Phospholipases A2: structure and function

Phospholipases A₂ (PLA₂) are an example of peripheral membrane proteins that must first bind to the phospholipid interface to allow phospholipid hydrolysis to occur. The interfacial (membrane) binding step plays a crucial role in the biological function of the enzyme and membrane affinity may be determined both by the phospholipid composition of the membrane and the properties of the interfacial binding surface of the protein. There are now three major categories of these enzymes, secreted PLA₂ (sPLA₂), cytosolic PLA₂ (cPLA₂) and Ca²⁺‐independent PLA₂ (iPLA₂). The structure and function of each category is discussed highlighting how both membrane binding and phospholipid substrate specificity may contribute to the overall functions of these enzymes.     

The Inactivation Mechanism of Human Group IIA Phospholipase A2 by Scalaradial

Secretory phospholipases A₂ (sPLA₂s) are implicated in the pathogenesis of several inflammation diseases, such as rheumatoid arthritis, septic shock, psoriasis, and asthma. Thus, an understanding of their inactivation mechanisms could be useful for the development of new classes of chemical selective inhibitors. In the marine environment, several bioactive terpenoids possess interesting anti‐inflammatory activity, often through covalent and/or noncovalent inactivation of sPLA₂. Herein, we report the molecular mechanism of human group IIA phospholipase A₂ (sPLA₂‐IIA) inactivation by Scalaradial (SLD), a marine 1,4‐dialdehyde terpenoid isolated from the sponge Cacospongia mollior and endowed with a significant anti‐inflammatory profile. Our results have been collected by a combination of biochemical approaches, advanced mass spectrometry, surface plasmon resonance, and molecular modeling. These suggest that SLD acts as a competitive inhibitor. Indeed, the sPLA₂‐IIA inactivation process seems to be driven by the noncovalent recognition process of SLD in the enzyme active site and by chelation of the catalytic calcium ion. In contrast, covalent modification of the enzyme by the SLD dialdehyde moiety emerges as only a minor side event in the ligand–enzyme interaction. These results could be helpful for the rational design of new PLA₂ inhibitors that would be able to selectively target the enzyme active site.     

Electronic storage of LC/ESI‐MS total ion current profiles of total lipid extracts and multidimensional lipidomic analysis of plasma lipoproteins

We have utilized the Hewlett‐Packard LC/ESI‐MS technology to store electronic records of total ion current profiles of molecular species of various natural and peroxidized phospholipids of native and auto‐oxidized plasma lipoproteins before and after digestion with secretory phospholipase A₂ (sPLA₂). The present report illustrates the successful utilization of the stored data in several originally un‐intended applications, including chemical, physico‐chemical and enzymatic alterations of the lipoprotein lipids. The inclusion of lipid samples from enzyme and control incubations adds a dynamic aspect to multidimensional lipidomic analysis of plasma lipoproteins.     

Hydrolysis of glycerophosphocholine epoxides by human group IIA,V, and X secretory phospholipases A2

This study was prompted by recent reports that epoxyeicosatrienoic (EET) and epoxyeicosatetraenoic (EEQ) acids accelerate tumor growth and metastasis by stimulation of angiogenesis, while eicosapentaenoic (EPA) and epoxydocosapentaenoic (EDP) acids inhibit angiogenesis, tumor growth, and metastasis. Cytochrome P450 epoxygenases convert arachidonic to EET, eicosapentaenoic acid to EEQ, and docosahexaenoic acid to EDP, which are found both in free form and esterified to glycerophosphocholine (GPC). Both free and esterified epoxy (EP) acids are also formed during lipid autoxidation. For biological activity, the GPC-EP requires hydrolysis, which we presumed could occur by sPLA₂s located in proximity of lipoproteins carrying the lipid epoxides. The plasma lipoproteins were isolated by ultracentrifugation and analyzed by LC/ESI-MS. The GPC-EPs were identified by reference to standards and to retention times of phospholipid masses. The GPC-EP monoepoxides (corrected for isobaric ether overlaps) in stored human LDL, HDL, HDL₃, or APHDL ranged from 0 to 1 nmol/mg protein, but during 4-h incubation at 37°C increased to 1–5 nmol/mg protein. An incubation of autoxidized LDL, HDL, or HDL₃ with 1 μg/ml of group V or X sPLA₂ resulted in complete hydrolysis of diacyl GPC epoxide esters. Group IIA sPLA₂ at 1 μg/ml failed to produce significant hydrolysis in 4 h, but at 2.5 μg/ml in 8 h yielded almost 80% hydrolysis, which represented complete diacyl GPC-EP hydrolysis. The present study shows that group IIA, V, and X sPLA₂s are capable of extensive hydrolysis of PtdCho epoxides of autoxidized plasma lipoproteins. Therefore, all three human sPLA₂s were potentially capable of inducing epoxide biological activity in vivo.     

