Decay Of Enzyme Activity Research Articles

Simultaneous Evaluation of β-Glucan and β-Glucanase Relationship during Different Mash Temperature Profiles

High wort β-glucan may contribute to brewery processing problems such as poor run-off, slow filtration, and unwanted haze. To investigate how β-glucanase impacts wort β-glucan throughout mashing, 10 different mashes were considered with varied temperature profiles, malt bills, and levels of malt modification. The European Brewing Congress (EBC) and Institute of Brewing (IoB) mashes were employed to compare the effects of mash conditions on enzyme activity and β-glucan content. Mashes were sampled periodically and evaluated for β-glucan concentration and β-glucanase activity using Megazyme kits adapted to the Gallery™ Plus BeerMaster Discrete Analyzer (Gallery). Enzyme activity quickly decayed in modified IoB mashes (average half-life 12.4 min) accompanied by logarithmic accumulation of wort β-glucan. IoB β-glucan percent extract ranged from 30.3% to 99.5%. In EBC mashes, a slow decay in enzyme activity was followed by an increased rate of decay after 30 min. The β-glucan concentration in well-modified samples remained steady while enzyme activity was appreciable, though it increased after 40 min. As a result, β-glucan percent extract remained relatively low, ranging from 11.9% to 34.3%. The β-glucanase activity at lower temperatures compensates for high malt β-glucan. Measuring wort β-glucan in an EBC mash is insufficient in predicting malt performance in other mash styles. Methods for β-glucan and β-glucanase analysis adapted for the Gallery autoanalyzer increased throughput, enabling analysis of the enzyme and substrate throughout mashing.

Coenzyme regeneration catalyzed by NADH oxidase from Lactococcus lactis

Lactococcus lactis was aerobically grown in a bioreactor to produce NADH oxidase, an enzyme used for NAD+ regeneration. The enzyme was isolated and purified from the cells that were harvested at the end of exponential phase of growth. The influence of temperature, pH and oxygen on enzyme activity was investigated. The enzyme was kinetically characterized at different pH values and in different buffers. It was found that Michaelis constants for oxygen are very low (the highest was 4.5μmoldm−3 at pH 8.0). NADH oxidase was tested as a regenerating enzyme in a model system of l-methionine oxidation catalyzed by l-phenylalanine dehydrogenase from Rhodococcus sp. When NADH oxidase from L. lactis was used for NAD+ regeneration, 100% l-methionine conversion was achieved, while without regeneration of NAD+ it was estimated to be about 28%. The operational stability of NADH oxidase was followed and it was found that enzyme activity decay occurs. The operational stability decay rate constant, kd, was estimated to be 8.0×10−5min−1.

Thermal and pH stabilities of partially purified polyphenol oxidase extracted from Solanum melongenas and Musa sapientum fruits

Enzyme activity depends largely on environmental conditions such as temperature and pH. The stability of polyphenol oxidase (PPO) extracted from Solanum melongenas and Musa sapientum fruits pre-incubated in varying thermal and pH conditions were carried out. Enzyme activity was measured by spectrophotometric methods. The reaction mixture contained 3.5 mL of 0.20 M phosphate buffer (pH = 6.8), 1.0 mL of 0.75 mM catechol, and 0.5 mL of enzyme solution. PPOS. melongenasand PPOM. sapientum gave different temperature and pH optima. The temperature-activity profile of PPOS. melongenas and PPOM. sapientum showed a strong positive correlation (r = 0.907363). At pH = 10.0, PPOM. sapientum activity represented 65.3% decay in enzyme activity, whereas PPOS. melongenas represented 79.3% decay in enzyme activity. PPOS.melongenas and PPOM. sapientum stability at pre-incubated temperatures of 20, 50 and 60°C and pH values of 3.5, 6.0 and 8.0 were measured. Residual activities of PPOS.melongenas and PPOM. sapientum showed a strong positive correlations under the same experimental thermal conditions, with exception at 20°C (r = 0.693375). Specifically, pre-incubation of PPOM.sapientum for t = 90 min at 60°C caused 18.4% decay in relative activity of PPOM. sapientum. At t = 90 min, pre-incubation of PPOM. sapientum, at pH = 3.5 caused decay in activity within the range of 30.8-36.1%, whereas PPOM. sapientum pre-incubated in pH = 6.0 and pH = 8.0 gave decay in activity within the range of (1.5-9.8%) and (2.7-6.5%) respectively. PPOS.melongenas and PPOM. sapientum showed relatively higher stabilities as the incubation pH tended towards alkaline conditions, whereas the two experimental temperatures (20 and 60°C) promoted destabilization. Key words: Polyphenol oxidase, temperature, pH, Solanum melongenas and Musa sapientum.

