Absence Of Phosphatidylethanolamine Research Articles

Indiicoccus explosivorum gen. nov., sp. nov., isolated from an explosives waste contaminated site.

A pink-pigmented, Gram-stain-positive, aerobic, coccoid-shaped bacterial strain, designated as S5-TSA-19T, was isolated from an explosives contaminated site in Panchkula, Haryana, India. The 16S rRNA gene sequencing blast analysis indicated that the strain is a member of the family Planococcaceae with the highest sequence similarity to Planomicrobium soli XN13T (96.1 %), followed by Planococcus maitriensis S1T (95.6 %), Planococcus plakortidis DSM 23997T (95.6 %), Planomicrobium flavidum ISL-41T (95.6 %), Planococcus rifietoensis M8T (95.5 %), Planococcus salinus LCB217T (95.5 %) and Planococcus maritimus DSM 17275T (95.5 %). Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences (based on a conserved set of 400 proteins) retrieved the strain in a distinct branch indicating a separate lineage within the family Planococcaceae. Strain S5-TSA-19T had a distinctive chemotaxonomic pattern comprising A4α type peptidoglycan based on l-Lys-d-Asp, iso-C15 : 0 as the major fatty acid, absence of phosphatidylethanolamine as a major lipid and MK-7 and MK-6 as the major menaquinones, differentiating it from the genera Planococcus and Planomicrobium, thus supporting the findings of molecular phylogeny. Further, strain S5-TSA-19T was able to biotransform hexahydro-1,3,5,-trinitro-1,2,5-triazine (RDX) into nitrite derivatives under aerobic conditions in 2-4 days, whereas the closest reference strains did not possess this property. On the basis of polyphasic taxonomic characterization and a phylogenomics approach, strain S5-TSA-19T is proposed as the type strain of a novel species in a novel genus for which the name Indiicoccus explosivorum gen. nov., sp. nov. is proposed (=JCM 31737T=KCTC 33871T=MTCC 12608T).

Atypical Binding Behavior of Epsin‐like Clathrin Adaptor 1 to Diacylglycerol Pyrophosphate

Epsin N‐terminal homology (ENTH) and AP‐180 N‐terminal homology (ANTH) domain proteins are membrane binding proteins known to interact with polyphosphoinositides (PIPs). They share function, structure and sequence similarity, and are grouped together as A/ENTH domain proteins. The ENTH domain contains a distinctive N‐terminal helix (Ho) region that plays a crucial role in membrane bending. This Ho helix is not usually found in ANTH domains. Epsin‐like Clathrin Adaptor 1 (ECA1) is one such A/ENTH protein that acts as an adaptor protein in clathrin mediated endocytosis. It interacts with clathrin via its C‐terminus, and PIPs via its N‐terminus. In addition to having ANTH domain sequence similarity, ECA1 also contains the distinctive Ho helix region that is generally found in the ENTH domain. ECA1 is therefore best characterized as an N‐ANTH domain protein with a unique combination of both ANTH and ENTH regions. ECA1 is known to play a role in stress signaling in plants and also binds to phosphatidic acid (PA) upon salt stress. Like proteins, membrane lipids undergo drastic changes during stress stimuli. PA is a lipid second messenger that rapidly and transiently increases under stress stimuli. Upon rise of PA levels another lipid, diacylglycerol pyrophosphate (DGPP) starts to accumulate. DGPP is phosphorylated PA. It is suggested to be synthesized at higher levels as a signal for PA attenuation during stress. We recently showed in vitro that ECA1‐PA ‐affinity is modulated as a function of membrane curvature stress. In this research, we investigate ECA1 affinity to DGPP. We hypothesize that the PA target ECA1 has affinity for its metabolite DGPP which is highly negatively charged and this affinity is variable at varying pH. We tested ECA1 affinity to DGPP using liposome binding assay along with other anionic lipids; i.e., phosphatidylserine, phosphatidylinositol (4)‐phosphate and phosphatidylinositol (4,5)‐bisphosphate in the presence and absence of phosphatidylethanolamine. Our results show striking evidence that protein‐lipid interactions are not simply based on electrostatics. We also show that higher charge density on membranes does not always correspond to higher protein affinity. In fact, DGPP showed lesser protein binding to ECA1 compared to PA although DGPP has a higher negative charge. This study brings us a step closer towards understanding the complexity of protein interactions with anionic lipids and opens the discussion to lipid homeostasis upon salt stress.Support or Funding InformationDepartment of Biological Science, Kent State University and NSF CHE 1412920This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vesicle Leakage Reflects the Target Selectivity of Antimicrobial Lipopeptides from Bacillus subtilis

