Kinetics of Ca2+-induced fusion of cardiolipin-phosphatidylcholine vesicles: correlation between vesicle aggregation, bilayer destabilization, and fusion

We have investigated the kinetics of Ca2+-induced aggregation and fusion of large unilamellar vesicles composed of an equimolar mixture of bovine heart cardiolipin and dioleoylphosphatidylcholine. Mixing of bilayer lipids was monitored with an assay based on resonance energy transfer (RET) and mixing of aqueous vesicle contents with the Tb/dipicolinate assay. The results obtained with either assay were analyzed in terms of a mass action kinetic model, providing separate rate constants for vesicle aggregation and for the fusion reaction proper. At different Ca2+ concentrations, either at 25 degrees C or at 37 degrees C, aggregation rate constants derived from the data obtained with the RET assay were the same as those derived from the Tb/dipicolinate data, indicating that mixing of bilayer lipids occurred only during vesicle aggregation events that resulted in mixing of aqueous contents as well. At 25 degrees C, identical fusion rate constants were obtained with either assay, indicating that at this temperature the probability of lipid mixing and that of aqueous contents mixing, occurring after vesicle aggregation, were the same. The fusion rate constants for the RET assay increased more steeply with increasing temperature than the fusion rate constants derived from the Tb/dipicolinate data. As a result, at 37 degrees C the tendency of the vesicles, after aggregation, to mix lipids was slightly higher than their tendency to mix aqueous contents. The aggregation rate constants increased steeply with Ca2+ concentrations increasing in a narrow range (9.5-11 mM), indicating that, in addition to a Ca2+-dependent charge neutralization on the vesicle surface, structural changes in the lipid bilayer are involved in the aggregation process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-induced fusion of phosphatidylserine vesicles: mass action kinetic analysis of membrane lipid mixing and aqueous contents mixing

We have investigated the initial kinetics of Ca2+-induced aggregation and fusion of phosphatidylserine large unilamellar vesicles at 3, 5 and 10 mM Ca2+ and 15, 25 and 35 degrees C, utilizing the Tb/dipicolinate (Tb/DPA) assay for mixing of aqueous vesicle contents and a resonance energy transfer (RET) assay for mixing of bilayer lipids. Separate rate constants for vesicle aggregation as well as deaggregation and for the fusion reaction itself were determined by analysis of the data in terms of a mass action kinetic model. At 15 degrees C the aggregation rate constants for either assay are the same, indicating that at this temperature all vesicle aggregation events that result in lipid mixing lead to mixing of aqueous contents as well. By contrast, at 35 degrees C the RET aggregation rate constants are higher than the Tb/DPA aggregation rate constants, indicating a significant frequency of reversible vesicle aggregation events that do result in mixing of bilayer lipids, but not in mixing of aqueous vesicle contents. In any conditions, the RET fusion rate constants are considerably higher than the Tb/DPA fusion rate constants, demonstrating the higher tendency of the vesicles, once aggregated, to mix lipids than to mix aqueous contents. This possibly reflects the formation of an intermediate fusion structure. With increasing Ca2+ concentrations the RET and the Tb/DPA fusion rate constants increase in parallel with the respective aggregation rate constants. This suggests that fusion susceptibility is conferred on the vesicles during the process of vesicle aggregation and not solely as a result of the interaction of Ca2+ with isolated vesicles. Aggregation of the vesicles in the presence of Mg2+ produces neither mixing of aqueous vesicle contents nor mixing of bilayer lipids.     

Determination of rate constants for the reaction of hydroxyl radicals with some purines and pyrimidines using sunlight

Sunlight mediated hydroxyl radical production from aqueous ferric perchlorate at low pH has been investigated using deoxyribose-thiobarbituric acid assay. The rate of production of hydroxyl radical was found to be dependent on the time of irradiation. Hydroxyl radical scavengers can compete with deoxyribose for hydroxyl radicals produced in the system leading to a decreased yield of thiobarbituric acid chromogen. The second-order rate constants of the added scavengers can be determined using a simple competition kinetic method. The rate constants for the reaction of hydroxyl radical with a number of purine and pyrimidine derivatives were determined using this method. The rate constants obtained (1-7 x 10(9) dm(3) mol(-1) s(-1)) were found to be in good agreement with those reported using pulse radiolysis technique. The rate constants of dimethyluracil, xanthosine, amino and methyl substituted pyrimidines, cytidine monophosphate and uridine monophosphate were also determined by this method. It is proposed that sunlight mediated production of hydroxyl radical coupled with deoxyribose-thiobarbituric acid assay is a simple and efficient method for the determination of rate constants for the reaction of hydroxyl radical with a wide range of biomolecules.     

