosphatidylserine-containing membranes, induce cooperativity. Thus, protein kinase C binds multiple phosphatidylserine molecules in the absence of interaction between potential binding sites. The method presented for determining the stoichiometry and cooperativity in the interaction of protein kinase C with phosphatidylserine is applicable to any multivalent molecule binding to monovalent ligands incorporated into lipid membranes.

Group IV cytosolic phospholipase A2 binds with high affinity and specificity to phosphatidylinositol 4,5-bisphosphate resulting in dramatic increases in activity.

The group IV cytosolic phospholipase A2 (cPLA2) exhibits a potent and specific increase in affinity for lipid surfaces containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) at physiologically relevant concentrations. Specifically, the presence of 1 mol% PtdIns(4,5)P2 in phosphatidylcholine vesicles results in a 20-fold increase in the binding affinity of cPLA2. This increased affinity is accompanied by an increase in substrate hydrolysis of a similar magnitude. The binding studies and kinetic analysis indicate that PtdIns(4,5)P2 binds to cPLA2 in a 1:1 stoichiometry. The magnitude of the effect of PtdIns(4,5)P2 is unique among anionic phospholipids and larger than that for other polyphosphate phosphatidylinositols. The effect of PtdIns(4,5)P2 on the activity of cPLA2 is at least an order of magnitude larger than the concomitant changes in the fraction of the enzyme associated with lipid membranes. Striking parallels between the interaction of cPLA2 with PtdIns(4,5)P2 and the interaction of the pleckstrin homology domain of phospholipase C delta 1 with PtdIns(4,5)2 combined with sequence analysis of cPLA2 lead us to propose the existence and location of a pleckstrin homology domain in cPLA2. We further show that the very nature of the interaction of proteins such as cPLA2 with multiple ligands incorporated into membranes follows a specific model which necessitates the use of an experimental methodology suitable for a membrane interface to allow for a meaningful analysis of the data.

Protein kinase C: an example of a calcium-regulated protein binding to membranes (review).

The location of the calcium-binding domain on protein kinase C is being addressed by mutational and structural studies. This work can be complemented by detailed studies of the properties of the binding of the enzyme to membranes. These binding studies have revealed a number of unique pieces of information about the properties of Ca(2+)-prompted membrane partitioning, including the fact that there is only one Ca(2+)-binding site which regulates the partitioning of the enzyme and that this site is located 0.3 nm from the membrane interface. Furthermore, the binding of protein kinase C to membranes has been shown to enhance the affinity of the enzyme for Ca(2+) by several orders of magnitude. We illustrate how contributions of the interactions of proteins with other molecules also affect the concentration of calcium required to affect membrane partitioning. Only when all of these factors are considered can a quantitative description of Ca(2+)-regulated protein binding to membranes be achieved. Thus conformational studies, together with classical thermodynamic studies, can provide a more detailed understanding of the functional, as well as, the structural, properties of amphitropic proteins.

Chemical specificity and physical properties of the lipid bilayer in the regulation of protein kinase C by anionic phospholipids: evidence for the lack of a specific binding site for phosphatidylserine.

The association of protein kinase C (PKC) with membranes was found not to be specific for phosphatidyl-L-serine (PS). In particular, a synthetic phospholipid, dansyl-phosphatidylethanolamine, proved to be fully functional in the association of PKC with lipid bilayers and in mediating the interaction of this enzyme with diacylglycerol. Dansyl-phosphatidylethanolamine was also able to activate the enzyme in a Ca2+-dependent fashion. Differences in the ability to bind and activate PKC observed for an array of anionic lipids were not larger than alterations caused by changes in acyl chain composition. Thus, although different lipids interact to different extents with PKC, there are no specific binding sites for the PS headgroup on the enzyme. We found that lipids with a greater tendency to form inverted phases increased the binding of PKC to bilayers. However, these changes in lipid structure cannot be considered separately from the miscibility of lipid components in the membrane. For pairs of lipids with similar acyl chains, the dependence on PS concentration is sigmoidal, while for dissimilar acyl chains there is much less dependence of binding on PS concentration. The results can be explained in terms of differences in the lateral distribution of components in the membrane.

Calcium-independent binding to interfacial phorbol esters causes protein kinase C to associate with membranes in the absence of acidic lipids.

