Progesterone-induced Acrosome Exocytosis Requires Sequential Involvement of Calcium-independent Phospholipase A2β (iPLA2β) and Group X Secreted Phospholipase A2 (sPLA2)

Phospholipase A₂ (PLA₂) activity has been shown to be involved in the sperm acrosome reaction (AR), but the molecular identity of PLA₂ isoforms has remained elusive. Here, we have tested the role of two intracellular (iPLA₂β and cytosolic PLA₂α) and one secreted (group X) PLA₂s in spontaneous and progesterone (P4)-induced AR by using a set of specific inhibitors and knock-out mice. iPLA₂β is critical for spontaneous AR, whereas both iPLA₂β and group X secreted PLA₂ are involved in P4-induced AR. Cytosolic PLA₂α is dispensable in both types of AR. P4-induced AR spreads over 30 min in the mouse, and kinetic analyses suggest the presence of different sperm subpopulations, using distinct PLA₂ pathways to achieve AR. At low P4 concentration (2 μm), sperm undergoing early AR (0–5 min post-P4) rely on iPLA₂β, whereas sperm undergoing late AR (20–30 min post-P4) rely on group X secreted PLA₂. Moreover, the role of PLA₂s in AR depends on P4 concentration, with the PLA₂s being key actors at low physiological P4 concentrations (≤2 μm) but not at higher P4 concentrations (∼10 μm).     

Group VIB Calcium-Independent Phospholipase A2 (iPLA2γ) Regulates Platelet Activation,Hemostasis and Thrombosis in Mice

In platelets, group IVA cytosolic phospholipase A₂ (cPLA₂α) has been implicated as a key regulator in the hydrolysis of platelet membrane phospholipids, leading to pro-thrombotic thromboxane A₂ and anti-thrombotic 12-(S)-hydroxyeicosatetranoic acid production. However, studies using cPLA₂α-deficient mice have indicated that other PLA₂(s) may also be involved in the hydrolysis of platelet glycerophospholipids. In this study, we found that group VIB Ca²⁺-independent PLA₂ (iPLA₂γ)-deficient platelets showed decreases in adenosine diphosphate (ADP)-dependent aggregation and ADP- or collagen-dependent thromboxane A₂ production. Electrospray ionization mass spectrometry analysis of platelet phospholipids revealed that fatty acyl compositions of ethanolamine plasmalogen and phosphatidylglycerol were altered in platelets from iPLA₂γ-null mice. Furthermore, mice lacking iPLA₂γ displayed prolonged bleeding times and were protected against pulmonary thromboembolism. These results suggest that iPLA₂γ is an additional, long-sought-after PLA₂ that hydrolyzes platelet membranes and facilitates platelet aggregation in response to ADP.     

Characterization of FKGK18 as Inhibitor of Group VIA Ca2+-Independent Phospholipase A2 (iPLA2β): Candidate Drug for Preventing Beta-Cell Apoptosis and Diabetes

Ongoing studies suggest an important role for iPLA₂β in a multitude of biological processes and it has been implicated in neurodegenerative, skeletal and vascular smooth muscle disorders, bone formation, and cardiac arrhythmias. Thus, identifying an iPLA₂βinhibitor that can be reliably and safely used in vivo is warranted. Currently, the mechanism-based inhibitor bromoenol lactone (BEL) is the most widely used to discern the role of iPLA₂β in biological processes. While BEL is recognized as a more potent inhibitor of iPLA₂ than of cPLA₂ or sPLA₂, leading to its designation as a “specific” inhibitor of iPLA₂, it has been shown to also inhibit non-PLA₂ enzymes. A potential complication of its use is that while the S and R enantiomers of BEL exhibit preference for cytosol-associated iPLA₂β and membrane-associated iPLA₂γ, respectively, the selectivity is only 10-fold for both. In addition, BEL is unstable in solution, promotes irreversible inhibition, and may be cytotoxic, making BEL not amenable for in vivo use. Recently, a fluoroketone (FK)-based compound (FKGK18) was described as a potent inhibitor of iPLA₂β. Here we characterized its inhibitory profile in beta-cells and find that FKGK18: (a) inhibits iPLA₂β with a greater potency (100-fold) than iPLA₂γ, (b) inhibition of iPLA₂β is reversible, (c) is an ineffective inhibitor of α-chymotrypsin, and (d) inhibits previously described outcomes of iPLA₂β activation including (i) glucose-stimulated insulin secretion, (ii) arachidonic acid hydrolysis; as reflected by PGE2 release from human islets, (iii) ER stress-induced neutral sphingomyelinase 2 expression, and (iv) ER stress-induced beta-cell apoptosis. These findings suggest that FKGK18 is similar to BEL in its ability to inhibit iPLA₂β. Because, in contrast to BEL, it is reversible and not a non-specific inhibitor of proteases, it is suggested that FKGK18 is more ideal for ex vivo and in vivo assessments of iPLA₂β role in biological functions.     

