Similarity of kinetics of three types of myeloperoxidase from human leukocytes and four types from HL-60 cells

Km values for H2O2 and Vmax values for three types of myeloperoxidase (MPO) from human leukocytes (MPO-I, -II, and -III) and four types from human myeloid leukemia HL-60 cells (MPO-IA, -IB, -II, and -III) were determined. Km values of human leukocyte MPOs decreased with increasing pH from 4.4 to 6.2 and increased with increasing NaCl concentration from 0.025 to 0.14 M. There was no significant difference among Km values of leukocyte MPO-I, -II, and -III. NaBr also showed a tendency similar to that of NaCl with regard to the effects of pH and halide concentration on Km values. However, Km values in the presence of NaBr were lower than those in the presence of NaCl. Effects of pH and NaCl concentration on Vmax values of MPO-I, -II, and -III were also examined. Vmax values of MPO-I, -II, and -III were higher at pH 4.9 and 5.4 and increased with increasing NaCl concentration. In addition, no difference was observed between Km values of leukocyte and those of HL-60 cells. MPO-IB, the half-molecular-weight enzyme of HL-60 cells, also had the same Km values as the others. Furthermore, inhibition of the activities of seven MPOs of leukocytes and HL-60 cells by H2O2 was similarly observed at concentrations above 1 mM at pH 5.4 but not at pH 4.4. These results indicate that there is no difference in the affinity to H2O2 among leukocyte MPO-I, -II, and -III and HL-60 cell MPO-IA, -IB, -II, and -III.     

The inhibitory effect of some new synthesized xanthates on mushroom tyrosinase activities

Three iso-alkyldithiocarbonates (xanthates), as sodium salts, C3H7OCS2Na (I), C4H9OCS2Na (II) and C5H11OCS2Na (III), were synthesized, by the reaction between CS2 with the corresponding iso-alcohol in the presence of NaOH, and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) from a commercial source of Agricus bisporus. 4-[(4-methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for the catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed and competitive inhibition for the three xanthates and also for cresolase and catecholase activities of MT. For cresolase activity, I and II showed a mixed inhibition pattern but III showed a competitive inhibition pattern. For catecholase activity, I showed mixed inhibition but II and III showed competitive inhibition. These new synthesized compounds are potent inhibitors of MT with K(i) values of 9.8, 7.2 and 6.1 microM for cresolase inhibitory activity, and also 12.9, 21.8 and 42.2 microM for catecholase inhibitory activity for I, II and III, respectively. They showed a greater inhibitory potency towards the cresolase activity of MT. Both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (alpha > 1) and this negative cooperativity increases with increasing length of the aliphatic tail in these compounds in both cresolase and catecholase activities. The cresolase inhibition is related to the chelating of the copper ions at the active site by a negative head group (S-) of the anion xanthate, which leads to similar values of K(i) for all three xanthates. Different K(i) values for catecholase inhibition are related to different interactions of the aliphatic chains of I, II and III with hydrophobic pockets in the active site of the enzyme.     

Myeloperoxidase is involved in H2O2-induced apoptosis of HL-60 human leukemia cells

