A comparison of results from antihuman globulin-graded reactions with the monocyte monolayer assay.

Blood transfusions are a common medical treatment. Risks arise when compatible blood is not available. This study assesses the correlation between antibody reaction strength at the antihuman globulin (AHG) phase of testing and the antibody clinical significance as predicted using the monocyte monolayer assay (MMA). Multiple examples of anti-K donor plasma samples were selected to sensitize K+k+ red blood cells (RBCs). Reactivity was confirmed by testing the sensitized K+k+ RBCs at saline-AHG. Antibody titers were determined by serial dilution using neat plasma. Sixteen samples were selected for the study based on comparable graded reactions with neat plasma (1+, 2+, 3+, and 4+) and similar titration endpoints. Each sample was used to sensitize the same Kk donor and then tested by monocytes to evaluate the clinical significance using the MMA, an in vitro procedure that mimics in vivo extravascular hemolysis to predict the survivability of incompatible transfused RBCs. The monocyte index (MI), i.e., the percentage of RBCs adhered, ingested, or both versus free monocytes, was calculated for each sample. Regardless of the reaction strength, all examples of anti-K were predicted to be clinically significant. While anti-K is known to be clinically significant, the immunogenicity rate of K ensures ample supply of antibody samples for inclusion in this project. This study demonstrates that in vitro antibody strength is highly subjective and variable. These results show no correlation between graded reaction strength at AHG and the predicted clinical significance of an antibody as assessed using the MMA.

Biological significance of MAPK, AKT and JAK-STAT protein activation by various erythropoietic factors in normal human early erythroid cells.

The aim of this study was to identify signal transduction pathways activated by erythropoietin (EpO) and erythropoietin co-stimulatory factors (kit ligand), insulin-like growth factor, thrombopoietin, interleukin 3 and granulocyte-macrophage colony-stimulating factor) in normal human bone marrow CD34(+) cells and d 11 erythroid burst forming unit derived glycophorin+ cells. The activation of these signal transduction pathways was further correlated with various biological effects such as (i) cell proliferation, (ii) inhibition of apoptosis, (iii) activation of adhesion and (iv) secretion of the matrix metalloproteinases (MMPs) MMP-9 and MMP-2, and vascular endothelial growth factor (VEGF). We found that in human CD34(+) cells and erythroblasts erythropoietic factors may activate similar but different signalling pathways, and that activation of each of the JAK-STAT, MAPK p42/44 or PI-3K-AKT axes alone is not sufficient either to stimulate cell proliferation or inhibit apoptosis, suggesting that these processes are regulated by orchestrated activation of multiple signalling cascades. Accordingly, we found that although cell proliferation was more related to simultaneous activation of JAK-STAT and MAPK p42/44, the effect on cell survival correlated with activation of PI-3K-AKT, MAPK p42/44 and JAK-STAT proteins. We also demonstrated that differentiating normal human erythroid cells lose their adhesive properties and secrete angiopoietic factors such as MMP-9, MMP-2 and VEGF, and we postulate that this secretion by early erythroid cells may play a role in their maturation and egress from the haematopoietic niches of the bone marrow.

The SDF-1-CXCR4 axis stimulates VEGF secretion and activates integrins but does not affect proliferation and survival in lymphohematopoietic cells.

To better define the role HIV-related chemokine receptor-chemokine axes play in human hematopoiesis, we investigated the function of the CXCR4 and CCR5 receptors in human myeloid, T- and B-lymphoid cell lines selected for the expression of these receptors (CXCR4(+), CXCR4(+) CCR5(+), and CCR5(+) cell lines). We evaluated the phosphorylation of MAPK p42/44, AKT, and STAT proteins and examined the ability of the ligands for these receptors (stromal-derived factor-1 [SDF-1] and macrophage inflammatory protein-1beta [MIP-1beta]) to influence cell growth, apoptosis, adhesion, and production of vascular endothelial growth factors (VEGF), matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in these cell lines. We found that A) SDF-1, after binding to CXCR4, activates multiple signaling pathways and that in comparison with the MIP-1beta-CCR5 axis, plays a privileged role in hematopoiesis; B) SDF-1 activation of the MAPK p42/44 pathway and the PI-3K-AKT axis does not affect proliferation and apoptosis but modulates integrin-mediated adhesion to fibronectin, and C) SDF-1 induces secretion of VEGF, but not of MMPs or TIMPs. Thus the role of SDF-1 relates primarily to the interaction of lymphohematopoietic cells with their microenvironment and does not directly influence their proliferation or survival. We conclude that perturbation of the SDF-1-CXCR4 axis during HIV infection may affect interactions of hematopoietic cells with the hematopoietic microenvironment.