Fluorescence anisotropy-based measurement of Pseudomonas aeruginosa penicillin-binding protein 2 transpeptidase inhibitor acylation rate constants

High-molecular-weight penicillin-binding proteins (PBPs) are essential integral membrane proteins of the bacterial cytoplasmic membrane responsible for biosynthesis of peptidoglycan. They are the targets of antibacterial β-lactam drugs, including penicillins, cephalosporins, and carbapenems. β-Lactams covalently acylate the active sites of the PBP transpeptidase domains. Because β-lactams are time-dependent inhibitors, quantitative assessment of the inhibitory activity of these compounds ideally involves measurement of their second-order acylation rate constants. We previously described a fluorescence anisotropy-based assay to measure these rate constants for soluble constructs of PBP3 (Anal. Biochem. 439 (2013) 37–43). Here we report the expression and purification of a soluble construct of Pseudomonas aeruginosa PBP2 as a fusion protein with NusA. This soluble PBP2 was used to measure second-order acylation rate constants with the fluorescence anisotropy assay. Measurements were obtained for mecillinam, which reacts specifically with PBP2, and for several carbapenems. The assay also revealed that PBP2 slowly hydrolyzed mecillinam and was used to measure the rate constant for this deacylation reaction.     

Scintillation proximity assay as a high-throughput method to identify slowly dissociating nonpeptide ligand binding to the GnRH receptor

Many nonpeptide antagonists of the gonadotropin-releasing hormone (GnRH) receptor, as well as other drug targets, possess a broad range of dissociation kinetic rate constants. Current methods to accurately define kinetic rate parameters such as K(on) and K(off) are time and labor intensive, prompting the development of a screening assay to identify slowly dissociating compounds for follow-up rate constant determination. The authors measured inhibition binding constants (K(i)) for GnRH receptor antagonists after 30 min and 10 h of incubation and observed several compounds with markedly decreased K(i) values over time (Ki(30 min)/Ki(10 h) > 6). They used scintillation proximity assay technology to perform these binding experiments because this homogeneous assay does not have a fixed termination end point as does filtration binding, permitting successive readings to be taken from the same assay plate over an extended period of time. They also used a quantitative method of kinetic rate analysis to confirm that a large disparity between a compound's K(i) value at 30 min and 10 h could identify compounds that dissociate slowly. Thus, the K(i) ratio can be used to screen for and select compounds to test using more quantitative, albeit lower throughput methods to accurately define kinetic rate constants.     

A kinetic method for determination of arylsulfatase activity

A new assay for arylsulfatase activity is described, which consists of direct kinetic measurements of pseudo-first-order rate constants by means of a spectrophotometric procedure. The assay is applicable for reactions occurring at different pH conditions and it can be used for a wide range of activities.     

A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy

Previously it has not been possible to determine the rate of deamination of cytosine in DNA at 37 degrees C because this reaction occurs so slowly. We describe here a sensitive genetic assay to measure the rate of cytosine deamination in DNA at a single cytosine residue. The assay is based on reversion of a mutant in the lacZ alpha gene coding sequence of bacteriophage M13mp2 and employs ung- bacterial strains lacking the enzyme uracil glycosylase. The assay is sufficiently sensitive to allow us to detect, at a given site, a single deamination event occurring with a background frequency as low as 1 in 200,000. With this assay, we determined cytosine deamination rate constants in single-stranded DNA at temperatures ranging from 30 to 90 degrees C and then calculated that the activation energy for cytosine deamination in single-stranded DNA is 28 +/- 1 kcal/mol. At 80 degrees C, deamination rate constants at six sites varied by less than a factor of 3. At 37 degrees C, the cytosine deamination rate constants for single- and double-stranded DNA at pH 7.4 are 1 x 10(-10) and about 7 x 10(-13) per second, respectively. (In other words, the measured half-life for cytosine in single-stranded DNA at 37 degrees C is ca. 200 years, while in double-stranded DNA it is on the order of 30,000 years.) Thus, cytosine is deaminated approximately 140-fold more slowly when present in the double helix. These and other data indicate that the rate of deamination is strongly dependent upon DNA structure and the degree of protonation of the cytosine. The data suggest that agents which perturb DNA structure or facilitate direct protonation of cytosine may induce deamination at biologically significant rates. The assay provides a means to directly test the hypothesis.     