Thermal and pH Stabilities of Partially Purified Polyphenol Oxidase Extracted from Solanum melongenas and Musa sapientum Fruits

Enzyme activity depends largely on environmental conditions such as temperature and pH. The stability of polyphenol oxidase (PPO) extracted from Solanum melongenas and Musa sapientum fruits preincubated in varying thermal and pH conditions were carried out. Enzyme activity was measured by spectrophotometric methods. The reaction mixture contained 3.5 mL of 0.20 M phosphate buffer (pH = 6.8), 1.0 mL of 0.75 mM catechol, and 0.5 mL of enzyme solution. PPO S. melongenas and PPO M. sapientum gave different temperature and pH optima. The temperature-activity profile of PPO S. melongenas and PPO M. sapientum showed a strong positive correlation ( r = 0.907363). At pH = 10.0, PPO M. sapientum activity represented 65.3% decay in enzyme activity, whereas PPO S. melongenas represented 79.3% decay in enzyme activity. PPOS.melongenas and PPO M. sapientum stability at pre-incubated temperatures of 20, 50 and 60°C and pH values of 3.5, 6.0 and 8.0 were measured. Residual activities of PPO S.melongenas and PPO M. sapientum showed a strong positive correlations under the same experimental thermal conditions, with exception at 20°C ( r = 0.693375). Specifically, pre-incubation of PPO M.sapientum for t = 90 min at 60°C caused 18.4% decay in relative activity of PPO M. sapientum . At t = 90 min, pre-incubation of PPO M. sapientum , at pH = 3.5 caused decay in activity within the range of 30.8-36.1%, whereas PPO M. sapientum pre-incubated in pH = 6.0 and pH = 8.0 gave decay in activity within the range of (1.5-9.8%) and (2.7-6.5%) respectively. PPO S.melongenas and PPO M. sapientum showed relatively higher stabilities as the incubation pH tended towards alkaline conditions, whereas the two experimental temperatures (20 and 60°C) promoted destabilization. Keywords : Polyphenol oxidase, temperature, pH, Solanum melongenas and Musa sapientum . African Journal of Biotechnology Vol. 12(38), pp. 5688-5694

Microbial Biomass Governs Enzyme Activity Decay during Aging of Worm‐Worked Substrates through Vermicomposting

Vermicomposting is the biooxidation and stabilization of organic matter involving the joint action of earthworms and microorganisms, thereby turning wastes into a valuable soil amendment called vermicompost. Studies have focused on the changes in the type of substrates available before and after vermicomposting, but little is known on how these changes take place, especially those changes related with maturation of vermicompost. This study investigated the effects of aging on the microbiological properties of fresh vermicompost produced from pig slurry by analyzing the substrate after the earthworms had left it. We incubated 16-wk-old vermicompost and sampled it after 15, 30, 45, and 60 d analyzing microbial biomass and activity (assessed as microbial biomass N and basal respiration respectively) and four enzymatic activities (beta-glucosidase, cellulase, protease, and alkaline phosphatase). Aging of vermicompost resulted in decreases of microbial biomass and activity. Three of the four enzymes analyzed also showed decrease. An initial increase followed by a rapid decrease in alkaline phosphatase was also recorded. High and significant correlations between microbial biomass and beta-glucosidase (r = 0.62, P < 0.001), cellulase (r = 0.56, P < 0.01), and protease (r = 0.82, P < 0.001) were found. Results suggest that there may be two steps involved in the aging dynamics of vermicompost with regards to extracellular enzyme activity; the first step was characterized by a decrease in microbial populations, which resulted in a reduction in the synthesis of new enzymes. The second step was the degradation of the pool of remaining enzymes. This dynamic does not seem to be affected by earthworms because similar decaying patterns of microbial biomass and activity were found in substrate where earthworms were present.

