A new system for washing ion-exchange paper disks used in radiochemical enzyme assays

A new device has been developed for the removal of radioactive substrate from ion-exchange paper disks, leaving ionized product bound to the paper. It has a number of important advantages over older methods of washing the ion-exchange paper: (1) It is much more thorough and efficient; background absorption of unionized material is reduced to relatively low levels. (2) Fragile media such as DEAE-substituted paper (DE81) are not subjected to the erosive effects of stirrers or agitators. (3) The disks remain organized throughout the sample application and washing procedure, eliminating need for time-consuming sorting of wet radioactive disks after washing. A larger number of assays can therefore be processed conveniently at one time. (4) Radioactive nucleosides and other adsorbable substrates are collected in a cartridge, eliminating the handling and sink-disposal of large volumes or excessive quantities of radioactive materials.     

Use of affinity elution chromatography for the microassay of adenosine 3':5'-monophosphate.

Using the principle of affinity elution chromatography, a microassay has been developed for the determination of picomolar and even smaller quantities of adenosine 3'.5' -monophosphate (cyclic AMP) in biological samples. In principle, cyclic AMP along with the other anions is adsorbed onto an anion exchange cellulose and subsequently, the cyclic nucleotide is specifically eluted with cyclic-AMP -dependent protein kinase. In the actual assay cyclic AMP is determined by competitive displacement of radiolabelled cyclic AMP by non-labelled cyclic nucleotide. The described procedure results in a linear dependence for the displacement of the radioactive nucleotide on cyclic AMP concentration and obviates the usual procedure of sample enrichment by lyophilization. The degree of interference of different nucleotides including cyclic GMP was found to be minimal and in this respect the use affinity elution chromatography was comparable to radioimmunoassay.     

Demonstration of Phosphoryl Group Transfer Indicates That the ATP-binding Cassette (ABC) Transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Exhibits Adenylate Kinase Activity

Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane-spanning adenosine 5′-triphosphate (ATP)-binding cassette (ABC) transporter. ABC transporters and other nuclear and cytoplasmic ABC proteins have ATPase activity that is coupled to their biological function. Recent studies with CFTR and two nonmembrane-bound ABC proteins, the DNA repair enzyme Rad50 and a structural maintenance of chromosome (SMC) protein, challenge the model that the function of all ABC proteins depends solely on their associated ATPase activity. Patch clamp studies indicated that in the presence of physiologically relevant concentrations of adenosine 5′-monophosphate (AMP), CFTR Cl⁻ channel function is coupled to adenylate kinase activity (ATP+AMP ⇆ 2 ADP). Work with Rad50 and SMC showed that these enzymes catalyze both ATPase and adenylate kinase reactions. However, despite the supportive electrophysiological results with CFTR, there are no biochemical data demonstrating intrinsic adenylate kinase activity of a membrane-bound ABC transporter. We developed a biochemical assay for adenylate kinase activity, in which the radioactive γ-phosphate of a nucleotide triphosphate could transfer to a photoactivatable AMP analog. UV irradiation could then trap the ³²P on the adenylate kinase. With this assay, we discovered phosphoryl group transfer that labeled CFTR, thereby demonstrating its adenylate kinase activity. Our results also suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for adenylate kinase activity. These biochemical data complement earlier biophysical studies of CFTR and indicate that the ABC transporter CFTR can function as an adenylate kinase.     

A simplified assay of enzymes catalyzing ATP-pyrophosphate exchange reactions.

The ATP-pyrophosphate exchange reaction is a widely used method for determination of the activity of enzymes forming enzyme-adenylate intermediates in the course of their catalytic action (1–4). For separation of the labeled ATP formed in the enzyme-catalyzed reaction from the labeled pyrophosphate, the adsorption of ATP on activated Norit is generally used. This method, however, is rather time consuming (considering the extensive washings required for the removal of pyrophosphate from Norit), and its reproducibility is highly dependent on pretreatment of charcoal with acid, Another method of separation applies anion-exchange paper (5). According to our experience, separation on DEAE-cellulose paper (Whatman DE 81) is not sufficient.     