We examined the mechanism of H(2)O(2)-induced cytotoxicity and its relationship to oxidation in human leukemia cells. The HL-60 promyelocytic leukemia cell line was sensitive to H(2)O(2), and at concentrations up to about 20-25 micrometer, the killing was mediated by apoptosis. There was limited evidence of lipid peroxidation, suggesting that the effects of H(2)O(2) do not involve hydroxyl radical. When HL-60 cells were exposed to H(2)O(2) in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN), we detected a 12-line electron paramagnetic resonance spectrum assigned to the POBN/POBN(.) N-centered spin adduct previously described in peroxidase-containing cell-free systems. Generation of this radical by HL-60 cells had the same H(2)O(2) concentration dependence as initiation of apoptosis. In contrast, studies with the K562 human erythroleukemia cell line, which is often used for comparison with the HL-60, and with high passaged HL-60 cells (spent HL-60) studied under the same conditions failed to generate POBN(.). Cellular levels of antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase did not explain the differences between these cell lines. Interestingly, the K562 and spent HL-60 cells, which did not generate the radical, also failed to undergo H(2)O(2)-induced apoptosis. Based on this we reasoned that the difference in H(2)O(2)-induced apoptosis might be due to the enzyme myeloperoxidase. Only the apoptosis-manifesting HL-60 cells contained appreciable immunoreactive protein or enzymatic activity of this cellular enzyme. When HL-60 cells were incubated with methimazole or 4-aminobenzoic acid hydrazide, which are inhibitors of myeloperoxidase, they no longer underwent H(2)O(2)-induced apoptosis. Hypochlorous acid stimulated apoptosis in both HL-60 and spent HL-60 cells, indicating that another oxidant generated by myeloperoxidase induces apoptosis and that it may be the direct mediator of H(2)O(2)-induced apoptosis. Taken together these observations indicate that H(2)O(2)-induced apoptosis in the HL-60 human leukemia cell is mediated by myeloperoxidase and is linked to a non-Fenton oxidative event marked by POBN(.).     

Nuclear localization of peptidylarginine deiminase V and histone deimination in granulocytes

Peptidylarginine deiminase (PAD) deiminates arginine residues in proteins to citrulline residues Ca(2+) dependently. There are four types of PADs, I, II, III, and V, in humans. We studied the subcellular distribution of PAD V in HL-60 granulocytes and peripheral blood granulocytes. Expression of green fluorescent protein-tagged PADs in HeLa cells revealed that PAD V is localized in the nucleus, whereas PAD I, II, and III are localized in the cytoplasm. PAD V deletion mutants indicated that the sequence residues 45-74 have a nuclear localization signal (NLS). A sequence feature of this NLS is a three-lysine residue cluster preceded by a proline residue and is not found in the three other PADs. Substitution of the lysine cluster by an alanine cluster abrogated the nuclear import activity. These results suggested that the NLS is a classical monopartite NLS. HL-60 granulocytes, neutrophils, and eosinophils stained with antibody specific for PAD V exhibited distinct positive signals in the nucleus. Subcellular fractionation of HL-60 granulocytes also showed the nuclear localization of the enzyme. When neutrophils were stimulated with calcium ionophore, protein deimination occurred in the nucleus. The major deiminated proteins were identified as histones H2A, H3, and H4. The implication of PAD V in histone modifications is discussed.     

The role of alkyl chain length in the inhibitory effect n-alkyl xanthates on mushroom tyrosinase activities

Sodium salts of four n-alkyl xanthate compounds, C2H5OCS2Na (I), C3H7OCS2Na (II), C4H9OCS2Na (III), and C6H13OCS2Na (IV) were synthesized and examined for inhibition of both cresolase and catecholase activities of mushroom tyrosinase (MT) in 10 mM sodium phosphate buffer, pH 6.8, at 293 K using UV spectrophotometry. 4-[(4-Methylbenzo)azo]-1,2-benzendiol (MeBACat) and 4-[(4-methylphenyl)azo]-phenol (MePAPh) were used as synthetic substrates for the enzyme for catecholase and cresolase reactions, respectively. Lineweaver-Burk plots showed different patterns of mixed, competitive or uncompetitive inhibition for the four xanthates. For the cresolase activity, I and II showed uncompetitive inhibition but III and IV showed competitive inhibition pattern. For the catecholase activity, I and II showed mixed inhibition but III and IV showed competitive inhibition. The synthesized compounds can be classified as potent inhibitors of MT due to their Ki values of 13.8, 11, 8 and 5 microM for the cresolase activity, and 1.4, 5, 13 and 25 microM for the catecholase activity for I, II, III and IV, respectively. For the catecholase activity both substrate and inhibitor can be bound to the enzyme with negative cooperativity between the binding sites (alpha > 1) and this negative cooperativity increases with increasing length of the aliphatic tail of these compounds. The length of the hydrophobic tail of the xanthates has a stronger effect on the Ki values for catecholase inhibition than for cresolase inhibition. Increasing the length of the hydrophobic tail leads to a decrease of the Ki values for cresolase inhibition and an increase of the Ki values for catecholase inhibition.     