Photodynamic Inactivation of the Na,K-ATPase Occurs via Different Pathways

The photodynamic, i.e., the light-induced, inactivation of the Na,K-ATPase in the presence of the sensitizer rose bengal was studied under different conditions. The shape of inactivation curves of the enzyme activity was analyzed as well as partial reactions of the pump cycle. Both experimental approaches showed the existence of two different time constants of inactivation of the ion pump, which reflect two pathways of a photodynamic modification. This is supported by the following observations: (1) The amplitude of the initial fast decay of enzyme activity was enhanced in the presence of D2O and reduced in the presence of the singlet oxygen scavenger imidazole. (Similar results were found for the SR Ca-ATPase.) (2) Contrary to the fast enzyme inactivation the slow process shows an inverse dose-rate behavior. (3) Inactivation of the partial reactions of Na+ -binding and of K+-binding to the membrane domain of the Na,K-ATPase showed only a single time constant, which corresponded to the slower time constant of enzyme inactivation. In the presence of high concentrations of singlet oxygen the fast time constant dominated the inactivation of the ATP-induced partial reaction for which the cytoplasmic domains of the enzyme play an important role. The data support the conclusion that fast inactivation is due to modification of the cytoplasmic domains and slow inactivation due to modifications of the membrane domain of the ion pumps.

Dynamics of penicillin G hydrolysis in an electro‐membrane reactor

AbstractAnalysis of penicillin G hydrolysis in a membrane reactor with membrane‐entrapped penicillin G acylase is performed using a mathematical model of the reactor system. An electric field imposed to the reactor is considered to enhance transport rates of reaction components and reaction rate. Diffusion, electrophoretic migration and electro‐osmotic flux across the membrane are considered. The analysis focuses on possible effects of the principal operational parameters (electric field intensity, inlet substrate concentration, membrane thickness) on reactor performance. Multiplicities of steady states are frequently encountered. The membrane reactor performance can be easily targeted towards the required reaction regime by applying a constant or periodically varying electric field to the system. The periodic alternation of the polarity of the electric field substantially increases the effectiveness factor of penicillin hydrolysis compared with the steady state operation. Proper adjustments of electric field intensity may also compensate for the decay in enzyme activity.© 2001 Society of Chemical Industry

Studies on the histidine residues in pigeonpea ( Cajanus cajan L.) urease

The pH dependence of pigeonpea urease catalysis reveals the presence of two ionizable groups of p K a values of 6.2±0.1 and 8.8±0.1, respectively. When urease was treated with excess diethylpyrocarbonate (DEP) at pH 6.8, a time-dependent exponential decay of its activity was observed and the pseudo-first order rate constant was proportional to the reagent concentration. The loss of activity was accompanied by a parallel increase in absorbance at 242 nm. Titration of urease with DEP revealed the presence of 12.9±0.1 ‘accessible’ histidine groups per hexameric pigeonpea urease. Hydroxylamine did not provide significant recovery of the DEP inactivated enzyme. Spectroscopic studies, circular dichroism and fluorescence show no effect of DEP on the gross conformation of urease. Irradiation of urease with visible light in the presence of small concentrations of the basic dyes like Rose Bengal or Methylene Blue at pH 6.8, brought about a time-dependent first-order decay of enzyme activity. Urea and acetohydroxamate (AHA) protect the enzyme against the inactivation by DEP or photo-oxidation. The inactivation reaction with the DEP or Rose Bengal was found to be linearly related to the blocking of 12 essential histidine groups per hexamer for complete inactivation. Since, each protein molecule is known to possess two catalytic units per hexamer hence, we propose that urease possesses at least one essential histidine per catalytic unit. The significance of these results is discussed.