Group VIA PLA2 (iPLA2β) is activated upstream of p38 mitogen-activated protein kinase (MAPK) in pancreatic islet β-cell signaling

Group VIA phospholipase A(2) (iPLA(2)β) in pancreatic islet β-cells participates in glucose-stimulated insulin secretion and sarco(endo)plasmic reticulum ATPase (SERCA) inhibitor-induced apoptosis, and both are attenuated by pharmacologic or genetic reductions in iPLA(2)β activity and amplified by iPLA(2)β overexpression. While exploring signaling events that occur downstream of iPLA(2)β activation, we found that p38 MAPK is activated by phosphorylation in INS-1 insulinoma cells and mouse pancreatic islets, that this increases with iPLA(2)β expression level, and that it is stimulated by the iPLA(2)β reaction product arachidonic acid. The insulin secretagogue D-glucose also stimulates β-cell p38 MAPK phosphorylation, and this is prevented by the iPLA(2)β inhibitor bromoenol lactone. Insulin secretion induced by d-glucose and forskolin is amplified by overexpressing iPLA(2)β in INS-1 cells and in mouse islets, and the p38 MAPK inhibitor PD169316 prevents both responses. The SERCA inhibitor thapsigargin also stimulates phosphorylation of both β-cell MAPK kinase isoforms and p38 MAPK, and bromoenol lactone prevents both events. Others have reported that iPLA(2)β products activate Rho family G-proteins that promote MAPK kinase activation via a mechanism inhibited by Clostridium difficile toxin B, which we find to inhibit thapsigargin-induced β-cell p38 MAPK phosphorylation. Thapsigargin-induced β-cell apoptosis and ceramide generation are also prevented by the p38 MAPK inhibitor PD169316. These observations indicate that p38 MAPK is activated downstream of iPLA(2)β in β-cells incubated with insulin secretagogues or thapsigargin, that this requires prior iPLA(2)β activation, and that p38 MAPK is involved in the β-cell functional responses of insulin secretion and apoptosis in which iPLA(2)β participates.     

Imaging decreased brain docosahexaenoic acid metabolism and signaling in iPLA2�� (VIA)-deficient mice

Ca²⁺-independent phospholipase A₂β (iPLA₂β) selectively hydrolyzes docosahexaenoic acid (DHA, 22:6n-3) in vitro from phospholipid. Mutations in the PLA2G6 gene encoding this enzyme occur in patients with idiopathic neurodegeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation, whereas mice lacking PLA2G6 show neurological dysfunction and neuropathology after 13 months. We hypothesized that brain DHA metabolism and signaling would be reduced in 4-month-old iPLA₂β-deficient mice without overt neuropathology. Saline or the cholinergic muscarinic M_1,3,5 receptor agonist arecoline (30 mg/kg) was administered to unanesthetized iPLA₂β^−/−, iPLA₂β^+/−, and iPLA₂β^+/+ mice, and [1-¹⁴C]DHA was infused intravenously. DHA incorporation coefficients k* and rates J_in, representing DHA metabolism, were determined using quantitative autoradiography in 81 brain regions. iPLA₂β^−/− or iPLA₂β^+/− compared with iPLA₂β^+/+ mice showed widespread and significant baseline reductions in k* and J_in for DHA. Arecoline increased both parameters in brain regions of iPLA₂β^+/+ mice but quantitatively less so in iPLA₂β^−/− and iPLA₂β^+/− mice. Consistent with iPLA₂β’s reported ability to selectively hydrolyze DHA from phospholipid in vitro, iPLA₂β deficiency reduces brain DHA metabolism and signaling in vivo at baseline and following M_1,3,5 receptor activation. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.     

