Cyclodextrins enhance recombinant phosphatidylinositol phosphate kinase activity

Inositol lipid kinases have been studied extensively in both plant and animal systems. However, major limitations for in vitro studies of recombinant lipid kinases are the low specific activity and instability of the purified proteins. Our goal was to determine if cyclodextrins would provide an effective substrate delivery system and enhance the specific activity of lipid kinases. For these studies, we have used recombinant Arabidopsis thaliana phosphatidylinositol phosphate kinase 1 (At PIPK1). At PIPK1 was produced as a fusion protein with glutathione-S-transferase and purified on glutathione-Sepharose beads. A comparison of lipid kinase activity using substrate prepared in alpha-, beta-, or gamma-cyclodextrin indicated that beta-cyclodextrin was most effective and enhanced lipid kinase activity 6-fold compared with substrate prepared in Triton X-100-mixed micelles. We have optimized reaction conditions and shown that product can be recovered from the cyclodextrin-treated recombinant protein, which reveals a potential method for automating the assay for pharmacological screening.     

Purification and characterization of bovine brain type I phosphatidylinositol kinase

Investigation into the phosphatidylinositol kinase activities in bovine brain has revealed the presence of a type I PtdIns kinase activity. This classification is based upon potent inhibition by neutral detergent and the production of a phosphatidylinositol phosphate that can be distinguished from phosphatidyl-inositol-4-phosphate [PtdIns(4)P] by thin-layer chromatography. The enzyme has been substantially purified and the activity is associated with an 85-kDa polypeptide on SDS/polyacrylamide gel electrophoresis. Analysis of the product confirms the identification of the enzyme as a type I PtdIns kinase. The purified kinase has been characterized with respect to substrate dependence (Mg2+, ATP, PtdIns), substrate presentation (pure lipid versus mixed micelle) and specificity [PtdIns versus PtdIns(4)P and phosphatidylinositol 4,5-bisphosphate].     

A fluorescence-based high-performance liquid chromatographic assay to determine acid ceramidase activity.

Acid ceramidase (N-acylsphingosine amidohydrolase) is the lysosomal enzyme required to hydrolyze the N-acyl linkage between the fatty acid and sphingosine moieties in ceramide. A deficiency of acid ceramidase activity results in the lipid storage disorder, Farber disease. This study reports a new assay method to detect acid ceramidase activity in vitro using Bodipy or lissamine rhodamine-conjugated ceramide (C12 ceramide; dodecanoylsphingosine). Using mouse kidney extracts as the source of acid ceramidase activity, this new method was compared with an assay using radioactive C12 ceramide (N-[(14)C]-dodecanoylsphingosine) as a substrate. The Bodipy C12 ceramide substrate provided data very similar to those of the radioactive substrate, but under the experimental conditions tested, it was significantly more sensitive. Using Bodipy C12 ceramide, femtomole quantities of the product, Bodipy dodecanoic acid, could be detected, providing an accurate measure of acid ceramidase activity as low as 0.1 pmol/mg protein/h. Acid ceramidase activities in skin fibroblasts and EBV-transformed lymphoblasts from Farber disease patients were around 7.8 and 10% of those in normal cells, respectively, confirming the specificity of this new assay method. Based on these results, we suggest that this fluorescence-based, high-performance liquid chromatographic technique is a reliable, rapid, and highly sensitive method to determine acid ceramidase activity, and that it could be useful wherever the in vitro detection of acid ceramidase activity is of importance.     