Molecular cloning,expression, and characterization of secretory phospholipase A<Subscript>2</Subscript> in tobacco

Phospholipase A₂ (PLA₂) activity was investigated in various tissues of tobacco (Nicotiana tabacum). PLA₂ activity in the flower was 15 times higher than that in the leaf, stem, and root. PLA₂ activity in the flower appears to have originated from both Ca²⁺-dependent and-independent PLA₂. A cDNA clone for protein with homology to animal secretory PLA₂ (sPLA₂), denoted as Nt PLA₂, was isolated from the tobacco flower. The cDNA of Nt PLA₂ encoded a mature protein of 127 amino acid residues with a putative signal peptide of 30 residues. The amino acid sequence for mature Nt PLA₂ contains 12 cysteines, a Ca²⁺ binding loop, and a catalytic domain that are commonly conserved in animal sPLA₂. The Nt PLA₂ mRNA was mainly expressed in the root and stem of tobacco. The recombinant Nt PLA₂ was expressed as a fusion protein with thioredoxin in Escherichia coli. From the bacterial cell lysate, the fusion protein was recovered in soluble form and cleaved by Factor Xa proteinase. Then the recombinant mature Nt PLA₂ was purified by ion exchange chromatography. It was discovered that the purified Nt PLA₂ essentially requires Ca²⁺, for the enzyme activity when the activity was determined using mixed-micellar phospholipid substrates with sodium cholate. The optimal activity of Nt PLA₂ was at pH 8–10 when PC was used as a substrate.     

Novel Very Long-Chain α-Methoxylated Δ5,9 Fatty Acids from the Sponge Asteropus niger Are Effective Inhibitors of Topoisomerases IB

The novel fatty acids (2R,5Z,9Z)‐2‐methoxy‐25‐methyl‐5,9‐hexacosadienoic acid ( 1a ) and (2R,5Z,9Z)‐2‐methoxy‐24‐methyl‐5,9‐hexacosadienoic acid ( 1b ) were isolated in 80 % purity from the Caribbean sponge Asteropus niger by chloroform/methanol extraction followed by solvent partitioning and silica gel column chromatography. The compounds were characterized by utilizing a combination of gas chromatography‐mass spectrometry, nuclear magnetic resonance, and circular dichroism. Acids 1a and 1b were not detected in the phospholipids (PtdCho and PtdIns) of the sponge, but rather as free FA and possibly in glycosylceramides. The mixtures of 1a and 1b displayed cytotoxicity towards THP‐1 and HepG2 cells with EC_50's between 41 and 35 μg/mL. Apoptosis was not the preferred mode of cell death induced by 1a – 1b in the THP‐1 cells. This implies other types of cytotoxicity mechanisms, such as membrane disruption and/or the inhibition (EC₅₀ = 1.8 μg/mL) of the human topoisomerase IB enzyme (hTopIB), with a mechanism of inhibition different from the one displayed by camptothecin (CPT). In a separate experiment, the mixture of 1a and 1b also displayed cytotoxicity towards ex vivo mouse splenocytes infected with Leishmania infantum amastigotes (IC₅₀ = 0.17 mg/mL) and free living promastigotes (IC₅₀ = 0.34 mg/mL). It was also found that the FA were inhibitory of the Leishmania topoisomerase IB (LTopIB) with an EC₅₀ = 5.1 μg/mL. Taken together, 1a and 1b represent a new class of FA with potential as TopIB inhibitors that preferentially inhibit hTopIB over LTopIB.     

Degumming rice bran oil using phospholipase‐A1

The efficacy of enzymatic degumming was assessed using the third generation phospholipase‐A₁, Lecitase®‐Ultra (EC 3.1.1.3) from Thermomyces lanuginosa/Fusarium oxysporum with different qualities of crude rice bran oil. The phosphorus content in the oil reduced to ～10 mg/kg from an initial level of 390 mg/kg after 2 h of incubation period at 50°C. However, in the solvent‐phase degumming, there was practically no phospholipid reduction at lower water content (2%) due to the poor contact between the highly nonpolar solvent and the aqueous phase (citric acid, NaOH, and enzyme solutions). Increasing the water content to 20% reduced the phosphorus level in the degummed‐oil to 71 mg/kg but did not match the performance of oil‐phase degumming. The degumming efficiency of Lecitase®‐Ultra was effective in oil‐phase and suitable for practical application. Solvent‐phase enzymatic degumming offers more benefits but needs greater efforts to overcome the challenges.     