Stromal-derived factor 1 and thrombopoietin regulate distinct aspects of human megakaryopoiesis.

The role of the chemokine binding stromal-derived factor 1 (SDF-1) in normal human megakaryopoiesis at the cellular and molecular levels and its comparison with that of thrombopoietin (TPO) have not been determined. In this study it was found that SDF-1, unlike TPO, does not stimulate alpha(IIb)beta(3)(+) cell proliferation or differentiation or have an antiapoptotic effect. However, it does induce chemotaxis, trans-Matrigel migration, and secretion of matrix metalloproteinase 9 (MMP-9) and vascular endothelial growth factor (VEGF) by these cells, and both SDF-1 and TPO increase the adhesion of alpha(IIb)beta(3)(+) cells to fibrinogen and vitronectin. Investigating the intracellular signaling pathways induced by SDF-1 and TPO revealed some overlapping patterns of protein phosphorylation/activation (mitogen-activated protein kinase [MAPK] p42/44, MAPK p38, and AKT [protein kinase B]) and some that were distinct for TPO (eg, JAK-STAT) and for SDF-1 (eg, NF-kappa B). It was also found that though inhibition of phosphatidyl-inositol 3-kinase (PI-3K) by LY294002 in alpha(IIb)beta(3)(+) cells induced apoptosis and inhibited chemotaxis adhesion and the secretion of MMP-9 and VEGF, the inhibition of MAPK p42/44 (by the MEK inhibitor U0126) had no effect on the survival, proliferation, and migration of these cells. Hence, it is suggested that the proliferative effect of TPO is more related to activation of the JAK-STAT pathway (unique to TPO), and the PI-3K-AKT axis is differentially involved in TPO- and SDF-1-dependent signaling. Accordingly, PI-3K is involved in TPO-mediated inhibition of apoptosis, TPO- and SDF-1-regulated adhesion to fibrinogen and vitronectin, and SDF-1-mediated migration. This study expands the understanding of the role of SDF-1 and TPO in normal human megakaryopoiesis and indicates the molecular basis of the observed differences in cellular responses. (Blood. 2000;96:4142-4151)

Differential MMP and TIMP production by human marrow and peripheral blood CD34(+) cells in response to chemokines.

As stromal cell-derived factor-1 (SDF-1), macrophage inflammatory protein-1alpha (MIP-1alpha), and interleukin-8 (IL-8) are implicated in the homing and mobilization of human hematopoietic progenitors (HPC), we hypothesized that these chemokines mediate the migration of HPC across subendothelial basement membranes by regulating production of matrix metalloproteinases (MMPs) and their natural tissue inhibitors (TIMPs). Assays for migration across reconstituted basement membrane (Matrigel) and chemotaxis were carried out using CD34(+) cells derived from normal human bone marrow (BM) and mobilized peripheral blood (PB). Secretion of MMPs and TIMPs was evaluated by zymography and reverse zymography and gene expression by RT-PCR. We found that an SDF-1 gradient increases the chemotaxis of BM and PB CD34(+) cells across Matrigel (BM > PB), which is blocked by inhibitors of MMPs (o-phenanthroline, rhTIMP-1, rhTIMP-2, and anti-MMP-9 and anti-MMP-2 antibodies) but enhanced by tumor necrosis factor-alpha (TNF-alpha), a strong stimulator of MMPs. Preincubation of these cells with SDF-1 stimulated the secretion of MMP-2 and MMP-9 in BM and PB CD34(+) cells but of TIMP-1 and TIMP-2 only in PB CD34(+) cells. Preincubation with MIP-1alpha and IL-8 also stimulated the secretion of MMP-9 and MMP-2 (BM > PB), but with respect to TIMPs, the effect was reversed (PB > BM), resulting in trans-Matrigel migration of BM but not of PB CD34(+) cells. We therefore propose that MMPs and TIMPs are involved in 1) SDF-1-induced chemotaxis of human HPC across subendothelial basement membranes, and 2) MIP-1alpha- and IL-8-stimulated migration of HPC.