Development of a Transcreener kinase assay for protein kinase A and demonstration of concordance of data with a filter-binding assay format

A Transcreener kinase fluorescence polarization (FP) assay has been developed for the serine/threonine kinase protein kinase A (PKA). The PKA Transcreener kinase assay is an homogenous, competitive antibody-based FP assay that uses Far Red Alexa Fluor 633-labeled adenosine 5' disphosphate (ADP) tracer and mouse monoclonal anti-ADP antibody. The Transcreener PKA assay was validated with both known PKA inhibitors and library compounds. The Transcreener PKA assay is resistant to low-wavelength (or common) fluorescent interference from small-molecule library compounds and generates IC50 results comparable with current radioactive filter-binding assay.     

Radiochemical assays for creatine kinase and arginine kinase using rapid ion exchange separations

Simple and specific radiochemical assays for the determination of creatine and arginine kinase activities in crude tissue extracts are described. Creatine kinase is assayed by incubation with radioactive creatine and subsequent determination of the radioactivity in creatine phosphate. Creatine and creatine phosphate are separated on DEAE-cellulose ion exchange papers. Arginine kinase is assayed simllarly by using radioactive arginine and separating it from arginine phosphate on short ion exchange columns. An assay for creatine kinase in the direction of creatine formation is also described.     

A high-throughput, nonisotopic, competitive binding assay for kinases using nonselective inhibitor probes (ED-NSIP)

A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of beta-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of beta-galactosidase (termed ED-SS). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3alpha, an enzyme previously screened in a radioactive kinase assay (i.e., measurement of [(33)P]-gamma-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC(50)) of 11 nM, which was similar to the IC(50) value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z' factors > 0.7) with high interassay precision (R(2) > 0.96). Interference of compounds with the beta-galactosidase signal readout was negligible. In conclusion, the DiscoveRx competitive kinase binding assay, termed ED-NSIP trade mark, provides a novel method for screening kinase inhibitors. The format is homogeneous, robust, and amenable to automation. Because there is no requirement for substrate-specific antibodies, the assay is particularly applicable to Ser/Thr kinase assay, in which difficulties in identifying a suitable substrate and antibody preclude development of nonisotopic assays. Although the nonselective kinase inhibitor, staurosporine, was used here, chemically conjugating the ED fragment to other small molecule enzyme inhibitors is also feasible, suggesting that the format is generally applicable to other enzyme systems.     

Development and comparison of nonradioactive in vitro kinase assays for NIMA-related kinase 2

NIMA (never in mitosis arrest)-related kinase 2 (Nek2) is a serine/threonine kinase required for centrosome splitting and bipolar spindle formation during mitosis. Currently, two in vitro kinase assays are commercially available: (i) a radioactive assay from Upstate Biotechnology and (ii) a nonradioactive fluorescence resonance energy transfer (FRET) assay from Invitrogen. However, due to several limitations such as radioactive waste management and lower sensitivity, a need for more robust nonradioactive assays would be ideal. Accordingly, we have developed four quantitative and sensitive nonradioactive Nek2 in vitro kinase assays: (i) a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) using peptides identified from a physiologically relevant protein substrate, (ii) DELFIA using Nek2 itself, (iii) a homogeneous time-resolved FRET assay termed LANCE, and (iv) A method of detecting phosphorylated products by HPLC. The DELFIA and LANCE assays are robust in that they generated more than 10-fold and 20-fold increases in signal-to-noise ratios, respectively, and are amenable to robotic high-throughput screening platforms. Validation of all four assays was confirmed by identifying a panel of small molecule ATP competitive inhibitors from an internal corporate library. The most potent compounds consistently demonstrated less than 100 nM activity regardless of the assay format and therefore were complementary. In summary, the Nek2 in vitro time-resolved FRET kinase assays reported are sensitive, quantitative, reproducible and amenable to high-throughput screening with improved waste management over radioactive assays.     

Radiochemical assays for adenylate kinase and AMP deaminase using polyethyleneimine-cellulose thin layers

Simple and specific radiochemical asays for adenylate kinase and AMP deaminase activities in crude tissue extracts are described. The radioactive substrate (AMP) is separated from the radioactive product (ADP or IMP) by chromatography on polyethyleneimine-cellulose thin layers. A rapid modification of the adenylate kinase assay is described in which samples of the reaction mixture are transferred directly to the polyethyleneimine-cellulose thin layer.     