Disturbed brain phospholipid and docosahexaenoic acid metabolism in calcium-independent phospholipase A(2)-VIA (iPLA(2)β)-knockout mice

Calcium-independent phospholipase A(2) group VIA (iPLA(2)β) releases docosahexaenoic acid (DHA) from phospholipids in vitro. Mutations in the iPLA(2)β gene, PLA2G6, are associated with dystonia-parkinsonism and infantile neuroaxonal dystrophy. To understand the role of iPLA(2)β in brain, we applied our in vivo kinetic method using radiolabeled DHA in 4 to 5-month-old wild type (iPLA(2)β(+/+)) and knockout (iPLA(2)β(-/-)) mice, and measured brain DHA kinetics, lipid concentrations, and expression of PLA(2), cyclooxygenase (COX), and lipoxygenase (LOX) enzymes. Compared to iPLA(2)β(+/+) mice, iPLA(2)β(-/-) mice showed decreased rates of incorporation of unesterified DHA from plasma into brain phospholipids, reduced concentrations of several fatty acids (including DHA) esterified in ethanolamine- and serine-glycerophospholipids, and increased lysophospholipid fatty acid concentrations. DHA turnover in brain phospholipids did not differ between genotypes. In iPLA(2)β(-/-) mice, brain levels of iPLA(2)β mRNA, protein, and activity were decreased, as was the iPLA(2)γ (Group VIB PLA(2)) mRNA level, while levels of secretory sPLA(2)-V mRNA, protein, and activity and cytosolic cPLA(2)-IVA mRNA were increased. Levels of COX-1 protein were decreased in brain, while COX-2 protein and mRNA were increased. Levels of 5-, 12-, and 15-LOX proteins did not differ significantly between genotypes. Thus, a genetic iPLA(2)β deficiency in mice is associated with reduced DHA metabolism, profound changes in lipid-metabolizing enzyme expression (demonstrating lack of redundancy) and of phospholipid fatty acid content of brain (particularly of DHA), which may be relevant to neurologic abnormalities in humans with PLA2G6 mutations.     

Proinflammatory Secreted Phospholipase A2 Type IIA (sPLA-IIA) Induces Integrin Activation through Direct Binding to a Newly Identified Binding Site (Site 2) in Integrins αvβ3, α4β1, and α5β1

Integrins are activated by signaling from inside the cell (inside-out signaling) through global conformational changes of integrins. We recently discovered that fractalkine activates integrins in the absence of CX3CR1 through the direct binding of fractalkine to a ligand-binding site in the integrin headpiece (site 2) that is distinct from the classical RGD-binding site (site 1). We propose that fractalkine binding to the newly identified site 2 induces activation of site 1 though conformational changes (in an allosteric mechanism). We reasoned that site 2-mediated activation of integrins is not limited to fractalkine. Human secreted phospholipase A2 type IIA (sPLA2-IIA), a proinflammatory protein, binds to integrins αvβ3 and α4β1 (site 1), and this interaction initiates a signaling pathway that leads to cell proliferation and inflammation. Human sPLA2-IIA does not bind to M-type receptor very well. Here we describe that sPLA2-IIA directly activated purified soluble integrin αvβ3 and transmembrane αvβ3 on the cell surface. This activation did not require catalytic activity or M-type receptor. Docking simulation predicted that sPLA2-IIA binds to site 2 in the closed-headpiece of αvβ3. A peptide from site 2 of integrin β1 specifically bound to sPLA2-IIA and suppressed sPLA2-IIA-induced integrin activation. This suggests that sPLA2-IIA activates αvβ3 through binding to site 2. sPLA2-IIA also activated integrins α4β1 and α5β1 in a site 2-mediated manner. We recently identified small compounds that bind to sPLA2-IIA and suppress integrin-sPLA2-IIA interaction (e.g. compound 21 (Cmpd21)). Cmpd21 effectively suppressed sPLA2-IIA-induced integrin activation. These results define a novel mechanism of proinflammatory action of sPLA2-IIA through integrin activation.     

Only One 3′-Hydroxyl Group Of ppp 5′A 2′p 5′A 2′p 5′A(2–5A) is Required for Activation of the Mouse 2–5A-Dependent Endonuclease

Abstract

The 3′-hydroxyl groups of each of the adenosines of 2–5A triraer (ppp5′A2′p5′A2′p5′A) were sequentially replaced by hydrogen through a phosphotriester synthetic approach. Biochemical evaluation of these analogs led to the conclusion that only the 3′-hydroxy group of the second adenosine is required for activation of RNase L.     