Interleukin-6 regulation of matrix metalloproteinase (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase (TIMP-1) expression in malignant non-Hodgkin's lymphomas.

We showed previously that human malignant non-Hodgkin's lymphomas (NHL) degrade extracellular matrix (ECM) components through the action of metalloproteinases and that elevated expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) correlated with a poor clinical outcome in patients with NHL. In the present study we sought to investigate whether there is any correlation between the expression of gelatinases (MMP-2 and MMP-9), TIMP-1, and the expression of cytokines and growth factors such as interleukin-1beta (IL-1beta), IL-6, IL-10, tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGFbeta), and basic fibroblast growth factor (bFGF) in human NHL. In lymphoma tissues obtained from 32 patients, elevated expression of IL-6 correlated significantly with elevated messenger RNA (mRNA) levels of MMP-9, MMP-2, and TIMP-1. Moreover, in human lymphoid cell lines of B- and T-cell origin (Raji, Jurkat, and NC-37), IL-6 stimulated production of MMP-9 and MMP-2 but not TIMP-1. In the Matrigel invasion assay IL-6 significantly upregulated transmigration of Raji and Jurkat cells, which in turn was inhibited by recombinant human TIMP-1 and anti-MMP-9 and MMP-2 antibodies. We postulate that IL-6 may play a role in the clinical aggressiveness of human NHL by stimulating MMP production.

Expression of matrix metalloproteinases (MMP-2 and -9) and tissue inhibitors of metalloproteinases (TIMP-1 and -2) in acute myelogenous leukaemia blasts: comparison with normal bone marrow cells.

We compared the expression of matrix metalloproteinases (MMP-2 and MMP-9) and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in bone marrow acute myelogenous leukaemia (AML) blasts and leukaemic cell lines (HEL, HL-60, K-562 and KG-1) with their expression in normal bone marrow cells. All AML samples and leukaemic cell lines tested expressed MMP-9 and/or MMP-2 mRNA and, accordingly, these gelatinases were secreted into media. Moreover, TIMP-1 and TIMP-2 mRNA and secreted proteins were demonstrated in all the AML samples. Although all the leukaemic cell lines expressed TIMP-1, the HL-60 cells also expressed TIMP-2. In contrast, normal steady-state bone marrow immature progenitor cells (CD34+ cells) did not express or secrete either MMP-2 or MMP-9, but more mature mononuclear cells from normal bone marrow expressed and secreted MMP-9. Also, normal bone marrow CD34+ cells and mononuclear cells expressed TIMP-1 and TIMP-2 mRNA, but these proteins were not detectable by reverse zymography. Furthermore, whereas bone marrow fibroblasts and endothelial cells secreted only latent MMP-2, the activated form of this enzyme was found in media conditioned by cells obtained from long-term cultures of normal and AML bone marrow adherent layers. Our finding of up-regulated production of gelatinases, TIMP-1 and TIMP-2 by leukaemic cells suggests that these proteins may be implicated in the invasive phenotype of AML.

Growth factors and cytokines upregulate gelatinase expression in bone marrow CD34(+) cells and their transmigration through reconstituted basement membrane.

