A scanning calorimetric study of the interaction of anthracyclines with neutral and acidic phospholipids alone and in binary mixtures

High sensitivity differential scanning calorimetry was employed to study the thermotropic behavior of multilamellar vesicles of neutral and acidic phospholipids and binary mixtures thereof in the presence of anthracycline antibiotics. Adriamycin and its lipophilic analogue, N-trifluoroacetyladriamycin-14-valerate (AD32) were investigated and compared to chlorpromazine and quinidine with respect to their ability to affect the pretransition and the main transition of the phospholipids suspended in physiological buffer. With liposomes of neutral dipalmitoylphosphatidylcholine the observed effects paralleled to some extent the corresponding octanol/buffer partition coefficients, with adriamycin being the least effective. Calorimetric measurements on liposomes prepared from pure dipalmitoylphosphatidylglycerol or from binary mixtures of dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylcholine showed that modulation of bilayer properties by adriamycin was greatly enhanced in the presence of negatively charged lipid headgroups presumably as a result of electrostatic interactions. AD32 interacted differently from adriamycin with the acidic bilayers at low drug concentrations, in a manner similar to that of its interaction with neutral bilayers. At high drug concentrations both adriamycin and AD32 produced transitions with multiple peaks not exhibited by chlorpromazine and quinidine which may be the result of a specific association of the anthracyclines with dipalmitoylphosphatidylglycerol. All four drugs produced only minor changes in the enthalpy of the main transition of the investigated lipids. The present findings are discussed in terms of their possible physiological relevance.     

Interaction of propranolol with model phospholipid membranes. Monolayer, spin label and fluorescent spectroscopy studies

The interaction of propranolol with model phospholipid membranes was studied using various experimental techniques. The partition coefficient of propranolol in the negatively charged membranes of vesicles prepared from phosphatidylserine and phosphatidic acid was found to be more than 20-times higher than in neutral phosphatidylcholine membranes. Preferential interaction of propranolol with acidic phospholipid membranes was confirmed using the monolayer compression isotherm technique and the spin-labelling method. Phosphatidylserine monolayers were markedly expanded even at a relatively low drug concentration (5 . 10(-6) M). In contrast, the effect of propranolol on phosphatidylcholine monolayers was much smaller, being detectable only at a higher concentration of the drug (1 . 10(-4) M). Spin-labeling experiments show that propranolol exerts marked ordering effect on bilayers prepared from acidic phospholipids and does not change the order parameter of phosphatidylcholine membranes. The dependence of the propranolol fluorescence spectrum on the polarity of the solvent allowed us to identify the intercalation region of the drug in the membrane. The fluorophore moiety of propranolol was found to be localized in the lipid polar head groups region of the bilayer. The role of electrostatic and hydrophobic effects in propranolol-membrane interaction is discussed and the effect of propranolol on the ordering of phospholipid bilayers is compared with the effects of other anesthetic-like molecules.     

Effects of local anesthetics on membrane properties. I. Changes in the fluidity of phospholipid bilayers.

The effect of the local anesthetic dibucaine on the solid to liquid-crystalline phase transition in phospholipid vesicles was studied by calorimetry and fluorescence polarization. The partition coefficient (greater than 3000) of dibucaine in the membranes of vesicles prepared from acidic phospholipids was more than 20 times higher than in neutral phospholipid membranes under the same conditions. Calorimetric measurements on vesicles prepared form acidic phospholipids (bovine brain phosphatidylserine; dipalmitoylphosphatidylglycerol) showed that dibucaine (1 with 10(-4) M) produced a significant reduction in the gel-liquid crystalline transition temperature (Tc). This fluidizing effect of dibucaine on acidic phospholipid membranes was even more marked in the presence of Ca2+. In contrast, dibucaine at the same concentration did not alter the Tc of neutral phospholipids (dipalmitoylphosphatidylcholine). Significant increase in the fluidity of neutral phospholipid membranes occurred only at higher dibucaine concentrations (2 with 10(-3) M). Measurements of the fluorescence polarization and lifetime of the probe, 1,6-diphenylhexatriene, in acidic phospholipid vesicles revealed that dibucaine (1 with 10(-4) M) caused an increase in the probe rotation rate indicating an increase in the fluidity of the phospholipid membranes. A good correlation was obtained between fluorescence polarization data on dibucaine-induced changes in membrane fluidity and calorimetric measurements on vesicles of the same type.     