Rates of dissociation of steroid and thyroid hormones from human serum albumin

A rapid filtration assay employing dextran-coated charcoal as acceptor particles for free hormone was used to measure rates of dissociation of steroid and thyroid hormones from human serum albumin. Modification of a previously described assay allowed measurements at 1-s intervals. Nevertheless, this still permitted only minimum estimates of the dissociation rate constants. The hormones studied were thyroxine, 3,5,3'-triiodothyronine, cortisol, corticosterone, testosterone, dihydrotestosterone, estradiol, progesterone, and aldosterone. The apparent dissociation rate constant of the thyroxine-albumin complex at 37 degrees C was 1.3 +/- 0.2 s-1 (t 1/2, 0.5 s). The apparent dissociation rate constants of the other hormone-albumin complexes at 37 degrees C generally exceeded 2 s-1 (t 1/2 less than 0.35 s). Apparent dissociation rate constants at 4 degrees C were only slightly lower. These findings indicate that steroid and thyroid hormones dissociate from albumin rapidly compared with the 1-s capillary transit times that characterize many tissues.     

A practical design of an elution system for preparative electrophoresis in polyacrylamide gel.

The previously applied direct spectrophotometric assay of β-lactamase activity toward cephalosporins was found readily applicable to penicillins too. The differential uv absorption spectra of various penicillins and their corresponding penicilloic acids were determined. The appropriate experimental conditions were examined and the spectrophotometric assay seems adequate for the study of several substrates in a mixture. Also this method was found highly suitable for computerized analysis of the kinetic data for the determination of Michaelis constants of the various penicillins. The use of the integrated form of the rate equation for the evaluation of the best estimates of Michaelis constants was found advantageous.     

Production and consumption of nitric oxide by denitrifying bacteria under anaerobic and aerobic conditions

Abstract: Pseudomonas aeruginosa, P. stutzeri and Azospirillum brasilense showed highest NO production rates and NO consumption rate constants when anaerobically grown cells were tested under anaerobic conditions. Aerobic assay conditions resulted in 20–75-fold lower NO production rates. NO consumption rate constants, however, decreased by less than a factor of four. NO consumption activity was observed even in aerobically grown P. aeruginosa , provided the assay was done under anaerobic conditions. Obviously, NO consumption was less O₂-sensitive than NO production so that compensation between production and consumption occurred at lower NO mixing ratios under aerobic than under anaerobic conditions.     

The deoxyribose method: a simple "test-tube" assay for determination of rate constants for reactions of hydroxyl radicals

Hydroxyl radicals, generated by reaction of an iron-EDTA complex with H2O2 in the presence of ascorbic acid, attack deoxyribose to form products that, upon heating with thiobarbituric acid at low pH, yield a pink chromogen. Added hydroxyl radical "scavengers" compete with deoxyribose for the hydroxyl radicals produced and diminish chromogen formation. A rate constant for reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. For a wide range of compounds, rate constants obtained in this way are similar to those determined by pulse radiolysis. It is suggested that the deoxyribose assay is a simple and cheap alternative to pulse radiolysis for determination of rate constants for reaction of most biological molecules with hydroxyl radicals. Rate constants for reactions of ATP, ADP, and Good's buffers with hydroxyl radicals have been determined by this method.     