The mechanism(s) underlying the release of stem/progenitor cells from bone marrow into the circulation is poorly understood. We hypothesized that matrix metalloproteinases (MMPs), especially gelatinases, which are believed to participate in the proteolysis of basement membranes and in the migration of leukocytes, may facilitate this process. First, we investigated whether CD34(+) stem/progenitor cells express gelatinases A (MMP-2) and/or B (MMP-9) and whether growth factors and cytokines (granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF], stem cell factor [SCF], macrophage colony-stimulating factor [M-CSF], interleukin-3 [IL-3], IL-6, IL-8, and tumor necrosis factor-alpha [TNF-alpha]) are able to modulate their expression. Next, we examined the transmigration of these stem/progenitor cells through reconstituted basement membrane (Matrigel) and its modulation by growth factors and cytokines. CD34(+) cells were obtained from steady-state bone marrow and peripheral blood (from leukapheresis products collected either in steady-state hematopoiesis or after mobilization with G-CSF plus chemotherapy or G-CSF alone). We found that peripheral blood CD34(+) cells, regardless of whether they were mobilized or not, strongly expressed both gelatinases (MMP-2 and MMP-9) in contrast to steady-state bone marrow CD34(+) cells, which did not. However, all the growth factors and cytokines tested could induce MMP-2 and MMP-9 secretion by the latter cells. Moreover, the stimulatory effects of G-CSF and SCF on both MMP-2 and MMP-9 secretion were found to be significantly higher in CD34(+) cells isolated from bone marrow than in those from peripheral blood. In addition TNF-alpha, GM-CSF, and IL-6 increased the secretion of a partially active form of MMP-2. Basal transmigration of bone marrow CD34(+) cells through Matrigel was lower than that of peripheral blood CD34(+) cells (P <.0001), but growth factors and cytokines increased it by 50% to 150%. Positive correlations were established between expression of gelatinases and CD34(+) cell migration (r >.9). The stimulatory effect of G-CSF was significantly greater on the migration of CD34(+) cells from bone marrow than on those from peripheral blood (P =.004). Moreover, CD34(+) cell migration was reduced to approximately 50% by antibodies to MMP-2 and MMP-9, tissue inhibitors of metalloproteinases (rhTIMP-1 and -2), and o-phenanthroline. TNF-alpha-induced gelatinase secretion and migration of CD34(+) cells and of clonogenic progenitors (colony-forming unit-granulocyte-macrophage [CFU-GM], burst-forming unit-erythroid [BFU-E], colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM], and colony-forming unit-megakaryocyte [CFU-MK]) were dose-dependent. Therefore, this study demonstrated that CD34(+) cells that are circulating in peripheral blood express both MMP-2 and MMP-9 and transmigrate through Matrigel. In contrast, CD34(+) cells from steady-state bone marrow acquire similar properties after exposure to growth factors and cytokines, which upregulate expression of gelatinases and transmigration of these cells when they enter the bloodstream. Hence, we suggest that growth factors and cytokines induce release of stem/progenitor cells from bone marrow into peripheral blood during mobilization, as well as during steady-state hematopoiesis, by signaling through gelatinase pathways.

Matrix Metalloproteinases in the Hematopoietic Microenvironment.

Matrix metalloproteinases (MMPs) are structurally and functionally related zinc-dependent endopeptidases capable of degrading the components of extracellular matrix (ECM) and basement membranes. MMPs participate in many physiological processes and have also been implicated in various pathological conditions including tumor invasion and metastasis. The functions of MMPs are known to be controlled by mechanisms leading to activation of their latent forms and through inhibition of both active and latent forms by natural tissue inhibitors of metalloproteinases (TIMPs). The complex relationships between MMPs and TIMPs within the bone marrow microenvironment during normal hematopoiesis as well as during leukemic growth and dissemination have not been extensively investigated. We reported that primary acute myelogenous leukemia (AML) blasts and leukemic KG-1 cells penetrate reconstituted basement membrane (Matrigel) in an in vitro invasion assay, secrete the gelatinases (MMP-2 and MMP-9) and express active MMP-2 on the cell surface. We also analyzed MMP/TIMP expression in normal bone marrow cells of the myeloid and stromal lineages and showed that MMP-2, MMP-9, TIMP-1 and TIMP-2 are produced in the bone marrow microenvironment. Furthermore, we examined the role of gelatinases in the transmigration of stem/progenitor cells from the bone marrow into peripheral blood. We found that steady-state bone marrow CD34(+) cells, unlike circulating peripheral blood CD34(+) cells, did not express MMP-2 and MMP-9 mRNA transcripts and proteins, and that various cytokines were able to upregulate expression of these MMPs in bone marrow CD34(+) cells and trans-Matrigel migration of these cells. Thus, we now have evidence that MMPs and TIMPs are constituents of the hematopoietic microenvironment although their roles in hematopoiesis have yet to be determined.