Group VIA Ca-independent phospholipase A2 (iPLA2β) and its role in β-cell programmed cell death

Activation of phospholipases A₂ (PLA₂s) leads to the generation of biologically active lipid mediators that can affect numerous cellular events. The Group VIA Ca²⁺-independent PLA₂, designated iPLA₂β, is active in the absence of Ca²⁺, activated by ATP, and inhibited by the bromoenol lactone suicide inhibitor (BEL). Over the past 10–15 years, studies using BEL have demonstrated that iPLA₂β participates in various biological processes and the recent availability of mice in which iPLA₂β expression levels have been genetically-modified are extending these findings. Work in our laboratory suggests that iPLA₂β activates a unique signaling cascade that promotes β-cell apoptosis. This pathway involves iPLA₂β dependent induction of neutral sphingomyelinase, production of ceramide, and activation of the intrinsic pathway of apoptosis. There is a growing body of literature supporting β-cell apoptosis as a major contributor to the loss of β-cell mass associated with the onset and progression of Type 1 and Type 2 diabetes mellitus. This underscores a need to gain a better understanding of the molecular mechanisms underlying β-cell apoptosis so that improved treatments can be developed to prevent or delay the onset and progression of diabetes mellitus. Herein, we offer a general review of Group VIA Ca²⁺-independent PLA₂ (iPLA₂β) followed by a more focused discussion of its participation in β-cell apoptosis. We suggest that iPLA₂β-derived products trigger pathways which can lead to β-cell apoptosis during the development of diabetes.     

Interfacial Kinetic and Binding Properties of Mammalian Group IVB Phospholipase A2 (cPLA2β) and Comparison with the Other cPLA2 Isoforms

The cytosolic (group IV) phospholipase A₂ (cPLA₂s) family contains six members. We have prepared recombinant proteins for human α, mouse β, human γ, human δ, human ϵ, and mouse ζ cPLA₂s and have studied their interfacial kinetic and binding properties in vitro. Mouse cPLA₂β action on phosphatidylcholine vesicles is activated by anionic phosphoinositides and cardiolipin but displays a requirement for Ca²⁺ only in the presence of cardiolipin. This activation pattern is explained by the effects of anionic phospholipids and Ca²⁺ on the interfacial binding of mouse cPLA₂β and its C2 domain to vesicles. Ca²⁺-dependent binding of mouse cPLA₂β to cardiolipin-containing vesicles requires a patch of basic residues near the Ca²⁺-binding surface loops of the C2 domain, but binding to phosphoinositide-containing vesicles does not depend on any specific cluster of basic residues. Human cPLA₂δ also displays Ca²⁺- and cardiolipin-enhanced interfacial binding and activity. The lysophospholipase, phospholipase A₁, and phospholipase A₂ activities of the full set of mammalian cPLA₂s were quantified. The relative level of these activities is very different among the isoforms, and human cPLA₂δ stands out as having relatively high phospholipase A₁ activity. We also tested the susceptibility of all cPLA₂ family members to a panel of previously reported inhibitors of human cPLA₂α and analogs of these compounds. This led to the discovery of a potent and selective inhibitor of mouse cPLA₂β. These in vitro studies help determine the regulation and function of the cPLA₂ family members.     

Calcium-independent phospholipase A2γ (iPLA2γ) and its roles in cellular functions and diseases

Calcium-independent phospholipase A₂γ (iPLA₂γ)/patatin-like phospholipase domain-containing lipase 8 (PNPLA8) is one of the iPLA₂ enzymes, which do not require Ca²⁺ ion for their activity. iPLA₂γ is a membrane-bound enzyme with unique features, including the utilization of four distinct translation initiation sites and the presence of mitochondrial and peroxisomal localization signals. This enzyme is preferentially distributed in the mitochondria and peroxisomes and is thought to be responsible for the maintenance of lipid homeostasis in these organelles. Thus, both the overexpression and the deletion of iPLA₂γ in vivo caused mitochondrial abnormalities and dysfunction. Roles of iPLA₂γ in lipid mediator production and cytoprotection against oxidative stress have also been suggested by in vitro and in vivo studies. The dysregulation of iPLA₂γ can therefore be a critical factor in the development of many diseases, including metabolic diseases and cancer. In this review, we provide an overview of the biochemical properties of iPLA₂γ and then summarize the current understanding of the in vivo roles of iPLA₂γ revealed by knockout mouse studies.     

