Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox.

High molecular weight homologues of gp91phox, the superoxide-generating subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, have been identified in human (h) and Caenorhabditis elegans (Ce), and are termed Duox for "dual oxidase" because they have both a peroxidase homology domain and a gp91phox domain. A topology model predicts that the enzyme will utilize cytosolic NADPH to generate reactive oxygen, but the function of the ecto peroxidase domain was unknown. Ce-Duox1 is expressed in hypodermal cells underlying the cuticle of larval animals. To investigate function, RNA interference (RNAi) was carried out in C. elegans. RNAi animals showed complex phenotypes similar to those described previously in mutations in collagen biosynthesis that are known to affect the cuticle, an extracellular matrix. Electron micrographs showed gross abnormalities in the cuticle of RNAi animals. In cuticle, collagen and other proteins are cross-linked via di- and trityrosine linkages, and these linkages were absent in RNAi animals. The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed cross-linking of free tyrosine ethyl ester. Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix.

X-ray absorption and resonance raman spectroscopy of human myeloperoxidase at neutral and acid pH.

Myeloperoxidase (MPO), an important enzyme in the oxygen-dependent host defense system of human polymorphonuclear leukocytes, utilizes hydrogen peroxide to catalyze the production of hypochlorous acid, an oxidizing bactericidal agent. While MPO shows significant sequence homology with other peroxidases and this homology is particularly striking among the active-site residues, MPO exhibits unusual spectral features and the unique ability to catalyze the oxidation of chloride ions. We have investigated the MPO active-site with X-ray absorption (XAS) and resonance Raman (RRS) spectroscopies at neutral pH and also at the physiological acidic pH (pH approximately 3) and have compared these results with those of horseradish peroxidase (HRP). At pH 7.5, XAS results show that the iron heme active site is 6-coordinate where the distal ligand is likely nitrogen or oxygen, but not sulfur. The heme is distorted compared to HRP, other peroxidases, and heme compounds, but at pH approximately 3, the distal ligand is lost and the heme is less distorted. RRS results under identical pH conditions show that the skeletal core-size sensitive modes and v3 are shifted to higher frequency at pH approximately 3 indicating a 6- to 5-coordination change of high spin ferric heme. In addition, a new band at 270 cm(-1) is observed at pH approximately 3 which is consistent with the loss of the sixth ligand. The higher symmetry of the heme at pH approximately 3 is reflected by a single v4 mode in the (RRS) spectrum. HRP also loses its loosely associated distal water at this pH, but little change in heme distortion is observed. This change suggests that loss of the distal ligand in MPO releases stress on the heme which may facilitate binding of chloride ion.

Isolation and identification of a protoheme IX derivative released during autolytic cleavage of human myeloperoxidase.

Myeloperoxidase (MPO) is a functionally important component of the normal human neutrophil host defense system. This enzyme possesses a dimeric structure composed of two heavy-subunit/light-subunit protomers, with a heme-like prosthetic group covalently linked to each heavy subunit. Although MPO exhibits unusual spectral and enzymatic properties, the nature of the prosthetic group and its mode of linkage with the apoenzyme have not been determined. In an earlier report (K.L. Taylor, J. Pohl, and J.M. Kinkade, Jr. (1992) J. Biol. Chem. 267, 25282-25288), characterization of the autolytic cleavage of MPO led to the proposal that the prosthetic group was covalently linked to the apoenzyme via a methionyl sulfonium bond with Met409. In the present study, we have demonstrated that autolytic cleavage of MPO, followed by protease digestion under nonreducing conditions, effects the release of a macrocycle with visible and Raman spectral properties consistent with that of a protoheme IX derivative. Mass spectrometric analysis, in conjunction with metabolic labeling studies and recent X-ray crystallographic data, have led to the structural assignment of this macrocycle as 1,5-dihydroxymethyl-3,8-dimethyl-4-vinyl-2-(2'-methylthio) ethenylporphine-6,7-dipropionic acid-iron complex. Based on the mechanism of methionyl sulfonium bond cleavage, this structure is consistent with our earlier proposal that the MPO prosthetic group is covalently linked to the enzyme via a methionyl sulfonium bond and suggests that this linkage occurs through a peripheral vinyl substituent.