The proofreading pathway of bacteriophage T4 DNA polymerase.

The base analog, 2-aminopurine (2AP), was used as a fluorescent reporter of the biochemical steps in the proofreading pathway catalyzed by bacteriophage T4 DNA polymerase. "Mutator" DNA polymerases that are defective in different steps in the exonucleolytic proofreading pathway were studied so that transient changes in fluorescence intensity could be equated with specific reaction steps. The G255S- and D131N-DNA polymerases can hydrolyze DNA, the final step in the proofreading pathway, but the mutator phenotype indicates a defect in one or more steps that prepare the primer-terminus for the cleavage reaction. The hydrolysis-defective D112A/E114A-DNA polymerase was also examined. Fluorescent enzyme-DNA complexes were preformed in the absence of Mg2+, and then rapid mixing, stopped-flow techniques were used to determine the fate of the fluorescent complexes upon the addition of Mg2+. Comparisons of fluorescence intensity changes between the wild type and mutant DNA polymerases were used to model the exonucleolytic proofreading pathway. These studies are consistent with a proofreading pathway in which the protein loop structure that contains residue Gly255 functions in strand separation and transfer of the primer strand from the polymerase active center to form a preexonuclease complex. Residue Asp131 acts at a later step in formation of the preexonuclease complex.

Mechanism of the oxidation of 3,5,3',5'-tetramethylbenzidine by myeloperoxidase determined by transient- and steady-state kinetics.

Earlier investigations of the oxidation of 3,5,3',5'-tetramethylbenzidine (TMB) using horseradish peroxidase and prostaglandin H-synthase have shown the formation of a cation free radical of TMB in equilibrium with a charge-transfer complex, consistent with either a two- or a one-electron initial oxidation. In this work, we exploited the distinct spectroscopic properties of myeloperoxidase and its oxidized intermediates, compounds I and II, to establish two successive one-electron oxidations of TMB. By employing stopped-flow techniques under transient-state and steady-state conditions, we also determined the rate constants for the elementary steps of the myeloperoxidase-catalyzed oxidation of TMB at pH 5.4 and 20 degrees C. The second-order rate constant for compound I formation from the reaction of native enzyme with H2O2 is 2.6 x 10(7) M-1 s-1. Compound I undergoes a one-electron reduction to compound II in the presence of TMB, and the rate constant for this reaction was determined to be (3.6 +/- 0.1) x 10(6) M-1 s-1. The spectral scans show that compound II accumulates in the steady state. The rate constant for compound II reduction to native enzyme by TMB obtained under steady-state conditions is (9.4 +/- 0.6) x 10(5) M-1 s-1. The results are applied to a new, more accurate assay for myeloperoxidase based upon the formation of the charge-transfer complex between TMB and its diimine final product.

Using 2-aminopurine fluorescence and mutational analysis to demonstrate an active role of bacteriophage T4 DNA polymerase in strand separation required for 3' --> 5'-exonuclease activity.