Kinetics of thermal deactivation of enzymes: a simple three parameters phenomenological model can describe the decay of enzyme activity, irrespectively of the mechanism.

Heat induced enzyme inactivation or protein denaturation is now well documented, due to progresses in methods, instruments and computation resources. Complex mechanisms, rather than the classic simple "one step - two states" model (still in use) are recognized in many cases, leading investigators to manipulate more or less complicated kinetic expressions describing the heat induced decay of enzyme activity.We show that the different kinetic expressions related to different mechanisms among the most frequently encountered can be arranged in a common simple three-parameters biexponential equation.This unifying simplification is of interest for people focusing attention to phenomenological rather than mechanistic description of the kinetics of heat induced enzyme deactivation. Moreover, the reduction in the number of parameters reduces the risk of cross-correlation and allows a better estimation of the apparent rate constants (which are in many cases the pertinent required information). It also illustrates the difficulty to make inference of mechanism from kinetics, since the same equation applies for a variety of mechanisms ("kinetic homeomorphism") - in particular, it stresses out the need of caution when reporting on existence of isoenzymes from deactivation kinetics.Application of this simple 3-parameters biexponential kinetic expression has been validated with a number of results in the Literature and current investigations in our laboratory. Two examples are given.

Recovery of α-Agarase Enzyme from Fermentation Broths by Membrane Crossflow Filtration

We describe the recovery of extracellular α-agarase enzyme from fermentation broths using a polypropylene hollow fiber filter with 0.5 μm pores normally used for plasma collection from donors. The permeate flux increased with inlet wall shear rate from 30 L/h·m2 at 1500 s−1 to 45 L/h·m2 at 3000 s−1. But the enzyme activity sieving coefficient (ratio of permeate to inlet activity) decreased with increasing shear rate and transmembrane pressure (TMP). Therefore, the enzyme flux transmitted was found to be maximum at a shear rate of 1500 s−1 and a TMP between 20 and 40 kPa. Since enzyme activity decay with time is less at low temperature, the recovered activity was found to be nearly independent of temperature between 5 and 37°C, the maximum occurring at 22°C. The superimposition of flow and pressure pulsations on the inlet and outlet lines of the filter with a modified roller pump produced permeate flux enhancements ranging from 44% at 1000 s−1 to 68% at 3000 s−1 for a TMP of 3 kPa.

2-Aminoisobutyric acid prevents the substrate-induced inactivation of ACC-synthase without inhibiting the enzyme

The substrate-dependent inactivation of 1-aminocyclopropane-1-carboxylate-synthase (ACC-S) has been studied with partially purified enzymes from sycamore cultured cells and winter squash tissue. For the enzyme from both sources the rate constants of inactivation were related to S-adenosyl- l-methionine (AdoMet) concentrations. At saturating concentrations of AdoMet, the t 1 2 was 20 min for sycamore and 32 min for winter squash enzyme. l-2-Aminoisobutyric acid (AIB), but not l-2-aminobytyric acid (ANB), strongly inhibited the AdoMet-dependent inactivation of both enzymes. At AdoMet concentrations corresponding to K m, AIB lengthened the half-life several times, and at concentrations slightly lower than K m AIB practically overcame the inactivation. Kinetic analysis showed that AIB did not affect the catalytic efficiency of the enzyme. The reciprocal plot of the inactivation constants against AdoMet concentrations revealed that AIB acts as a competitive inhibitor of the substrate-induced decay of enzyme activity.