The post-translational processing of myeloperoxidase is regulated by the availability of heme.

Myeloperoxidase (MPO) is a hemoprotein that is synthesized in the lumen of the endoplasmic reticulum (ER) as a single-chain precursor and undergoes a complex series of post-translational modifications prior to packaging into azurophilic granules. We and others have previously observed that treatment of human myeloid leukemic cells with succinylacetone (SA), a potent inhibitor of 5-aminolevulinic acid dehydratase (ALA-D), and hence of heme biosynthesis, resulted in loss of MPO enzyme activity, inhibition of the appearance of mature MPO, and accumulation of enzymatically unreactive, but immunoreactive, MPO in the ER. The present study using HL-60 cells was undertaken to establish the nature and specificity of the inhibition by SA and to identify and quantify the biochemical changes in the post-translational pathway of MPO processing. Dose-response studies showed that SA (250 microM) did not affect cell viability or growth up to 72 h, but resulted in inhibition of ALA-D activity (> 93%) and decreased cellular levels of both heme and MPO (approximately 25% of control). There were no effects on the level of total cellular protein or on the activities of lactate dehydrogenase or several other nonheme enzymes colocalized with MPO in azurophilic granules. Northern blot analyses confirmed the nontoxic nature of the conditions and indicated there was no effect on transcription of MPO mRNA. The kinetics of processing in the presence and absence of 250 microM SA were determined using pulse-chase and Percoll density gradient centrifugation methods, followed by identification and quantification of MPO species by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The initial rate of disappearance of precursor MPO was identical for control and SA-treated cells and, after a lag of 2-3 h, there was a fourfold decrease in the rate of appearance of mature MPO in SA-treated cells. In the presence of SA, precursor apoMPO remained in the ER, did not undergo proteolytic processing and, compared to control cells, about 50% was degraded. The disruption in MPO processing was reversible by the addition of exogenous heme. We conclude that the availability of heme is important in the complex maturation of MPO that occurs in the ER, events which precede exit from this compartment and subsequent proteolytic processing and transport to the azurophilic granule.

Changes in the localization of catalase during differentiation of neutrophilic granulocytes.

The nature of the compartmentalization of catalase in human myeloid cells is an unresolved issue. Using a rabbit polyclonal antibody specific for catalase, indirect immunocytofluorescence of immature leukemic promyelocytes (HL-60 cells) showed a pattern of small, sharp, punctate staining in the cytoplasm of all cells, while mature neutrophils showed a larger diffuse, flocculent pattern of cytoplasmic staining. Differential centrifugation of nitrogen cavitates of HL-60 cells indicated that the putative catalase-containing compartment was relatively fragile compared with the compartment(s) that contained myeloperoxidase (MPO), beta-hexosaminidase, beta-glucuronidase, and lysosomal alpha-mannosidase activities. Parallel studies using dimethylsulfoxide (DMSO)-induced HL-60 cells and mature neutrophils showed that, in the course of differentiation, there was an apparent shift in the localization of catalase from the granule fraction to the cytosolic fraction. Percoll-sucrose density gradient centrifugation of HL-60 cell cavitates showed a catalase-containing compartment with a mean peak density (1.05 g/mL) significantly lower than that of the major myeloperoxidase-containing compartment (1.08 g/mL); in mature neutrophils, catalase activity comigrated with lactate dehydrogenase (LDH) activity. Catalase in isolated fractions was protected from proteolysis in the absence, but not in the presence, of 0.1% Triton X-100. Digitonin titration experiments confirmed the compartmentalized nature of catalase in immature HL-60 cells and were consistent with a cytosolic localization in mature neutrophils. Ultrastructural localization of catalase by Protein A-gold immunocytochemistry demonstrated four to six catalase-containing compartments in all HL-60 cell profiles. In mature neutrophils, catalase was localized primarily in the cytoplasmic matrix, although in fewer than 2% of the cell profiles, one to two catalase-containing compartments were observed. The changes in catalase localization that occur during myeloid differentiation appear to be similar to the changes that occur during erythroid and megakaryocytic differentiation, and may have potential clinical significance in the classification of acute leukemia and in the development of drug resistance.