A continuous, nonradioactive assay for histone acetyltransferases

Histone acetyltransferases (HATs) catalyze the acetyl-group transfer from acetyl-CoA to the epsilon-amino group of specific lysine residues within core histone proteins. HATs and other chromatin-remodeling enzymes have been recently shown to regulate gene activation within specific loci. To facilitate mechanistic studies, we have developed two continuous, nonradioactive assays for the prototypical GCN5 HAT. The CoASH generated in the HAT reactions was continuously measured by using a coupled enzyme system with either alpha-ketoglutarate dehydrogenase or pyruvate dehydrogenase. The CoASH-dependent oxidation of alpha-ketoglutarate or pyruvate is accompanied by the reduction of NAD to NADH, which was measured spectrophotometrically at 340 nm. The steady-state rate constants with substrates acetyl-CoA and a synthetic peptide (corresponding to the first 20 amino acids of H3 histone) were determined. The resulting rate constants were not significantly different between the two coupled assays, providing strong validation of these methods. Rate constants were also determined using the commonly employed radioactive filter-binding assay and compared. The 1.5- to 5-fold lower values obtained in the radioactive end-point assay are discussed in terms of the technical problems and limitations of this assay. The coupled assays should be widely applicable since the production of CoASH is common to all HAT enzymes, regardless of protein substrate.     

Acetylcholine receptor-controlled ion translocation caused by phenyltrimethylammonium and nereistoxin: simple estimation of equilibrium constants of the minimal model

The rate of slow Li+ influx and the fraction of active form of acetylcholine receptor (AChR) of Electrophorus electricus membrane vesicles at equilibrium between the active and desensitized forms of the receptor were measured in the presence of various concentrations of phenyltrimethylammonium (PTA) and nereistoxin (NTX), by a simple filtration assay and flame emission spectroscopy. The equilibrium constants of these ligands in the minimal model, which accounts for the AChR-mediated ion flux, were estimated simply from these two measurements, since the equilibrium constants for acetylcholine (ACh) and carbamylcholine (Carb) estimated from two kinetic measurements agreed well with those estimated from five sophisticated kinetic measurements of AChR-mediated ion fluxes. PTA showed high potency but not high efficacy, and showed inhibition when large doses were applied. NTX showed both low potency and low efficacy and acted as an inhibitor when it was added with Carb. The apparent dissociation constants of these three agonists evaluated from the minimal model and the equilibrium constants agreed with those obtained by assay of inhibition of radiolabeled ligand binding.     

Acyl-CoA-binding protein from cow. Binding characteristics and cellular and tissue distribution.

Hydroxyl radicals (OH.) in free solution react with scavengers at rates predictable from their known second-order rate constants. However, when OH. radicals are produced in biological systems by metal-ion-dependent Fenton-type reactions scavengers do not always appear to conform to these established rate constants. The detector molecules deoxyribose and benzoate were used to study damage by OH. involving a hydrogen-abstraction reaction and an aromatic hydroxylation. In the presence of EDTA the rate constant for the reaction of scavengers with OH. was generally higher than in the absence of EDTA. This radiomimetic effect of EDTA can be explained by the removal of iron from the detector molecule, where it brings about a site-specific reaction, by EDTA allowing more OH. radicals to escape into free solution to react with added scavengers. The deoxyribose assay, although chemically complex, in the presence of EDTA appears to give a simple and cheap method of obtaining rate constants for OH. reactions that compare well with those obtained by using pulse radiolysis.     

Kinetic consequences of covalent linkage of DNA binding polyamides

Polyamides composed of N-methylpyrrole (Py) and N-methylimidazole (Im) subunits can bind in the minor groove of DNA at predetermined sequences with subnanomolar affinity and high specificity. Covalent linkage of polymer subunits using a gamma-aminobutyric acid linker has been shown to increase both the affinity and specificity of polyamides. Using a fluorescence detected stopped-flow assay, we have studied the differences in association and dissociation kinetics of a series of polyamides representing unlinked, hairpin and cyclic analogues of the four ring polyamide ImPyPyPy-beta-Dp. Whereas the large differences seen in the equilibrium association constants between the unlinked and covalently linked polyamides are primarily due to higher association rate constants, discrimination between matched and mismatched sites by each polyamide can be ascribed in large part to differences in their dissociation rate constants. The consequences of this kinetic behavior for future design are discussed.     

