The importance of hydroperoxide activation for the detection and assay of mammalian 5-lipoxygenase

Sulfhydryl reagents such as dithiothreitol stabilized human leukocyte 5-lipoxygenase (5-LO) during purification. During enzyme assay, however, these reagents led to irreproducible or unexpectedly low activity. This inconsistency in the assay was eliminated by inclusion of hydroperoxyeicosatetraenoic acids (1-5 microM) during the reaction which effected a 10-20-fold stimulation of 5-LO activity. Structural studies indicated that an intact hydroperoxy function, and a long-chain fatty acyl moiety were required for 5-LO stimulation. These data suggest that human leukocyte 5-LO is activated by hydroperoxy fatty acids, and that this results in a requirement for exogenous hydroperoxide in the presence of sulfhydryl reagents.     

A versatile high-throughput screen for inhibitors of lipid kinase activity: development of an immobilized phospholipid plate assay for phosphoinositide 3-kinase gamma

The family of phosphoinositide 3-kinases (PI3K) regulates fundamental cellular responses such as proliferation, apoptosis, motility, and adhesion. In particular, the PI3K gamma isoform plays a critical role in the control of cell migration. Despite the attractiveness of PI3-kinases as drug targets, drug discovery efforts have been hampered by the lack of appropriate lipid kinase assay formats suitable for high-throughput screening. The authors report the development of a simple and robust 384-well plate assay that is based on(33) P-phosphate transfer from radiolabeled [gamma(33) P]ATP to phosphatidylinositol immobilized on Maxisorp plates. The established assay format for PI3K gamma was easily adapted to the automated screening platform and was successfully employed for high-throughput screening. Enzymatic and inhibition characteristics of recombinant human PI3K gamma determined with the plate assay are in very good agreement with previously reported values determined in other assay formats. Maximal catalytic activity of PI3K gamma was observed at pH 7.0. The apparent K(m) value for ATP using a 1:1 mixture of phosphatidylinositol and phosphatidylserine was determined to be 7.3 microM (6.0-8.6 microM, 95% confidence interval [CI]). IC(50) values for known PI3-kinase inhibitors were determined to be 1.45 nM (1.17-1.80 nM, 95% CI) for wortmannin and estimated from partial inhibition data to be 1400, 2830, and 21,400 nM for quercetin, LY294002, and staurosporine, respectively. This novel assay approach allows for screening of inhibitors of lipid kinases in high-throughput mode and thereby may facilitate the identification of novel inhibitory structures for drug development.     

Modulation of human 5-lipoxygenase activity by membrane lipids

Mammalian 5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) to leukotrienes, potent inflammatory mediators. 5-LO is activated by a Ca(2+)-mediated translocation to membranes, and demonstrates the characteristic features of interfacially activated enzymes, yet the mechanism of membrane binding of 5-LO is not well understood. In an attempt to understand the mechanism of lipid-mediated activation of 5-LO, we have studied the effects of a large set of lipids on human recombinant 5-LO activity, as well as mutual structural effects of 5-LO and membranes. In the presence of 0.35 mM phosphatidylcholine (PC) and 0.2 mM Ca(2+), there was substrate inhibition at >100 microM AA. Data analysis at low AA concentrations yielded the following: K(m) approximately 103 microM and k(cat) approximately 56 s(-1). 5-LO activity was supported by PC more than by any other lipid tested except for a cationic lipid, which was more stimulatory than PC. Binding of 5-LO to zwitterionic and acidic membranes was relatively weak; the extent of binding increased 4-8 times in the presence of Ca(2+), whereas binding to cationic membranes was stronger and essentially Ca(2+)-independent. Polarized attenuated total reflection infrared experiments implied that 5-LO binds to membranes at a defined orientation with the symmetry axis of the putative N-terminal beta-barrel tilted approximately 45 degrees from the membrane normal. Furthermore, membrane binding of 5-LO resulted in dehydration of the membrane surface and was paralleled with stabilization of the structures of both 5-LO and the membrane. Our results provide insight into the understanding of the effects of membrane surface properties on 5-LO-membrane interactions and the interfacial activation of 5-LO.     