The fluorescence of 2-aminopurine deoxynucleotide positioned in a 3'-terminal mismatch was used to evaluate the pre-steady state kinetics of the 3' --> 5' exonuclease activity of bacteriophage T4 DNA polymerase on defined DNA substrates. DNA substrates with one, two, or three preformed terminal mispairs simulated increasing degrees of strand separation at a primer terminus. The effects of base pair stability and local DNA sequence on excision rates were investigated by using DNA substrates that were either relatively G + C- or A + T-rich. The importance of strand separation as a prerequisite to the hydrolysis of a terminal nucleotide was demonstrated by using a unique mutant DNA polymerase that could degrade single-stranded but not double-stranded DNA, unless two or more 3'-terminal nucleotides were unpaired. Our results led us to conclude that the reduced exonuclease activity of this mutant DNA polymerase on duplex DNA substrates is due to a defect in melting the primer terminus in preparation for the excision reaction. The mutated amino acid (serine substitution for glycine at codon 255) resides in a critical loop structure determined from a crystallographic study of an amino-terminal fragment of T4 DNA polymerase. These results suggest an active role for amino acid residues in the exonuclease domain of the T4 DNA polymerase in the strand separation step.

Kinetic and spectral properties of pea cytosolic ascorbate peroxidase.

Sufficient highly purified native pea cytosolic ascorbate peroxidase was obtained to characterize some of its kinetic and spectral properties. Its rate constant for compound I formation from reaction with H2O2 is 4.O x 10(7) M-1 s-1, somewhat faster than is typical for peroxidases. Compound I has the typical optical spectrum of an iron(IV)-porphyrin-pi-cation radical, despite considerable homology with yeast cytochrome c peroxidase. The rate constant for compound I reduction by ascorbate is extremely fast (8.0 x 10(7) M-1 S-1 at pH 7.8), again in marked contrast to the behavior of the yeast enzyme. The pH-rate profile for compound I formation indicates a pKa value of 5.0 for a group affecting the active site reaction.

Kinetics of oxidation of tyrosine and dityrosine by myeloperoxidase compounds I and II. Implications for lipoprotein peroxidation studies.

The oxidation of lipoproteins is considered to play a key role in atherogenesis, and tyrosyl radicals have been implicated in the oxidation reaction. Tyrosyl radicals are generated in a system containing myeloperoxidase, H2O2, and tyrosine, but details of this enzyme-catalyzed reaction have not been explored. We have performed transient spectral and kinetic measurements to study the oxidation of tyrosine by the myeloperoxidase intermediates, compounds I and II, using both sequential mixing and single-mixing stopped-flow techniques. The one-electron reduction of compound I to compound II by tyrosine has a second order rate constant of (7.7 +/- 0.1) x 10(5) M-1 s-1. Compound II is then reduced by tyrosine to native enzyme with a second order rate constant of (1.57 +/- 0.06) x 10(4) M-1 s-1. Our study further revealed that, compared with horseradish peroxidase, thyroid peroxidase, and lactoperoxidase, myeloperoxidase is the most efficient catalyst of tyrosine oxidation at physiological pH. The second order rate constant for the myeloperoxidase compound I reaction with tyrosine is comparable with that of its compound I reaction with chloride: (4.7 +/- 0.1) x 10(6) M-1 s-1. Thus, although chloride is considered the major myeloperoxidase substrate, tyrosine is able to compete effectively for compound I. Steady state inhibition studies demonstrate that chloride binds very weakly to the tyrosine binding site of the enzyme. Coupling of tyrosyl radicals yields dityrosine, a highly fluorescent stable compound that had been identified as a possible marker for lipoprotein oxidation. We present spectral and kinetic data showing that dityrosine is further oxidized by both myeloperoxidase compounds I and II. The second order rate constants we determined for dityrosine oxidation are (1.12 +/- 0.01) x 10(5) M-1 s-1 for compound I and (7.5 +/- 0.3) x 10(2) M-1 s-1 for compound II. Therefore, caution must be exercised when using dityrosine as a quantitative index of lipoprotein oxidation, particularly in the presence of myeloperoxidase and H2O2.

Transient and steady-state kinetics of the oxidation of scopoletin by horseradish peroxidase compounds I, II and III in the presence of NADH.