The mechanism of interaction of phorbol esters with conventional protein kinase Cs was addressed by examining the direct binding of this class of activators to protein kinase C beta II. Binding measurements reveal that the major role of phorbol esters is to increase the affinity of protein kinase C for membranes by several orders of magnitude. The relative increase depends linearly on the mole fraction of phorbol esters in membranes, with the potency illustrated by the finding that 1 mol% phorbol 12-myristate 13-acetate (PMA) increases protein kinase C's membrane association by approximately 4 orders of magnitude. For comparison, diacylglycerol (DG), which also activates protein kinase C by increasing the enzyme's membrane affinity, is 2 orders of magnitude less effective than PMA in altering protein kinase C's membrane affinity. The remarkably high-affinity interaction with phorbol esters allowed us to measure the direct binding of protein kinase C to PMA in neutral membranes and, thus, to evaluate the effect of Ca2+ on the phorbol ester interaction in the absence of Ca2+ effects on the enzyme's interaction with acidic lipids. Changing the Ca2+ concentration over 5 orders of magnitude had no effect on the direct interaction of protein kinase C with PMA immobilized in phosphatidylcholine membranes. Thus, the Ca(2+)-binding site for membrane association and the phorbol ester-binding site do not interact allosterically. Lastly, a method that does not have the limitations of the Scatchard plot for analysis of amphitropic proteins was used to determine the dissociation constant of protein kinase C from phorbol esters: expressed relative to membrane lipids, the dissociation constant is 1.5 x 10(-5) mol %. In summary, our data reveal that (1) the direct binding of protein kinase C to phorbol esters, in the absence of interactions with acidic lipids, provides a major contribution to the free energy change involved in the association of protein kinase C with membranes and (2) this interaction is not regulated by Ca2+.

Mechanism of interaction of protein kinase C with phorbol esters. Reversibility and nature of membrane association.

A variety of approaches have been employed to demonstrate that the interaction of protein kinase C beta II with phorbol ester-containing membranes is reversible, is not accompanied by significant insertion of the protein into the hydrophobic core of the membrane, and is qualitatively similar to the interaction with diacylglycerol (DG). First, we show that under conditions when protein kinase C is bound with equal affinity to membranes containing either DG or phorbol myristate acetate (PMA), increasing ionic strength causes a similar reduction in membrane binding. The similar sensitivity to ionic strength indicates that the forces mediating the binding of protein kinase C to PMA are not significantly different from those mediating the binding to DG. At sufficiently high concentrations of PMA and relatively low concentrations of phosphatidylserine, the binding of protein kinase C to membranes became markedly less sensitive to ionic strength, suggesting that under these conditions direct non-electrostatic interactions with PMA dominate over electrostatic interactions with the lipid headgroups. Importantly, regardless of the strength of the interaction with PMA, protein kinase C exchanges between vesicle surfaces: protein kinase C bound first to phorbol ester-containing multilamellar vesicles exchanged to large unilamellar vesicles upon addition of an excess surface area of the latter. Lastly, the enzyme's intrinsic tryptophan fluorescence was not quenched by bromines located at various positions in the hydrophobic core of the membrane. In contrast, the enzyme's tryptophan fluorescence was significantly quenched by probes positioned at the membrane surface. In summary, our results are consistent with protein kinase C binding reversibly to PMA- or DG-containing membranes primarily via interactions at the membrane interface.

Characterization of the calcium-binding site that regulates association of protein kinase C with phospholipid bilayers.

Association of calcium-dependent isotypes of protein kinase C (PKC) with a phospholipid bilayer is regulated by a single Ca(2+)-binding site. The dependence of PKC association with phosphatidylserine-containing membranes on the concentration of Ca2+ is linear in the submicro- to submillimolar range. The Ca(2+)-regulated association of PKC with the membrane is sensitive to the factors that alter the diffuse double-layer potential produced by anionic lipids such as phosphatidylserine (PS). This indicates that the Ca(2+)-binding site on the membrane-bound enzyme senses a higher concentration of Ca2+ than is present in bulk solution. This is a consequence of the accumulation of Ca2+ in the layer adjacent to the plane of the membrane by the double-layer potential. Calculations based on the Gouy-Chapman-Stern theory of the diffuse double layer yielded a unique value of the Ca2+ dissociation constant for the Ca(2+)-PKC-bilayer complex equal to approximately 700 nM. The soluble form of the enzyme has a 3.5 order of magnitude lower affinity for Ca2+. The free energy of interaction between the Ca(2+)- and PS-binding sites is large (approximately 5 kcal/mol). In contrast, the interaction between the diacylglycerol-binding site and either the Ca(2+)- or PS-binding site appears to be weak.

Mechanism of activation of protein kinase C: roles of diolein and phosphatidylserine.