Cyclic lipopeptides act against a variety of plant pathogens and are thus highly efficient crop-protection agents. Some pesticides contain Bacillus subtilis strains that produce lipopeptide families, such as surfactins (SF), iturins (IT), and fengycins (FE). The antimicrobial activity of these peptides is mainly mediated by permeabilizing cellular membranes. We used a fluorescence-lifetime based leakage assay to examine the effect of individual lipid components in model membranes on lipopeptide activity. Leakage induction by FE was strongly inhibited by cholesterol (CHOL) as well as by phosphatidylethanolamine (PE) and -glycerol (PG) lipids. Already moderate amounts of CHOL increased the tolerable FE content in membranes by an order of magnitude to 0.5 FE per PC + CHOL. This indicates reduced FE-lipid demixing and aggregation, which is known to be required for membrane permeabilization and explains the strong inhibition by CHOL. Ergosterol (ERG) had a weak antagonistic effect. This confirms results of microbiological tests and agrees with the fungicidal activity and selectivity of FE. SF is known to be much less selective in its antimicrobial action. In line with this, liposome leakage by SF was little affected by sterols and PE. Interestingly, PG increased SF activity and changed its leakage mechanism toward all-or-none, suggesting more specific, larger, and/or longer-lived defect structures. This may be because of the reduced energetic cost of locally accumulating anionic SF in an anionic lipid matrix. IT was found largely inactive in our assays. B. subtilis QST713 produces the lipopeptides in a ratio of 6 mol SF: 37 mol FE: 57 mol IT. Leakage induced by this native mixture was inhibited by CHOL and PE, but unaffected by ERG and by PG in the absence of PE. Note that fungi contain anionic lipids, but little PE. Hence, our data explain the strong, fungicidal activity and selectivity of B. subtilis QST713 lipopeptides.

Lipids and Topological Rules of Membrane Protein Assembly: BALANCE BETWEEN LONG AND SHORT RANGE LIPID-PROTEIN INTERACTIONS

The N-terminal six-transmembrane domain (TM) bundle of lactose permease of Escherichia coli is uniformly inverted when assembled in membranes lacking phosphatidylethanolamine (PE). Inversion is dependent on the net charge of cytoplasmically exposed protein domains containing positive and negative residues, net charge of the membrane surface, and low hydrophobicity of TM VII acting as a molecular hinge between the two halves of lactose permease (Bogdanov, M., Xie, J., Heacock, P., and Dowhan, W. (2008) J. Cell Biol. 182, 925-935). Net neutral lipids suppress the membrane translocation potential of negatively charged amino acids, thus increasing the cytoplasmic retention potential of positively charged amino acids. Herein, TM organization of sucrose permease (CscB) and phenylalanine permease (PheP) as a function of membrane lipid composition was investigated to extend these principles to other proteins. For CscB, topological dependence on PE only becomes evident after a significant increase in the net negative charge of the cytoplasmic surface of the N-terminal TM bundle. High negative charge is required to overcome the thermodynamic block to inversion due to the high hydrophobicity of TM VII. Increasing the positive charge of the cytoplasmic surface of the N-terminal TM hairpin of PheP, which is misoriented in PE-lacking cells, favors native orientation in the absence of PE. PheP and CscB also display co-existing dual topologies dependent on changes in the charge balance between protein domains and the membrane lipids. Therefore, the topology of both permeases is dependent on PE. However, CscB topology is governed by thermodynamic balance between opposing lipid-dependent electrostatic and hydrophobic interactions.