The effect of dimerization on the heat ability of soluble enzymes

The balance equation describing the deactivation of an enzyme in soluble form under perfect stirring conditions when the enzyme undergoes a dimerization process was written and analytically solved in dimensionless form. The effect of four relevant parameters on the concentration of active enzyme sites under the assumption that a typical steam-heating scheme is followed was described and discussed. Dimerization always brings about faster decay of enzyme activity, and this effect is enhanced by elapsing time and by small ratios of the time scale associated with enzyme decay to that associated with heat transfer.

The inhibitory effect of factor J on the alternative complement pathway

Factor J (FJ) is a cationic glycoprotein with inhibitory activity in C1, the first component of the classical complement pathway. This study demonstrates that FJ is able to regulate the activity of the alternative complement pathway. FJ inhibits the generation of fluid-phase and cell-bound alternative pathway C3 convertase, C3b,Bb (C3-cleaving enzyme). Thus, FJ interferes with the generation of alternative pathway C3 convertase when sheep erythrocytes bearing antibody and activated C3 and C4 (EAC4b,3b) are incubated with the individual complement components, factors B, D, and P. FJ accelerates the decay of C3 convertase with a time course similar to that of factor H, and when both regulators are present together, the decay of enzyme activity is faster than when they are added separately. Furthermore, FJ is able to inhibit the cleavage of C3 by factor B in a fluid-phase assay. FJ prevents the initiation of alternative pathway activation in "more stabilized systems" with well known activators of alternative pathway C3 convertase such as C3 nephritic factor (an autoantibody against alternative pathway C3 convertase), cobra venom factor, and rabbit erythrocytes. In these systems, FJ has no effect on C3 convertase stabilized by rabbit erythrocytes or cobra venom factor. In contrast, FJ promotes the dissociation of C3 convertase stabilized by C3 nephritic factor, but with much lower efficiency than in preventing initiation. Direct interaction of FJ with individual components of C3 convertase was shown by a solid-phase binding assay using plates coated with C3, C3b, B, Bb, or FJ. FJ inhibitory activity in the alternative pathway can be modulated by polyanions like heparin. FJ-mediated inhibition in the alternative complement pathway can be modified by surface interactions, as occurs during alternative pathway C3 convertase activation. Thus, when FJ is adsorbed by and eluted from hydroxylapatite and reverse-phase columns, its inhibitory effect on more stabilized systems is lost. This loss of inhibitory activity is fully reversed when FJ is rechromatographed on heparin-Sepharose or Sepharose columns. Taking into account these data, FJ may be included in the group of highly charged molecules that inhibit the activation of classical and alternative complement pathways (i.e. eosinophil major basic protein, protamine, and heparin).

Effect of sodium and nucleotide on the stability of solubilized calcium-free calcium-ATPase from scallop sarcoplasmic reticulum

In membranous scallop sarcoplasmic reticulum, the alkali metal cations Na+ and K+ and nucleotide together promote dimer formation by the Ca(2+)-free Ca-ATPase and stabilize the enzyme activity [Kalabokis, V. N., Bozzola, J. J., Castellani, L., & Hardwicke, P. M. D. (1991) J. Biol. Chem. 266, 22044-22050]. The dependence of stabilization of the Ca(2+)-free membranous scallop Ca-ATPase on Na+ concentration does not show saturation and may involve several superimposed effects. In order to assess the contribution of dimer toward stabilization, i.e., determine the relative importance of intra- and intermolecular effects on stabilization, the influence of varying Na+ concentration and nucleotide on the decay of enzyme activity of the Ca(2+)-free detergent-solubilized Ca-ATPase was studied. Loss of enzyme activity on removal of Ca2+ with EGTA was associated with loss of capacity for phosphorylation by ATP, a Ca(2+)-dependent function. Stabilization of the soluble Ca(2+)-free enzyme by Na+ showed major differences from that seen with the membranous enzyme. The extent of stabilization of the Ca(2+)-free soluble enzyme by Na+ showed clear saturation with increasing Na+ concentration. In contrast to the Ca(2+)-free membranous enzyme, which is inactivated at pH 7.0 with biphasic first-order kinetics, loss of enzymatic function by the solubilized Ca-ATPase at pH 6.92, 0 degrees C, followed monophasic first-order kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of histidine residues and mechanism of action of isocitrate lyase of castor seedling endosperm