Liposomes Targeting P21 Activated Kinase-1 (PAK-1) and Selective for Secretory Phospholipase A2 (sPLA2) Decrease Cell Viability and Induce Apoptosis in Metastatic Triple-Negative Breast Cancer Cells

P21 activated kinases (or group I PAKs) are serine/threonine kinases whose expression is altered in prostate and breast cancers. PAK-1 activity is inhibited by the small molecule “Inhibitor targeting PAK-1 activation-3” (IPA-3), which has selectivity for PAK-1 but is metabolically unstable. Secretory Group IIA phospholipase A₂ (sPLA₂) expression correlates to increased metastasis and decreased survival in many cancers. We previously designed novel liposomal formulations targeting both PAK-1 and sPLA_2, called Secretory Phospholipase Responsive liposomes or SPRL-IPA-3, and demonstrated their ability to alter prostate cancer growth. The efficacy of SPRL against other types of cancers is not well understood. We addressed this limitation by determining the ability of SPRL to induce cell death in a diverse panel of cells representing different stages of breast cancer, including the invasive but non-metastatic MCF-7 cells, and metastatic triple-negative breast cancer (TNBC) cells such as MDA-MB-231, MDA-MB-468, and MDA-MB-435. We investigated the role of sPLA₂ in the disposition of these liposomes by comparing the efficacy of SPRL-IPA-3 to IPA-3 encapsulated in sterically stabilized liposomes (SSL-IPA-3), a formulation shown to be less sensitive to sPLA₂. Both SSL-IPA-3 and SPRL-IPA-3 induced time- and dose-dependent decreases in MTT staining in all cell lines tested, but SPRL-IPA-3-induced effects in metastatic TNBC cell lines were superior over SSL-IPA-3. The reduction in MTT staining induced by SPRL-IPA-3 correlated to the expression of Group IIA sPLA₂. sPLA₂ expression also correlated to increased induction of apoptosis in TNBC cell lines by SPRL-IPA-3. These data suggest that SPRL-IPA-3 is selective for metastatic TNBC cells and that the efficacy of SPRL-IPA-3 is mediated, in part, by the expression of Group IIA sPLA₂.     

Anaerobic lipoxygenase activity from Chlorella pyrenoidosa responsible for the cleavage of the 13‐hydroperoxides of linoleic and linolenic acids

An enzyme from the alga Chlorella pyrenoidosa, previously identified as a hydroperoxide lyase (HPLS), cleaves the 13‐hydroperoxide derivatives of linoleic and linolenic acids into a volatile C5 fragment and a C13 oxo‐product, 13‐oxo‐9(Z),11(E)tridecadienoic acid (13‐OTA). Gas chromatography/mass spectrometry (GC/MS) headspace analysis of the volatile products indicated the formation of pentane when the substrate was the 13‐hydroperoxide derivative of linoleic acid, whereas a more complex mixture of hydrocarbons was formed when the 13‐hydroperoxide derivative of linolenic acid was the substrate. Analysis of the nonvolatile products by GC/MS and liquid chromatography/mass spectrometry (LC/MS) indicated the formation of 13‐OTA along with the 13‐ketone derivative. This enzymatic activity was inhibited by oxygen but was restored with nitrogen. The enzymatic cleavage activity was coincidental in purified fractions with lipoxygenase activity that produced the 13‐ and 9‐hydroperoxide derivatives of linolenic acid. The results suggest that the enzymatic cleavage activity in Chlorella pyrenoidosa was not a consequence of hydroperoxide lyase activity as previously thought, but was due to anaerobic lipoxygenase activity. This enzyme fraction was purified by (NH₄)₂ SO₄ precipitation, gel filtration, and hydrophobic interaction chromatography. The purified enzyme has an approximate MW of 120 KDa and maximum activity at pH 8.0.     

Characterization of Secretory Phospholipase A<Subscript>2</Subscript> with Phospholipase A<Subscript>1</Subscript> Activity in Tobacco, <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> (L.)