Scopoletin, a naturally occurring fluorescent component of some plants and a proven plant growth inhibitor, is a known reactant with peroxidase. However, the kinetics of the elementary steps of the reaction have never been investigated, nor has the quantitative effect of interfering substances ever been explored in detail, despite the fact that scopoletin is widely used in a peroxidase assay for H2O2. In this work, we employed both transient-state and steady-state methods to determine the second-order rate constants for the oxidation of scopoletin by the horseradish peroxidase (HRP) intermediate compounds I and II: (3.7 +/- 0.1) x 10(6) M-1 s-1 and (8.5 +/- 0.5) x 10(5) M-1 s-1 at 20 degrees C, pH 6.0 and ionic strength of 0.1 M. We investigated the possible inhibitory effect of NADH on the reaction of scopoletin with HRP and also the effect of scopoletin on the NADH reaction. In the presence of NADH the rate constant for the reaction between HRP-I and scopoletin decreased slightly to (2.8 +/- 0.1) x 10(6) M-1 s-1. Thus, although NADH is also a peroxidase substrate, it cannot compete effectively for the oxidized forms of the enzyme. On the other hand, scopoletin stimulates the oxidation of NADH by the HRP/H2O2 system, apparently by forming a phenoxyl radical which then oxidizes NADH to NAD. radicals. We present spectral evidence showing that in the aerobic reaction between HRP and NADH at pH 7.0 (without exogenously added H2O2) HRP-II is the dominant enzyme intermediate with HRP-III also detectable. Addition of scopoletin to the HRP/NADH system leads to a biphasic reaction in which HRP-II and HRP-III disappear. The rate constants for both phases are linearly dependent on scopoletin concentration. We attribute the faster phase to the HRP-II reaction with scopoletin with a rate constant of (6.2 +/- 0.1) x 10(5) M-1 s-1 and the slower phase to the HRP-III reaction with scopoletin with rate constant (5.0 +/- 0.4) x 10(4) M-1 s-1. Our present work not only provides rate constants for the oxidation of scopoletin by HRP-I, II and III but also elucidates the interactions that possibly occur physiologically during NADH oxidation in the presence of scopoletin.

Chlorination of taurine by myeloperoxidase. Kinetic evidence for an enzyme-bound intermediate.

The chlorination of taurine by the myeloperoxidase-H2O2-Cl- system was investigated under steady state conditions. By systematically varying the pH and the concentrations of H2O2,Cl-, and taurine such that chloride inhibition and the unwanted formation of inactive compound II intermediate are minimized, rate data were found to fit a mechanism involving an enzyme-bound chlorinating intermediate. The mechanism we propose is as follows. [formula: see text] The kinetic parameters determined at pH 4.7 are: k1 = (3.3 +/- 0.2) x 10(7) M-1 S-1, k2 = (2.8 +/- 1.2) x 10(6) M-1 S-1, and k3 = (4.4 +/- 0.2) x 10(5) M-1 S-1. The rate constant for compound I formation (k1) is of the same order of magnitude as the value (1.8 x 10(7) M-1 S-1) obtained using transient state techniques in a previous study by our group. The value of k3 is 2 orders of magnitude greater than the non-enzymatic reaction between HOCl and taurine at the same pH. The results of this study indicate that the chlorination reaction mediated by the myeloperoxidase system in vivo may involve an enzyme intermediate species rather than free HOCl. Not only does this mechanism offer the advantage of substrate specificity but also of speed compared to the non-enzymatic reaction. This mechanism can also explain how the indiscriminate oxidation reactions by HOCl are prevented in the leukocyte. The fast formation of taurine monochloramine, a relatively non-toxic and stable compound compared to HOCl, is consistent with the proposed role of taurine in the neutrophil, that of protecting certain targets including myeloperoxidase from the attack by potent chlorinated oxidants.

Spectral and kinetic studies on the formation of myeloperoxidase compounds I and II: roles of hydrogen peroxide and superoxide.

