RESUMEN
A correlation of the logarithmic values of the in vitro dissolution rate, G, and the apparent solubility, S, was evaluated in phosphate and ammonium acetate buffer at an initial pH of 7. The dissolution rates were determined with a newly designed and build miniaturized rotating disk equipment, as well as with a traditional rotating disk apparatus. The two apparatuses gave the same correlation pattern of logG and logS. Thirteen diverse drug substances from all of the classes in the Biopharmaceutics Classification System (BCS) were used for the correlation in the phosphate buffer system, with the results from the miniaturized apparatus only. A coefficient of determination, R2, of 0.982 was found if bases formulated as hydrochloride salts were excluded in the correlation. The miniaturized equipment is used for rapid screening of the dissolution rate, approximately 10 min for one run, and consumes small amounts of substance (about 5 mg) and dissolution media. All quantifications were performed by using reversed phase high-performance liquid chromatography (RPHPLC) with a diode array detector (DAD), integrated with the miniaturized rotating disk equipment.
RESUMEN
A correlation of the logarithmic values of the in vitro dissolution rate, G, and apparent solubility, S, was made for seven different drug substances from all of the classes in the Biopharmaceutics Classification System (BCS), in four different phosphate buffers. The effect of inorganic salts added as sodium chloride, sodium nitrate, sodium phosphate and sodium sulfate in the buffer media was investigated for the correlation. Triethanolammonium acetate buffer was also included in the study of the correlation of logG vs. logS. The pH was 7.0 ± 0.1 in all of the buffers to mimic a pH condition in intestinal fluids. The dissolution rate was determined with a newly constructed miniaturized rotating disk equipment, which enables fast determinations and consumes only minute quantities of substance (about 5 mg). The solubility was determined by conventional shake-flask methodology, using 1.5 mL solution volumes. All quantifications were performed with reversed phase high-performance liquid chromatography (RP-HPLC) and diode array detection (DAD). The different inorganic anions seemed to affect the solubility more than the dissolution rate. The phosphate and nitrate ions decreased the solubility for amines compared to the chloride ion. The best correlations of logG and logS were however obtained with a triethanolammonium acetate buffer. The good correlation (R2 = 0.991) may be sufficient in initial screening of drug solubility, based on dissolution rates in aqueous buffer media.
RESUMEN
The glandular kallikrein family is composed of structurally related serine proteases. Studies show that the mouse family encompasses at least 14 highly conserved functional genes, but of these only the tissue kallikarein has a human ortholog. In man, the tissue kallikrein display high sequence similarity with prostate specific antigen and human glandular kallikrein 2, suggesting that they evolved after the separation of primates and rodents. A phylogenetic study of the genes encoding glandular kallikreins in species evolutionarily located between rodents and man may reveal interesting details on how the gene family evolved, which in turn could yield information about the function of the proteins. Therefore, we have initiated a study of the glandular kallikreins of the cotton-top tamarin (Saguinus oedipus), a New World Monkey. Here, we report the cloning and nucleotide sequence of one of these, the tissue kallikrein gene. The gene of 4.4 kb is composed of five exons, and the structure is 90% similar to that of the orthologous human gene. It gives rise to a polypeptide of 261 amino acids, including a signal peptide of 17 residues, a pro-piece of 7 residues, and the mature protein of 237 residues with an estimated molecular mass of 26.3 kD. The similarity to the human prostate specific antigen and human glandular kallikrein 2 genes is 73% and 72%, respectively, including introns and flanking regions. The lower similarity to these genes compared with the human tissue kallikrein gene indicates that they, or a progenitor to them, arose in primates prior to the separation of New and Old World monkeys. Genomic Southern blots also show that the cotton-top tamarin genome encompasses at least one more glandular kallikrein gene.
Asunto(s)
Saguinus/genética , Calicreínas de Tejido/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Mapeo Restrictivo , Alineación de Secuencia , Análisis de Secuencia de ADN , Calicreínas de Tejido/químicaRESUMEN
The serine protease cathepsin G is synthesized during the promyelomonocytic stage of neutrophil and monocyte differentiation. After processing, including removal of an amino-terminal propeptide from the catalytically inactive proform, the active protease acquires a mature conformation and is stored in azurophil granules. To investigate the importance of the proform-conformation for targeting to granules, a cDNA encoding a double-mutant form of human preprocathepsin G lacking functional catalytic site and amino-terminal prodipeptide (CatG/Gly201/triangle upGly19Glu20) was constructed, because we were not able to stably express a mutant lacking only the propeptide. Transfection of the cDNA to the rat basophilic leukemia RBL-1 and the murine myeloblast-like 32D cl3 cell lines resulted in stable, protein-expressing clones. In contrast to wild-type proenzyme, CatG/Gly201/triangle upGly19Glu20 adopted a mature conformation cotranslationally, as judged by the early acquisition of affinity to the serine protease inhibitor aprotinin, appearing before the carboxyl-terminal processing and also in the presence of the Golgi-disrupting agent brefeldin A. The presence of a mature amino-terminus was confirmed by amino-terminal radiosequencing. As with wild-type proenzyme, CatG/Gly201/triangle upGly19Glu20 was proteolytically processed carboxyl-terminally and glycosylated with asparagine-linked carbohydrates that were converted into complex forms. Furthermore, it was targeted to granules, as determined by subcellular fractionation. Our results show that the initial proform-conformation is not critical for intracellular sorting of human cathepsin G. Moreover, we demonstrate that double-mutant cathepsin G can achieve a mature conformation before carboxyl-terminal processing of the proform.