Heterogeneity of peroxidase-positive granules in normal human an Chédiak-Higashi neutrophils.

Studies have demonstrated significant heterogeneity in neutrophil granule morphology and physical density. This study evaluated the heterogeneity morphometrically, morphologically, cytochemically, and biochemically. Intact human peripheral blood neutrophils collected from normal volunteers and a patient with Chédiak-Higashi syndrome (CHS) and isolated normal neutrophil granules were processed for ultrastructural morphology and peroxidase staining. Intact cells, nuclei, and granule profiles were analyzed by computer-assisted planimetry. Peroxidase-positive granules (PPG) represented about 40% of normal neutrophil granules and covered the entire spectrum of granule size. PPG in the least-dense fractions of isolated granules were significantly smaller than in higher-density fractions. PPG in low- and intermediate-density fractions differed from high-density fraction by moderate to strong vicinal glycol staining with Thiéry's periodate-thiocarbohydrazide-silver proteinate method. Differing ratios of % beta-glucuronidase/% myeloperoxidase (MPO) across granule fractions indicated PPG heterogeneity. Morphometric analysis of neutrophils treated with 1 microM calcium ionophore A23187 did not show significant differences in PPG size or number. Biochemically analyzed MPO in these cells was preserved, although the number of peroxidase-negative granules (PNG) and levels of vitamin B12-binding protein were markedly decreased. In CHS, about 20% of granules were PPG. Analysis of CHS neutrophils revealed the persistence of microgranules similar to normals. PNG number and volume fractions of PPG and TG were not different from normals. Complex heterogeneity of normal PPG was quantitated using morphometry and appeared preserved in ionophore-treated cells but was uniquely modified in CHS.

Unique autolytic cleavage of human myeloperoxidase. Implications for the involvement of active site MET409.

Myeloperoxidase (MPO) is a functionally important component of the normal human neutrophil host defense system. This enzyme possesses a dimeric structure composed of two heavy subunit (55-63 kDa)/light subunit (10-15 kDa) protomers, each of which is associated with a heme-like prosthetic group. In addition, MPO species of approximately 38 and 22 kDa have been reported by many different investigators, but their nature and mode of origin are not understood. In the present study, we demonstrate that when MPO is heated under nonreducing, denaturing conditions, these two species are produced via a novel autolytic cleavage of the heavy subunit. The 38-kDa species was isolated by fast-protein liquid chromatography and identified by sequencing as the carboxyl-terminal portion of the heavy subunit, and the cleavage was shown to occur exclusively between Met409 and Pro410. In order to further characterize this unusual cleavage reaction, the 22-kDa species was digested with endoproteinase Asp-N, and the peptide corresponding to its carboxyl terminus was isolated and analyzed by sequencing and mass spectrometry. These data indicated that during cleavage of the heavy subunit, Met409 was converted to homoserine lactone. Thus, the cleavage appeared to formally resemble the cyanogen bromide-dependent cleavage of Met-X peptide bonds. Recent x-ray crystallographic data for canine MPO have indicated that Met409 is in close proximity to the heme-like prosthetic group of MPO. Our studies suggest that interaction of Met409 with this group leads to the formation of a methionyl sulfonium derivative which undergoes intramolecular rearrangement with subsequent peptide cleavage under nonreducing conditions. This derivative may be, at least in part, responsible for the unusual spectral characteristics and enzymatic properties of the enzyme. The primary structure of the 22-kDa MPO species is also reported, and direct evidence is provided for asparagine-linked oligosaccharide moieties at two of the three predicted glycosylation sites.

Expression of recombinant myeloperoxidase using a baculovirus expression system.