We studied the roles of lipid concentration, phosphatidylserine (PS), and diolein (DG) contents, as well as Ca2+ concentration, on the partitioning of protein kinase C (PKC) between aqueous and membrane environments as well as the relationship of this partitioning to the activation of the enzyme. Physiological concentrations of 1 mol % DG increased the apparent binding constant of PKC to the 3:1 PC/PS membrane 500 times. This increase was proportional to the mol % DG. Over 50% PKC was bound to that membrane at micromolar concentrations of Ca2+ and physiologically relevant total concentration of lipid only when 1 mol % DG was included. PKC bound either to PS alone or to PS and DG was enzymatically competent; however, the rate of phosphorylation was doubled in the presence of 1 mol % diolein. The dependence of PKC binding on the mol % PS was highly sigmoidal. The Hill coefficient was in the range of 4-6, with the higher values found at the lower lipid concentrations. These results suggest that the observed apparent cooperativity is due, at least in part, to the change in dimensionality when PKC binds to the membrane.

Critical cell volume and shape of bovine erythrocytes.

The relationship between erythrocyte shape and the critical cell volume was investigated. Agents able to increase the critical cell volume induced three main stable shapes of erythrocytes: discocytic, stomatocytic, and echinocytic. The absence of correlation between shape and critical cell volume under isoosmotic conditions suggests that relative differences between the surface areas of the inner and the outer leaflet of the cell membrane do not influence the critical volume of a cell.

The effect of arsenate and vanadate ions on the critical cell volume of bovine erythrocytes.

Arsenate, used as an inhibitor of glycolysis, decreases the critical cell volume of erythrocytes, whereas orthovanadate, used as an accelerator of dephosphorylation of phosphatidyl-inositol-4,5-biphosphate, exerts an opposite effect. The ATP-dependent changes of the critical cell volume do not depend on the phosphorylation state of phosphatidylinositol. An interaction of the membrane skeleton with the lipid-protein matrix has been proposed as a regulation mechanism of the critical cell volume of erythrocytes.

Electrostatics and reduction of dimensionality produce apparent cooperativity when basic peptides bind to acidic lipids in membranes.

The binding of pentalysine to phospholipid vesicles depends in a sigmoidal manner on the mole fraction of acidic lipid in the vesicles. A simple analysis demonstrates that this apparent cooperativity is probably due to both the reduction of dimensionality that occurs when the first basic residue binds to an acidic lipid in the membrane and the Boltzmann accumulation of the peptide in the electrostatic diffuse double layer produced by the charged lipids.

Binding of basic peptides to acidic lipids in membranes: effects of inserting alanine(s) between the basic residues.

We studied the binding of peptides containing five basic residues to membranes containing acidic lipids. The peptides have five arginine or lysine residues and zero, one, or two alanines between the basic groups. The vesicles were formed from mixtures of a zwitterionic lipid, phosphatidylcholine, and an acidic lipid, either phosphatidylserine or phosphatidylglycerol. Measuring the binding using equilibrium dialysis, ultrafiltration, and electrophoretic mobility techniques, we found that all peptides bind to the membranes with a sigmoidal dependence on the mole fraction of acidic lipid. The sigmoidal dependence (Hill coefficient greater than 1 or apparent cooperativity) is due to both electrostatics and reduction of dimensionality and can be described by a simple model that combines Gouy-Chapman-Stern theory with mass action formalism. The adjustable parameter in this model is the microscopic association constant k between a basic residue and an acidic lipid (1 less than k less than 10 M-1). The addition of alanine residues decreases the affinity of the peptides for the membranes; two alanines inserted between the basic residues reduces k 2-fold. Equivalently, the affinity of the peptide for the membrane decreases 10-fold, probably due to a combination of local electrostatic effects and the increased loss of entropy that may occur when the more massive alanine-containing peptides bind to the membrane. The arginine peptides bind more strongly than the lysine peptides: k for an arginine residue is 2-fold higher than for a lysine residue. Our results imply that a cluster of arginine and lysine residues with interspersed electrically neutral amino acids can bind a significant fraction of a cytoplasmic protein to the plasma membrane if the cluster contains more than five basic residues.

Binding of peptides with basic residues to membranes containing acidic phospholipids.