Physiological pH and Acidic Phospholipids Contribute to Substrate Specificity in Lipidation of Atg8

Yeast Atg8 and its mammalian homolog LC3 are ubiquitin-like proteins involved in autophagy, a primary pathway for degradation of cytosolic constituents in vacuoles/lysosomes. Whereas the lipid phosphatidylethanolamine (PE) was identified as the sole in vivo target of their conjugation reactions, in vitro studies showed that the same system can mediate the conjugation of these proteins with phosphatidylserine as efficiently as with PE. Here, we show that, in contrast to PE conjugation, the in vitro phosphatidylserine conjugation of Atg8 is markedly suppressed at physiological pH. Furthermore, the addition of acidic phospholipids to liposomes also results in the preferential formation of the Atg8-PE conjugate. We have successfully captured authentic thioester intermediates, allowing us to elucidate which step in the conjugation reaction is affected by these changes in pH and membrane lipid composition. We propose that these factors contribute to the selective formation of Atg8-PE in the cell.

The impact of environmental perturbation on microbial community structure and dynamics: Factors affecting growth of Clonothrix fusca in groundwater

In a previous work temporal succession of two sheathed, filamentous methanotrophic γ-Proteobacteria, Crenothrix polyspora and Clonothrix fusca, was observed by analyzing the microbial community of an artesian well. Although a correlation between physical-chemical environmental parameters and growth of these microorganisms was suggested, the link with the microbial physiology remained elusive. In this study we demonstrate that phosphatidylethanolamine and cardiolipin, generally abundant in most γ-Proteobacteria, could not be detected in C. fusca membranes by thin-layer chromatography. The absence of cardiolipin was supported by results of enzymatic assay of cardiolipin synthase activity and by Southern blot analysis that failed to detect cls homologous sequences in the genomic DNA of this microorganism. The absence of phosphatidylethanolamine and cardiolipin might account for requirement of millimolar concentrations of magnesium and calcium for growth, and for massive over-growth of this microorganism during the winter season when the water levels of magnesium and calcium increased in the artesian well.

Conformational changes in a bacterial multidrug transporter are phosphatidylethanolamine-dependent

LmrP is an electrogenic H(+)/drug antiporter that extrudes a broad spectrum of antibiotics. Five carboxylic residues are implicated in drug binding (Asp142 and Glu327) and proton motive force-mediated restructuring (Asp68, Asp128 and Asp235). ATR-FTIR (Attenuated Total Reflection - Fourier Transform Infrared) and tryptophan quenching experiments revealed that phosphatidylethanolamine (PE) is required to generate the structural intermediates induced by ionization of carboxylic residues. Surprisingly, no ionization-induced conformational changes were detectable in the absence of PE, suggesting either that carboxylic acid residues do not ionize or that ionization does not lead to any conformational change. The mean pKa of carboxylic residues evaluated by ATR-FTIR spectroscopy was 6.5 for LmrP reconstituted in PE liposomes, whereas the pKa calculated in the absence of PE was 4.6. Considering that 16 of the 19 carboxylic residues are located in the extramembrane loops, the pKa values obtained in the absence and in the presence of PE suggest that the interaction of the loop acid residues with the membrane interface depends on the lipid composition.

Unbalanced phospholipid composition of Escherichia coli membranes affects PPHO promoter activity

The activity of phosphatase PPHO or arabinose PBAD promoters of Escherichia coli has been studied as dependent on the content of zwitterionic phosphatidylethanolamine (PE) and anionic phospholipids in membranes. In the absence of PE or upon a significant decrease in the content of anionic phospholipids, the activity of the PPHO promoter (but not that of the PBAD promoter) was inhibited. Since the PPHO promoter belongs to the Pho regulon, a member of the family of two-component regulatory systems of signal transduction that have membrane sensors, the regulation of gene expression by phospholipids is presumed to be accomplished through a membrane sensor.