Production of the superoxide adduct of myeloperoxidase (compound III) by stimulated human neutrophils and its reactivity with hydrogen peroxide and chloride.

Examination of the spectra of phagocytosing neutrophils and of myeloperoxidase present in the medium of neutrophils stimulated with phorbol myristate acetate has shown that superoxide generated by the cells converts both intravacuolar and exogenous myeloperoxidase into the superoxo-ferric or oxyferrous form (compound III or MPO2). A similar product was observed with myeloperoxidase in the presence of hypoxanthine, xanthine oxidase and Cl-. Both transformations were inhibited by superoxide dismutase. Thus it appears that myeloperoxidase in the neutrophil must function predominantly as this superoxide derivative. MPO2 autoxidized slowly (t 1/2 = 12 min at 25 degrees C) to the ferric enzyme. It did not react directly with H2O2 or Cl-, but did react with compound II (MP2+ X H2O2). MPO2 catalysed hypochlorite formation from H2O2 and Cl- at approximately the same rate as the ferric enzyme, and both reactions showed the same H2O2-dependence. This suggests that MPO2 can enter the main peroxidation pathway, possibly via its reaction with compound II. Both ferric myeloperoxidase and MPO2 showed catalase activity, in the presence or absence of Cl-, which predominated over chlorination at H2O2 concentrations above 200 microM. Thus, although the reaction of neutrophil myeloperoxidase with superoxide does not appear to impair its chlorinating ability, the H2O2 concentration in its environment will determine whether the enzyme acts primarily as a catalase or peroxidase.     

The Novel Recognition Site in the C-Terminal Heparin-Binding Domain of Fibronectin by Integrin α4β1 Receptor on HL-60 Cells

The hematopoietic cell recognition sites of human fibronectin (FN) are the Arg–Gly–Asp–Ser (RGDS) sequence recognized by widely distributed integrin receptor α5β1 and the type III connecting segment (III CS) containing two cell-binding sites, designated CS1 and CS5, that are recognized by the α4β1 receptor. The C-terminal heparin-binding domain of FN (Hep II) has recently been demonstrated to support adhesion of α4β1-dependent melanoma cells [A. P. Mould and M. J. Humphries (1991)EMBO J.10, 4089–4095]. Previously we demonstrated that this region of FN mediated binding of FN to HL-60 cells (acute promyelocytic leukemia cell line) by direct interaction independently of RGD and CS1 [H. Fujitaet al.,(1995)Exp. Cell Res.217, 484–488]. In this study we have characterized a novel site in the Hep II region for binding to HL-60 cells. α4β1 and α5β1 were expressed on HL-60 cells, while α2β1 and α3β1 were not present, as shown by flow cytometry using monoclonal antibodies specific for the different integrins. Anti-α4β1 (P4C2) and anti-β1 (JB1a) antibodies inhibited binding of a 29-kDa dispase-digestive fragment of FN to HL-60 cells. This fragment contains the C-terminal heparin-binding domain of FN but lacks CS1 and CS5. Only the peptide representing the sequence from Val¹⁸⁶⁶to Arg¹⁸⁸⁰, designated E1, inhibited the binding of the 29-kDa fragment to HL-60 cells. The active region of this peptide was a sequence of Thr–Asp–Ile–Asp–Ala–Pro–Ser (TAI- DAPS), which is homologous to Leu–Asp–Val–Pro–Ser (LDVPS) derived from the active site of CS1. Furthermore, labeled E1 peptide directly bound to HL-60 cells. The anti-α4β1 antibody (P4C2) inhibited this interaction. These results indicate that the site of binding to hematopoietic cells is present in the Hep II region of FN and the definition of the chemical structure of FN clarifies a fundamental mechanism of cell invasion of the extracellular matrix.     