Development of a microplate-based, electrophoretic fluorescent protein kinase a assay: comparison with filter-binding and fluorescence polarization assay formats

A microplate-based electrophoretic assay has been developed for the serine/threonine kinase protein kinase A (PKA). The ElectroCapture PKA assay developed uses a positively charged, lissamine-rhodamine-labeled kemptide peptide substrate for the kinase reaction and Nanogen's ElectroCapture HTS Workstation and 384-well laminated membrane plates to electrophoretically separate the negatively charged phosphorylated peptide product from the kinase reaction mix. After the electrophoretic separation, the amount of rhodamine-labeled phosphopeptide product was quantified using a Tecan Ultra384 fluorescence reader. The ElectroCapture PKA assay was validated with both known PKA inhibitors and library compounds. The pK(iapp) results obtained in the ElectroCapture PKA assay were comparable to those generated with current radioactive filter-binding assay and antibody-based competitive fluorescence polarization PKA assay formats.     

Studies on pig liver mevalonate-5-diphosphate decarboxylase

A procedure in which three sequential enzymes of cholesterol biosynthesis, mevalonate kinase (ATP: (R)-mevalonate 5-phosphotransferase, EC 2.7.1.36), phosphomevalonate kinase (ATP: (R)-5-phosphomevalonate phosphotransferase, EC 2.7.4.2) and mevalonate-5-diphosphate decarboxylase (ATP: (R)-5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33), from pig liver, could be purified in the one operation is described. Mevalonate kinase and phosphomevalonate kinase were utilized for the enzymic synthesis of mevalonate 5-diphosphate (both 1-14C-labelled and unlabelled), the substrate for mevalonate-5-diphosphate decarboxylase, using excess free ATP4-. A radioactive assay for the enzyme, based on the release of 14CO2 from [1-14C]mevalonate-5-diphosphate, was developed. The assay allowed reassessment of the metal and nucleotide specificity of the decarboxylase. ATP could be partially replaced by GTP and ITP, but no activity was observed with CTP, UTP or TTP. Apparent activation of the enzyme by ATP4- was observed as found for mevalonate kinase (C.S. Lee and W.J. O'Sullivan (1983) Biochim. Biophys. Acta 747, 215-224) and phosphomevalonate kinase (C.S. Lee and W.J. O'Sullivan (1985) Biochim. Biophys. Acta 839, 83-89). The presence of 1 mM excess free ATP4-, above that complexed as the substrate MgATP2-, decreased the Km for MgATP2- from 0.45 mM to 0.15 mM. MgADP- was shown to act as a competitive inhibitor with respect to MgATP2-.     

Solid-phase methods for sequencing nucleic acids. VII. Chemical degradation of synthetic DNA-RNA hybrid fragments using CCS and DE 81 anion-exchange papers

Synthetic oligo(ribo-deoxyribo)nucleotides were analyzed and characterized by different solid-phase chemical degradation procedures, 5'- and 3'-end labelled mixed fragments were degraded by a slightly modified DNA cleavage procedure using 1 and 10% piperidine for the chain scission reaction and CCS anion-exchange paper. Besides the normal degradation products obtained by the usual modification and strand cleavage reactions of both deoxy- and ribonucleotide residues, additional bands were identified in the sequence patterns resulting from the hydrolysis of the RNA moiety induced by piperidine. Since both degradation reactions cleave the backbone of the mixed DNA-RNA fragments differently and produce nucleotide components with different charges, the degradation products do not interfere and can be resolved by gel electrophoresis on polyacrylamide. In addition, 3'-end labelled DNA-RNA oligomers were degraded by a RNA cleavage procedure using DE 81 anion-exchange paper as solid support. The combination of all three degradation methods allows to confirm the nucleotide sequence.     

Radioisotopic assay of femtomole quantities of total adenine nucleotides,ATP plus ADP,and AMP

AMP is converted to ATP by incubating overnight with pyruvate kinase, phosphoenolpyruvate and adenylate kinase in th prensence of endogenous ATP (ADP) as primer. In a subsequent incubation in the presence of pyruvate kinase, phosphoenolpyruvate, radioactive glucose and hexokinase. ATP and ADP are estimated together by coupling their recycling to the formation of glucose 6-phosphate. The latter is separated by precipitation using 76% (v/v) acetone for radioactivity measurement in the same Eppendorf tube. The sensitivity of these simple procedures matches or exceeds those of luciferase methods of nucleotide determination.     

