Modulation of the tumor disposition of vinca alkaloids by PSC 833 in vitro and in vivo

PSC 833, a nonimmunosuppressive cyclosporin, is able to inhibit the efflux of antitumor drugs mediated by P-glycoprotein (P-gp). The purpose of the present study is to compare the effect of PSC 833 on the tumor disposition of [3H]vincristine ([3H]VCR) and [3H]vinblastine ([3H]VBL) in in vitro and in vivo experiments from a pharmacokinetic point of view. In in vitro experiments, the effect of PSC 833 was investigated on the cellular uptake of [3H]VCR and [3H]VBL by HCT-15 and COLO 205, human colorectal tumor cell lines with extensive and minimal expression of P-gp, respectively. PSC 833 (2 microM) increased the cellular uptake of [3H]VCR and [3H]VBL by HCT-15 cells, but not that by COLO 205 cells, 8- and 6-fold, respectively, without affecting the initial influx rates. In addition, 2 microM PSC 833 reduced the efflux of [3H]VCR from HCT-15 cells to a level comparable with that from COLO 205 cells. Furthermore, the effect of PSC 833 on the tumor disposition of intravenously administered [3H]VCR and [3H]VBL was studied in tumor inoculated mice. Infusion of PSC 833 (10 microg/hr/mouse) increased the HCT-15 tumor disposition of [3H]VBL and [3H]VCR in vivo to a level comparable with that observed in vitro. These findings demonstrate that PSC 833 enhances the tumor disposition of vinca alkaloids by inhibition of P-gp-mediated efflux not only in vitro but also in vivo in a solid tumor model.     

Selective and reversible effects of vinca alkaloids on Trypanosoma cruzi epimastigote forms: blockage of cytokinesis without inhibition of the organelle duplication

We previously reported excessive apoptosis and high levels of tumor necrosis factor-alpha (TNF-alpha) in the bone marrows of patients with myelodysplastic syndromes (MDS), using histochemical techniques. The present studies provide further circumstantial evidence for the involvement of TNF-alpha in apoptotic death of the marrow cells in MDS. Using our newly developed in situ double-labeling technique that sequentially employs DNA polymerase (DNA Pol) followed by terminal deoxynucleotidyl transferase (TdT) to label cells undergoing apoptosis, we have characterized DNA fragmentation patterns during spontaneous apoptosis in MDS bone marrow and in HL60 cells treated with TNF-alpha or etoposide (VP16). Clear DNA laddering detected by gel electrophoresis in MDS samples confirmed the unique length of apoptotic DNA fragments (180-200 bp). Surprisingly, however, phenotypically heterogeneous population of MDS cells as well as the homogenous population of HL60 cells showed three distinct labeling patterns after double labeling--only DNA-Pol reaction, only TdT reaction, and a combined DNA Pol + TdT reaction, albeit in different cohorts of cells. Each labeling pattern was found at all morphological stages of apoptosis. MDS mononuclear cells, during spontaneous apoptosis in 4 hr cultures, showed highest increase in double-labeled cells (DNA Pol + TdT reaction). Interestingly, this was paralleled by TNF-alpha-induced apoptosis in HL60 cells. In contrast, VP16 treatment of HL60 cells led to increased apoptosis in cells showing only TdT reaction. The double-labeling technique was applied to normal bone marrow and peripheral blood mononuclear cells after treatment with known endonucleases that specifically cause 3' recessed (BamHI), 5' recessed (PstI), or blunt ended (DraI) double-stranded DNA breaks. It was found that the DNA-Pol reaction in MDS and HL60 cells corresponds to 3' recessed DNA fragments, the TdT reaction to 5' recessed and/or blunt ended fragments, and a combined "DNA Pol + TdT reaction" corresponds to a copresence of 3' recessed with 5' recessed and/or blunt ended fragments. Clearly, therefore, apoptotic DNA fragments, in spite of a unique length, may have differently staggered ends that could be cell (or tissue) specific and be selectively triggered by different inducers of apoptosis. The presence of TNF-alpha-inducible apoptotic DNA fragmentation pattern in MDS supports its involvement in these disorders and suggests that anti-TNF-alpha (or anticytokine) therapy may be of special benefit to MDS patients, where no definitive treatment is yet available.     