Inhibition of PhoE translocation across Escherichia coli inner-membrane vesicles by synthetic signal peptides suggests an important role of acidic phospholipids in protein translocation

To obtain insight into the mechanism of precursor protein translocation across membranes, the effect of synthetic signal peptides and other relevant (poly)peptides on in vitro PhoE translocation was studied. The PhoE signal peptide, associated with inner membrane vesicles, caused a concentration-dependent inhibition of PhoE translocation, as a result of a specific interaction with the membrane. Using a PhoE signal peptide analog and PhoE signal peptide fragments, it was demonstrated that the hydrophobic part of the peptide caused the inhibitory effect, while the basic amino terminus is most likely important for an optimal interaction with the membrane. A quantitative analysis of our data and the known preferential interaction of synthetic signal peptides with acidic phospholipids in model membranes strongly suggest the involvement of negatively charged phospholipids in the inhibitory interaction of the synthetic PhoE signal peptide with the inner membrane. The important role of acidic phospholipids in protein translocation was further confirmed by the observation that other (poly)peptides, known to have both a high affinity for acidic lipids and hydrophobic interactions with model membranes, also caused strong inhibition of PhoE translocation. The implication of these results with respect to the role of signal peptides in protein translocation is indicated.     

Role of specific acidic lipids on the reconstitution of Na(+)-dependent amino acid transport in proteoliposomes derived from Ehrlich cell plasma membranes

The effect of acidic phospholipids on the activity of a Na(+)-dependent amino acid transporter (A system) from Ehrlich ascites cell plasma membranes was examined. Plasma membranes were solubilized in cholate/urea and reconstituted with Ba2(+)-precipitated asolectin (soybean phospholipid free of anionic phospholipids) replenished with different acidic phospholipids. In the absence of added acidic phospholipids, transport activity was very low. However, three acidic lipids [cardiolipin greater than phosphatidic acid (PA) greater than phosphatidylinositol] were capable of restoring transport activity (in the order given) to proteoliposomes made from Ba2(+)-precipitated asolectin, while other acidic phospholipids (phosphatidylserine and phosphatidylglycerol) were much less active in this respect. For restoration of optimal activity, PA containing at least one unsaturated fatty acyl moiety, particularly in the beta position, was required. PA containing only saturated fatty acids in the beta and gamma positions was largely inactive. No difference in restoration of function was observed on varying the saturated fatty acyl chain length in PA from 10 carbons to 18 carbons. The specific effects of PA on the A-system transporter were not shared by the Na(+)-independent amino acid exchange system (L system) or the glucose transport system. Treatment with poly(ethylene glycol) 8000 was shown to reduce the nonspecific permeability of the reconstituted proteoliposomes and to enhance Na(+)-dependent amino acid transport.     

Dual mechanism of activation of plant plasma membrane Ca2+-ATPase by acidic phospholipids: evidence for a phospholipid binding site which overlaps the calmodulin-binding site

The effect of phospholipids on the activity of isoform ACA8 of Arabidopsis thaliana plasma membrane (PM) Ca2+-ATPase was evaluated in membranes isolated from Saccharomyces cerevisiae strain K616 expressing wild type or mutated ACA8 cDNA. Acidic phospholipids stimulated the basal Ca2+-ATPase activity in the following order of efficiency: phosphatidylinositol 4-monophosphate > phosphatidylserine > phosphatidylcholine approximately = phosphatidylethanolamine approximately = 0. Acidic phospholipids increased V(max-Ca2+) and lowered the value of K(0.5-Ca2+) below the value measured in the presence of calmodulin (CaM). In the presence of CaM acidic phospholipids activated ACA8 by further decreasing its K(0.5-Ca2+) value. Phosphatidylinositol 4-monophosphate and, with lower efficiency, phosphatidylserine bound peptides reproducing ACA8 N-terminus (aa 1-116). Single point mutation of three residues (A56, R59 and Y62) within the sequence A56-T63 lowered the apparent affinity of ACA8 for phosphatidylinositol 4-monophosphate by two to three fold, indicating that this region contains a binding site for acidic phospholipids. However, the N-deleted mutant Delta74-ACA8 was also activated by acidic phospholipids, indicating that acidic phospholipids activate ACA8 through a complex mechanism, involving interaction with different sites. The striking similarity between the response to acidic phospholipids of ACA8 and animal plasma membrane Ca2+-ATPase provides new evidence that type 2B Ca2+-ATPases share common regulatory properties independently of structural differences such as the localization of the terminal regulatory region at the N- or C-terminal end of the protein.     