Monoclonal antibodies directed against gonococcal protein I vary in bactericidal activity

Monoclonal antibodies (Mab) with specificity for protein I (PI) from Neisseria gonorrhoeae (GC) were examined for bactericidal activity. Mab 4G5 (gamma 3), ID3 (gamma 2a), and 1G6 (gamma 2a) bound to surface-exposed epitopes on PI of GC strain R11 (IA serotype) as assessed by co-agglutination and 125I protein A uptake. Mab 2H1 (gamma 3) that were directed against IB serotype strains and Mab 2E9 (gamma 2a) were negative in co-agglutination and protein A uptake assays and served as controls for some experiments. Only 4G5 and 1D3 were bactericidal for R11 when presensitized organisms were incubated in 10% absorbed, pooled normal human serum (PNHS) or 10% hypogammaglobulinemic serum (H gamma S) despite binding of nearly equivalent numbers of 4G5, 1D3, and 1G6 to R11 during presensitization, as assessed by 125I-protein A uptake. These Mab activated complement to a similar extent on GC R11, leading to deposition of 56.4 X 10(3), 61.9 X 1093), and 47.1 X 10(3) molecules of C3/organism during incubation in 10% C8-deficient serum. Deposition occurred almost exclusively via the classical complement pathway. Measurement of complement component C9 binding to R11 during incubation in H gamma S showed 35,700 molecules of C9/organism with 4G5, 32,600 C9/organism with 1D3, and surprisingly, 29,600 C9/organism with 1G6. Eight thousand four hundred molecules of C9/organism bound to 2E9-coated organisms, 6000 C9/organism to 2H1-coated bacteria, and 3600 C9/organism to nonpresensitized organisms. The C5b-9 complex deposited by 4G5 had a different sedimentation profile by sucrose density gradient analysis from the C5b-9 complex deposited by 1G6, consistent with a different molecular configuration of the bound complex. Mab 1G6 and 1D3, but not 2E9 or 2H1, were able to compete with 125I-4G5 for binding to GC R11. A Mab (2E6) directed against protein III of GC competed weakly with 125I-4G5 for binding to GC R11. Mab 1G6, but not 1D3, blocked 4G5-dependent killing in a dose-related fashion. Both 4G5 and IG6 reacted weakly with native PI of GC R11 by immunoblotting, but neither Mab recognized the 34,800 m.w. fragment of PI generated by trypsin and chymotrypsin treatment of outer membranes. In contrast, 2E9 reacted strongly by immunoblot with both native and cleaved PI of GC R11, suggesting binding to buried determinants of PI. These experiments show that Mab directed against identical or closely associated, surface-exposed epitopes on gonococcal PI differ markedly in bactericidal activity, despite leading to deposition of nearly equivalent numbers of C3 and C9 molecules per organism.     

Monoclonal antibodies against various forms of the adenylyl cyclase catalytic subunit and associated proteins

Monoclonal antibodies against partially purified adenylyl cyclase from bovine brain cortex were raised in mice. Three types of antibody were obtained. Type 1 was specific for the calmodulin-sensitive enzyme. Type II also recognized this enzyme, but recognized the calmodulin-insensitive enzymes from a variety of species and tissues as well. Type I antibodies precipitated their antigens in both the native and denatured forms, while type II strongly favored the denatured forms. Type III antibodies precipitated adenylyl cyclase activity, but as shown by Western blot analysis, were directed against 38-kDa and 45-kDa glycoproteins. The 38-kDa protein was identified as synaptophysin.     