Kinetic analysis of secretory protein traffic and characterization of golgi to plasma membrane transport intermediates in living cells

Quantitative time-lapse imaging data of single cells expressing the transmembrane protein, vesicular stomatitis virus ts045 G protein fused to green fluorescent protein (VSVG-GFP), were used for kinetic modeling of protein traffic through the various compartments of the secretory pathway. A series of first order rate laws was sufficient to accurately describe VSVG-GFP transport, and provided compartment residence times and rate constants for transport into and out of the Golgi complex and delivery to the plasma membrane. For ER to Golgi transport the mean rate constant (i.e., the fraction of VSVG-GFP moved per unit of time) was 2.8% per min, for Golgi to plasma membrane transport it was 3.0% per min, and for transport from the plasma membrane to a degradative site it was 0.25% per min. Because these rate constants did not change as the concentration of VSVG-GFP in different compartments went from high (early in the experiment) to low (late in the experiment), secretory transport machinery was never saturated during the experiments. The processes of budding, translocation, and fusion of post-Golgi transport intermediates carrying VSVG- GFP to the plasma membrane were also analyzed using quantitative imaging techniques. Large pleiomorphic tubular structures, rather than small vesicles, were found to be the primary vehicles for Golgi to plasma membrane transport of VSVG-GFP. These structures budded as entire domains from the Golgi complex and underwent dynamic shape changes as they moved along microtubule tracks to the cell periphery. They carried up to 10,000 VSVG-GFP molecules and had a mean life time in COS cells of 3.8 min. In addition, they fused with the plasma membrane without intersecting other membrane transport pathways in the cell. These properties suggest that the post-Golgi intermediates represent a unique transport organelle for conveying large quantities of protein cargo from the Golgi complex directly to the plasma membrane.     

Growth inhibition of K-ras-expressing tumours by a new vinca alkaloid, conophylline, in nude mice

Conophylline, a new vinca alkaloid isolated from the plant Ervatamia microphylla induced normal flat morphology in K-ras-NRK and K-ras-NIH cell lines, and lowered the increased uptake of 2-deoxyglucose in K-ras-NRK cells. Conophylline inhibited the growth of K-ras-NRK cells, but this inhibition was reversible. The alkaloid also inhibited the growth of K-ras-NRK and K-ras-NIH3T3 tumours transplanted into nude mice. On the other hand, it showed no effect on survival of the mice loaded with L1210 leukaemia. Thus, conophylline is a new antitumour vinca alkaloid that induced normal phenotypes in ras-expressing cells.     

Selective inhibition of T-cell-dependent immune responses by bisbenzylisoquinoline alkaloids in vivo

The bisbenzylisoquinoline (BBI) alkaloids, chondocurine, tetrandrine, isotetrandrine and cepharanthine, were tested for immunosuppressive activity in mice. A plaque-forming cell (PFC) response to a T-cell-dependent antigen, sheep red blood cell, was significantly suppressed by a 7 day treatment of chondocurine or tetrandrine at 1 mg/kg/day and of isotetrandrine at 50 mg/kg/day, but not suppressed by cepharanthine treatment. The suppressive effect of chondocurine was greater when it was given after immunization rather than before or concurrently. However, it did not affect the PFC response to a T-cell-independent antigen, lipopolysaccharide. A delayed-type hypersensitivity was also suppressed by chondocurine treatment. There was no significant change in lymphocyte number and proportion of T-cell subsets in the BBI alkaloid-treated mice. These data suggest that there is selective inhibition by chondocurine and tetrandrine of the T-cell-dependent immune reactions.     

Evidence for distal tubular inhibition of calcium efflux by nisoldipine in the SHR rat

Nisoldipine, a calcium channel blocking agent, is known to have antihypertensive, renal tubular and hemodynamic effects. The present studies were designed to examine the effects of this drug on the renal tubular transport of calcium in 12 saline-loaded SHR rats. Calcium-45 was infused into three different nephron segments: early proximal, late proximal and early distal sites with or without nisoldipine. Calcium efflux averaged 93.6 +/- 4.9 and 90.5 +/- 8.7% after early and late proximal administration, respectively, indicating that the proximal tubule and the loop of Henle are highly efficient in transporting calcium out of the tubule. In distal nephron segments, calcium transport was limited to 41.1 +/- 4.8% of the amount delivered to these tubules. Nisoldipine inhibited the efflux of simultaneously infused calcium. This apparent inhibitory effect occurred predominantly in distal nephron segments where the drug reduced calcium efflux from 41.1 +/- 4.8 to 22.5 +/- 2.7%, indicating a 45.3% reduction in net calcium reabsorption. The results are consistent with the interpretation that nisoldipine-induced reduction in the tubular efflux of calcium was secondary to a direct inhibition of voltage-sensitive, L-type calcium channels or to a blunting of the rate of phosphorylation of channel proteins by protein kinase C in the distal tubular epithelial cells.     