5′-O-Dephosphorylated 2′,5′-oligoadenylate (2-5A) with 8-methyladenosine at the 2′-terminus activates human RNase L

Human ribonuclease L (RNase L), an interferon-induced endoribonuclease, becomes enzymatically active after binding to 2-5A. The 5′-phosphoryl group of 2-5A is reportedly necessary for the conformational change leading to RNase L activation. However, we found that 5′-O-dephosphorylated 2-5A tetramer analogs with 8-methyladenosine at the 2′-terminus were more effective as an activator of RNase L than the parent 2-5A tetramer. Introduction of 8-methyladenosine is thought to induce a dramatic shift of 2-5A in the binding site of RNase L.     

Combinatorial role of two G-quadruplexes in 5′ UTR of transforming growth factor β2 (TGFβ2)

Albeit most studies demonstrate the inhibitory role of G-quadruplex in the 5′ Untranslated Region (5′ UTR), our previous report depicted its completely contrasting activating role in the 5′ UTR of transforming growth factor β2 (TGFβ2) mRNA. Therefore, we screened the 5′ UTR of TGFβ2 manually and identified a second putative G-quadruplex sequence. Our in vitro experiments encompassing CD and UV spectroscopy confirmed the ability of this sequence to form a G-quadruplex and in cellulo studies further indicated its activating role in modulation of TGFβ2 gene expression. Our study suggests that these two 5′ UTR G-quadruplexes most probably operate additively to substantially increase gene expression of TGFβ2. Neither of the two G-quadruplex alone is sufficient enough to drastically augment protein production. Both G-quadruplexes are essential for increasing protein output. To the best of our knowledge, our study is the first report showcasing the combinatorial role of two G-quadruplexes in the 5′ UTR of an mRNA.     

A Convenient Synthesis of 9-(5′-DEOXY-β-D-Allofuranosyl)-Adenine (5′-Homoadenosine)

Abstract

The synthesis of 5′-homoadenosine, a chain extended analogue of adenosine, has been developed by coupling the appropriately protected deoxyallofuranose derivative with adenine.     

A Convergent Regiospecific Synthesis of the Lariat-Trinucleotides and A2′p 5′G and A2′p 5′G 3′p 5′C from A O4-2-Nitrophenyl) -Uridine Building Block

Abstract

Total synthesis of title compounds 1_ and 2_ from a common intermediate 7 is reported using the phosphotriester-phosphiteamidite approach. Appropriate NMR evidence has been presented in support of the regiospecific synthesis of target molecules in addition to enzymatic analysis. Present work clearly shows that the NMR evidence is mandatory to establish the isomeric purity of branched RNA molecules; enzymatic or/and electrophoretic analysis alone as tools for confirmation of branched RNA structures can be misleading.     

Smooth muscle-specific expression of calcium-independent phospholipase A2β (iPLA2β) participates in the initiation and early progression of vascular inflammation and neointima formation

Whether group VIA phospholipase A(2) (iPLA(2)β) is involved in vascular inflammation and neointima formation is largely unknown. Here, we report that iPLA(2)β expression increases in the vascular tunica media upon carotid artery ligation and that neointima formation is suppressed by genetic deletion of iPLA(2)β or by inhibiting its activity or expression via perivascular delivery of bromoenol lactone or of antisense oligonucleotides, respectively. To investigate whether smooth muscle-specific iPLA(2)β is involved in neointima formation, we generated transgenic mice in which iPLA(2)β is expressed specifically in smooth muscle cells and demonstrate that smooth muscle-specific expression of iPLA(2)β exacerbates ligation-induced neointima formation and enhanced both production of proinflammatory cytokines and vascular infiltration by macrophages. With cultured vascular smooth muscle cell, angiotensin II, arachidonic acid, and TNF-α markedly induce increased expression of IL-6 and TNF-α mRNAs, all of which were suppressed by inhibiting iPLA(2)β activity or expression with bromoenol lactone, antisense oligonucleotides, and genetic deletion, respectively. Similar suppression also results from genetic deletion of 12/15-lipoxygenase or inhibiting its activity with nordihydroguaiaretic acid or luteolin. Expression of iPLA(2)β protein in cultured vascular smooth muscle cells was found to depend on the phenotypic state and to rise upon incubation with TNF-α. Our studies thus illustrate that smooth muscle cell-specific iPLA(2)β participates in the initiation and early progression of vascular inflammation and neointima formation and suggest that iPLA(2)β may represent a novel therapeutic target for preventing cardiovascular diseases.     