A cDNA encoding protein with homology to plant secretory phospholipase A₂ (sPLA₂), denoted as Nt1 PLA₂, was isolated from tobacco (Nicotiana tabacum). The cDNA encodes a mature protein of 118 amino acid residues with a putative signal peptide of 29 residues. The mature form of Nt1 PLA₂ has 12 cysteines, Ca²⁺ binding loop and catalytic site domain that are commonly conserved in plant sPLA₂s. The recombinant Nt1 PLA₂ was expressed as a fusion protein with thioredoxin in E. coli BL21 cells and was purified by an ion exchange chromatography after digestion of the fusion proteins by Factor Xa protease to obtain the mature form. Interestingly, Nt1 PLA₂ could hydrolyze the ester bond at the sn-1 position of glycerophospholipids as well as at the sn-2 position, when the activities were determined using mixed-micellar phospholipids with sodium cholate. Both activities for the sn-1 and -2 positions of glycerophospholipids required Ca²⁺ essentially, and maximal activities were found in an alkaline region when phosphatidylcholine, phosphatidylglycerol or phosphatidylethanolamine was used as a substrate. The level of Nt1 PLA₂ mRNA was detected at a higher level in tobacco flowers than stem, leaves and roots, and was induced by salicylic acid.     

Biochemical Characterization and Mechanistic Analysis of the Levoglucosan Kinase from Lipomyces starkeyi

Levoglucosan kinase (LGK) catalyzes the simultaneous hydrolysis and phosphorylation of levoglucosan (1,6‐anhydro‐β‐d ‐glucopyranose) in the presence of Mg²⁺–ATP. For the Lipomyces starkeyi LGK, we show here with real‐time in situ NMR spectroscopy at 10 °C and pH 7.0 that the enzymatic reaction proceeds with inversion of anomeric stereochemistry, resulting in the formation of α‐d ‐glucose‐6‐phosphate in a manner reminiscent of an inverting β‐glycoside hydrolase. Kinetic characterization revealed the Mg²⁺ concentration for optimum activity (20–50 mm ), the apparent binding of levoglucosan (K_m=180 mm ) and ATP (K_m=1.0 mm ), as well as the inhibition by ADP (K_i=0.45 mm ) and d ‐glucose‐6‐phosphate (IC₅₀=56 mm ). The enzyme was highly specific for levoglucosan and exhibited weak ATPase activity in the absence of substrate. The equilibrium conversion of levoglucosan and ATP lay far on the product side, and no enzymatic back reaction from d ‐glucose‐6‐phosphate and ADP was observed under a broad range of conditions. 6‐Phospho‐α‐d ‐glucopyranosyl fluoride and 6‐phospho‐1,5‐anhydro‐2‐deoxy‐d ‐arabino‐hex‐1‐enitol (6‐phospho‐d ‐glucal) were synthesized as probes for the enzymatic mechanism but proved inactive with the enzyme in the presence of ADP. The pyranose ring flip ⁴C₁→¹C₄ required for 1,6‐anhydro‐product synthesis from d ‐glucose‐6‐phosphate probably presents a major thermodynamic restriction to the back reaction of the enzyme.     

Metabolism of epoxidized phosphatidylcholine by phospholipase A2 and epoxide hydrolase

The isolation and measurement of phospholipid epoxides as major peroxidation products in biomembrane preparations prompted an investigation of enzymatic mechanisms which may be responsible for their elimination. Analysis of microsomal epoxide hydrolase and phospholipase A₂ activity against a phospholipid epoxide commonly encountered in tissues indicated it to be a poor substrate for epoxide hydrolase, but rapidly hydrolyzed by phospholipase A₂. Microsomal and purified phospholipase A₂ preparations hydrolyzed the phospholipid epoxide at rates 2-fold greater than were observed with a monoenoic phospholipid from which the epoxide would be derived. The product fatty acid epoxide,cis-9,10-epoxystearic acid, was rapidly hydrated by microsomal and cytosolic epoxide hydrolase. On the basis of earlier reports demonstrating increased phospholipase activity against oxidized phospholipids, and on the results of the present study, a model for the metabolism of oxidized membrane phospholipids is proposed.     