Kinetic studies on the inhibition of isopeptidase T by ubiquitin aldehyde

Isopeptidase T (IPaseT) can hydrolyze isopeptide bonds of polyubiquitin (polyUb) chains, simple C-terminal derivatives of Ub, and certain peptides. We recently reported that IPaseT is regulated by ubiquitin (Ub); while submicromolar Ub activates, higher concentrations inhibit this enzyme [Stein et al. (1995) Biochemistry 34, 12616]. To explain these observations, we proposed a model for IPaseT involving two binding sites for Ub. According to the model, the two sites are adjacent to one another and are the extended active site that binds two Ub moieties of a polyUb chain. The "activation site" binds the Ub that donates Lys to the isopeptide bond. The "inhibition site" is adjacent and binds the Ub that donates the C-terminal Gly to the isopeptide bond. We now report that the interaction of IPaseT with the C-terminal aldehyde of Ub (Ub-H) is also modulated by Ub. In the absence of Ub, Ub-H inhibits IPaseT with a Ki of 2.3 nM, while at 0.6 microM Ub, where the "activation site" is occupied, Ki is less than 0.1 nM. At high Ub concentrations, where both the "activation" and "inhibition" sites are occupied, IPaseT cannot bind Ub-H. We also determined the kinetics of inhibition of IPaseT by Ub-H. In the absence of Ub, a two-step mechanism is followed. In the first step, Ub-H slowly combines with IPaseT to form a relatively weak complex (K1 = 260 nM) that slowly isomerizes to the final, stable complex that accumulates in the steady-state (k2 = 2 x 10(-3) s-1; k-2 = 0.02 x 10(-3) s-1). In contrast, Ub-activated IPaseT is inhibited by Ub-H through a three-step process. In the first step, Ub-H rapidly combines with IPaseT to form a complex (K1 = 10 nM) that slowly isomerizes to a second, more stable complex (k2 = 18 x 10(-3) s-1; k-2 = 1.5 x 10(-3) s-1). In the third step, the second complex converts to the final complex (k3 = 1.5 x 10(-3) s-1; k-3 < 0.2 x 10(-3) s-1). To unify the results of this study with our previous results on catalysis, we propose that binding of Ub either to catalytic transition states or to tetrahedral inhibition intermediates liberates more free energy than binding of Ub to the reactant state of IPaseT and that IPaseT can utilize this binding energy to stabilize both of these tetrahedral species. The overall effect is a Ub-induced increase in catalytic efficiency or inhibitory potency.     

The treatment of disseminated malignant melanoma with special reference to the role of interferons, vinca alkaloids and tamoxifen

Treatment of quiescent NIH3T3 cells with PDGF BB results in the transient activation and hyperphosphorylation of the protein-tyrosine kinase, c-Src. These effects correlate with novel serine and tyrosine phosphorylations in the N-terminal non-catalytic region of the molecule, which contains an SH3 and SH2 domain. In this study, a site of PDGF-induced tyrosine phosphorylation was mapped to Tyr 138 in the SH3 domain; Tyr 138 is exposed on the SH3 peptide binding surface. This same site is phosphorylated in vitro by the PDGF receptor when purified baculovirus-expressed c-Src is complexed with the activated receptor. Phosphorylation of Tyr 138 required association of c-Src with the PDGF receptor via its SH2 domain. When a c-Src Phe 138 mutant was stably expressed in Src- mouse fibroblasts, it was activated to the same extent as wild type c-Src following PDGF stimulation, indicating that phosphorylation of this site is not required for PDGF-mediated activation. However, Tyr 138 phosphorylation was found to diminish SH3 domain peptide ligand binding ability in vitro.     