MEK-1/2 inhibition prevents 5-lipoxygenase translocation in N-formylpeptide-challenged human neutrophils

In order to elucidate the role of mitogen-activated protein kinase kinase (MEK-1/2) in 5-lipoxygenase (5-LO) activation we studied the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced 5-LO translocation in human blood neutrophils (PMNs). In non-primed, Ca(2+)-repleted PMNs, fMLP consistently stimulated MEK-1/2 phosphorylation, but induced 5-LO translocation and product formation (430+/-128 pmol; SEM, n=13) only in 13 of 18 PMN preparations from different healthy donors. In fMLP-responsive cells, the MEK-1/2 inhibitor PD098059 (50 microM) attenuated MEK phosphorylation and abolished 5-LO activation at the translocation step. The fMLP-mediated 5-LO product formation was also sensitive to MEK inhibition by U0126 and to p38 inhibition by SB203580. But in contrast to PD098059, U0126 at 10 microM and SB203580 at 20-50 microM impaired 5-LO activity in the cell-free assay setting, suggesting direct actions of higher concentrations of U0126 and SB203580 on 5-LO apart from MEK and p38 inhibition, respectively. These data show that fMLP initiates 5-LO product formation in non-primed, Ca(2+)-repleted human blood PMNs from healthy donors, and that MEK signaling is pivotal, but not sufficient for 5-LO activation in response to the receptor agonist fMLP.     

High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity

The molecular chaperone heat-shock protein 90 (HSP90) plays a key role in the cell by stabilizing a number of client proteins, many of which are oncogenic. The intrinsic ATPase activity of HSP90 is essential to this activity. HSP90 is a new cancer drug target as inhibition results in simultaneous disruption of several key signaling pathways, leading to a combinatorial approach to the treatment of malignancy. Inhibitors of HSP90 ATPase activity including the benzoquinone ansamycins, geldanamycin and 17-allylamino-17-demethoxygeldanamycin, and radicicol have been described. A high-throughput screen has been developed to identify small-molecule inhibitors that could be developed as therapeutic agents with improved pharmacological properties. A colorimetric assay for inorganic phosphate, based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green, was used to measure the ATPase activity of yeast HSP90. The Km for ATP determined in the assay was 510+/-70 microM. The known HSP90 inhibitors geldanamycin and radicicol gave IC(50) values of 4.8 and 0.9 microM respectively, which compare with values found using the conventional coupled-enzyme assay. The assay was robust and reproducible (2-8% CV) and used to screen a compound collection of approximately 56,000 compounds in 384-well format with Z' factors between 0.6 and 0.8.     

Monocyte-derived soluble protein confers 5-lipoxygenase activity Ca2+-dependent

5-Lipoxygenase (5-LO) is a Ca2+-stimulated enzyme that initializes the formation of proinflammatory leukotrienes from arachidonic acid (AA). In this report, we demonstrate that a soluble protein of the monocytic cell line Mono Mac 6 confers 5-LO activity Ca2+-dependent in vitro. Thus, in broken cell preparations of human polymorphonuclear leukocytes (PMNL) and rat basophilic leukemia (RBL)-1 cells, 5-LO converted AA (>20 microM) in the absence of Ca2+, whereas Ca2+ was absolutely required for 5-LO activity in broken cell preparations of MM6 cells. 5-LO partially purified from MM6 cells was substantially active in the absence of Ca2+. Recombination experiments revealed that the cytosolic fraction of MM6 cells contains a factor that suppresses the activity of partially purified 5-LO from PMNL, RBL-1, and MM6 cells in the absence but not in the presence of Ca2+. Further characterization showed that this factor is a 80-100 kDa heat-sensitive protein.     