Stimulation of polyprenyl 4-hydroxybenzoate transferase activity by sodium cholate and 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate

Polyprenyl 4-hydroxybenzoate transferase (Coq2p) plays a central role in ubiquinone biosynthesis. Coq2p mediates the conjugation of 4-hydroxybenzoate, the benzoquinone ring precursor, with the completed side chain. The activity is most easily assayed by measuring the rate of incorporation of 4-hydroxybenzoate as radiolabeled substrate into polyprenyl 4-hydroxybenzoate. The in vitro assay requires addition of a detergent into the reaction mixture to activate enzyme activity, and Triton X-100 is used for this purpose in the routine assay. We have found that both 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate and sodium cholate, but not sodium deoxycholate, lysophosphatidyl choline, or octylglucoside, significantly stimulate the activity over that measured with Triton X-100. High-performance liquid chromatography analysis of lipid extracts revealed that the increase of specific activity resulted in a similar increase in reaction product, this effect is due not merely to a better lipid extraction but also to the actual stimulation of enzyme activity. With our improved method, we were able to measure Coq2p activity with much greater sensitivity in both fresh and frozen/thawed mitochondria and in crude homogenates obtained from cultured cells. Our method will simplify evaluation of Coq2p activity in scarce biological materials, such as cells obtained from human tissue biopsies, and thus it will facilitate the biochemical characterization of ubiquinone deficiencies.     

A novel high-throughput screening format to identify inhibitors of secreted acid sphingomyelinase

Secreted extracellular acid sphingomyelinase (sASM) activity has been suggested to promote atherosclerosis by enhancing subendothelial aggregation and retention of low-density lipoprotein (LDL) with resultant foam cell formation. Compounds that inhibit sASM activity, at neutral pH, may prevent lipid retention and thus would be expected to be anti-atherosclerotic. With the goal of identifying novel compounds that inhibit sASM at pH 7.4, a high-throughput screen was performed. Initial screening was run using a modification of a proven system that measures the hydrolysis of radiolabeled sphingomyelin presented in detergent micelles in a 96-well format. Separation of the radiolabeled aqueous phosphorylcholine reaction product from uncleaved sphingomyelin lipid substrate was achieved by chloroform/methanol extraction. During the screening campaign, a novel extraction procedure was developed to eliminate the use of the hazardous organic reagents. This new procedure exploited the ability of uncleaved, radiolabeled lipid substrate to interact with hydrophobic phenyl-sepharose beads. A comparison of the organic-based and the bead-based extraction sASM screening assays revealed Z' factor values ranging from 0.7 to 0.95 for both formats. In addition, both assay formats led to the identification of sub- to low micromolar inhibitors of sASM at pH 7.4 with similar IC(50) values. Subsequent studies demonstrated that both methods were also adaptable to run in a 384-well format. In contrast to the results observed at neutral pH, however, only the organic extraction assay was capable of accurately measuring sASM activity at its pH optimum of 5.0. The advantages and disadvantages of both sASM assay formats are discussed.     

Synaptic vesicle ceramide kinase. A calcium-stimulated lipid kinase that co-purifies with brain synaptic vesicles

Much current work on the mechanism of neurosecretion has focused on proteins specific to neural secretory vesicles (synaptic vesicles). We report a calcium-stimulated lipid kinase that co-purifies with rat brain synaptic vesicles. This enzyme activity is found only in membrane fractions that contain synaptic vesicle markers. Based on identification of the lipid product as ceramide 1-phosphate and on the finding that ceramide kinase activity co-purifies with synaptic vesicles, the enzyme is proposed to be a ceramide kinase. Kinase activity is stimulated by micromolar concentrations of calcium. Calcium increases the apparent Vmax of the reaction with little effect on the Km for ceramide. The vesicular localization of this enzyme, the requirement for ATP, and the stimulation of enzyme activity by micromolar calcium suggest that ceramide phosphorylation may be associated with neurotransmitter release.     

Reconstitution of ATP- and cytosol-dependent transport of de novo synthesized ceramide to the site of sphingomyelin synthesis in semi-intact cells