Verapamil inhibition of enzymatic product efflux leads to improved detection of beta-galactosidase activity in lacZ-transfected cells

BACKGROUND: We studied the usefulness of nuclear DNA patterns and argyrophylic nucleolar organizer regions (AgNORs) for evaluating the malignant potential of colorectal cancers, which is increasingly being regarded as important in predicting patients' prognosis and for their appropriate postoperative management. METHODS: We measured these two factors in curatively resected specimens of 91 colorectal cancer cases, which were followed up for 1,549 +/- 788 days postoperatively. Ploidy pattern was either diploid or aneuploid, and AgNORs score was either low (LS) or high (HS). Thus, we classified our cases into Group I (diploid, LS). Group II (aneuploid, LS), Group III (diploid, HS), and Group IV (aneuploid, HS). Postoperative survival curves in the cases belonging to these groups were analyzed. RESULTS: Survival rates in Groups I and II were significantly higher than those in Group IV. Correlation between subgroups and clinicopathological factors such as average age, histologic type, depth of invasion, and histologic stage were observed. Incidence of lymph node metastasis at the time of operation and that of postoperative recurrence were higher in group IV than that in groups I and II. CONCLUSIONS: Measurement of DNA ploidy patterns and AgNORs score were found to be useful in evaluating malignant potential of colorectal cancers.     

A society of goal-oriented agents for the analysis of living cells

This paper presents a new model for the segmentation and analysis of living cells. A multi-agent model has been developed for this application. It is based on a generic agent model, which is composed of different behaviors: perception, interaction and reproduction. The agent is further specialized to accomplish a specific goal. Different goals are defined from the different components of the cell images. The specialization specifies the parameters of the behaviors for the achievement of the agent's goal. From these goal-oriented agents, a society is defined, and it evolves dynamically as the agents are created and deleted. An internal manager is integrated in the agent to control the behavior's execution. It makes use of an event-driven scheme to manage the behavior priorities. The present design is mainly oriented toward image segmentation, however, it includes some features on tracking and motion analysis.     

Kinetic characterization of CYP2E1 inhibition in vivo and in vitro by the chloroethylenes

Trans- and cis-1,2-dichloroethylene (DCE) isomers inhibit their own metabolism in vivo by inactivation of the metabolizing enzyme, presumably the cytochrome P450 isoform, CYP2E1. In this study, we examined cytochrome P450 isoform-specific inhibition by three chloroethylenes, cis-DCE, trans-DCE, and trichloroethylene (TCE), and evaluated several kinetic mechanisms of enzyme inhibition with physiological models of inhibition. Trans-DCE was more potent than cis-DCE, and both were much more effective than TCE in inhibiting CYP2E1. The kinetics of in vitro loss of p-nitrophenol hydroxylase (pNP-OH) activity (a marker of CYP2E1) in microsomal incubations and of the in vivo gas uptake results were most consistent with a mechanism in which inhibition of the metabolizing enzyme (CYP2E1) was presumed to be related to interaction of a reactive DCE metabolite with remaining substrate-bound, active CYP2E1. The kinetics of inhibition by TCE, a weak inhibitor in vitro, were very different from that of the dichloroethylenes. With TCE, parent compound concentrations influenced enzyme loss. Trans-DCE was a more potent inhibitor of CYP2E1 than cis-DCE based on both in vivo and in vitro studies. Quantitative differences in the inhibitory properties of the 1,2-DCE isomers may be due to the different stability of epoxides formed from bioactivation by CYP2E1. Epoxide intermediates of DCE metabolism, reacting by water addition, would yield dialdehyde, a potent cross-linking reagent.     

Dual-function reporter protein for analysis of gene expression in living cells

The firefly luciferase (Luc) protein and the jellyfish green fluorescent protein (GFP) are two commonly used molecular reporters that can be detected noninvasively in living cells. The properties that make GFP or Luc useful for a particular experimental application are quite distinct. A recombinant protein with both fluorescent and bioluminescent characteristics might take advantage of the strengths of both reporters. An expression vector encoding a chimeric protein in which GFP was tethered to Luc through a 19-amino acid linker was prepared and characterized. Western blotting with antibodies specific for either GFP or Luc showed that a protein of appropriate size was expressed in transfected cells. Fluorescence microscopy revealed bright green fluorescence from transfected cells, indicating proper formation of the GFP chromophore. Luc enzymatic activity in protein extracts from transfected cells showed that Luc was fully functional. The treatment of living cell cultures stably expressing the GFP-Luc fusion protein with the protein translation-inhibitor cycloheximide (Chx) was used to show that the half-life for Luc protein activity was approximately 2 h at 37 degrees C. The utility of this dual-function reporter protein was shown by the identification of single living cells expressing the chimeric protein within a population by fluorescence microscopy, followed by quantification of Luc activity from the same living cells.     