N-Retinylidene-phosphatidylethanolamine Is the Preferred Retinoid Substrate for the Photoreceptor-specific ABC Transporter ABCA4 (ABCR)

ABCA4, a member of the family of ATP binding cassette (ABC) proteins found in rod and cone photoreceptors, has been implicated in the transport of retinoid compounds across the outer segment disk membrane following the photoactivation of rhodopsin. Mutations in the ABCA4 gene are responsible for Stargardt macular dystrophy and related retinal degenerative diseases that cause a loss in vision. To identify the retinoid substrate that interacts with ABCA4, we have isolated ABCA4 from rod outer segment disk membranes on an immunoaffinity matrix and analyzed retinoid compounds that bind to ABCA4 using high performance liquid chromatography and radiolabeling methods. When all-trans-retinal was added to ABCA4 in the presence of phosphatidylethanolamine, approximately 0.9 mol of N-retinylidene-phosphatidylethanolamine and 0.3 mol of all-trans-retinal were bound per mol of ABCA4 with an apparent K(d) of 2-5 microm. ATP and GTP released these retinoids from ABCA4, whereas ADP, GDP, and nonhydrolyzable derivatives, adenosine 5'-(beta,gamma-imido)triphosphate and guanosine 5'-(beta,gamma-imido)triphosphate, were ineffective. One mole of N-retinyl-phosphatidylethanolamine, the reduced form of N-retinylidene-phosphatidylethanolamine, bound per mol of ABCA4, whereas 0.3 mol of all-trans-retinal were bound in the absence of phosphatidylethanolamine. No binding of all-trans-retinol to ABCA4 was observed. Our results indicate that ABCA4 preferentially binds N-retinylidene-phosphatidylethanolamine with high affinity in the absence of ATP. Our studies further suggest that ATP binding and hydrolysis induces a protein conformational change that causes N-retinylidene-phosphatidylethanolamine to dissociate from ABCA4.

Effect of membrane phospholipid composition and charge of the signal peptide of Escherichia coli alkaline phosphatase on efficiency of its secretion.

The secretion of the Escherichia coli alkaline phosphatase with a different charge of signal peptide due to replacement of positively charged Lys(-20) has been studied depending on the phospholipid composition of the membranes and the activity of the translocational ATPase--protein SecA. Changing the signal peptide charge, along with a change in phospholipid composition, has been shown to reduce the efficiency of secretion. In the absence of phosphatidylethanolamine the membrane contains anionic phospholipids only, and the dependence of secretion on the signal peptide charge decreases. The dependence of secretion on membrane phospholipid composition and the signal peptide charge is also determined by the activity of SecA protein. If SecA is inactivated by sodium azide, then the dependence of secretion on anionic phospholipids increases; on the contrary, higher content of anionic phospholipids (in the absence of phosphatidylethanolamine) decreases the dependence of secretion on the SecA activity. The results suggest a direct interaction of positively charged signal peptide with negatively charged membrane phospholipids under initiation of secretion and also interdependent contribution of the signal peptide charge, anionic phospholipids, and translocational ATPase to secretion.

Topology of polytopic membrane protein subdomains is dictated by membrane phospholipid composition.

In Escherichia coli, the major cytoplasmic domain (C6) of the polytopic membrane protein lactose permease (LacY) is exposed to the opposite side of the membrane from a neighboring periplasmic domain (P7). However, these domains are both exposed on the periplasmic side of the membrane in a mutant of E.coli lacking phosphatidylethanolamine (PE) wherein LacY only mediates facilitated transport. When purified LacY was reconstituted into liposomes lacking PE or phosphatidylcholine (PC), C6 and P7 were on the same side of the bilayer. In liposomes containing PE or PC, C6 and P7 were on opposite sides of the bilayer. Only the presence of PE in the liposomes restored active transport function of LacY as opposed to restoration of only facilitated transport function in the absence of PE. These results were the same for LacY purified from PE-containing or PE-lacking cells, and are consistent with the topology and function of LacY assembled in vivo. Therefore, irrespective of the mechanism of membrane insertion, the subdomain topological orientation and function of LacY are determined primarily by membrane phospholipid composition.