Development of a simple assay protocol for high-throughput screening of Mycobacterium tuberculosis glutamine synthetase for the identification of novel inhibitors

Mycobacterium tuberculosis glutamine synthetase (GS) is an essential enzyme involved in the pathogenicity of the organism. The screening of a compound library using a robust high-throughput screening (HTS) assay is currently thought to be the most efficient way of getting lead molecules, which are potent inhibitors for this enzyme. The authors have purified the enzyme to a >90% level from the recombinant Escherichia coli strain YMC21E, and it was used for partial characterization as well as standardization experiments. The results indicated that the Km of the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The Km for ADP, arsenate, and Mn2+ were 2 microM, 5 microM, and 25 microM, respectively. When the components were adjusted according to their Km values, the activity remained constant for at least 3 h at both 25 degrees C and 37 degrees C. The Z' factor determined in microplate format indicated robustness of the assay. When the signal/noise ratios were determined for different assay volumes, it was observed that the 200-microl volume was found to be optimum. The DMSO tolerance of the enzyme was checked up to 10%, with minimal inhibition. The IC50 value determined for L-methionine S-sulfoximine on the enzyme activity was 3 mM. Approximately 18,000 small molecules could be screened per day using this protocol by a Beckman Coulter HTS setup.     

Stimulation of calcium transport of sarcoplasmic reticulum vesicles by the calcium complex of ethylene glycol bis(beta-aminoethyl ether)-N,N',-tetraacetic acid

The calcium ion dependence of calcium transport by isolated sarcoplasmic reticulum vesicles from rabbit skeletal muscle has been investigated by means of the Calcium-stat method, in which transport may be measured in the micromolar free calcium ion concentration range, in the absence of calcium buffers. At pH 7.2 and 20 degrees C, ATP, in the range 1 to 10 mM, decreased [Ca2+]0.5 from 2.0 microM to 0.3 microM and decreased Vmax of oxalate-supported transport from 0.5 to 1.3 mumol min-1 mg-1. Simultaneous measurements of transport and of ATPase activity in the range 0.8 to 10 microM free Ca2+ showed a ratio of 2.1 calcium ions translocated/molecule of ATP hydrolyzed. Transport, in the presence of 5 mM ATP, ceased when calcium ion concentration fell to 0.6 to 1.2 microM, whilst ATPase activity of 90 nmol of ATP hydrolyzed min-1 mg-1 persisted. The data obtained by the Calcium-stat method differed from those described previously using calcium buffers, in that they showed lower apparent affinities of the transport site for calcium ions, more marked sigmoidal behavior, an effect of ATP concentration on Ca2+ concentration dependence and lower ATPase activity in the absence of transport. The calcium complex of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (CaEGTA) had no effect when transport was stimulated maximally at saturating free Ca2+ concentrations. However, at calcium ion levels below [Ca2+]0.5, 70 microM CaEGTA stimulated transport to rates of 20 to 45% of Vmax. Half-maximal stimulation of transport occurred at 19 microM CaEGTA. CaEGTA, 50 microM, decreased [Ca2+]0.5, determined at 5 mM ATP, from 1.3 microM to 0.45 microM. It is proposed that a ternary complex, E . Ca2+ . EGTA4-, is formed as an intermediate species during CaEGTA-stimulated calcium transport by sarcoplasmic reticulum membranes and stimulates the calcium pump at limiting free Ca2+ ion concentration.     

Fluorescent assay for polymerization of purified bacterial FtsZ cell-division protein

Septum formation in Escherichia coli is a complex cascade of interactions among cell-division proteins. The tubulin-like FtsZ division protein localizes to the division site and serves a cytoskeletal role during septum formation. A novel fluorescent-based 96-well format filter assay has been developed to measure the polymerization of FtsZ. A mixture of monomers and aggregates (38 to approximately 200 KDa in range) of purified wild-type FtsZ and a fluorescently tagged derivative of FtsZ protein in stoichiometric ratio passes through a 0.2-microm filter membrane, while polymerized FtsZ is retained on the filter. Addition of the SulA protein to the assay leads to rapid disassembly of existing FtsZ polymers, demonstrating its natural regulatory effect on FtsZ under the assay conditions. This assay is sensitive (requiring 2 microM FtsZ or less) and facilitates high-throughput screening of factors affecting FtsZ polymerization.     