Transport of ceramide synthesized at the endoplasmic reticulum to the Golgi compartment, where sphingomyelin (SM) synthase exists, was reconstituted within semi-intact Chinese hamster ovary cells. When [(3)H]ceramide that had been produced from [(3)H]sphingosine at 15 degrees C in perforated cells was chased at 37 degrees C, [(3)H]ceramide-to-[(3)H]SM conversion occurred in a cytosol-dependent manner. In various aspects (i.e. kinetics, ATP dependence, and temperature dependence), [(3)H]ceramide-to-[(3)H]SM conversion in perforated cells was consistent with that in intact cells. The cytosol from LY-A strain, a Chinese hamster ovary cell mutant defective in endoplasmic reticulum-to-Golgi transport of ceramide, did not support [(3)H]ceramide-to-[(3)H]SM conversion in perforated wild-type cells, whereas the wild-type cytosol rescued the conversion in perforated LY-A cells. Brefeldin A-treated cells, in which the endoplasmic reticulum and the Golgi apparatus were merged, no longer required cytosol for conversion of [(3)H]ceramide to [(3)H]SM. These results indicated that the assay of [(3)H]ceramide-to-[(3)H]SM conversion in semi-intact cells is a faithful in vitro assay for the activity of cytosol-dependent transport of ceramide and that LY-A cells are defective in a cytosolic factor involved in ceramide transport. In addition, conversion of [(3)H]ceramide to [(3)H]glucosylceramide in semi-intact cells was little dependent on cytosol, suggesting that ceramide reached the site of glucosylceramide synthesis by a cytosol-independent (or less dependent) pathway.     

A phosphoprotein from the archaeon Sulfolobus solfataricus with protein-serine/threonine kinase activity

Sulfolobus solfataricus contains a membrane-associated protein kinase activity that displays a strong preference for threonine as the phospho-acceptor amino acid residue. When a partially purified detergent extract of the membrane fraction from the archaeon S. solfataricus that had been enriched for this activity was incubated with [gamma-(32)P]ATP, radiolabeled phosphate was incorporated into roughly a dozen polypeptides, several of which contained phosphothreonine. One of the phosphothreonine-containing proteins was identified by mass peptide profiling as the product of open reading frame [ORF] sso0469. Inspection of the DNA-derived amino acid sequence of the predicted protein product of ORF sso0469 revealed the presence of sequence characteristics faintly reminiscent of the "eukaryotic" protein kinase superfamily. ORF sso0469 therefore was cloned, and its polypeptide product was expressed in Escherichia coli. The recombinant protein formed insoluble aggregates that could be dispersed using urea or detergents. The solubilized polypeptide phosphorylated several exogenous proteins in vitro, including casein, myelin basic protein, and bovine serum albumin. Mutagenic alteration of amino acids predicted to be essential for catalytic activity abolished or severely reduced catalytic activity. Phosphorylation of exogenous substrates took place on serine and, occasionally, threonine. This new archaeal protein kinase displayed no catalytic activity when GTP was substituted for ATP as the phospho-donor substrate, while Mn(2+) was the preferred cofactor.     

A novel assay for monoacylglycerol hydrolysis suitable for high-throughput screening

A simple assay for monoacylglycerol hydrolysis suitable for high-throughput screening is described. The assay uses [(3)H]2-oleoylglycerol as substrate, with the tritium label in the glycerol part of the molecule and the use of phenyl sepharose gel to separate the hydrolyzed product ([(3)H]glycerol) from substrate. Using cytosolic fractions derived from rat cerebella as a source of hydrolytic activity, the assay gives the appropriate pH profile and sensitivity to inhibition with compounds known to inhibit hydrolysis of this substrate. The assay could also be adapted to a 96-well plate format, using C6 cells as the source of hydrolytic activity. Thus the assay is simple and appropriate for high-throughput screening of inhibitors of monoacylglycerol hydrolysis.     

Development and validation of a competitive AKT serine/threonine kinase fluorescence polarization assay using a product-specific anti-phospho-serine antibody.