Thermal effect of carbamates based polymer on the TiO2 growth

We investigated the growth of TiO₂ on poly((tetrahydropyran‐2‐yl N‐(2‐methacryloxyethyl) carbamate)‐co‐(methyl 4‐(3‐methacryloyloxypropoxy) cinnamate) (THP‐polymer) using thermal heating, octyl isocyanate (OIC), and glutaraldehyde. It is found that TiO₂ can be grown on surfaces terminated with ? NH₂ and ? O? groups from aqueous solution. However, TiO₂ did not deposit on ? CH₃ terminated surfaces, due to the low surface energy of these surfaces. Fourier transform infrared spectroscopy and thermogravimetric analysis data showed that the ? THP functional group can be removed and the surface functional group converted to ? NH₂ by heating the material over 180°C. OIC can then be immobilized on the surface after heating, changing the surface functional group from ? NH₂ to ? CH₃. As TiO₂ can be deposited from solution on ? NH₂ terminated, but not ? CH₃ terminated surfaces, THP‐polymer can be used to switch the surface properties by thermal activation and subsequent reaction with OIC. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Determination of Kinetics and the Crystal Structure of a Novel Type 2 Isopentenyl Diphosphate: Dimethylallyl Diphosphate Isomerase from Streptococcus pneumoniae

Isopentenyl diphosphate isomerase (IDI) is a key enzyme in the isoprenoid biosynthetic pathway and is required for all organisms that synthesize isoprenoid metabolites from mevalonate. Type 1 IDI (IDI‐1) is a metalloprotein that is found in eukaryotes, whereas the type 2 isoform (IDI‐2) is a flavoenzyme found in bacteria that is completely absent from human. IDI‐2 from the pathogenic bacterium Streptococcus pneumoniae was recombinantly expressed in Escherichia coli. Steady‐state kinetic studies of the enzyme indicated that FMNH₂ (K_M =0.3 μM ) bound before isopentenyl diphosphate (K_M =40 μM ) in an ordered binding mechanism. An X‐ray crystal structure at 1.4 Å resolution was obtained for the holoenzyme in the closed conformation with a reduced flavin cofactor and two sulfate ions in the active site. These results helped to further approach the enzymatic mechanism of IDI‐2 and, thus, open new possibilities for the rational design of antibacterial compounds against sequence‐similar and structure‐related pathogens such as Enterococcus faecalis or Staphylococcus aureus.     

Hydrolysis of Phosphatidylcholine-Isoprostanes (PtdCho-IP) by Peripheral Human Group IIA,V and X Secretory Phospholipases A<Subscript>2</Subscript> (sPLA<Subscript>2</Subscript>)

Biologically active F- and E/D-type-prostane ring isomers (F₂-IP and E₂/D₂-IP, respectively) are produced in situ by non-enzymatic peroxidation of arachidonic acid esterified to GroPCho (PtdCho-IP) and are universally distributed in tissue lipoproteins and cell membranes. Previous work has shown that platelet-activating factor acetylhydrolases (PAF-AH) are the main endogenous PLA₂ involved in degradation of PtdCho-IP. The present study shows that the PtdCho-IP are also subject to hydrolysis by group IIA, V and X secretory PLA₂, which also have a wide peripheral tissue distribution. For this demonstration, we compared the LC/MS profiles of PtdCho-IP of auto-oxidized plasma lipoproteins after incubation for 1–4 h (37 °C) in the absence or presence of recombinant human sPLA₂ (1–2.5 µg/ml). In the absence of exogenously added sPLA₂ the total PtdCho-IP level after 4 h incubation reached 15.9, 21.6 and 8.7 nmol/mg protein of LDL, HDL and HDL₃, respectively. In the presence of group V or group X sPLA₂ (2.5 µg/ml), the PtdCho-IP was completely hydrolyzed in 1 h, while in the presence of group IIA sPLA₂ (2.5 µg/ml) the hydrolysis was less than 25% in 4 h, although it was complete after 8–24 h incubation. This report provides the first demonstration that PtdCho-IP are readily hydrolyzed by group IIA, V and X sPLA₂. A co-location of sPLA₂ and the substrates in various tissues has been recorded. Thus, the initiation of interaction and production of isoprostanes in situ are highly probable.     

Effect of <Emphasis Type="Italic">Fabp1/Scp</Emphasis>-<Emphasis Type="Italic">2/Scp</Emphasis>-<Emphasis Type="Italic">x</Emphasis> Ablation on Whole Body and Hepatic Phenotype of Phytol-Fed Male Mice