Microdomains of GPI-anchored proteins in living cells revealed by crosslinking

There is some discussion as to whether glycosyl-phosphatidylinositol(GPI)-anchored proteins occur in microdomains in the cell membrane. These putative microdomains have been implicated in processes such as sorting in polarized cells and signal transduction. Complexes enriched in GPI-anchored proteins, cholesterol and glycosphingolipids have been isolated from cell membranes by using non-ionic detergents: these complexes were thought to represent a clustered arrangement of GPI-anchored proteins. However, results obtained when clustering of GPI-anchored proteins induced by antibodies or by detergents was prevented support the idea of a dispersed surface distribution of GPI-anchored proteins at steady state. Here we use chemical crosslinking to show that membrane microdomains of a GPI-anchored protein exist at the surface in living cells. This clustering is specific for the GPI-anchored form, as two transmembrane forms bearing the same ectodomain do not form oligomers. Depletion of membrane cholesterol causes the clustering of GPI-anchored proteins to break up, whereas treatment of cells with detergent substantially increases the size of the complexes. We find that in living cells these GPI-anchored proteins reside in microdomains consisting of at least 15 molecules, which are much smaller than those seen after detergent extraction.     

Inhibition of vinblastine efflux mediated by P-glycoprotein by grapefruit juice components in caco-2 cells

The authors describe a resource-allocation model developed in the Medical Care Clinical Center at the Baltimore Veterans Affairs Medical Center (a part of the VA Maryland Health Care System) and implemented in 1989. This model is a computer-based system that tracks the workload of each of the clinical center's specialty sections (e.g., cardiology) and calculates each section's workload as a percentage of the total clinical center workload. As the basis of this calculation, six activities of each section are tracked by the model (e.g., inpatient attending physicians' rotations; inpatient consultations; etc.) to determine what percentage of each activity of the entire clinical center was provided by each section. Each of these percentages is then recalculated according to a weighted average based on the relative value of the activity to the department; these averages are revised periodically as needed. The model provides an incentive for the specialty sections to increase productivity by generating competition among sections for physician salary support. Communication among all concerned at the clinical center and its associated medical school and teaching hospital has been the key to success in implementing the model, which is periodically reviewed and has been revised several times after meetings with section chiefs and division heads. The authors are confident that the use of the model has been at least partly responsible for increased productivity of clinical center physicians, especially in the areas of visits per physician and funded VA research dollars per physician. Perhaps equally important is the future potential of the model. Because of its simplicity and because it is generally seen to be fair and effective, it will continue to be used to reward activities most important to the clinical center, especially now that the center operates under a fully capitated system, and in this way wil help ensure the financial viability of the center.     

Kinetic analysis of the in vitro inhibition, aging, and reactivation of brain acetylcholinesterase from rat and channel catfish by paraoxon and chlorpyrifos-oxon

In rats, the phosphorothionate insecticide parathion exhibits greater toxicity than chlorpyrifos, while in catfish the toxicities are reversed. The in vitro inhibition of brain acetylcholinesterase (AChE) by the active metabolites of the insecticides and the rates at which these inhibitor-enzyme complexes undergo reactivation/ aging were investigated in both species. Rat AChE was more sensitive to inhibition than catfish AChE as demonstrated by greater bimolecular rate constants (ki) in rats than in catfish. In both species, chlorpyrifos-oxon yielded higher ki's than paraoxon. The higher association constant (KA) of chlorpyrifos-oxon than paraoxon in both species and the lack of significant differences in the phosphorylation constants (kp) suggest that association of the inhibitor with AChE is the principal factor in the different potencies between these two inhibitors. In catfish, the ki of chlorpyrifos-oxon was 22-fold greater than that of paraoxon, while in rats it was 9-fold greater, suggesting that target site sensitivity is an important factor in the higher toxicity of chlorpyrifos to catfish but not in the higher toxicity of parathion to rats. No spontaneous reactivation of phosphorylated catfish AChE occurred and there were no differences in the first oder aging constants (ka) between compounds. For phosphorylated rat AChE, there were no differences in the first order reactivation constants (kr) but the ka for chlorpyrifos-oxon was significantly greater than that for paraoxon. This difference suggests that the steric positioning of the diethyl phosphate in the esteratic site is not the same between the two compounds, leading to differences in aging.     