Study of interaction of export initiation domain of Escherichia coli mature alkaline phosphatase with membrane phospholipids during secretion.

The efficiency of secretion of alkaline phosphatase from Escherichia coli depending on the primary structure of its N-terminal region and the content of zwitterionic phospholipid phosphatidylethanolamine and anionic phospholipids in membranes has been studied in this work to establish the peculiarities of interaction of mature protein during its secretion with membrane phospholipids. It has been shown that the effect of phosphatidylethanolamine but not anionic phospholipids on the efficiency of alkaline phosphatase secretion is determined by the primary structure of its N-terminal region. The absence of phosphatidylethanolamine appreciably reduces the efficiency of secretion of wild type alkaline phosphatase and its mutant forms with amino acid substitutions in positions +5+6 and +13+14. In contrast, secretion of the protein withamino acid substitutions in positions +2+3, significantly decreased as a result of such mutation, in the presence of phosphatidylethanolamine, reaches the level of wild type protein secretion in the absence of phosphatidylethanolamine. The results suggest an interaction of the N-terminal region of the mature protein under its translocation across the membrane with phosphatidylethanolamine.

FtsY Binds to the Escherichia coli Inner Membrane via Interactions with Phosphatidylethanolamine and Membrane Proteins

Targeting of many polytopic proteins to the inner membrane of prokaryotes occurs via an essential signal recognition particle-like pathway. FtsY, the Escherichia coli homolog of the eukaryotic signal recognition particle receptor alpha-subunit, binds to membranes via its amino-terminal AN domain. We demonstrate that FtsY assembles on membranes via interactions with phosphatidylethanolamine and with a trypsin-sensitive component. Both interactions are mediated by the AN domain of FtsY. In the absence of phosphatidylethanolamine, the trypsin-sensitive component is sufficient for binding and function of FtsY in the targeting of membrane proteins. We propose a two-step mechanism for the assembly of FtsY on the membrane similar to that of SecA on the E. coli inner membrane.

Phospholipid-assisted Refolding of an Integral Membrane Protein: MINIMUM STRUCTURAL FEATURES FOR PHOSPHATIDYLETHANOLAMINE TO ACT AS A MOLECULAR CHAPERONE

Escherichia coli-derived phosphatidylethanolamine (PE) or PE with fully saturated fatty acids was able to correct in vitro a defect in folding in the lipid-dependent epitope 4B1 of lactose permease (LacY) resulting from in vivo assembly in the absence of PE. PE plasmalogen, PE with two unsaturated fatty acids, and lyso-PE, which all do not favor bilayer organization, did not support proper refolding. Proper refolding occurred when these latter lipids were mixed with a bilayer-forming lipid (phosphatidylglycerol), which alone could not support refolding. L-Phosphatidylserine (PS; natural diastereomer) did support proper refolding. PE derivatives of increasing degrees of methylation were progressively less effective in supporting refolding, with phosphatidylcholine being completely ineffective. Therefore, the properties of nonmethylated aminophospholipids capable of organization into a bilayer configuration are essential for the recovery of the native state of epitope 4B1 after misassembly in vivo in the absence of PE. Neither D-PS (sn-glycero-1-phosphate backbone) nor P-D-S (D-serine in the head group) is competent in supporting proper refolding unless used in binary mixtures with phosphatidylglycerol. The detailed characterization of phospholipid-assisted refolding reported here further supports a specific rather than nonspecific role for PE in structural maturation of lactose permease in vivo (Bogdanov, M., and Dowhan, W. (1998) EMBO J. 17, 5255-5264).

The effect of membrane composition on the hemostatic balance.