Enhancement of opiate binding by various molecular forms of phosphatidylserine and inhibition by other unsaturated lipids.

A study was undertaken on the possible involvement of phospholipids on stereospecific opiate binding to a rat brain membrane fraction comprised mainly of synaptic membranes. The addition of acidic phospholipids such as phosphatidylserine, phosphoinositides, and phosphatidic acid significantly enhanced opiate binding. With the exception of phosphatidylserine, when the acidic phospholipids contained a polyunsaturated acyl group, they were actually inhibitory, along with neutral phospholipids derived from brain. Both the C18:0, C18:1 form (derived from myelin) and the C18:0, C22:6 form of phosphatidylserine (derived from synaptic membranes) produced as much as a 45% enhancement in opiate binding. Unsaturated fatty acids were highly inhibitory, the degree of inhibition being related to the degree of unsaturation. Both phospholipase A and C were inhibitory; and the inhibitory effect of A could not be prevented by albumin or overcome with the addition of phosphatidylserine. With the use of the cross-linking agent, dinitrodifluorobenzene, it could be demonstrated that the phosphatidylserine of synaptic membranes appeared to be preferentially associated with membrane protein. The enhancement of opiate binding by phosphatidylserine diminished with increasing degree of cross-linking.     

Binding of LL-37 to model biomembranes: insight into target vs host cell recognition

Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X=0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.     

Calmodulin-independent inhibition of platelet phospholipase A2 by calmodulin antagonists

We tested the effects of calmodulin, two types of calmodulin antagonists, and various phospholipids on the phospholipase A2 activities of intact platelets, platelet membranes, and partially purified enzyme preparations. Trifluoperazine, chlorpromazine (phenothiazines) and N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7), at concentrations which antagonize the effects of calmodulin, significantly inhibited thrombin- and Ca2+ ionophore-induced production of arachidonic acid metabolites by suspensions of rabbit platelets and Ca2+-induced arachidonic acid release from phospholipids of membrane fractions, but not phospholipase A2 activity in purified enzyme preparations. The addition of acidic phospholipids, but not calmodulin, stimulated phospholipase A2 activity in purified enzyme preparations while decreasing its Km for Ca2+. The dose-response and kinetics of inhibition by calmodulin antagonists of acidic phospholipid-activated phospholipase A2 activity in purified preparations were similar to those of Ca2+-induced arachidonic acid release from membrane fractions. Calmodulin antagonists were also found to inhibit Ca2+ binding to acidic phospholipids in a similar dose-dependent manner. Our results suggest that the platelet phospholipase A2 is the key enzyme involved in arachidonic acid mobilization in platelets and is regulated by acidic phospholipids in a Ca2+-dependent manner and that calmodulin antagonists inhibit phospholipase A2 activity via an action on acidic phospholipids.     

Electrogenesis from an ATPase-ATP-sodium pseudo pump.

The short circuit current and the open circuit voltage responses of membranes to ATP, which have been attributed to membrane ATPase acting as a sodium pump, have been reproduced not only in a lipid membrane containing solubilized ATPase but also in membranes formed of the phospholipids contained in ATPase. The response is greatest with cardiolipin, but occurs with other acidic phospholipids. This observation of electrogenesis without hydrolysis is a surface phenomenon probably due to the alignment of ATP on the phospholipid by ion association at its interface with the water phase. The finding constitutes a precaution for interpreting studies of membrane Na-K-ATPase or for its incorporation into an artificial membrane. The substances necessary for electrogenesis are present at the mitochondrial membrane, and the particular orientation of the ATP on the phospholipids in vitro suggests a role for this ion association in the function of Na-K-ATPase.     

Electrogenesis from an ATPase-ATP-sodium pseudo pump

Summary The short circuit current and the open circuit voltage responses of membranes to ATP, which have been attributed to membrane ATPase acting as a sodium pump, have been reproduced not only in a lipid membrane containing solubilized ATPase but also in membranes formed of the phospholipids contained in ATPase. The response is greatest with cardiolipin, but occurs with other acidic phospholipids. This observation of electrogenesis without hydrolysis is a surface phenomenon probably due to the alignment of ATP on the phospholipid by ion association at its interface with the water phase. The finding constitutes a precaution for interpreting studies of membrane Na–K-ATPase or for its incorporation into an artificial membrane. The substances necessary for electrogenesis are present at the mitochondrial membrane, and the particular orientation of the ATP on the phospholipids in vitro suggests a role for this ion association in the function of Na–K-ATPase.     