Irradiation of isocitrate lyase of castor seedling endosperm with visible light in the presence of a small concentration of Rose Bengal at pH 6.8 brings about a time-dependent exponential decay of enzyme activity. The first-order rate constant of the inactivation reaction increases with pH in the range pH 6–8 and becomes constant at higher pH values. When the enzyme is treated with diethyl pyrocarbonate (DEPC), a time-dependent exponential decay of its activity is observed and the pseudo first-order rate constant is proportional to the reagent concentration. The loss of activity is accompanied by a parallel increase in absorbance at 240 nm. The latter corresponds to modification of two histidine residues per monomeric subunit of the enzyme for complete inactivation. An analysis of the kinetic data shows that only one of these histidine residues is involved in the catalytic activity. Control experiments ruled out the involvement of residues other than histidine in inactivation by photooxidation and DEPC. Succinate protects the enzyme strongly against inactivation by both procedures but glyoxylate does not. A probable mechanism of action of isocitrate lyase is proposed and discussed in the light of these and earlier results.

Regulation by 1,4-diamines of the ornithine decarboxylase activity induced by ornithine in perifused tumor cells

Ornithine decarboxylase (ODC) activity of Ehrlich carcinoma cells was increased more than 36-fold after being maintained for 3.5 hr in vitro in a special chamber which allowed continuous perifusion with 0.5 mM ornithine; if incubated in vitro without perifusion the ODC activity was, of course, only 9-fold by the same concentration of ornithine. Ornithine withdrawal from the perifusion medium resulted in a decay of enzyme activity observed after 90 min; this decay was prevented by addition of 55 μM pyridoxal to the medium. The 1,4-diamines putrescine, spermidine, spermine, agmatine, histamine, serotonin, tryptamine, chlorpheniramine and harmaline at 55 μM strongly suppressed ODC induction by 0.5 mM ornithine in perifused Ehrlich ascites cells. Methyl derivatives also behave as strong inhibitors of ODC induction. On the contrary, N-acetylation paralleled with a decrease in the inhibition capacity: 55 μM N-acetyl putrescine, N-acetyl serotonin or N-ω-acetylhistamine suppressed ODC induction by ornithine in 66, 64 and 19%, respectively. The addition to the perifusion medium of the same concentrations of 1,3-diamines (1,3-diaminopropane, 1,3-diamino-2-propanol or the alkaloid gramine) as well as 1,5-diamines (1,5-diaminopentane and the antihistaminic doxylamine or cimetidine) failed to suppress the induction of ODC activity by ornithine. Interestingly, 1,4-benzenediamine, which strongly inhibits ODC activity when the induced enzyme is assayed in its presence, did not suppress the induction of the enzyme when both 0.5 mM ornithine and 55,u;M 1,4-benzenediamine were present in the perifusion medium. The inhibitory capacity in down-regulating ODC is not due to differences in the diamine uptake by the cells. The results suggest that the N—N distance (6 Å) and the charge of one amino group are important chemical characteristics for regulatory effects.

Simulation of peptide processing, compartmentation and release in neurosecretory cells

A theoretical framework is presented which mathematically describes the transport of secretory granules in neurons and the enzymatic processing of a prohormone and subsequent intermediates within such granules. The transport system represents the synthesis of individual granules, the migration of the granules among various “diffusionally”-connected compartments, and the release of granules from designated compartmental release sites. The processing of prohormone is represented by a multiple-site cleavage reaction with separate rate constants for each cleavage site on each peptide fragment. The effects of neuron growth, processing enzyme activity decay, and changes in release probability with granule age are taken into account. Computer models were constructed based on these underlying assumptions. Simulations are used to illustrate how the distribution of peptide fragments contained in a neuron will depend on interactions of peptide processing kinetics and cellular transport and storage processes. The models provide a tool for testing how multiple cell-biological variables will interact to control the chemical mixture secreted from a peptidergic neuron.