The phospholipid composition requirements for optimal prothrombin activation and factor Va inactivation by activated protein C (APC) anticoagulant were examined. Vesicles composed of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) supported factor Va inactivation relatively well. However, optimal factor Va inactivation still required relatively high concentrations of phosphatidylserine (PS). In addition, at a fixed concentration of phospholipid, PS, and APC, vesicles devoid of PE never attained a rate of factor Va inactivation achievable with vesicles containing PE. Polyunsaturation of any vesicle component also contributed significantly to APC inactivation of factor Va. Thus, PE makes an important contribution to factor Va inactivation that cannot be mimicked by PS. In the absence of polyunsaturation in the other membrane constituents, this contribution was dependent upon the presence of both the PE headgroup per se and unsaturation of the 1,2 fatty acids. Although PE did not affect prothrombin activation rates at optimal PS concentrations, PE reduced the requirement for PS approximately 10-fold. The Km(app) for prothrombin and the Kd(app) for factor Xa-factor Va decreased as a function of increasing PS concentration, reaching optimal values at 10-15% PS in the absence of PE but only 1% PS in the presence of PE. Fatty acid polyunsaturation had minimal effects. A lupus anticoagulant immunoglobulin was more inhibitory to both prothrombinase and factor Va inactivation in the presence of PE. The degree of inhibition of APC was significantly greater and much more dependent on the phospholipid composition than that of prothrombinase. Thus, subtle changes in the phospholipid composition of cells may control procoagulant and anticoagulant reactions differentially under both normal and pathological conditions.

Enhancement of Human Protein C Function by Site-directed Mutagenesis of the γ-Carboxyglutamic Acid Domain

This study reports properties of site-directed mutants of human protein C that display enhanced calcium and/or membrane binding properties. Mutants containing the S11G modification all showed increased affinity for membranes at saturating calcium concentration. Ser-11 is unique to human protein C, whereas all other vitamin K-dependent proteins contain glycine. This site is located in a compact region of the protein, close to a suggested membrane contact site. Additional changes of H10Q or S12N resulted in proteins with lower calcium requirement for membrane contact but without further increase in membrane affinity at saturating calcium. Mutations Q32E and N33D did not, by themselves, alter membrane affinity to a significant degree. These mutations were included in other mutant proteins and may contribute somewhat to higher function in these mutants. This family of mutants helped discriminate events that are necessary for protein-membrane binding. These include calcium binding to the free protein and subsequent protein-membrane contact. Depending on conditions of the assay used, the mutants displayed increased activity of the corresponding activated protein C (APC) derivatives. The degree of enhanced activity (up to 10-fold) was dependent on the concentration of phospholipid and quality of phospholipid (+/- phosphatidylethanolamine) used in the assay. This was expected, because APC is active in its membrane-associated form, which can be regulated by changes in either the protein or phospholipid. As expected, the largest impact of the mutants occurred at low phospholipid concentration and in the absence of phosphatidylethanolamine. The anticoagulant activity of all proteins was stimulated by protein S, with the greatest impact on the enhanced mutants. Whereas plasma containing Factor V:R506Q was partially resistant to all forms of APC, the enhanced variants were more active than normal APC. Protein C variants with enhanced function present new reagents for study of coagulation and may offer improved materials for biomedical applications.

A Phospholipid Acts as a Chaperone in Assembly of a Membrane Transport Protein

A mutant of Escherichia coli lacking phosphatidylethanolamine (PE) and a monoclonal antibody (mAb 4B1) directed against a conformationally sensitive epitope (4B1) of lactose permease were used to establish a novel role for a phospholipid in the assembly of a membrane protein. Epitope 4B1 is readily detectable in spheroplasts and right-side-out membrane vesicles from PE-containing but not from PE-deficient cells expressing lactose permease. Lactose permease from PE-containing membranes, but not from PE-deficient membranes, subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis is also recognized by mAb 4B1. If total E. coli phospholipids or PE (but not phosphatidylcholine, phosphatidylglycerol, or cardiolipin) are blotted on nitrocellulose sheets (Eastern blot) prior to transfer of proteins from SDS-polyacrylamide gels, the permease from PE-deficient cells regains its recognition by mAb 4B1. Therefore, PE is required during assembly to form epitope 4B1, but, once formed, sufficient "conformational memory" is retained in the permease to either retain or reform this epitope in the absence of PE. Lactose permease lacking epitope 4B1 can be induced to form the epitope if partially denatured and then renatured in the presence of PE specifically. These results establish for the first time a role for PE as a molecular chaperone in the assembly of the lactose permease.