Liver fatty acid binding protein (Fabp1) and sterol carrier protein‐2/sterol carrier protein‐x (SCP‐2/SCP‐x) genes encode proteins that enhance hepatic uptake, cytosolic transport, and peroxisomal oxidation of toxic branched‐chain fatty acids derived from dietary phytol. Since male wild‐type (WT) mice express markedly higher levels of these proteins than females, the impact of ablating both genes (TKO) was examined in phytol‐fed males. In WT males, high phytol diet alone had little impact on whole body weight and did not alter the proportion of lean tissue mass (LTM) versus fat tissue mass (FTM). TKO conferred on dietary phytol the ability to induce weight loss as well as reduce liver weight, FTM, and even more so LTM. Concomitantly TKO induced hepatic lipid accumulation, preferentially threefold increased phospholipid (PL) at the expense of decreased triacylglycerol (TG) and total cholesterol. Increased PL was associated with upregulation of membrane fatty acid transport/translocase proteins (FATP 2,4), cytosolic fatty acid/fatty acyl‐CoA binding proteins (FABP2, ACBP), and the rate limiting enzyme in PL synthesis (Gpam). Decreased TG and cholesterol levels were not attributable to altered levels in respective synthetic enzymes or nuclear receptors. These data suggest that the higher level of Fabp1 and Scp2/Scpx gene products in WT males was protective against deleterious effects of dietary phytol, but TKO significantly exacerbated phytol effects in males.     

Discovery of α‐Substituted Imidazole‐4‐acetic Acid Analogues as a Novel Class of ρ1 γ‐Aminobutyric Acid Type A Receptor Antagonists with Effect on Retinal Vascular Tone

The ρ‐containing γ‐aminobutyric acid type A receptors (GABA_ARs) play an important role in controlling visual signaling. Therefore, ligands that selectively target these GABA_ARs are of interest. In this study, we demonstrate that the partial GABA_AR agonist imidazole‐4‐acetic acid (IAA) is able to penetrate the blood–brain barrier in vivo; we prepared a series of α‐ and N‐alkylated, as well as bicyclic analogues of IAA to explore the structure–activity relationship of this scaffold focusing on the acetic acid side chain of IAA. The compounds were prepared via IAA from l ‐histidine by an efficient minimal‐step synthesis, and their pharmacological properties were characterized at native rat GABA_ARs in a [³H]muscimol binding assay and at recombinant human α₁β₂γ_2S and ρ₁ GABA_ARs using the FLIPR? membrane potential assay. The (+)‐α‐methyl‐ and α‐cyclopropyl‐substituted IAA analogues ((+)‐ 6 a and 6 c , respectively) were identified as fairly potent antagonists of the ρ₁ GABA_AR that also displayed significant selectivity for this receptor over the α₁β₂γ_2S GABA_AR. Both 6 a and 6 c were shown to inhibit GABA‐induced relaxation of retinal arterioles from porcine eyes.     

Trametes versicolor laccase immobilized poly(glycidyl methacrylate) based cryogels for phenol degradation from aqueous media

This study aims removal of phenols in wastewater by enzymatic oxidation method. In this study, Trametes versicolor laccase was covalently immobilized onto a cryogel matrix by the nucleophilic attack of amino groups of laccase to epoxy groups of matrix. Glycidyl methacrylate was chosen as functional monomer to prepare poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) [p(HEMA‐co‐GMA)] cryogels. The enzyme immobilized matrix was characterized by FTIR, SEM, and swelling tests. The effect of pH, reaction time, temperature, substrate concentration, enzyme concentration, and storage period on immobilized enzyme activity was determined and compared with those of free enzyme. The model substrate was 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid (ABTS). Lineweaver‐Burk plots were used to calculate K_m and V_m values. K_m values were 165.1 and 156.0 µM while V_m values were 55.2 µM min^?1 and 1.57 µM min^?1 for free and immobilized laccase, respectively. Immobilized enzyme was determined to retain 82.5% and 72.0% of the original activity, respectively, after 6 consecutive use and storage period of 4 weeks. The free enzyme retained only 24.0% of its original activity following the same storage period. Lastly, decomposition products resulting from enzymatic oxidation of a model phenolic compound (3,5‐dinitrosalicylic acid) in aqueous solution were identified by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41981.     

Potent Inhibition of Acid Sphingomyelinase by Phosphoinositide Analogues

The different mammalian sphingomyelinases are involved in cell regulation, apoptosis and inflammatory events. Recent reports suggest pharmacological potential especially for inhibitors of the acid sphingomyelinase. Phosphatidyl inositol‐3,5bisphosphate (PtdIns3,5P₂) is the most potent selective acid sphingomyelinase inhibitor known to date. In the present study, we synthesized analogues of PtdIns3,5P₂ for initial structure–activity‐relationship (SAR) studies. We identified an inhibitor that is easy to synthesize, that has superior chemical and biophysical properties when compared to PtdIns3,5P₂ and that should be stable against virtually all phospholipases. Last but not least, the new inhibitor partially protected cells from dexamethasone‐induced cell death.