Asunto(s)
Catepsinas/metabolismo , Gránulos Citoplasmáticos/metabolismo , Precursores Enzimáticos/metabolismo , Conformación Proteica , Animales , Antibacterianos/farmacología , Aprotinina/metabolismo , Sitios de Unión , Transporte Biológico , Brefeldino A , Células COS , Catepsina G , Catepsinas/química , Ciclopentanos/farmacología , ADN Complementario/genética , Precursores Enzimáticos/química , Glicosilación , Aparato de Golgi/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Hexosaminidasas/farmacología , Humanos , Leucemia Basofílica Aguda/patología , Macrólidos , Ratones , Mutagénesis Sitio-Dirigida , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Serina Endopeptidasas , Especificidad por Sustrato , Transfección , Células Tumorales CultivadasRESUMEN
The hematopoietic neutral serine proteases leukocyte elastase and cathepsin G are synthesized as inactive precursors, but become activated by removal of an amino-terminal dipeptide and are stored in granules. Moreover, the pro forms of elastase and cathepsin G show carboxyl-terminal prodomains of 20 and 11 amino acids, respectively, which are not present in the mature enzymes. To investigate mechanisms of processing, activation, and granular targeting, we have utilized transgenic expression of myeloid serine proteases in the rat basophilic/mast cell line RBL-1 (Gullberg, U., Lindmark, A., Nilsson, E., Persson, A.-M., and Olsson, I. (1994) J. Biol. Chem. 269, 25219-25225). Leukocyte elastase was stably expressed in RBL-1 cells, and the translation products were characterized by biosynthetic labeling followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography. Processing of a main pro form of 34 kDa into mature 31- and 29-kDa forms was demonstrated. Translocation of mature forms to granule-containing fractions was shown by subcellular fractionation experiments. The processed forms were enzymatically active, judging by the occurrence of amino-terminal processing demonstrated by radiosequence analysis, the acquisition of affinity for the protease inhibitor aprotinin, and the appearance of elastase activity in transfected RBL cells. To investigate the function of the carboxyl-terminal prodomains, deletion mutants of leukocyte elastase and cathepsin G lacking the carboxyl-terminal extension were constructed and transfected into RBL cells. Our results show that as full-length proteins, the deletion mutants were converted to active enzymes and transferred to granules with kinetics similar to that of wild-type enzymes. We conclude that human leukocyte elastase and cathepsin G are converted into enzymatically active forms when expressed in RBL cells and targeted for storage in granules; the carboxyl-terminal prodomains are necessary neither for enzymatic activation nor for targeting to granules in RBL cells.
Asunto(s)
Catepsinas/metabolismo , Compartimento Celular , Gránulos Citoplasmáticos/metabolismo , Precursores Enzimáticos/metabolismo , Leucocitos/enzimología , Elastasa Pancreática/metabolismo , Células Madre/metabolismo , Secuencia de Aminoácidos , Aprotinina/metabolismo , Secuencia de Bases , Basófilos/metabolismo , Transporte Biológico , Catepsina G , Fraccionamiento Celular , Activación Enzimática , Humanos , Elastasa de Leucocito , Mastocitos/metabolismo , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Relación Estructura-ActividadRESUMEN
The cDNA of the human wild-type p53 tumor suppressor gene was constitutively overexpressed in the leukemic cell line K562 (which lacks detectable amounts of p53 protein) in order to investigate the consequences for growth and differentiation. Several stable clones were established by transfection of the expression vector pc53SN3. Expression of p53 protein was characterized by biosynthetic labeling and immunoprecipitation with the monoclonal antibodies pAb 1801 (reacting with wild-type and mutant human p53), pAb 240 (reacting with mutant human p53) and pAb 1620 (reacting with wild-type human p53). All clones which were 1801+, 240-, 1620- or 1801+, 240-, 1620+ were defined as "wild-type-like p53-expressing" clones. Our results show that expression of p53 protein is compatible with continuous proliferation of K562 cells. The growth characteristics of wild-type-like p53-expressing clones did not differ from that of control clones. However, the former were more sensitive than p53-negative control clones to growth inhibition by tumor necrosis factor (TNF), a cytokine with a potential role in growth and differentiation of myeloid leukemic cells. In addition, a 2- to 4-fold increase of the amount of hemoglobin, a marker of erythroid differentiation, was observed when wild-type-like p53 protein-expressing clones were incubated with TNF. This suggests that differentiation is the mechanism responsible for the increased TNF sensitivity of these clones. Our results support a role for p53 in mediating growth inhibitory and differentiation inducing signals by TNF.