Characteristics of monoclonal antibody binding with the C domain of human angiotensin converting enzyme

Binding of a panel of eight monoclonal antibodies (mAbs) with the C domain of angiotensin converting enzyme (ACE) to human testicular ACE (tACE) (corresponding to the C domain of the somatic enzyme) was studied and the inhibition of the enzyme by the mAb 4E3 was found. The dissociation constants of complexes of two mAbs, IB8 and 2H9, with tACE were 2.3 +/- 0.4 and 2.5 +/- 0.4 nM, respectively, for recombinant tACE and 1.6 +/- 0.3 nM for spermatozoid tACE. Competition parameters of mAb binding with tACE were obtained and analyzed. As a result, the eight mAbs were divided into three groups, whose binding epitopes did not overlap: (1) 1E10, 2B11, 2H9, 3F11, and 4E3; (2) 1B8 and 3F10; and (3) IB3. A diagram demonstrating mAb competitive binding with tACE was proposed. Comparative analysis of mAb binding to human and chimpanzee ACE was carried out, which resulted in revealing of two amino acid residues, Lys677 and Pro730, responsible for binding of three antibodies, 1E10, 1B8, and 3F10. It was found by mutation of Asp616 located close to Lys677 that the mAb binding epitope 1E10 contains Asp616 and Lys677, whereas mAbs 1B8 and 3F10 contain Pro730.     

Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid.

Myeloperoxidase catalyses the conversion of H2O2 and Cl- to hypochlorous acid (HOCl). It also reacts with O2- to form the oxy adduct (compound III). To determine how O2- affects the formation of HOCl, chlorination of monochlorodimedon by myeloperoxidase was investigated using xanthine oxidase and hypoxanthine as a source of O2- and H2O2. Myeloperoxidase was mostly converted to compound III, and H2O2 was essential for chlorination. At pH 5.4, superoxide dismutase (SOD) enhanced chlorination and prevented formation of compound III. However, at pH 7.8, SOD inhibited chlorination and promoted formation of the ferrous peroxide adduct (compound II) instead of compound III. We present spectral evidence for a direct reaction between compound III and H2O2 to form compound II, and for the reduction of compound II by O2- to regenerate native myeloperoxidase. These reactions enable compound III and compound II to participate in the chlorination reaction. Myeloperoxidase catalytically inhibited O2- -dependent reduction of Nitro Blue Tetrazolium. This inhibition is explained by myeloperoxidase undergoing a cycle of reactions with O2-, H2O2 and O2-, with compounds III and II as intermediates, i.e., by myeloperoxidase acting as a combined SOD/catalase enzyme. By preventing the accumulation of inactive compound II, O2- enhances the activity of myeloperoxidase. We propose that, under physiological conditions, this optimizes the production of HOCl and may potentiate oxidant damage by stimulated neutrophils.     

Biosynthesis of properdin

Properdin (P) is synthesized by the human promyelocytic cell line, HL-60, after differentiation with DMSO. The secreted P was physiochemically indistinguishable from purified plasma P. It was polymerized and able to bind to C3IBb-Sepharose but not to C3i-Sepharose. No extracellular precursor was present. The intracellular form, detected between 1 and 4 h after labeling, was similar but had a slightly lower Mr. It also bound reversibly to C3iBb-Sepharose, and polymers could be demonstrated by cross-linking. Pulse-chase experiments suggested the existence of an earlier, but undetectable, intracellular precursor(s). This form could not be immunoprecipitated even when harsh solubilization conditions and/or antibodies against reduced and denatured P were utilized. Studies with endoglycosidases F and H and tunicamycin indicated that the detectable intracellular precursor contains high mannose N-linked carbohydrate that is processed to the complex form before secretion. The sugars are not required for polymerization, secretion, or functional activity, or responsible for the electrophoretic heterogeneity. Polymerization of P is therefore an early intracellular event, perhaps carefully controlled to prevent anomalous aggregation.     