Myeloperoxidase (MPO) is a glycosylated heme-containing enzyme present in the azurophilic granules of normal human polymorphonuclear neutrophils. This enzyme plays a major role in the microbicidal activity of the host defense system by catalyzing the formation of the potent oxidant, hypochlorous acid. Although the amino acid sequence of MPO has been deduced from the cDNA, the structural basis for the observed heterogeneity of this enzyme is not known. Furthermore, the nature of the prosthetic group and its mode of linkage to the apoprotein has not been determined. To address questions regarding the structural features of MPO, which arise during the complex posttranslational processing of this enzyme, we utilized a baculovirus system to express MPO in Sf9 insect cells. Two glycosylated, single-chain precursor species of MPO were observed: an 84 kDa species that was secreted and a 74 kDa species that was cell-associated. This is the first report of an expression system in which a cell-associated MPO precursor undergoes posttranslational proteolytic processing.

Ultrastructural, immunochemical, and cytochemical study of myeloperoxidase in myeloid leukemia HL-60 cells following treatment with succinylacetone, an inhibitor of heme biosynthesis.

Myeloperoxidase (MPO) is a heme-containing glycoprotein found in the primary granules (or azurophilic granules) of human polymorphonuclear leukocytes. In the present study, cultured myeloid leukemia HL-60 cells were exposed for 0-72 h to 250 microM 4,6-dioxoheptanoic acid (succinylacetone, SA), a specific inhibitor of heme biosynthesis, and the effects were evaluated using ultrastructural, immunochemical, and cytochemical methods. En bloc peroxidase staining of glutaraldehyde-fixed cells was accomplished with a 30-min exposure to 3,3'-diaminobenzidine (DAB) tetrahydrochloride. Ultrastructural examination revealed that peroxidase reactivity in the endoplasmic reticulum (ER) was relatively unchanged for 8 h and decreased between 12 and 24 h; however, ER lacked DAB-reactive peroxidase at 48-72 h. Peroxidase reactivity in the ER reappeared within 4 h after removal of SA. Seventy-two hours after exposure to SA the number of condensed cytoplasmic granules stained with DAB was significantly decreased, and many of the granules had a "target" appearance with a central DAB-reactive dense core. Staining of mitochondria was observed with overnight exposure to DAB and persisted in HL-60 cells treated 72 h with SA. Mitochondrial and nuclear morphology appeared unaltered. Immunostaining of MPO in thin sections of paraformaldehyde/glutaraldehyde-fixed unosmicated HL-60 cells, embedded in Lowicryl K4M, was accomplished with sequential exposure to an affinity-purified monospecific rabbit antibody to HL-60-MPO and protein A conjugated to 5- or 10-nm colloidal gold. Compared to untreated control HL-60 cells, cells exposed to SA for 48 h exhibited comparable to increased immunoreactive MPO in the ER, despite the absence of heme-dependent peroxidase reactivity. The data indicate that SA inhibits formation of enzymatically active MPO and that in the presence of SA, the ER contains a form(s) of MPO that lacks enzymatic reactivity.

Distinct chromatographic forms of human hemi-myeloperoxidase obtained by reductive cleavage of the dimeric enzyme. Evidence for subunit heterogeneity.

The enzyme myeloperoxidase (MPO) is a functionally important glycoprotein of neutrophilic granulocytes and occurs in three major isoforms (forms 1, 2, and 3) that are dimeric structures composed of two heavy subunit-light subunit protomers, each of which is associated with a chlorine-like prosthetic group. In the present study, highly purified MPO isoforms were obtained from the cells of a single normal donor, and each protein was subjected to reductive alkylation under nondenaturing conditions. The resulting enzymatically active protomers were separated from unreacted dimer using gel filtration chromatography. Use of a fast protein liquid chromatography cation exchange system with a Mono S matrix revealed heterogeneity of the protomers, and allowed essentially complete resolution of the protomers of MPO form 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the two resolved protomeric species under reducing conditions revealed small but reproducible differences in the Mr of their heavy subunits (59,000 and 57,000). Treatment with either endo-beta-N-acetylglucosaminidase or peptide N-glycohydrolase F reduced the Mr of each heavy subunit by approximately 3000 but did not change their relative electrophoretic mobilities. Heavy and light subunits were prepared from each of the MPO isoforms by reductive alkylation under conditions that allowed full retention of the prosthetic group with the heavy subunit. Reverse-phase chromatography and amino-terminal sequencing showed that each MPO isoform contained one major species of light subunit and several minor species. No differences in peroxidatic activity or inhibition by salicylhydroxamic acid were observed among any of the MPO isoforms or resolved protomers, but the latter were considerably more heat labile than dimeric forms of the enzyme and a monomeric form isolated from HL-60 cells. This is the first report of the isolation and partial characterization of distinct protomers from a single isoform of human MPO and suggests that the structure of MPO is more complex than considered previously.