A Solid-Phase Synthesis of 2′,5′-Linked Oligoadenylates (2-5A)

Abstract

2′,5′-Oligoadenylate 5′-triphosphates (2-5A) as products of 2-5A synthetase and activators of ribonuclease L (RNase L), are mediators in one of the mechanisms of interferon′s antiviral action. Upon activation, RNase L inhibits protein synthesis due to the degradation of RNAs. This activity of 2-5A could possibly find an application in virus or cancer chemotherapy, but two major barriers prevent the use of 2′,5′-linked oligoadenylates as therapeutic agents. The 2-5A is readily degraded by a 2′,5′ phosphodiesterase and as a highly negatively charged molecule, is not readily taken up by cells. One possible solution to this latter limitation might be found in chemical modifications of the 2-5A structure. Many analogues of 2-5A have been already obtained with modified base, ribose or phosphate moieties. While these have provided some important information about the enzyme- activator interactions, the cell permeability problem still remains unsolved. One of the major obstacles in this study is lack of a convenient method of synthesis of 2′,5′ ribonucleotides of widely varying structure.     

Studies of the Pharmacokinetics and Toxicology of 2′,3′-Dideoxy-β-L-5-fluorocytidine (β-L-FddC) and 2′,3′-Dideoxy-β-L-cytidine (β-L-ddC) In Vivo; and Synthesis and Antiviral Evaluations of 2′,3′-Dideoxy-β-L-5-azacytidine

Abstract

The pharmacokinetics and toxicology of 2′,3′-dideoxy-β-L-5-fluorocytidine (β-L-FddC) and 2′,3′-dideoxy-β-L-cytidine (β-L-ddC) in mice was investigated. In addition, 2′,3′-dideoxy-β-L-5-azacytidine (β-L-5-aza-ddC) and its α-L-anomer (α-L-5-aza-ddC) were synthesized by coupling the silylated 5-azacytosine derivative with 1-O-acetyl-5-O-(tert-butyldimethylsilyl)-2,3-dideoxy-L-ribofuranose, followed by separation of the α-and β-anomers and were evaluated in vitro against HBV and HIV. β-L-5-aza-ddC was found to show significant anti-HBV activity at approximately the same level as 2′,3′-dideoxy-β-D-cytidine (ddC), which is a known anti-HBV agent. β-L-5-aza-ddC was not cytotoxic to L1210, P388, S-180, and CCRF-CEM cells up to a concentration of 100 μ. Conversely, the α-L-anomer was not active against HBV at the same concentration.     

Substrate Efflux Propensity Is the Key Determinant of Ca2+-independent Phospholipase A-β (iPLAβ)-mediated Glycerophospholipid Hydrolysis

The A-type phospholipases (PLAs) are key players in glycerophospholipid (GPL) homeostasis and in mammalian cells; Ca²⁺-independent PLA-β (iPLAβ) in particular has been implicated in this essential process. However, the regulation of this enzyme, which is necessary to avoid futile competition between synthesis and degradation, is not understood. Recently, we provided evidence that the efflux of the substrate molecules from the bilayer is the rate-limiting step in the hydrolysis of GPLs by some secretory (nonhomeostatic) PLAs. To study whether this is the case with iPLAβ as well, a mass spectrometric assay was employed to determine the rate of hydrolysis of multiple saturated and unsaturated GPL species in parallel using micelles or vesicle bilayers as the macrosubstrate. With micelles, the hydrolysis decreased with increasing acyl chain length independent of unsaturation, and modest discrimination between acyl positional isomers was observed, presumably due to the differences in the structure of the sn-1 and sn-2 acyl-binding sites of the protein. In striking contrast, no significant discrimination between positional isomers was observed with bilayers, and the rate of hydrolysis decreased with the acyl chain length logarithmically and far more than with micelles. These data provide compelling evidence that efflux of the substrate molecule from the bilayer, which also decreases monotonously with acyl chain length, is the rate-determining step in iPLAβ-mediated hydrolysis of GPLs in membranes. This finding is intriguing as it may help to understand how homeostatic PLAs are regulated and how degradation and biosynthesis are coordinated.