A competitive fluorescence polarization (FP) assay has been developed for the serine/threonine kinase, AKT. The FP assay has been formatted in a 384-well microtiter plate and automated using a pipeting workstation with performance suitable for high-throughput screening. The assay design utilizes a fluorescent phosphorylated peptide complexed to a product-specific anti-phospho-serine antibody. When unlabeled substrate is phosphorylated, by the kinase, the product competes with the fluorescent phosphorylated peptide for the antibody. The fluorescent phosphorylated peptide is then released from the antibody into solution resulting in a loss in polarization signal. Seven fluorescent phosphorylated peptides and 19 antibodies were evaluated for this assay. RARTSpSFAEPGK-Fl peptide and anti-phospho-GSK-3alpha Ser21 antibody gave the best affinity and change in polarization signal. The apparent kinetic constants were calculated for the FP assay and were consistent with reported values. The FP assay was validated with known inhibitors and the results compared to a radioactive Flashplate transfer assay, utilizing [(33)P]ATP and a biotinylated substrate, also developed in our laboratory. The IC(50) values generated were comparable between the two methods suggesting the competitive FP assay and Flashplate assay have similar sensitivities and abilities to identify inhibitors during screening.     

A direct method for the simultaneous measurement of ceramide and phospholipase D activity

Both ceramide and phospholipase D (PLD) have important roles in a variety of signal transduction pathways. Recent evidence suggests that ceramide is a novel second messenger with specific biological effects. Publications in this field have increased rapidly in the last few years. However, a method to directly and rapidly measure cermide production has been lacking. Herein, we report on a novel, inexpensive, direct and rapid assay for the measurement of ceramide and the simultaneous measurement of PLD activity. This method uses labeling of cells with [(14)C]myristic acid and a TLC solvent of ethyl acetate/acetic acid/trimethylpentane. This method avoids the loss of radioactivity and variability due to changes in DAG kinase activity that are associated with the commonly-used DAG kinase assay.     

Substrate specificity of human ceramide kinase

Previous studies in our laboratory have established ceramide kinase (CERK) as a critical mediator of eicosanoid synthesis. To date, CERK has not been well characterized in vitro. In this study, we investigated the substrate specificity of CERK using baculovirus-expressed human CERK (6 x His) and a newly designed assay based on mixed micelles of Triton X-100. The results indicate that the ability of CERK to recognize ceramide as a substrate is stereospecific. A minimum of a 12 carbon acyl chain was required for normal CERK activity, and the 4-5 trans double bond was important for substrate recognition. A significant discrimination by CERK was not observed between ceramides with long saturated and long unsaturated fatty acyl chains. Methylation of the primary hydroxyl group resulted in a loss of activity, confirming that CERK produces ceramide-1-phosphate versus ceramide-3-phosphate. In addition, methylation of the secondary hydroxyl group drastically decreased the phosphorylation by CERK. These results also indicated that the free hydrogen of the secondary amide group is critical for substrate recognition. Lastly, the sphingoid chain was also required for substrate recognition by CERK. Together, these results indicate a very high specificity for substrate recognition by CERK, explaining the use of ceramide and not sphingosine or diacylglycerol as substrates.     

A rapid assay for assessment of sphingosine kinase inhibitors and substrates

Sphingosine kinases (SphKs) catalyze the transfer of phosphate from adenosine triphosphate (ATP) to sphingosine to generate sphingosine 1-phosphate (S1P), an important bioactive lipid molecule that mediates a diverse range of cell signaling processes. The conventional assay of SphK enzymatic activity uses [γ-³²P]ATP and sphingosine as substrates, with the radiolabeled S1P product recovered by organic extraction, displayed by thin layer chromatography, and quantified by liquid scintillation counting. Although this assay is sensitive and accurate, it is slow and labor-intensive; thus, it precludes the simultaneous screening of more than a few inhibitor compounds. Here we describe a 96-well assay for SphKs that is rapid and reproducible. Our method, which takes advantage of the limited solubility of S1P, detects radioactive S1P adhering to the plate by scintillation proximity counting. Our procedure obviates extraction into organic solvents, postreaction transfers, and chromatography. Furthermore, our assay enables assessment of both inhibitors and substrates, and it can detect endogenous SphK activity in cell and tissue extracts. The SphK kinetic parameter, K_m, and the K_i values of inhibitors determined with our assay and the conventional assay were indistinguishable. These results document that our assay is well-suited for the screening of chemical libraries of SphK inhibitors.     