The ontogeny of a 57-Kd cationic antimicrobial protein of human polymorphonuclear leukocytes: localization to a novel granule population.

The ontogeny of a 57-Kd cationic antimicrobial protein (CAP57) that has substantial similarities to bactericidal permeability increasing protein (BPI) has been determined immunocytochemically. CAP57 was detected in the granules of mature peripheral blood neutrophils. However, it was absent from other cells of the peripheral blood: eosinophils, red blood cells (RBCs), and mononuclear cells. In human bone marrow, CAP57 was confined to the neutrophilic series. The earliest stage of development of the myeloid cells at which CAP57 was demonstrated was the promyelocyte. Double immunofluorescent labeling showed that CAP57 was detected in cells positive for myeloperoxidase. The absence of lactoferrin in certain cells (promyelocytes) containing CAP57 indicated that CAP57 was synthesized and packaged in a population of granules prior to the development of granules that contain lactoferrin. CAP57 could not be demonstrated in HL60 cells either by enzyme-linked immunosorbent assay (ELISA) or by immunocytochemistry. However, the presence of another granule-associated cationic antimicrobial protein of molecular weight 37 Kd (CAP37) was readily detected in undifferentiated HL60 cells. Amino acid sequence analysis showed that CAP57 and BPI were identical. Further indication of the identity between CAP57 and BPI was that monoclonal anti-CAP57 antibodies cross reacted with BPI. Sucrose density-gradient centrifugations showed CAP57 was confined to a granule population that exhibited a buoyant density intermediate of the previously described light and heavy azurophil granules. Further resolution of the individual azurophil granule populations by Percoll density-gradient centrifugation revealed that CAP57 was most concentrated in the density range of 1.093 to 1.100 g/cc. These results strongly suggest the unique finding that CAP57 may be associated with a heretofore unreported granule type.

Assembly of dimeric myeloperoxidase during posttranslational maturation in human leukemic HL-60 cells.

Myeloperoxidase is a major protein component of the azurophilic granules (specialized lysosomes) of normal human neutrophils and serves as part of a potent bactericidal system in the host defense function of these cells. In normal, mature cells, myeloperoxidase occurs exclusively as a dimer of Mr 150,000 while in immature leukemia cells, there are both monomeric (Mr 80,000) as well as dimeric species. Like other lysosomal enzymes, myeloperoxidase is synthesized as a larger glycosylated precursor (Mr 91,000) that undergoes processing through single-chain intermediates (Mr 81,000 and 74,000) to yield mature heavy (Mr 60,000) and light (Mr 15,000) subunits. To study the assembly of dimeric myeloperoxidase, azurophilic granules were isolated from either unlabeled or pulse-labeled ([35S]methionine/cysteine) HL-60 cells, and myeloperoxidase was extracted and separated into monomeric and dimeric forms by FPLC gel filtration chromatography. Steady-state levels of dimeric and monomeric myeloperoxidase were found to account for 67% and 33%, respectively, of the total peroxidase activity and were correlated with the levels of associated heme as measured by absorption at 430 nm. Labeled myeloperoxidase polypeptides were immunoprecipitated using a monospecific rabbit antibody and were identified and quantitated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography and liquid scintillation counting. After a 2-h pulse, labeled myeloperoxidase species of Mr 74,000 and 60,000 were found in fractions coeluting with the monomeric form of myeloperoxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of retinoic acid-induced differentiation of human leukemia (HL-60) cells by serum factors and sphinganine.