Miniaturizable homogenous time-resolved fluorescence assay for carboxypeptidase B activity

An epitope-unmasking, homogeneous time-resolved fluorescence (HTRF) assay has been developed for measuring carboxypeptidase B (CPB) activity in a miniaturized high-throughput screening format. The enzyme substrate (biotin-RYRGLMVGGVVR-OH) is cleaved by CPB at the C terminus, causing release of the C-terminal Arg residue. The product (biotin-RYRGLMVGGVV-OH) is recognized specifically by a monoclonal antibody (G2-10) which is labeled with Eu(3+)-cryptate ([Eu(3+)]G2-10 mAb), and the complex is detected by fluorescence resonance energy transfer using streptavidin labeled with allophycocyanin ([XL665]SA). The CPB HTRF assay is readily adapted from 96- to 1536-well format as a robust (Z(')>0.5) assay for high-throughput screening.     

A conformational change of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled sarcoplasmic reticulum Ca2+-ATPase upon ATP binding to the catalytic site

Sarcoplasmic reticulum vesicles were modified with a fluorescent thiol reagent, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine. One mol of readily reactive thiols per mol of the Ca2+-ATPase was labeled without a loss of the catalytic activity. The fluorescence of the label increased by 8% upon binding of Ca2+ to the high affinity sites of the enzyme. This fluorescence enhancement probably reflects a conformational change responsible for Ca2+-induced enzyme activation. Upon addition of ATP to the Ca2+-activated enzyme, the fluorescence decreased by 15%. This fluorescence drop and formation of the phosphoenzyme intermediate were determined under the same conditions with a stopped-flow apparatus and a rapid quenching system. The amplitude of the fluorescence drop thus determined was saturated with 3 microM ATP. This shows that the fluorescence drop was caused by ATP binding to the catalytic site. In contrast, the rate of the fluorescence drop was not saturated even with 50 microM ATP. The fluorescence drop coincided with phosphoenzyme formation at 0.5 or 3 microM ATP, but it became much faster than phosphoenzyme formation when the ATP concentration was raised to 100 microM. These results indicate that the ATP-induced fluorescence drop reflects a conformational change in the enzyme.ATP complex. The fluorescence drop was accompanied by a red spectrum shift, which suggests that the label was exposed to a more hydrophilic environment. The electrophoretic analysis of the tryptic digest of the labeled enzyme (10.9 kDa) showed that almost all of the label was located on the 5.2-kDa fragment which includes the carboxyl terminus and the putative ATP-binding domain. The sequencing of the two major labeled peptides, which were isolated from the thermolytic digest of the labeled enzyme, revealed that the labeled site in either of these peptides was Cys674. It seems likely that the label bound to this Cys674 could be involved in the observed fluorescence changes.     

Development of a high-throughput screening method for LIM kinase 1 using a luciferase-based assay of ATP consumption

Kinases are attractive drug targets because of the central roles they play in signal transduction pathways and human diseases. Their well-formed adenosine triphosphate (ATP)-binding pockets make ideal targets for small-molecule inhibitors. For drug discovery purposes, many peptide-based kinase assays have been developed that measure substrate phosphorylation using fluorescence-based readouts. However, for some kinases these assays may not be appropriate. In the case of the LIM kinases (LIMK), an inability to phosphorylate peptide substrates resulted in previous high-throughput screens (HTS) using radioactive labeling of recombinant cofilin protein as the readout. We describe the development of an HTS-compatible assay that measures relative ATP levels using luciferase-generated luminescence as a function of LIMK activity. The assay was inexpensive to perform, and proof-of-principle screening of kinase inhibitors demonstrated that compound potency against LIMK could be determined; ultimately, the assay was used for successful prosecution of automated HTS. Following HTS, the secondary assay format was changed to obtain more accurate measures of potency and mechanism of action using more complex (and expensive) assays. The luciferase assay nonetheless provides an inexpensive and reliable primary assay for HTS that allowed for the identification of LIMK inhibitors to initiate discovery programs for the eventual treatment of human diseases.     