There are clusters of basic amino acids on many cytoplasmic proteins that bind transiently to membranes (e.g., protein kinase C) as well as on the cytoplasmic domain of many intrinsic membrane proteins (e.g., glycophorin). To explore the possibility that these basic residues bind electrostatically to monovalent acidic lipids, we studied the binding of the peptides Lysn and Argn (n = 1-5) to bilayer membranes containing phosphatidylserine (PS) or phosphatidylglycerol (PG). We made electrophoretic mobility measurements using multilamellar vesicles, fluorescence and equilibrium binding measurements using large unilamellar vesicles, and surface potential measurements using monolayers. None of the peptides bound to vesicles formed from the zwitterionic lipid phosphatidylcholine (PC) but all bound to vesicles formed from PC/PS or PC/PG mixtures. None of the peptides exhibited specificity between PS and PG. Each lysine residue that was added to Lys2 decreased by one order of magnitude the concentration of peptide required to reverse the charge on the vesicle; equivalently it increased by one order of magnitude the binding affinity of the peptides for the PS vesicles. The simplest explanation is that each added lysine binds independently to a separate PS with a microscopic association constant of 10 M-1 or a free energy of approximately 1.4 kcal/mol. Similar, but not identical, results were obtained with the Argn peptides. A simple theoretical model combines the Gouy-Chapman theory (which accounts for the nonspecific electrostatic accumulation of the peptides in the aqueous diffuse double layer adjacent to the membrane) with mass action equations (which account for the binding of the peptides to greater than 1 PS). This model can account qualitatively for the dependence of binding on both the number of basic residues in the peptides and the mole fraction of PS in the membrane.

Peptides that mimic the pseudosubstrate region of protein kinase C bind to acidic lipids in membranes.

The cytoplasmic form of protein kinase C (PKC) is inactive, probably because the pseudosubstrate region in its regulatory domain blocks the substrate-binding site in its kinase domain. Calcium ions cause a translocation to the membrane: maximum activation requires a negative lipid such as phosphatidylserine (PS) and the neutral lipid diacylglycerol (DAG) but the mechanism by which PS and DAG activate PKC is unknown. Pseudosubstrate region 19-36 of PKC-beta has six basic and one acidic amino acids and region 19-29 has five basic and no acidic amino acids. Since any binding of basic residues in the pseudosubstrate region to acidic lipids in the membrane should stabilize the active form of PKC, we studied how peptides with amino acids equivalent to residues 19-36 and 19-29 of PKC-beta bound to phospholipid vesicles. We made equilibrium dialysis, filtration, and electrophoretic mobility measurements. The fraction of bound peptide is a steep sigmoidal function of the mol fraction of negative lipid in the membrane, as predicted from a simple theoretical model that assumes the basic residues provide identical independent binding sites. The proportionality constant between the number of bound peptides/area and the concentration of peptide in the bulk aqueous phase is 1 micron for a membrane with 25% negative lipid formed in 0.1 M KCl. Equivalently, the association constant of the peptide with the membrane is 10(4) M-1, or the net binding energy is 6 kcal/mol. Thus the interaction of basic residues in the pseudosubstrate region with acidic lipids in the membrane could provide 6 kcal/mol free energy towards stabilizing the active form of PKC.

The effect of ATP on the order and the mobility of lipids in the bovine erythrocyte membrane.

The order and the mobility of the lipids in the membrane were measured by the ESR method for the erythrocytes with both normal and modified intracellular concentration of ATP. The lipid order did not depend on the ATP level, but the lipid mobility was affected by the intracellular ATP concentration. The lipid mobility was higher in the cells with a larger concentration of ATP.

Effect of the level of ATP and of the state of spectrin on osmotic properties of bovine erythrocytes.

The effect of the intracellular level of ATP and of the state of spectrin on the critical cell volume of bovine erythrocyte was studied. The state of spectrin was changed by thermal denaturation, which for the bovine red cell took place at similar temperature as for the human erythrocyte. The increase of the ATP level and the spectrin denaturation increased the critical cell volume, while metabolic starvation decreased it. The changes of the ATP level did not influence the critical volume after the denaturation of spectrin. The results suggest that the ATP-dependent effect on the critical cell volume was caused by an alteration of the membrane extensibility due to the change of the membrane skeleton-lipid bilayer interaction(s).

Elastic properties of the erythrocyte membrane and the critical cell volume of erythrocytes.

The results on elastic membrane area extension during hemolysis, reported by Richieri and Mel (Richieri, G.V. and Mel, H.C. (1985) Biochim. Biophys. Acta 813, 41-50), are discussed. Careful analysis of their data leads to the conclusion, that the differences in osmolarity, as found in the experiment, were insufficient to cause the reported values of elastic changes in erythrocyte volume (17-22%) and of membrane area extension (11-14%). The recalculated values of the elastic extensions of membrane area are not different from those measured by the micropipet method (i.e. 3-4%).

Effect of DIDS on osmotic properties of bovine erythrocytes.

The effect of DIDS on osmotic properties of bovine erythrocytes was studied. The isoosmotic volume, the amount of intracellular solutes, and the osmotically non-active volume were not influenced by DIDS. An increase of osmotic fragility of erythrocytes upon DIDS treatment was evident and identical both in NaCl and in NaCl + KCl hypotonic solutions. These results suggest that the critical cell volume decreases. The link between the effect of DIDS on the membrane skeleton extractability and on the osmotic fragility was postulated.