Fluorescence-based,high-throughput DNA polymerase assay

The commonly used DNA polymerase assay is based on the detection of incorporated radiolabeled nucleotides in a DNA elongation reaction. It is laborious, radioactive, and can be highly variable. Here we report a nonradioactive fluorescence-based assay. The method consists of Cydye-labeled nucleotides, biotinylated primer, and a streptavidin-coated microplate. The assay is found to have sensitivity and dynamic range comparable to the classical radioactive method. Moreover, it has the advantages of being simple, stable, nonradioactive, and suitable for high-throughput applications. We have also found that, to ensure efficient measurement of the enzyme activity, the template DNA used in this method should have a sequence that avoids the incorporation of the fluorescence-labeled nucleotide in a consecutive way.     

Determination of a large number of kinase activities using peptide substrates,P81 phosphocellulose paper arrays and phosphor imaging

To perform phosphoproteomics and signal transduction studies, a number of protein kinase activities and levels must be simultaneously analyzed in different cell samples and correlated with phosphoprotein patterns to obtain conclusions with regard to the regulation of kinase networks. We describe here a miniaturized format of the classical phosphocellulose (P81) paper binding assay with which up to 594 kinase reactions can be simultaneously analyzed. Kinase peptide substrates possessing a minimum of three consecutive basic residues were subjected to phosphorylation in 96-well plates and aliquots of the phosphorylation reactions were spotted on arrays printed on P81 papers. Phosphorylation levels were quantified using a storage phosphor system imager. The versatility of the procedure was validated by analyzing casein kinase 2, protein kinase C, and p34cdc2/cyclin B in cell extracts and testing the effect of known inhibitors and activators on kinase activities. This improved, miniaturized version of the classical P81 paper method combines simplicity, high sensitivity, high reproducibility, high reliability, and optimal Z factors and takes into account possible sources of background signals. We discuss the possibility of automation and the advantages over other methods.     

Determination of the phosphorylation sites of calmodulin catalyzed by casein kinase 2

Calmodulin is specifically phosphorylated by casein kinase 2 (CK 2), but not by casein kinase 1, A kinase, or C kinase. In the present report, the stoichiometry of the phosphorylation of calmodulin by CK 2 in the presence and absence of polylysine and its phosphorylation sites were examined. In the absence of polylysine, the radioactive phosphate incorporated into calmodulin by CK 2 was only 0.01 mol/mol and the phosphorylation occurred at Ser-101. In the presence of polylysine, 1.2 mol of radioactive phosphate was incorporated into 1 mol of calmodulin. In this case, Thr-79 in addition to Ser-101 was phosphorylated, but Ser-81 was not. The sequence around the phosphorylated Thr is Asp-Thr(P)-Asp-Ser-Glu-Glu-Glu-.     

A luminescence-based test for determining ornithine decarboxylase activity

A sensitive chemiluminescence-based method for the assay of ornithine decarboxylase (ODC) has been developed. This method, which permits the detection of putrescine (the product of ODC) at a picomolar range, can be used to determine ODC activity in cellular extracts. Extracts are incubated with ornithine and spotted onto p81 phosphocellulose paper strips. After drying, the papers are washed with ammonium hydroxide to remove contaminants, which may interfere with the assay. Putrescine is next eluted from the paper by shaking in an elution buffer containing magnesium sulfate. Partially purified hog kidney diamine oxidase is then used to oxidize putrescine in the eluate. The hydrogen peroxide formed during the oxidation is determined by chemiluminescence using luminol and peroxidase. This simple analytical method has the sensitivity of conventional assays based on the use of radioactive ornithine.     