Antibodies to inactive conformations of glyceraldehyde-3-phosphate dehydrogenase inactivate the apo-and holoforms of the enzyme

Polyclonal antibodies produced after the immunization of a rabbit with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus were used to isolate two types of antibodies interacting with different non-native forms of the antigen. Type I antibodies were purified using Sepharose-bound apo-GAPDH that was treated with glutaraldehyde to stabilize the enzyme in the tetrameric form. Type II antibodies were isolated using immobilized denatured monomers of the enzyme. It was shown that the type I antibodies bound to the native holo- and apoforms of the enzyme with the ratio of one antibody molecule per GAPDH tetramer. While interacting with the native holoenzyme, the type I antibodies induce a time-dependent decrease in its activity by 80-90%. In the case of the apoenzyme, the decrease in the activity constitutes only 25%, this indicating that only one subunit of the tetramer is inactivated. Differential scanning calorimetry experiments showed that the formation of the complex between both forms of the enzyme and the type I antibodies resulted in a shift of the maximum of the thermal capacity curves (T(m) value) to lower temperatures. The extremely stable holoenzyme was affected to the greatest extent, the shift of the T(m) value constituting approximately 20 degrees C. We assume that the formation of the complex between the holo- or apo-GAPDH and the type I antibody results in time-dependent conformational changes in the enzyme molecule. Thus, the antibodies induce the structural rearrangements yielding the conformation that is identical to the structure of the antigen used for the selection of the antibodies (i.e., inactive). The interaction of the antibodies with the apo-GAPDH results in the inactivation of the subunit directly bound to the antibody. Virtually complete inactivation of the holoenzyme by the antibodies is likely due to the transmission of the conformational changes through the intersubunit contacts. The type II antibodies, which were selected using the immunosorbent with unfolded enzyme form, do not affect the activity of native holo- and apo-GAPDH, but prevent the reactivation of the denatured GAPDH, binding the denatured forms of the enzyme.     

Myeloperoxidases of human myeloid leukemia cells HL-60 grown in culture and in nude mice

Human myeloid leukemia HL-60 cells were grown either in suspension culture or in nude mice. The two types of myeloperoxidase, the small and the large type, in crude extracts of these cells were analyzed by sucrose density gradient centrifugation. The proportions of the small and the large myeloperoxidase varied markedly depending on the growth conditions of cells. In cells in culture, the small myeloperoxidase amounted to 80% of the total myeloperoxidase, whereas in the solid tumors it amounted to only about 30% of the total. Both in cultured cells and solid tumors, 40% of the total myeloperoxidase was found in the soluble fraction and the rest in the granule fraction. However, adult and fetal blood granulocytes contained only the large myeloperoxidase, which was mainly recovered in the granule fraction. Antiserum prepared against purified large myeloperoxidase of HL-60 solid tumors reacted with the small myeloperoxidase as well as the large enzyme of HL-60 cells in culture. The antiserum also precipitated myeloperoxidase of adult and fetal blood granulocytes. An Ouchterlony double immunodiffusion test also revealed their immunological identity.     

Formation of DNA adducts in HL-60 cells treated with the toluene metabolite p-cresol: a potential biomarker for toluene exposure

We have examined DNA adduct formation in myeloperoxidase containing HL-60 cells treated with the toluene metabolite p-cresol. Treatment of HL-60 cells with the combination of p-cresol and H(2)O(2) produced four DNA adducts 1: (75.0%), 2: (9.1%), 3: (7.0%) and 4: (8.8%) and adduct levels ranging from 0.3 to 33.6 x 10(-7). The levels of DNA adducts formed by p-cresol were dependent on concentrations of p-cresol, H(2)O(2) and treatment time. In vitro incubation of p-cresol with myeloperoxidase and H(2)O(2) produced three DNA adducts 1: (40.5%), 2: (28.4%) and 3: (29.7%) with a relative adduct level of 0.7x10(-7). The quinone methide derivative of p-cresol (PCQM) was prepared by Ag(I)O oxidation. Reaction of calf thymus DNA with PCQM produced four adducts 1: (18.5%), 2: (36.4%), 3: (29.0%) and 5: (16.0%) with a relative adduct level 1.6x10(-7). Rechromatography analyses indicates that DNA adducts 1-3 formed in HL-60 cells treated with p-cresol and after myeloperoxidase activation of p-cresol were similar to those formed by reaction of DNA with PCQM. This observation suggests that p-cresol is activated to a quinone methide intermediate in each of these activation systems. Taken together, these results suggest PCQM is the reactive intermediate leading to the formation of DNA adducts in HL-60 cells treated with p-cresol. Furthermore, the DNA adducts formed by PCQM may provide a biomarker to assess occupational exposure to toluene.     