Real-time assay method of lipid extraction activity

Intracellular lipid translocation is mediated by lipid transfer proteins and their functional impairments cause severe disorder in lipid metabolism. However, molecular mechanisms of protein-mediated lipid transfer remain unclear since conventional assay methods could not observe elementary processes in the lipid transfer reaction, such as lipid bilayer binding and lipid uptake. In this study, we found that ceramide extraction mediated by a ceramide trafficking protein (CERT) could be detected as decreasing the response of surface plasmon resonance (SPR). Based on this finding, we developed a novel real-time assay method that enables quantitative evaluation of the ceramide extraction activity of CERT, using the SPR technique. Performing this SPR-based assay using ceramide-embedded and ceramide-free lipid bilayers as ligands allows for the exclusive investigation of ceramide uptake processes, differentiating them from other CERT-membrane binding events. Furthermore, mutagenesis experiments of CERT using this SPR-based assay clearly elucidated whether an amino acid residue plays a role in the ceramide uptake process or the lipid bilayer binding process. This SPR-based assay method can separately evaluate the lipid extraction activity and lipid bilayer binding activity of the lipid transfer proteins, and provide more detailed information about lipid transfer phenomena.     

Polysaccharide kinase activity of human milk IgG antibodies

A small fraction of human milk IgG antibodies is shown to possess polysaccharide kinase activity for the first time. Unlike all known kinases, IgG antibodies can use as phosphate donor not only [gamma-(32)P]ATP, but also directly [(32)P]ortho-phosphate. Human milk IgGs therefore possess high affinity to ortho-phosphate (K(m) = 9-71 microM), which is a more effective substrate than ATP. IgG antibodies possessing polysaccharide kinase activity are yet another example of natural abzymes possessing not hydrolytic, but synthetic enzymatic activity.     

Phosphatidate Kinase,A Novel Enzyme in Phospholipid Metabolism (Characterization of the Enzyme from Suspension-Cultured Catharanthus roseus Cells)

Phosphatidate kinase (adenosine 5[prime]-triphosphate:phosphatidic acid phosphotransferase), a novel enzyme of phospholipid metabolism, was detected recently in the plasma membranes of suspension-cultured Catharanthus roseus cells and purified (J.B. Wissing, H. Behrbohm [1993] Plant Physiol 102: 1243-1249). In the present work the properties of phosphatidate kinase are described. The enzyme showed a pH optimum of 6.1 and an isoelectric point of 4.8, and was rather stable in the presence of its substrates. Although the kinase accepted both ATP and GTP, with Km values of about 12 and 18 [mu]M, respectively, the only lipid substrate was phosphatidic acid; neither lysophosphatidic acid nor any other lipid tested was phosphorylated. With 32P- and 14C-labeled diacylglycerol pyrophosphate, the product of the enzyme, it was shown that the kinase catalyzes a reversible reaction. The activity of the extracted enzyme depended on the presence of surfactants such as Triton X-100 or [beta]-octylglucoside, whereas deoxycholate was strongly inhibitory. Kinetic analysis with Triton X-100/phosphatidate mixed micelles performed according to the "surface dilution" kinetic model showed saturation kinetics with respect to both bulk and surface concentration of phosphatidate. The interfacial Michaelis constant for phosphatidate was determined as 0.6 mol %.     

Ceramide in nitric oxide inhibition of glioma cell growth. Evidence for the involvement of ceramide traffic

The treatment of C6 glioma cells with the nitric oxide donor, PAPANONOate ((Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate), resulted in a dose-dependent inhibition of cell proliferation. This was associated to a rapid and significant increase of ceramide levels and was mimicked by treatments that augment cellular ceramide. Metabolic experiments with radioactive sphingosine, serine, and choline showed that nitric oxide strongly reduced the utilization of ceramide for the biosynthesis of both sphingomyelin and glucosylceramide. Nevertheless, nitric oxide did not modify the activity of different enzymes of ceramide metabolism. The possibility that nitric oxide impairs the availability of ceramide for sphingolipid biosynthesis was then investigated. The metabolism of N-hexanoyl-[(3)H]sphingosine demonstrated that nitric oxide did not affect the biosynthesis of N-hexanoyl-[(3)H]sphingolipids but inhibited the metabolic utilization of long chain [(3)H]ceramide, synthesized in the endoplasmic reticulum (ER) from the recycled [(3)H]sphingosine. Moreover, results obtained with fluorescent ceramides, brefeldin A, ATP depletion, as well as in a ceramide transport assay indicate that nitric oxide impairs the traffic of ceramide from ER to Golgi apparatus. All this supports that, in glioma cells, the modulation of ceramide traffic can contribute to the regulation of its intracellular levels and participate in the nitric oxide-activated signaling pathway involved in the control of cell proliferation.     