L-Phenylalanine Ammonia-Lyase from Sunflower Hypocotyls: Modulation by Cinnamic Acids

Summary Trans -cinnamic acid and other metabolites of the phenylpropanoid pathway, namely p -coumaric, caffeic, ferulic and sinapic acids, inhibited the increase in extractable phenylalanine ammonia-lyase (PAL) activity present in sunflower hypocotyls in response to light-sucrose and excision-light-sucrose treatments. Otherwise, in vitro inhibitors of PAL activity, such as D-phenylalanine, and 2-aminooxyacetic and phenylpropiolic acids, superinduced extractable PAL activity and prevented the enzyme activity decay typically observed in the light-sucrose induction system. In both inducing treatments, delayed transfer of hypocotyl tissue to t -cinnamic acid caused a rapid decline in PAL activity, while a superinduction effect was observed upon transfer of the tissue to phenylpropiolic acid. These results indicate that in sunflower hypocotyls cinnamic acid(s) could act as in vivo modulator(s) of PAL, affecting the mechanisms responsible for both the increase and decrease in enzyme activity.

Rapid up- and down-regulation of hexokinase II in rat skeletal muscle in response to altered contractile activity

An up to 14-fold increase in total hexokinase activity induced by low-frequency stimulation in rat fast-twitch muscle was followed by a rapid decay in enzyme activity after cessation of stimulation. In vivo labeling revealed that these alterations were related to rapid changes in [ 35S]methionine incorporation into hexokinase II. A recovery period of 15 h after cessation of stimulation was sufficient to normalize the approximately 30-fold elevated [ 35S]methionine incorporation.

Sensitivity of immunoaffinity-purified porcine 5-lipoxygenase to inhibitors and activating lipid hydroperoxides

The requirement for hydroperoxide activation and the effect of inhibitors from different structural classes on 5-lipoxygenase activity were determined on the immunoaffinity-purified enzyme from porcine leukocytes. The 5-lipoxygenase activity was measured using a continuous spectrophotometric assay monitoring the increase in conjugated diene formation (A 235) upon incubation of the enzyme with arachidonic acid. Under standard assay conditions, the reaction progress curves showed little or no lag phase, with a rapid first-order decay in enzyme activity ( T 1 2 = 0.7 to 1.1 min) . Both the initial rate of the reaction and total product formation were stimulated by the addition of ATP, Ca 2+ and phosphatidylcholine (PC). PC (24 μg/ml) was also found to increase the recovery of radiolabeled arachidonic acid from the assay mixture and thus part of the stimulation may be due to an increase in substrate availability and reduction of surface adsorption effects. The requirement of hydroperoxides for the initiation of the reaction was shown by the induction of 0.1 to 1-min lag phases using NaBH 4 or glutathione peroxidase and by the reduction in lag times by 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and 13-hydroperoxyoctadecadienoic acid (13-HPOD). The following compounds were evaluated as inhibitors of the 5-lipoxygenase reaction and caused a 50% decrease in product accumulation ( ic 50) at the indicated concentrations: quercetin, L-651,896, L-656,224, MTPPH and L-651,392 (0.3-0.5 μM); diphenyldisulfide (2–5 μM); phenidone (5–10 μM); AA861 (4–10 μM) and BW 755C (4–15 μM). In addition, the presence of inhibitors extended the initial lag phase of the reaction and increased the dependence of the initiation of the reaction on exogenous lipid hydroperoxides. The inhibition by phenidone was accompanied by a 2-fold increase in the rate of enzyme inactivation, whereas other compounds such as AA861 and L-656,224 did not show this effect. The results indicate that the presence of inhibitors can modify the kinetics of 5-lipoxygenase at the levels of the initiation of the reaction and the rate of enzyme inactivation, with variations depending on the structural class of the inhibitor and the concentration of lipid hydroperoxides.