Purification of human procollagen type III N-proteinase from placenta and preparation of antiserum.

Procollagen type III N-proteinase, of Mr about 70,000, was detected in human placental tissue and purified from this source more than 5800-fold. It was found to be a glycoprotein, which was bound to both concanavalin A-Ultrogel and heparin-Sepharose affinity columns. Binding to a type III pN-collagen-Sepharose affinity column was used as the final step in purification. The purified enzyme accepted only native type III procollagen or [14C]carboxymethylated type III pN-collagen as its substrate; type I, type II and type IV procollagen and heat-denatured type III pN-collagen were not cleaved by the enzyme. Antibodies against this purified enzyme protein raised in rabbits demonstrated a high inhibitory effect on the enzyme activity. Immunoblotting of the denatured protein and immunoelectrophoresis of the native enzyme showed only one major antigenic component, again with an Mr of about 70,000. The antibodies cross-reacted with the enzyme preparation from foetal-calf aorta smooth-muscle cells.     

Isolation and characterization of an unprocessed extracellular myeloperoxidase in HL-60 cell cultures

Extracellular myeloperoxidase of human myeloid leukemia HL-60 cells was purified to homogeneity from its culture supernatant by ammonium sulfate fractionation, CM-Sepharose column chromatography, and monoclonal antibody-Sepharose affinity column chromatography. The yield of enzyme activity was 38% that of the ammonium sulfate fraction. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified preparation gave a single band of approximately 84 kDa. Analysis of protein blot with antibodies specific for the light and heavy chains of myeloperoxidase indicated that the enzyme contained a light and a heavy chain in a single polypeptide. The amino-terminal amino acid sequence of the enzyme began at amino acid residue 155 of the 745-amino acid sequence predicted from myeloperoxidase cDNA, indicating that the enzyme consisted of 591 amino acids. Sucrose density gradient centrifugation of the enzyme showed that the enzyme was a monomeric form. In pulse-chase experiments on HL-60 cells with [35S]methionine, pulse-labeled myeloperoxidase precursors were shown to be processed to a light chain and a heavy chain of cellular enzyme. During a 3-day chase period, newly formed processed monomeric enzyme was converted to a dimeric form.     

Mitoxantrone resistance in HL-60 leukemia cells: reduced nuclear topoisomerase II catalytic activity and drug-induced DNA cleavage in association with reduced expression of the topoisomerase II beta isoform.

Mitoxantrone-resistant variants of the human HL-60 leukemia cell line are cross-resistant to several natural product and synthetic antineoplastic agents. The resistant cells (HL-60/MX2) retain sensitivity to the Vinca alkaloids vincristine and vinblastine, drugs that are typically associated with the classical multidrug resistance phenotype. Mitoxantrone accumulation and retention are equivalent in the sensitive and resistant cell types, suggesting that mitoxantrone resistance in HL-60/MX2 cells might be associated with an alteration in the type II DNA topoisomerases. We discovered that topoisomerase II catalytic activity in 1.0 M NaCl nuclear extracts from the HL-60/MX2 variant, as measured by the decatenation of Crithidia fasciculata kinetoplast DNA, was reduced 4- to 5-fold compared to that in the parental HL-60 cells. Total cellular topoisomerase II activity in HL-60/MX2 cells was only 50% lower than that in HL-60 cells, however, because the "cytosolic fraction" of the HL-60/MX2 nuclear preparation contained high levels of decatenating activity. Antisera to calf thymus topoisomerase II defined a distinctive immunoreactive pattern of topoisomerase II proteins in crude nuclear extracts from the HL-60/MX2 cells. Both alpha (170 kDa) and beta (180 kDa) forms of topoisomerase II were detected in the HL-60 cell extracts, but only the alpha form was detected in extracts from HL-60/MX2 cells. This finding was associated with the appearance of a new 160-kDa immunoreactive species in nuclear extracts from HL-60/MX2 but not HL-60 cells. Studies were designed to minimize the proteolytic degradation of the topoisomerase II enzymes by extraction of whole cells with hot SDS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and subunit structure of rat-liver phosphoprotein phosphatase, whose molecular weight is 260000 by gel filtration (phosphatase IB)