Phosphatidylethanolamine augments factor VIIa-tissue factor activity: enhancement of sensitivity to phosphatidylserine.

The effect of phosphatidylethanolamine (PE) on the activity of the factor VIIa-tissue factor complex (fVIIa-TF) has been examined with respect to plasma clotting activity and activation of factor X (fX) in a purified system. Vesicles prepared by relipidating membrane-anchored TF (dcTF; TF1-244, lacking the C-terminal cytoplasmic tail) into phospholipid vesicles containing 6 mol % phosphatidylserine (PS) and increasing levels of PE up to 40 mol % (the balance consisting of phosphatidylcholine) were found to progressively shorten TF-initiated clotting in normal human plasma to levels comparable to those observed using dcTF relipidated with cephalin. The shortened clotting times were at least in part due to the ability of PE-containing membranes to better support the activation of fX by the fVIIa.TF complex, as vesicles with increased PE content yielded progressively higher initial rates of fX activation. Surprisingly, PE substantially altered the sensitivity of fX activation to low levels of PS, yielding near-maximal rates of activation at only 3 mol % PS compared to 15-20 mol % PS required in the absence of PE. The effect of PE was not synergistic with that of PS since PE did not increase fX activation rates at high levels of PS (20 mol %). Examination of the kinetic parameters for fX activation revealed that the majority of the effect of PE was in decreasing the apparent Km for fX.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of the hormone-sensitive triacylglycerol lipase from adipose tissue by phosphatidylethanolamine

The activity of a pigeon adipose tissue hormone-sensitive triacylglycerol lipase preparation was increased from 2- to 5-fold by the presence of phosphatidylethanolamine in assays with three different methods of preparing triolein substrates. Phosphatidylethanolamine from egg yolk produced the greatest stimulation of lipase activity; the stimulation was concentration-dependent but was not time-dependent. A comparable increase in triacylglycerol lipase activity due to phosphatidylethanolamine was also observed with enzyme preparations from chicken and rat adipose tissue. Phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin, Triton X-100 and sodium dodecyl sulfate all inhibited enzyme activity. Phosphatidylethanolamine had no effect on acid lipase activity in the pigeon adipose tissue preparation. Preincubation of the pigeon adipose tissue lipase with ATP, cyclic AMP and protein kinase resulted in a 2.15-fold activation of hydrolase activity determined in the absence of phosphatidylethanolamine. In contrast, non-activated and protein kinase-activated forms of the lipase were characterized as having very nearly the same activity in assays with substrate preparations containing phosphatidylethanolamine. The phosphatidylethanolamine-dependent stimulation of lipase activity was characterized kinetically as being due to an increase in maximal velocity. The modulation of the adipose tissue hormone-sensitive lipase activity by phospholipids could be involved in the hormonal regulation of lipolysis.

Circulating phospholipids in interstitial inflammatory fluid of the rabbit.

Using a sponge implantation model in the rabbit, we have examined the distribution and fatty acid composition of extravascular phospholipids in interstitial inflammatory fluid. Inflammatory fluid contained less phosphatidylserine and -inositol and a complete absence of phosphatidylethanolamine as compared to autologous plasma. Inflammatory fluid phospholipids contained an increased amount of oleic acid, decreased linoleic acid, and undetectable levels of arachidonic acid, despite unaltered levels of these fatty acids in other extravascular lipid classes as compared to plasma. Potential metabolic consequences of these findings are discussed.