Enhancement of opiate binding by various molecular forms of phosphatidylserine and inhibition by other unsaturated lipids

A study was undertaken on the possible involvement of phospholipids on stereospecific opiate binding to a rat brain membrane fraction comprised mainly of synaptic membranes. The addition of acidic phospholipids such as phosphatidylserine, phosphoinositides, and phosphatidic acid significantly enhanced opiate binding. With the exception of phosphatidylserine, when the acidic phospholipids contained a polyunsaturated acyl group, they were actually inhibitory, along with neutral phospholipids derived from brain. Both the C_18:0, C_18:1 form (derived from myelin) and the C_18:0, C_22:6 form of phosphatidylserine (derived from synaptic membranes) produced as much as a 45% enhancement in opiate binding. Unsaturated fatty acids were highly inhibitory, the degree of inhibition being related to the degree of unsaturation. Bot phospholipase A and C were inhibitory; and the inhibitory effect of A could not be prevented by albumin or overcome with the addition of phosphatidylserine. With the use of the cross-linking agent, dinitrodifluorobenzene, it could be demonstrated that the phosphatidylserine of synaptic membranes appeared to be preferentially associated with membrane protein. The enhancement of opiate binding by phosphatidylserine diminished with increasing degree of cross-linking.     

Interaction of propranolol with model phospholipid membranes Monolayer,spin label and fluorescence spectroscopy studies

The interaction of propranolol with model phospholipid membranes was studied using various experimental techniques. The partition coefficient of propranolol in the negatively charged membranes of vesicles prepared from phosphatidylserine and phosphatidic acid was found to be more than 20-times higher than in neutral phosphatidylcholine membranes. Preferential interaction of propranolol with acidic phospholipid membranes was confirmed using the monolayer compression isotherm technique and the spin-labeling method. Phosphatidylserine monolayers were markedly expanded even at a relatively low drug concentration ($\text{5 · 10}{}^{\mathrm{?6}}$ M). In contrast, the effect of propranolol on phosphatidylcholine monolayers was much smaller, being detectable only at a higher concentration of the drug ($\text{1 · 10}{}^{\mathrm{?4}}$ M). Spin-labeling experiments show that propranolol exerts marked ordering effect on bilayers prepared from acidic phospholipids and does not change the order parameter of phosphatidylcholine membranes. The dependence of the propranolol fluorescence spectrum on the polarity of the solvent allowed us to identify the intercalation region of the drug in the membrane. The fluorophore moiety of propranolol was found to be localized in the lipid polar head groups region of the bilayer. The role of electrostatic and hydrophobic effects in propranolol-membrane interaction is discussed and the effect of propranolol on the ordering of phospholipid bilayers is compared with the effects of other anesthetic-like molecules.     

SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane.

Translocation of preproteins across the Escherichia coli inner membrane requires acidic phospholipids. We have studied the translocation of the precursor protein proOmpA across inverted inner membrane vesicles prepared from cells depleted of phosphatidylglycerol and cardiolipin. These membranes support neither translocation nor the translocation ATPase activity of the SecA subunit of preprotein translocase. We now report that inner membrane vesicles which are depleted of acidic phospholipids are unable to bind SecA protein with high affinity. These membranes can be restored to translocation competence by fusion with liposomes containing phosphatidylglycerol, suggesting that the defect in SecA binding is a direct effect of phospholipid depletion rather than a general derangement of inner membrane structure. Reconstitution of SecY/E, the membrane-embedded domain of translocase, into proteoliposomes containing predominantly a single synthetic acidic lipid, dioleoylphosphatidylglycerol, allows efficient catalysis of preprotein translocation.     

Interaction of annexin VI with membranes: highly restricted dissipation of clustered phospholipids in membranes containing phosphatidylethanolamine.