The conversion of myeloperoxidase to compounds I and II in the presence of H2O2 has been reinvestigated in order to explain the abnormal stoichiometry of compound I formation and the fast spontaneous decay of compound I to compound II. Rapid-scan studies show that at least a 20-fold excess of H2O2 is required to obtain a good spectrum of relatively pure compound I; a further increase in H2O2 concentration causes compound I to be reduced to compound II, which is a very stable intermediate. Compound I formation is reversible, with an apparent second-order forward rate constant of (1.8 +/- 0.1) x 10(7) M-1 s-1 and a reverse rate constant of 58 +/- 4 s-1, giving a constant of 3.2 microM for the dissociation of compound I to native enzyme and H2O. This reversibility is one factor that can explain the large excess of H2O2 required to form compound I. The apparent second-order rate constant for compound II formation from compound I and H2O2 is (8.2 +/- 0.2) x 10(4) M-1 s-1. We confirm pH dependence studies, which suggest that the formation of compounds I and II is controlled by a residue in the enzyme with a pKa of about 4.0. Excess H2O2 is also converted to O2 via catalase activity of the enzyme. However, we do not consider this a dominant pathway because it fails to account for the fast spontaneous reduction of compound I to compound II. The time courses for both the decay of compound I and the formation of compound II are biphasic.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of acetaminophen with myeloperoxidase intermediates: optimum stimulation of enzyme activity.

The myeloperoxidase-H2O2 system constitutes an effective physiological defense mechanism because of its ability to oxidize Cl- to HOCl, a powerful oxidizing agent, via the enzyme intermediate compound I. Two other oxidized intermediates, compounds II and III, are inactive in generation of HOCl. Acetaminophen, a safe drug at therapeutic doses but toxic at higher doses, was found to react with the oxidized intermediates of myeloperoxidase. Using steady-state kinetics on the chlorination of monochlorodimedon to measure the activity of myeloperoxidase, our study reveals that acetaminophen stimulates the chlorinating activity of the enzyme and optimum stimulation is achieved at about 30 microM. Increasing the concentration further causes a decline in the chlorination rate. The increase in enzyme activity at lower acetaminophen concentrations is accounted for by the increased turnover of compounds II and III to native enzyme, while the decline at higher acetaminophen concentrations is explained by the competition of acetaminophen with Cl- for compound I. Rapid scan and transient state kinetic results on the reaction of compound II and acetaminophen show that: (i) compound II does not pass through any other intermediate when acetaminophen reduces it back to native enzyme; and (ii) a simple binding interaction before enzyme reduction is involved. An apparent dissociation constant of 1.3 +/- 0.3 x 10(-4) M and a first-order rate constant for reduction of 37 +/- 4 s-1 were determined at 25 degrees C. Conventional spectral scans of the reaction between compound III and acetaminophen indicate that compound III goes back to native enzyme without any detectable intermediate. The rate of this reaction levels off at higher acetaminophen concentration. Rapid scans reveal that the reduction of compound I to compound II is faster in the presence of acetaminophen. Since the therapeutic concentrations of acetaminophen in man range approximately from 50 to 150 microM, the results of this study indicate that stimulation of myeloperoxidase activity is achieved within the safe dosage of the drug.

The activity of mammalian peroxidases (lactoperoxidase and myeloperoxidase) and their compounds III toward 2-t-butyl-4-methoxyphenol (butylated hydroxyanisole) and its dimer (2,2'-dihydroxy-3,3'-di-t-butyl-5,5'-dimethoxydiphenyl).

Spectral evidence is presented which shows that butylated hydroxyanisole (BHA) and its dimer act as electron donors for lactoperoxidase (LPO) and myeloperoxidase (MPO) by two different pathways: peroxidative and oxidative. LPO compound II and MPO compound II are converted to native enzymes in their reactions with BHA without detectable intermediates. This confirms a normal peroxidatic oxidation of this commonly used antioxidant. We also report spectral data indicating the reductions of peroxidase compound III to the native state in reactions with BHA (LPO, MPO) or with di-BHA (LPO). This oxidative reaction has significant physiological relevance, ensuring return of peroxidases to the native state for re-entry into the normal peroxidatic cycle or into halogenating reactions.