Comparison of miniaturized time-resolved fluorescence resonance energy transfer and enzyme-coupled luciferase high-throughput screening assays to discover inhibitors of Rho-kinase II (ROCK-II)

Kinases are important drug discovery targets for a wide variety of therapeutic indications; consequently, the measurement of kinase activity remains a common high-throughput screening (HTS) application. Recently, enzyme-coupled luciferase-kinase (LK) format assays have been introduced. This format measures luminescence resulting from metabolism of adenosine triphosphate (ATP) via a luciferin/luciferase-coupled reaction. In the research presented here, 1536-well format time-resolved fluorescence resonance energy transfer (TR-FRET) and LK assays were created to identify novel Rho-associated kinase II (ROCK-II) inhibitors. HTS campaigns for both assays were conducted in this miniaturized format. It was found that both assays were able to consistently reproduce the expected pharmacology of inhibitors known to be specific to ROCK-II (fasudil IC50: 283 +/- 27 nM and 336 +/- 54 nM for TR-FRET and LK assays, respectively; Y-27632 IC50: 133 +/- 7.8 nM and 150 +/- 22 nM for TR-FRET and LK assays, respectively). In addition, both assays proved robust for HTS efforts, demonstrating excellent plate Z' values during the HTS campaign (0.84 +/- 0.03; 0.72 +/- 0.05 for LK and TR-FRET campaigns, respectively). Both formats identified scaffolds of known and novel ROCK-II inhibitors with similar sensitivity. A comparison of the performance of these 2 assay formats in an HTS campaign was enabled by the existence of a subset of 25,000 compounds found in both our institutional and the Molecular Library Screening Center Network screening files. Analysis of the HTS campaign results based on this subset of common compounds showed that both formats had comparable total hit rates, hit distributions, amount of hit clusters, and format-specific artifact. It can be concluded that both assay formats are suitable for the discovery of ROCK-II inhibitors, and the choice of assay format depends on reagents and/or screening technology available.     

An enzymatic assay for poly(ADP-ribose) polymerase-1 (PARP-1) via the chemical quantitation of NAD(+): application to the high-throughput screening of small molecules as potential inhibitors

The enzyme poly(adenosine 5'-diphosphate (ADP)-ribose) polymerase (PARP-1) catalyzes the formation of (ADP)-ribose polymers on a variety of protein acceptors in a NAD+ -dependent manner. While PARP-1 is activated by DNA damage and plays a critical role in cellular survival mechanisms, its overactivation leads to a depletion of NAD+/ATP energy stores and ultimately to necrotic cell death. Due to this dual role of PARP in the cell, small-molecule inhibitors of the PARP family of enzymes have been widely investigated for use as potentiators of anticancer therapies and as inhibitors of neurodegeneration and ischemic injuries. Unfortunately, standard assays for PARP inhibition are not optimal for the high-throughput screening of compound collections or combinatorial libraries. Described herein is a highly sensitive, inexpensive, and operationally simple assay for the rapid assessment of PARP activity that relies on the conversion of NAD+ into a highly fluorescent compound. We demonstrate that this assay can readily detect PARP inhibitors in a high-throughput screen using 384-well plates. In addition, the assay can be used to determine IC50 values for PARP inhibitors that have a range of inhibitory properties. As existing PARP assays utilize specialized reagents such as radiolabeled/biotinylated NAD+ or antibodies to poly(ADP-ribose), the chemical quantitation method described herein offers a highly sensitive and convenient alternative for rapidly screening compound collections for PARP inhibition.     