Kinetic analysis of transcytosis of epidermal growth factor in Madin-Darby canine kidney epithelial cells

OBJECTIVE: Treatment outcome in sexually abused preschool children was evaluated 6 and 12 months after treatment. METHOD: Forty-three sexually abused preschool children and their parents were evaluated 6 and 12 months after completion of either Cognitive-Behavioral Therapy for Sexually Abused Preschoolers (CBT-SAP) or nondirective supportive therapy (NST). Parents completed the Child Behavior Checklist, Child Sexual Behavior inventory, and Weekly Behavior Report to measure a variety of symptoms in their children. RESULTS: Repeated-measures analyses indicated that there were significant group by time interactions on several outcome measures from the beginning of the study to the end of the 12-month follow-up period, with the CBT-SAP group exhibiting significantly more improvement over time than the NST group. Clinical findings also indicated the superior effectiveness of CBT-SAP over NST in reducing sexually inappropriate behavior. CONCLUSIONS: Findings support the superior efficacy of CBT-SAP over NST in maintaining symptom reduction in the year after treatment completion. The importance of using cognitive-behavioral interventions for sexually inappropriate behaviors and including nonoffending parents in the treatment of sexually abused preschool children is discussed.     

Molecular mechanisms involved in the inhibition of neutrophil locomotion by tumor cells

A chronic myelogenous leukemia cell line (K562) releases a factor of about 8 kD which we have named K562-inhibitory factor (K562-IF) because it inhibits neutrophil locomotion. This factor has potent anti-inflammatory activity in mice, associated with an inhibition of neutrophil function including not only random locomotion and fMetLeuPhe- or serum-induced locomotion but also adherence and zymosan-induced chemiluminescence and degranulation. In contrast, K562-IF does not affect the oxidative burst induced by soluble compounds such as fMetLeuPhe and phorbol esters. Analysis of the mechanism of action of K562-IF on neutrophils showed that it involves an adherence protein, mainly CR3 (the receptor of complement fraction iC3b). Neither, CR3 expression nor its up-regulation were altered, whereas the function of CR3 was depressed, i.e., it failed to cap upon neutrophil stimulation and did not bind iC3b. One unexplained finding is that K562-IF inhibits actin polymerization induced by fMetLeuPhe but not by activation of the Fc-gamma receptor III. Studies are underway to establish whether K562 cells are representative of other malignant cells with regard to the production of neutrophil inhibitors.     

Kinetic analysis of the induction period in lipoxygenase catalysis

The dioxygenation of 50 microM linoleate at 0.1 microM (13S)-hydroperoxylinoleate, 240 microM O2, pH 10, and 25 degrees C, catalyzed by varying amounts of soybean lipoxygenase-1, was studied with rapid kinetic techniques. The aim was to assess the effect of transient redistributions of the Fe(II) and Fe(III) enzyme forms on the shape of the reaction progress curves. Reactions initiated with iron(II) lipoxygenase show an initial increase in rate, the "kinetic lag phase" or "induction period". The duration of this induction period varies from approximately 1 s at [lipoxygenase] > 20 nM to 5 s at [lipoxygenase] = 3 nM. At [lipoxygenase] < 2 nM, the duration of the induction period in these curves is inversely proportional to [lipoxygenase]. The integrated steady-state rate equation for the single fatty acid binding site model of lipoxygenase catalysis [Schilstra et al. (1992) Biochemistry 31, 7692-7699] also shows an induction period whose duration is inversely proportional to [lipoxygenase]. These observations, in combination with non-steady-state numerical simulations, lead to the conclusion that, at [lipoxygenase] < 2 nM, pre-steady-state redistributions of enzyme intermediates occur fast with respect to the rate at which the concentrations of substrates and products change. At higher lipoxygenase concentrations, the pre-steady-state redistributions contribute significantly to the induction period. From a nonlinear least-squares fit to the steady-state rate equation of data obtained at lipoxygenase concentrations of 0.5 and 1 nM, it was calculated that 1% of the linoleate radicals that are formed after hydrogen abstraction dissociate from the active site before enzymic oxygen insertion has occurred.