Rapid protein kinase assay using phosphocellulose-paper absorption.

Protein kinase (EC 2.7.1.37) catalyzes the phosphorylation of serine and threonine residues of a number of proteins. Histone is widely used as an acceptor substrate in measuring the activity of this enzyme isolated from a variety of sources. We have devised a rapid procedure for resolving phosphohistone from ATP and its metabolites based on the specific absorption of phosphorylated histone onto phosphocellulose paper. Using [γ-³²P]ATP as the phosphoryl donor, aliquots of the protein kinase assay mixture are applied to phosphocellulose-paper disks that are then immersed in water which elutes [γ-³²P]ATP and metabolites. After brief organic solvent extraction and drying, bound radioactivity is measured by liquid scintillation spectrometry.     

Platelet membrane phosphatidylinositol kinase activity. Triton X-100 effects provide evidence for intramicellar reaction.

Phosphatidylinositol (PI) kinase activity of platelet membranes was solubilized and partially purified by anion-exchange chromatography to measure the initial enzymatic rates. Kinetic studies were performed in the presence of Triton X-100 to obtain mixed micelles. The partially purified enzyme exhibited a Michaelian behaviour towards ATP, with a Km of 58 microM. The enzymatic rates were dependent upon Triton concentrations. Upon increasing its concentration, this detergent exhibited an activating effect followed by an inhibitory one. The optimal micellar Triton concentration was proportionnal to the PI concentration used in the assay. Conversely, the behaviour of the enzyme towards PI was dependent upon the Triton concentration. However, when PI concentration was expressed as its surfacic concentration within the micelles, the activity became independent of the detergent concentration, and a Km value of 0.09 mol/mol was estimated. Therefore, in vitro phosphorylation of phosphatidylinositol by PI kinase is rate-limited by an intramicellar reaction, and provides a study model for the in vivo reaction.     

Lipid kinase activity of antibodies from milk of clinically healthy human mothers

We have shown recently that polyclonal human milk sIgA contains a subfraction of antibodies (Abs) tightly bound to unusual minor milk lipids containing sialic acid. Here, we show that a small subfraction of milk IgG is tightly bound to the similar or the same minor lipids. The ability of small fractions of sIgA and IgG from human milk to phosphorylate selectively two minor lipids in the presence of [gamma-(32)P]nucleoside triphosphates was shown here for the first time to be an intrinsic property of these antibodies. In contrast to known kinases, antibodies with lipid kinase activity can transfer phosphoryl group to lipids not only from ATP but also from other different nucleotides (dATP, GTP, dGTP, UTP, TTP) with comparable efficiencies (30-100%). To our knowledge, there are no examples of enzymes using orthophosphate as a substrate of phosphorylation reactions. An extremely unusual property of lipid kinase Abs is their high affinity for orthophosphate (K(m)=1.6-5.6 microM) and capability to phosphorylate minor lipids using [(32)P]orthophosphate as donor of phosphate group. The relative specific activity and affinity of abzymes for orthophosphate and ATP depend significantly on donor milk. However, the levels of Ab-dependent phosphorylation of lipids for all Abs in the case of ATP (100%) and orthophosphate (60-80%) as substrates are comparable. The first example of natural abzymes with synthetic activity was milk sIgA with protein kinase activity. Most probably, lipid kinase sIgA and IgG of human milk are the second example of Abs with synthetic activity.