Evaluating the utility of the HTRF Transcreener ADP assay technology: A comparison with the standard HTRF assay technology

The HTRF (homogeneous time-resolved fluorescence) Transcreener ADP assay is a new kinase assay technology marketed by Cis-Bio International (Bagnols-Cèze, France). It measures kinase activity by detecting the formation of ADP using a monoclonal antibody and HTRF detection principles. In this article, we compare this technology with a standard HTRF kinase assay using EGFR [L858R/T790M] mutant enzyme as a case study. We demonstrate that the HTRF Transcreener ADP assay generated similar kinetic constants and inhibitor potency compared with the standard HTRF assay. However, the smaller dynamic window and lower Z′ factor of the HTRF Transcreener ADP assay make this format less preferable for high-throughput screening. Based on the assay principle, the HTRF Transcreener ADP assay can detect both kinase and ATPase activities simultaneously. The ability to probe ATPase activity opens up new avenues for assaying kinases with intrinsic ATPase activity without the need to identify substrates, and this can speed up the drug discovery process. However, caution must be exercised because any contaminating ATPase activity will result in an invalid assay. The inability to tolerate high concentrations of ATP in the assay will also limit the application of this technology, especially in compound mechanistic studies such as ATP competition. Overall, the HTRF Transcreener ADP assay provides a new alternative tool to complement existing assay technologies for drug discovery.     

Development of a fluorescence-based enzyme assay of human 5-lipoxygenase

Leukotrienes are important mediators in a number of inflammatory diseases and therefore are a target of several therapeutic approaches. The first committed step in the synthesis of leukotrienes is the conversion of arachidonic acid to leukotriene A(4) (LTA(4)) in two successive reactions catalyzed by 5-lipoxygenase (5-LOX). Assays to measure 5-LOX activity typically have been low throughput and time consuming. In this article, we describe a fluorescence assay that is amenable to high-throughput screening in a 384-well microplate format. The fluorescent signal is measured during oxidation of 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) by human 5-LOX. The assay has been found to reliably identify small molecule inhibitors of human 5-LOX. The IC(50) values of several 5-LOX inhibitors in this new assay are comparable to those determined in a standard spectrophotometric assay that measures the formation of the 5(S)-hydroperoxyeicosatetraenoic acid (5-HpETE) product. In addition, we demonstrate the use of the assay in a high-throughput screen of the Pfizer compound collection to identify inhibitors of 5-LOX.     

Partial purification and characterization of phospholipid N-methyltransferases from murine thymocyte microsomes

Significant amounts of phospholipid N-methyltransferase activity in murine thymocytes were found to be distributed on the plasma membrane. The enzyme activity had an optimum pH of 9. The presence of divalent cations, Mg2+ (10 mM) or Ca2+ (1 mM), and EGTA separately in the assay had only a small effect on the enzyme activity. However, addition of both 10 mM Mg2+ and 1 mM Ca2+ increased the enzyme activity. The presence of two enzymes for each conversion of phosphatidylethanolamine (PE) to phosphatidylmonomethylethanolamine (PME) and PME to phosphatidylcholine (PC) was suggested by the result of the determination of the incorporated radioactivity into PME, phosphatidyldimethylethanolamine (PDE) and PC; the apparent Km values for S-adenosyl-L-methionine were 20 and 400-500 microM for the conversion of PE to PME and for the conversion of PME to PC they were 5 microM and 40 microM. S-Adenosyl-L-homocysteine (AdoHcy), a known inhibitor of enzymatic methylation, competitively inhibited [14C]methyl incorporation into total lipid. The apparent Ki value for AdoHcy was 44.7 microM. Two phospholipid N-methyltransferases were partially purified by extraction with sodium deoxycholate, gel filtration on Sephadex G-75, and affinity column chromatography on AdoHcy-Sepharose. One enzyme, mainly catalyzing the formation of PME, was purified approximately 1548-fold and the other catalyzing the formation of PDE and PC, was purified approximately 629- to 703-fold. However, the former still contained a little activity for PDE and PC formation and the latter contained a little activity for PME formation. In these partially purified phospholipid N-methyltransferase preparations, little contaminating protein O-carboxylmethyltransferase activity was observed; however, significant PC-phospholipase A2 activity was detected. This result may suggest that phospholipid N-methyltransferases associate with phospholipase A2 in the thymocyte plasma membrane.     