Association of annexin VI with membranes induced extensive clustering of acidic phospholipids as detected by self-quenching of fluorescent-labeled acidic phospholipids [Bazzi, M.D., & Nelsestuen, G.L. (1991) Biochemistry 30, 7961]. The present study examined the rates of protein-induced clustering of acidic phospholipids in membranes containing 10-15% fluorescent-labeled phosphatidic acid dispersed in phosphatidylcholine (PC) or phosphatidylethanolamine (PE). Both membranes supported similar levels of protein-induced fluorescence quenching. With membranes containing PC, protein-membrane association and fluorescence quenching were rapid, and were virtually complete within seconds after the reagents were mixed. Membranes containing PE exhibited rapid protein-membrane association, but showed a fluorescence quenching that was several orders of magnitude slower than membranes containing PC. Calcium chelation resulted in rapid dissociation of protein-membrane complexes. Subsequent recovery of the fluorescence signal of both membranes was virtually complete, but the rate of fluorescence recovery was very different. The recovery was rapid in membranes containing PC, while PE-containing membranes showed slow recovery that approached the rate at which the fluorescent-labeled phosphatidic acid exchanged between vesicles. Thus, the presence of PE appeared to severely restrict dissipation of clustered phospholipids in membranes. Membranes containing PE, N-methyl-PE, N,N-dimethyl-PE, and PC showed successive increases in the rates of fluorescence quenching and recovery, suggesting that hydrogen bonding between head groups was the basis for this property. If the restricted dissipation of phosphatidic acid in PE membranes is a general property, the relative mobility of membrane components and even diffusion on interior cell membranes may be greatly influenced by this phenomenon.     

Role of phospholipids in the structure and function of the thyrotropin receptor.

Phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine interact with 125I-thyrotropin and inhibit its binding to thyroid plasma membranes; phosphatidylcholine is not similarly effective. The interaction has been monitored by column chromatography on Sephadex G-100 which shows, for example, that 125I-labeled thyrotropin forms an adduct with phosphatidylinositol but not with phosphatidylcholine. Formation of the 125I-labeled thyrotropin-phosphatidylinositol adduct is dependent on the phosphatidylinositol concentration but can be reversed by both unlabeled thyrotropin and excess membranes. The efficacy of the phospholipid interaction and the phospholipid inhibition of thyrotropin binding to thyroid membranes is paralleled by changes in fluorescence and fluorescence polarization imposed on the 5-dimethylamino-1-naphthalene sulfonate (dansyl) derivative of thyrotropin. These changes are reversed by unlabeled thyrotropin but not by prolactin, placental lactogen, or growth hormone; similar changes are not observed when phospholipids are incubated with dansylated growth hormone, prolactin, and placental lactogen. Monovalent potassium, sodium, and lithium salts neither prevent nor reverse the formation of the phospholipid-dansyl-thyrotropin adduct; these results contrast with the effects of the same salts on the formation of ganglioside adducts with dansyl-thyrotropin. Despite their ability to interact witw 125I-thyrotropin in solution, neither phosphatidylinositol, phosphatidylserine, nor phosphatidylethanolamine, when incorporated in a liposome, binds the 125I-labeled ligand. These same phospholipids have no effect on ganglioside binding of 125I-labeled thyrotropin when gangliosides are incorporated in a liposome. These phospholipids do, however, modulate the expression of the glycoprotein component of the thyrotropin receptor when it is imbedded in a liposome. The phosphatidylinositol in this case serves as a negative modulator, both by decreasing the incorporation of the glycoprotein component of the receptor into the liposome and by inhibiting the binding activity of the glycoprotein component which is incorporated. Speculation is offered as to a possible role of the phospholipids in the message transmission process which would be consistent with current studies demonstrating a direct interaction of acidic phospholipids with thyrotropin. The effect of phospholipids on liposomes containing the glycoprotein component of the thyrotropin receptor raises the possibility that phospholipids and, in particular, phosphatidylinositol, may also play a role in regulating the insertion and expression of this receptor component in thyroid plasma membranes.     

Binding of LL-37 to model biomembranes: Insight into target vs host cell recognition

Pursuing the molecular mechanisms of the concentration dependent cytotoxic and hemolytic effects of the human antimicrobial peptide LL-37 on cells, we investigated the interactions of this peptide with lipids using different model membranes, together with fluorescence spectroscopy for the Trp-containing mutant LL-37(F27W). Minimum concentrations inhibiting bacterial growth and lipid interactions assessed by dynamic light scattering and monolayer penetration revealed the mutant to retain the characteristics of native LL-37. Although both LL-37 and the mutant intercalated effectively into zwitterionic phosphatidylcholine membranes the presence of acidic phospholipids caused augmented membrane binding. Interestingly, strongly attenuated intercalation of LL-37 into membranes containing both cholesterol and sphingomyelin (both at X = 0.3) was observed. Accordingly, the distinction between target and host cells by LL-37 is likely to derive from i) acidic phospholipids causing enhanced association with the former cells as well as ii) from attenuated interactions with the outer surface of the plasma membrane of the peptide secreting host, imposed by its high content of cholesterol and sphingomyelin. Our results further suggest that LL-37 may exert its antimicrobial effects by compromising the membrane barrier properties of the target microbes by a mechanism involving cytotoxic oligomers, similarly to other peptides forming amyloid-like fibers in the presence of acidic phospholipids.     