The human cell line HL-60 was used to investigate the role of protein kinase C in the regulation of retinoic acid-induced maturation of promyelocytic leukemia cells by growth and differentiation factors found in serum. Cells grown in serum-containing medium differentiated less than cells in serum-free medium due to several factors, including albumin binding of retinoic acid. Addition of an inhibitor (sphinganine) of protein kinase C, an enzyme that participates in cellular responses to many serum factors, facilitated the retinoic acid-induced differentiation. Cells treated with both retinoic acid and sphinganine produced more superoxide when stimulated by formylmethionylleucylphenylalanine; hence, this combination generated a more functional population of cells. The ability of sphinganine to promote retinoic acid-induced differentiation suggests that retinoic acid therapy might be improved by the concurrent use of a modulator of protein kinase C activity.

Differential effects of long-chain (sphingoid) bases on the monocytic differentiation of human leukemia (HL-60) cells induced by phorbol esters, 1 alpha, 25-dihydroxyvitamin D3, or ganglioside GM3.

Conditions were developed to prolong the ability of sphinganine, a potent inhibitor of protein kinase C, to block the phorbol ester-induced adherence of HL-60 cells beyond 24 h. The loss of inhibition after this time seen previously (A.H. Merrill, Jr., A.M. Sereni, V.L. Stevens, Y.A. Hannun, R.M. Bell, and J.M. Kinkade, Jr., J. Biol. Chem., 261: 12610-12615, 1986), which appeared to be due to metabolism of this long-chain base, was overcome by supplying sphinganine daily. After 4 days, phorbol myristate acetate-induced adherence was inhibited approximately 50% by sphinganine. Sphinganine significantly decreased the expression of nonspecific esterase induced by phorbol myristate acetate in the nonadherent cells, indicating that other aspects of maturation besides adherence were blocked. The effects of daily sphinganine treatments on the monocytic differentiation induced by 1 alpha-25-dihydroxyvitamin D3 or ganglioside GM3 were also investigated. The increases in nonspecific esterase expression, nitroblue tetrazolium reduction, and morphological maturation caused by either agent were unaffected by the long-chain base. In addition, the changes in several cell surface antigens caused by 1 alpha,25-dihydroxyvitamin D3 were unaltered by sphinganine. Although phorbol esters, 1 alpha,25-dihydroxyvitamin D3, and ganglioside GM3 all induce the maturation of HL-60 cells along the monocytic lineage, the finding that sphinganine only affected the differentiation initiated by phorbol esters, in which protein kinase C clearly is a major regulator, suggests that this enzyme does not play a major role in these other pathways of differentiation.

Monensin disruption of neutrophil granule genesis.

The Na+/H+ ionophore monensin (M) has been used widely to study intracellular pH gradients and acidic subcellular compartments. In the present study, cultured myeloid leukemia HL60 cells, directly sampled bone marrow cells, and peripheral blood neutrophils were exposed to 1-5 microM monensin for 0.5-20 hours. The effects were evaluated using ultrastructural, cytochemical, and biochemical methods. In HL60 cells and marrow promyelocytes treated with monensin, progressive vacuolation of the trans then the cis Golgi was observed. These vacuoles lacked diaminobenzidine (DAB) reactive peroxidase, high iron diamine (HID) reactive sulfated glycoconjugates, and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) reactive vicinal glycol containing complex carbohydrates, but some cis Golgi elements retained osmium zinc iodide reactive reducing groups. The number of normal intensely stained HID reactive granules decreased and an incomplete granule that was DAB-positive/HID-negative, PA-TCH-SP-negative with flocculent matrix density increased in frequency as a function of time and concentration of monensin. Treatment of HL60 cells with monensin markedly reduced 35SO4 incorporation but myeloperoxidase labeling and activity per cell remained constant, although it shifted to lower density granule fractions consistent with the persistent DAB staining of endoplasmic reticulum and synthesis of a DAB-positive, HID-negative granule in intact HL60 cells. The Golgi complex of monensin-treated myelocytes and segmented neutrophils was also vacuolated. A subpopulation of preformed primary granules in promyelocytes, myelocytes, and segmented neutrophils appeared to increase in size and peripheral or central electron lucency. These selective effects of monensin indicate that granule components may be packaged into DAB-positive organelles that are deficient in trans Golgi-derived elements (HID- and PA-TCH-SP-negative) and that some preformed primary granules contain a monensin sensitive Na+/H+ gradient.