An optimized ATP/PP(i)-exchange assay in 96-well format for screening of adenylation domains for applications in combinatorial biosynthesis

We report a new format for measuring ATP/[(32)P]pyrophosphate exchange in a higher throughput assay of adenylation domains (A-domains) of non-ribosomal peptide synthetases. These enzymes are key specificity determinants in the assembly line biosynthesis of non-ribosomal peptides, an important class of natural products with an activity spectrum ranging from antibiotic to antitumor activities. Our assay in 96-well format allows the rapid measurement of approximately 1000 data points per week as a basis for precise assessment of the kinetics of A-domains. The assay also allows quantitative high-throughput screening of the substrate specificity of A-domains identifying alternative, promiscuous substrates. We show that our assay is able to give high quality data for the T278A mutant of the A-domain of the tyrocidine synthetase module TycA with a 330-fold lower k(cat)/K(M). The large dynamic range of this assay will be useful for the screening of libraries of mutant A-domains. Finally we describe and evaluate a procedure for the high-throughput purification of A-domains in 96-well format for the latter purpose. Our approach will be of utility for mechanistic analysis, substrate profiling and directed evolution of the A-domains, to ultimately enable the combinatorial biosynthesis of non-natural analogues of non-ribosomal peptides that may have potential as alternative drug candidates.     

Catalytic properties of inositol trisphosphate kinase: activation by Ca2+ and calmodulin

Inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) is an important second-messenger molecule that mobilizes Ca2+ from intracellular stores in response to the occupancy of receptor by various Ca2+-mobilizing agonists. The fate of Ins-1,4,5-P3 is determined by two enzymes, a 3-kinase and a 5-phosphomonoesterase. The first enzyme converts Ins-1,4,5-P3 to Ins-1,3,4,5-P4, whereas the latter forms Ins-1,4-P2. Recent studies suggest that Ins-1,3,4,5-P4 might modulate the entry of Ca2+ from an extracellular source. In the current report, we describe the partial purification of the 3-kinase [approximately 400-fold purified, specific activity = 0.12 mumol/(min.mg)] from the cytosolic fraction of bovine brain and studies of its catalytic properties. We found that the 3-kinase activity is significantly activated by the Ca2+/calmodulin complex. Therefore, we propose that Ca2+ mobilized from endoplasmic reticulum by the action of Ins-1,4,5-P3 forms a complex with calmodulin, and that the Ca2+/calmodulin complex stimulates the conversion of Ins-1,4,5-P3, an intracellular Ca2+ mobilizer, to Ins-1,3,4,5-P4, an extracellular Ca2+ mobilizer. A rapid assay method for the 3-kinase was developed that is based on the separation of [3-32P]Ins-1,3,4,5-P4 and [gamma-32P]ATP by thin-layer chromatography. Using this new assay method, we evaluated kinetic parameters (Km for ATP = 40 microM, Km for Ins-1,4,5-P3 = 0.7 microM, Ki for ADP = 12 microM) and divalent cation specificity (Mg2+ much greater than Mn2+ greater than Ca2+) for the 3-kinase. Studies with various inositol polyphosphates indicate that the substrate-binding site is quite specific to Ins-1,4,5-P3. Nevertheless, Ins-2,4,5-P3 could be phosphorylated at a velocity approximately 1/20-1/30 that of Ins-1,4,5-P3.     

1-Oleoyl-2-acetylglycerol stimulates 5-lipoxygenase activity via a putative (phospho)lipid binding site within the N-terminal C2-like domain

5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2+ ions and phospholipids that both act via the N-terminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca2+ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 x g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca2+, OAG caused no stimulation of 5-LO. Also, phospholipids or cellular membranes abolished the effects of OAG. As found previously for Ca2+, OAG renders 5-LO activity resistant against inhibition by glutathione peroxidase activity, and this effect of OAG is reversed by phospholipids. Intriguingly, a 5-LO mutant lacking tryptophan residues (Trp-13, -75, and -102) important for the binding of the 5-LO C2-like domain to phospholipids was not stimulated by OAG. We conclude that OAG directly stimulates 5-LO by acting at a phospholipid binding site located within the C2-like domain.