1. Phosphoprotein phosphatase IB is a form of rat liver phosphoprotein phosphatase, distinguished from the previously studied phosphoprotein phosphatase II [Tamura et al. (1980) Eur. J. Biochem. 104, 347-355] by earlier elution from DEAE-cellulose, by higher molecular weight on gel filtration (260000) and by lower activity toward phosphorylase alpha. This enzyme was purified to apparent homogeneity by chromatography on DEAE-cellulose, aminohexyl--Sepharose-4B, histone--Sepharose-4B, protamine--Sepharose-4B and Sephadex G-200. 2. The molecular weight of purified phosphatase IB was 260000 by gel filtration and 185000 from S20,W and Stokes' radius. Using histone phosphatase activity as the reference for comparison, the phosphorylase phosphatase activity of purified phosphatase IB was only one-fifth that of phosphatase II. 3. Sodium dodecyl sulfate gel electrophoresis revealed that phosphatase IB contains three types of subunit, namely alpha, beta and gamma, whose molecular weights are 35000, 69000 and 58000, respectively. The alpha subunit is identical to the alpha subunit of phosphatase II. While the beta subunit is also identical or similar to the beta subunit of phoshatase II, the gamma subunit appears to be unique to phosphatase IB. 4. When purified phosphatase IB was treated with 2-mercaptoethanol at -20 degrees C, the enzyme was dissociated to release the catalytically active alpha subunit. Along with this dissociation, there was a 7.4-fold increase in phosphorylase phosphatase activity; but histone phosphatase activity increased only 1.6-fold. The possible functions of the gamma subunit are discussed in relation to this activation of enzyme.     

Ethylenediamine-palladium(II) complexes with pyridine and its derivatives: synthesis, molecular structure and initial antitumor studies.

The synthesis of four mononuclear palladium complexes of general formula [Pd(en)Cl(L)]NO3 (en = ethylenediamine; L = pyridine (I), 4-methylpyridine (II), 4-hydroxypyridine (III) or 4-aminopyridine (IV) has been achieved. The structure of these compounds was studied by elemental analysis, IR, far-IR and 1H NMR; complex I was analyzed by X-ray diffraction. The crystal of [Pd(en)(pyridine)Cl]NO3 is monoclinic, space group P21/c (a = 7.990(2), b = 16.058(3), c = 9.846(2) A, beta = 103.81(3) degrees, Z = 4, R = 0.067, Rw = 0.066). The Pd(II) atom exhibits an approximately square planar coordination with bond lengths in the range 2.017-2.042 A for Pd-N and 2.320 A for Pd-Cl. In order to determine the donor strength of the aromatic pyridine ligands, the stability constants of binary complex ML2+ (M = [Pd(en) (H2O)2]2+; L = pyridine, 4-Me-pyridine, 4-OH-pyridine and 4-NH2-pyridine) were determined by potentiometric pH titration in aqueous solution (T = 25 degrees C, I = 0.1 mol l-1 NaNO3). The results show that the stability constants of the binary complexes systematically increase with increasing pKa of the pyridines. The above four palladium complexes, [Pt(en)(pyridine)Cl]NO3 and cis-diamminedichloroplatinum (II) (cis-DDP) were assayed for cytotoxicity in vitro against the human leukemia cell line HL-60, and compounds I, II, III and cis-DDP show significant cytotoxic activity against HL-60.