Protein bound to mitochondrial DNA from tumor and normal tissues of humans and animals.

Stable mitochondrial (mt) DNA-protein complexes have been reported repeatedly in the last decade. We have found that the amount of proteins bound to mt DNA was increased in human HL-60 promyelocytic and chronic myelocytic leukemia cells. Mt of human tumor cells and bovine and rate liver cells were isolated by differential centrifugation. The DNA was purified by SDS-NaCl precipitation of protein, alcohol precipitation, and sucrose density gradient centrifugation. Mt DNA-bound protein obtained by enzymatic digestion of the DNA and purified by gel exclusion and reverse-phase HPLC chomatographies and SDS-polyacrylamide gel electrophoresis, showed a major protein band at 70 kD and a minor band at 35 kD. Hydrolysis of the mtDNA-protein complex in formic acid yielded DNA bases and peptides by HPLC on a C18 reverse-phase column. Tumor cell mtDNA contained 5-10 times more bound protein than mtDNA from normal tissue.

Inhibition of protein kinase C by defensins, antibiotic peptides from human neutrophils.

Defensins, human neutrophil peptide (HNP) antibiotics, potently inhibited phospholipid/Ca2+ protein kinase (protein kinase C, PKC) and phosphorylation of endogenous proteins from rat brains catalyzed by the enzyme. Of the three defensin peptides, HNP-2 appeared to be more potent than HNP-1 and HNP-3. Kinetic studies indicated that defensins inhibited PKC noncompetitively with respect to phosphatidylserine (a phospholipid cofactor), Ca2+ (an activator), ATP (a phosphoryl donor) and histone H1 (a substrate protein) with Ki values ranging from 1.2 to 1.7 microM. Defensins, unlike polymyxin B (another peptide inhibitor of PKC), did not inhibit the binding of [3H]phorbol 12,13-dibutyrate to PKC; however, defensins, like polymyxin B, inhibited the PKC activity stimulated by 12-O-tetradecanoylphorbol-13-acetate. Defensins had little or no effect on myosin light chain kinase (a calmodulin/Ca2+-dependent protein kinase) and the holoenzyme or catalytic subunit of cyclic AMP-dependent protein kinase, indicating a specificity of action of defensins. It is suggested that defensins, among the most potent peptide inhibitors of PKC so far identified, may have profound effects on functions of neutrophils and other mammalian cells, in addition to their well-recognized antimicrobial activities.

Peroxidase-containing microgranules in human neutrophils: physical, morphological, cytochemical, and secretory properties.

A microgranule fraction, isolated from human neutrophils by using a novel high-resolution Percoll density gradient system contained granules with the lowest density and diameter when compared with 12 other isopycnic granule fractions. Ultrastructurally, from 34% to 50% of the microgranules showed homogeneous diaminobenzidine (DAB) staining under conditions for localizing peroxidase reactivity. The presence of myeloperoxidase (MPO) was further confirmed by biochemical and spectral analysis and immunodiffusion methods. Periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) intensely stained vicinal glycols in the matrix of greater than 97% microgranules in contrast to the weak or absent staining seen in larger primary granules. Directly sampled segmented neutrophils contained small DAB- and PA-TCH-SP-positive granules, which often appeared in clusters. These DAB-positive microgranules selectively remained within the cells after stimulation of exocytosis with the calcium ionophore A23187. The enriched DAB-positive microgranule fraction recovered from A23187-treated cells also contained lysozyme and beta-glucuronidase but lacked vitamin B12 binding protein activity. A similar small, DAB- and PA-TCH-SP-positive granule type was also identified in normal promyelocytes and was the predominant or only granule type observed in leukemic or preleukemic myeloid cells from four patients. This study demonstrates a unique subpopulation of MPO-containing microgranules in normal and leukemic human myeloid cells that are distinguished from (other) primary granules by their extremely low density, small size, content of complex carbohydrates, and resistance to secretion.