Linear and Non Linear Competition Plots in the Deoxyribose Assay for Determination of Rate Constants for Reaction of non Steroidal Antiinflammatory Drugs with Hydroxyl Radicals

Performing the deoxyribose (DR) assay for determination of the rate constants for reaction of non steroidal antiinflammatory drugs with hydroxyl radicals led to some unusual competition plots. The molecules from the arylpropionic family of drugs: ibuprofen, flurbiprofen, ketoprofen and naproxen produced the linear relationship. However, acemetacin, diclofenac Na, flufenamic acid, indometacin, niflumic acid, tolmetin Na and sulindac presented non linear competition plots manifesting at relatively low drug concentrations. This effect was corrected by increasing DR concentrations from 2.8 mM to 15 mM. The modification did not affect rate constants values for those derivatives which already presented a linear plot at 2.8 mM, but allowed to calculate rate constants for other compounds. It is suggested that the experimental conditions have to be adapted particularly for those derivatives with a relatively high. rate constant for reaction with the radical species. The oxicam derivatives (tenoxicam and piroxicam) presented another kind of deviation that revealed a prooxidant effect in this system: non linear plots were also observed at relatively low drug concentrations, but in the opposite direction than for the other molecules. This last effect was independent of DR concentration but could be corrected by increasing ascorbate concentration in the system.     

Fluorescent intercalator displacement replacement (FIDR) assay: determination of relative thermodynamic and kinetic parameters in triplex formation--a case study using triplex-forming LNAs

Triplex forming oligonucleotides (TFOs) are the most commonly used approach for site-specific targeting of double stranded DNA (dsDNA). Important parameters describing triplex formation include equilibrium binding constants (K(eq)) and association/dissociation rate constants (k(on) and k(off)). The 'fluorescent intercalator displacement replacement' (FIDR) assay is introduced herein as an operationally simple approach toward determination of these parameters for triplexes involving TC-motif TFOs. Briefly described, relative rate constants are determined from fluorescence intensity changes upon: (i) TFO-mediated displacement of pre-intercalated and fluorescent ethidium from dsDNA targets (triplex association) and (ii) Watson-Crick complement-mediated displacement of the TFO and replacement with ethidium (triplex dissociation). The assay is used to characterize triplexes between purine-rich dsDNA targets and TC-motif TFOs modified with six different locked nucleic acid (LNA) monomers, i.e. conventional and C5-alkynyl-functionalized LNA and α-L-LNA pyrimidine monomers. All of the studied monomers increase triplex stability by decreasing the triplex dissociation rate. LNA-modified TFOs form more stable triplexes than α-L-LNA-modified counterparts owing to slower triplex dissociation. Triplexes modified with C5-(3-aminopropyn-1-yl)-LNA-U monomer Z are particularly stable. The study demonstrates that three affinity-enhancing features can be combined into one high-affinity TFO monomer: conformational restriction of the sugar ring, expansion of the pyrimidine π-stacking surface and introduction of an exocyclic amine.     

Tropomyosin inhibits the rate of actin polymerization by stabilizing actin filaments

Tropomyosin inhibition of the rate of spontaneous polymerization of actin is associated with binding of tropomyosin to actin filaments. Rate constants determined by using a direct electron microscopic assay of elongation showed that alpha alpha- and alpha beta-tropomyosin have a small or no effect on the rate of elongation at either end of the filaments. The most likely explanation for the inhibition of the rate of polymerization of actin in bulk samples is that tropomyosin reduces the number of filament ends by mechanical stabilization of the filaments.     

Emerging challenges in ligand discovery: new opportunities for chromatographic assay

Ligand discovery initiatives are facing interesting challenges as ever-increasing numbers of proteins are entering screening programs. As an answer to steady pressure to improve performance in drug discovery, ligand discovery can expect to play an expanded role in generating small molecules as probes to help uncover the function of novel proteins. Chromatographic assay formats can offer new entry points into standard interaction characterization (binding and rate constants) as well as powerful, scaleable methods for compound screening. This review presents recent advancements in chromatographic assay technology, with a particular focus on frontal affinity chromatography as a platform technology for interaction analysis.     

Emerging challenges in ligand discovery: new opportunities for chromatographic assay

Ligand discovery initiatives are facing interesting challenges as ever-increasing numbers of proteins are entering screening programs. As an answer to steady pressure to improve performance in drug discovery, ligand discovery can expect to play an expanded role in generating small molecules as probes to help uncover the function of novel proteins. Chromatographic assay formats can offer new entry points into standard interaction characterization (binding and rate constants) as well as powerful, scaleable methods for compound screening. This review presents recent advancements in chromatographic assay technology, with a particular focus on frontal affinity chromatography as a platform technology for interaction analysis.