Membrane regulation of the chromosomal replication activity of E.coli DnaA requires a discrete site on the protein.

The capacity of DnaA protein to initiate DNA synthesis at the chromosomal origin is influenced profoundly by the tightly bound nucleotides ATP and ADP. Acidic phospholipids can catalyze the conversion of inactive ADP-DnaK protein into the active ATP form. Proteolytic fragments of the nucleotide form of DnaA protein were examined to determine regions of the protein critical for functional interaction with membranes. A 35 kDa chymotryptic and 29 kDa tryptic fragment retained the tightly bound nucleotide. The fragments, whose amino-termini are within three residues of each other, but differ at their carboxyl ends, showed strikingly different behavior when treated with acidic phospholipids. The larger chymotryptic fragment released the bound nucleotide in the presence of acidic, but not neutral phospholipids. In contrast, the smaller tryptic fragment was inert to both forms of phospholipids. Acidic membranes, but not those composed of neutral phospholipids, protect from tryptic digestion a small portion of the segment that constitutes the difference between the 29 and 35 kDa fragments. The resulting 30 kDa tryptic fragment, which possesses this protected region, interacts functionally with acidic membranes to release the bound effector nucleotide. Inasmuch as the anionic ganglioside GM1, a compound structurally dissimilar to acidic glycerophospholipids, efficiently releases the nucleotide from DnaA protein, an acidic surface associated with a hydrophobic environment is the characteristic of the membrane that appears crucial for regulatory interaction with DnaA protein.     

Membrane regulation of the chromosomal replication activity of E. coli DnaA requires a discrete site on the protein.

The capacity of DnaA protein to initiate DNA synthesis at the chromosomal origin is influenced profoundly by the tightly bound nucleotides ATP and ADP. Acidic phospholipids can catalyze the conversion of inactive ADP-DnaA protein into the active ATP form. Proteolytic fragments of the nucleotide form of DnaA protein were examined to determine regions of the protein critical for functional interaction with membranes. A 35 kDa chymotryptic and 29 kDa tryptic fragment retained the tightly bound nucleotide. The fragments, whose amino-termini are within three residues of each other, but differ at their carboxyl ends, showed strikingly different behavior when treated with acidic phospholipids. The larger chymotryptic fragment released the bound nucleotide in the presence of acidic, but not neutral phospholipids. In contrast, the smaller tryptic fragment was inert to both forms of phospholipids. Acidic membranes, but not those composed of neutral phospholipids, protect from tryptic digestion a small portion of the segment that constitutes the difference between the 29 and 35 kDa fragments. The resulting 30 kDa tryptic fragment, which possesses this protected region, interacts functionally with acidic membranes to release the bound effector nucleotide. Inasmuch as the anionic ganglioside GM1, a compound structurally dissimilar to acidic glycerophospholipids, efficiently releases the nucleotide from DnaA protein, an acidic surface associated with a hydrophobic environment is the characteristic of the membrane that appears crucial for regulatory interaction with DnaA protein.     

Inhibition of the phosphatase activity of the red cell membrane Ca2+ pump by acidic phospholipids.

The effect of phospholipids was tested on the p-nitrophenylphosphatase activity of the Ca2+ pump. Acidic phospholipids like phosphatidylserine and phosphatidylinositol inhibited the phosphatase activity, while neutral phospholipids like phosphatidylcholine did not. This result contrasts sharply with the known activating effect of acidic phospholipids on the Ca2(+)-ATPase activity of the pump. It is known that the phosphatase activity of the Ca2+ pump can be elicited either by calmodulin and Ca2+ or by ATP and Ca2+. Unlike calmodulin, acidic phospholipids failed to stimulate the phosphatase activity. Furthermore, calmodulin-activated phosphatase was completely inhibited by acidic phospholipids. Maximal inhibition of the ATP-activated phosphatase was only 70%. Inhibition by acidic phospholipids was non-competitive regarding to calmodulin, suggesting that acidic phospholipids and calmodulin do not bind to the same domain of the pump. The presence of Ca2+ was essential for the inhibition, and the apparent affinity for Ca2+ for this effect was increased by acidic phospholipids. Results are consistent with the idea that acidic phospholipids stabilize an enzyme-Ca complex lacking phosphatase activity.