Protein kinase C inhibition by sphingoid long-chain bases: effects on secretion in human neutrophils.

Sphingoid long-chain bases (sphinganine and sphingosine) have recently been shown to inhibit protein kinase C both in vitro [Y. Hannun et al. (1986) J. Biol. Chem. 261, 12604-12609] and in intact human neutrophils, in which they block activation of the superoxide-generating respiratory burst [E. Wilson et al. (1986) J. Biol. Chem. 261, 12616-12623]. In the present study we have used sphingosine to investigate the pathways for agonist-induced secretion of neutrophil granule contents. Induction of secretion of the specific granule component lactoferrin by a variety of agonists [phorbol 12-myristate-13-acetate (PMA), formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore A23187] was completely inhibited by sphingosine with an ED50 of 6 to 10 microM. PMA-induced secretion of lysozyme (present in both the azurophilic and specific granules) was completely blocked with an ED50 of 10 microM, whereas fMLP-induced secretion was only about 50% inhibited. Secretion of the azurophilic granule proteins beta-glucuronidase and myeloperoxidase was activated by fMLP and A23187, but not by PMA, and was not affected by sphingosine. The use of A23187 in the presence of sphingosine allowed differentiation between calcium activation of protein kinase C-dependent versus-independent pathways. The effect of sphingosine was not mediated by neutralizing intracellular acidic compartments, since treatment of neutrophils with inhibitory concentrations of sphingosine did not significantly alter the uptake of labeled methylamine. We conclude that at least two mechanisms participate in the regulation of specific and azurophilic granule secretion, respectively: a protein kinase C-dependent pathway and a calcium-dependent pathway which does not involve protein kinase C.

Biochemical and ultrastructural effects of monensin on the processing, intracellular transport, and packaging of myeloperoxidase into low and high density compartments of human leukemia (HL-60) cells.

The biosynthesis of myeloperoxidase in human promyelocytic leukemia HL-60 cells was studied by pulse-chase and immunoprecipitation methods and separation of subcellular organelles using Percoll density gradient fractionation. These studies revealed that in control and monensin (1 microM) treated cells, more than 85% of the total immunoprecipitable radiolabeled myeloperoxidase was present predominantly in precursor form (Mr 91,000) and resided in lower density compartments after an initial 3-h labeling period. Using biochemical and ultrastructural techniques, the lower density regions of the gradient were found to contain elements of the endoplasmic reticulum and the Golgi complex. Following a 16-h chase period, about 70% of the radiolabeled myeloperoxidase in untreated cells was found predominantly in denser regions of the gradient and was present mainly in the form of the mature large subunit (Mr 63,000). These dense regions were shown to contain azurophilic granules by means of the distribution of beta-glucuronidase and myeloperoxidase activities and by electron microscopy. Processing of myeloperoxidase and its deposition into dense granules were blocked by monensin treatment. Following a 16-h chase period in the presence of monensin, approximately 80% of the radiolabeled myeloperoxidase continued to reside in lower density compartments and was predominantly in precursor (Mr 91,000) and intermediate (Mr 81,000 and 74,000) forms. Only about 10% of the radiolabeled myeloperoxidase was associated with dense azurophilic granules. Monensin treatment produced large, Golgi-derived vacuoles which were isolated using Percoll density centrifugation and identified by electron microscopy. These vacuoles were found to be essentially devoid of peroxidase activity and pulse-labeled, newly synthesized radiolabeled myeloperoxidase species. The effects of monensin on transport and processing were reversible after a 3-h exposure and 16-h chase period in the absence of monensin. Taken together, these data indicate that maturation of myeloperoxidase is closely linked to its deposition into dense azurophilic granules via a monensin-sensitive process(es). The lower density compartments within which immature myeloperoxidase species accumulate in the presence of monensin appear to be functionally related to or associated with Golgi or endoplasmic reticulum structures distinct from the large monensin-induced vacuoles.