Contribution of Akt and endothelial nitric oxide synthase to diazoxide-induced late preconditioning

The opening of mitochondrial ATP-sensitive K+ (mitoK(ATP)) channels has a significant role in delayed ischemic preconditioning, and nitric oxide (NO) is a well-known trigger for its activation. However, the source of NO remains unknown. Phosphorylation of endothelial NO synthase (eNOS) increases NO production and reduces apoptosis through the Akt signaling pathway. To elucidate the Akt signaling pathway involved in the opening and antiapoptotic effect of mitoKATP channel during delayed pharmacological preconditioning, the mitoKATP channel opener diazoxide (DE, 7 microg/kg i.p.) alone or DE plus Nomega-nitro-L-arginine methyl ester (L-NAME, 30 microg/kg i.v.), an inhibitor of NOS, or wortmannin (WTN, 15 microg/kg i.v.), an inhibitor of phosphatidylinositol 3'-kinase (PI3 kinase), was administered to wild-type (WT) or eNOS(-/-) mice during DE treatment. Twenty-four hours later, hearts were isolated and subjected to 40 min ischemia and 30 min reperfusion (I/R). The effect of DE and other interventions on hemodynamic, terminal dUTP nick-end labeling staining and biochemical changes during I/R was assessed in mouse hearts. Treatment with DE resulted in a 2.2-fold increase in phosphorylation of Akt and a significant increase in eNOS and inducible NOS (iNOS) proteins. Akt is upstream of NOS and the mitoKATP channel as simultaneous pretreatment of WTN with DE abolished phosphorylation of Akt, which was not affected by L-NAME and 5-hydroxydecanoate. In hearts treated with DE, cardiac function was significantly improved after I/R, and apoptosis was also significantly decreased. WTN abolished the antiapoptotic effect of DE. Similarly, S-methylisothiourea, a specific iNOS inhibitor, when given to eNOS(-/-) mice that were pretreated with DE completely abolished the beneficial effects of DE on reduction of apoptotic death. DE was partially effective in eNOS(-/-) mice against the ischemic injury. It is concluded that DE activates Akt through the PI3 kinase signaling pathway and iNOS and eNOS is downstream of Akt.     

Development of solid-phase enzyme-linked immunosorbent assays for the determination of epidermal growth factor receptor and pp60c-src tyrosine protein kinase activity.

Solid-phase ELISAs for the determination of EGF receptor (EGF-R) and pp60c-src tyrosine protein kinase activity are described. The methods were developed and optimized using purified recombinant EGF-R intracellular domain (ICD) and pp60c-src tyrosine protein kinases. A standardized assay that utilizes poly (GluNa-Tyr)4:1 as substrate and a monoclonal antiphosphotyrosine antibody for detection is described. Assay conditions for both enzymes were optimized with respect to substrate and ELISA plate-coating condition, divalent metal ion preferences, enzyme concentration, apparent kinetic constants for ATP, and reaction linearity. Following standardization, a number of reference tyrosine protein kinase inhibitors were tested in the ELISAs and compared to results obtained using solution-phase radioactive tyrosine protein kinase assays, which are based on the transfer of 32P from [gamma-32P]ATP to synthetic substrate. To enable a comprehensive comparison, IC50 values obtained in the ELISA were compared with values obtained in radioactive assays using both the holo-EGF-R and EGF-R ICD kinases. No substantial qualitative differences between these assays were seen. For many routine tyrosine protein kinase assays, semiquantitative or qualitative measurement of TPK activity is adequate. For such purposes, the ELISAs would be an attractive alternative to radioactive assays.     

Glycosphingolipid glycosyltransferase assay using sephadex G-50 chromatography in aqueous phase

An assay method for glycosphingolipid glycosyltransferase activity using simple Sephadex G-50 gel permeation chromatography in an aqueous solvent has been developed. An acceptor glycosphingolipid and a donor radioactive nucleotide sugar were incubated with an enzyme source. The reaction mixture was loaded onto a Sephadex G-50 column previously equilibrated with 0.3% (w/v) Triton X-100, 0.1 M sodium chloride, and 0.02% (w/v) sodium azide. The radiolabeled reaction product was eluted by the same solvent in the excluded volume and was collected directly into a liquid scintillation vial, separated from other radioactive compounds. This assay method was utilized to determine the activity of cytidine 5'-monophosphate-N-acetylneuraminic acid:GM3 ganglioside sialyltransferase, which catalyzes the synthesis of GD3 ganglioside, and proved to be as reliable and sensitive as previously published assay procedures. In addition, this assay can be carried out in less time and is simpler than previously reported procedures.