Asunto(s)
Genes p53/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Factor de Necrosis Tumoral alfa/farmacología , Anticuerpos Monoclonales/inmunología , Apoptosis , Secuencia de Bases , Butiratos/farmacología , Ácido Butírico , Diferenciación Celular , División Celular/efectos de los fármacos , Expresión Génica/genética , Hemoglobinas/biosíntesis , Humanos , Datos de Secuencia Molecular , Transfección/genética , Tretinoina/farmacología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The azurophil granules of neutrophil granulocytes contain neutral proteases such as leukocyte elastase and cathepsin G. These are synthesized as inactive precursors, but following proteolytic processing, they are stored in granules as active enzymes. We describe the establishment of a transgenic cellular model for expression of the human myeloid serine protease cathepsin G. The cDNA for preprocathepsin G was stably expressed in the rat basophilic/mast cell line RBL-1 and the translation product was characterized by use of biosynthetic labeling followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography. Conversion into complex form of an asparagine-linked carbohydrate unit of approximately 3.5 kDa was shown, as judged by the products obtained upon treatment with endoglycosidase H and N-glycanase. Proteolytic processing of 32.5-kDa procathepsin G into a 31-kDa form, within 1-2 h after synthesis, was demonstrated by pulse-chase experiments. Further processing into a 30-kDa form also occurred to a minor extent. The processed forms were enzymatically active, as judged by affinity for the serine protease inhibitors diisopropylfluorophosphate and aprotinin. Translocation of processed forms of cathepsin G to high density fractions, indicating targeting of the protease to granules, was demonstrated by subcellular fractionation. The weak base NH4Cl was shown to delay the processing and enzymatic activation of cathepsin G, whereas the monovalent ionophore monensin completely inhibited both events. Our data demonstrate that human cathepsin G transfected to rat RBL-1 cells, is proteolytically processed into enzymatically active forms and that subcellular transfer to granular organelles occurs. As the processing of transgenic human cathepsin G corresponds to that of endogenous protease of myeloid cells, the model should provide new unique possibilities to further characterize the activation and granular targeting of myeloid serine proteases.
Asunto(s)
Catepsinas/metabolismo , Procesamiento Proteico-Postraduccional , Transfección , Amidohidrolasas/farmacología , Cloruro de Amonio/farmacología , Animales , Secuencia de Bases , Transporte Biológico , Catepsina G , Catepsinas/genética , Humanos , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa/farmacología , Mastocitos/metabolismo , Datos de Secuencia Molecular , Monensina/farmacología , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Ratas , Serina Endopeptidasas , Células Tumorales CultivadasRESUMEN
Two separate tumor necrosis factor (TNF) receptors of approximately 55 kDa (TNF-R55) and 75 kDa (TNF-R75) have been identified. The role of protein kinase A activation by dibutyryl cAMP (dbcAMP) and of protein kinase C activation with phorbol myristate acetate (PMA) for transcriptional and posttranscriptional regulation of the two receptors was investigated in promyelocytic HL-60 cells. Incubation with dbcAMP or the adenylate cyclase agonist forskolin caused an increase in the level of TNF-R75 mRNA while TNF-R55 mRNA was unaffected. The half-life of transcripts for both TNF-R55 and TNF-R75 was unaffected as judged by disappearance of mRNA after inhibition of transcription with actinomycin D. Thus the transcription of the TNF-R75 gene seemed to be enhanced by activation of protein kinase A. This enhancement was not dependent on de novo protein synthesis. Incubation with PMA did not affect the mRNA level of any of the TNF receptors. Both TNF-R55 and TNF-R75 mRNA showed a prolonged half-life after incubation with the inhibitor of protein synthesis cycloheximide, indicating superinduction of the genes. Our results demonstrate that the two TNF receptors can be regulated differently at the transcriptional level and that both transcriptional and posttranscriptional regulation occurs.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Procesamiento Postranscripcional del ARN , Receptores del Factor de Necrosis Tumoral/biosíntesis , Transcripción Genética , Secuencia de Bases , Bucladesina/farmacología , Membrana Celular/metabolismo , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cicloheximida/farmacología , Cartilla de ADN , Activación Enzimática , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Cinética , Leucemia Promielocítica Aguda , Datos de Secuencia Molecular , Oligonucleótidos Antisentido , Reacción en Cadena de la Polimerasa , Proteína Quinasa C/metabolismo , Procesamiento Postranscripcional del ARN/efectos de los fármacos , ARN Mensajero/biosíntesis , ARN Mensajero/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
The processing of the neutral proteases cathepsin G and neutrophil elastase, normally synthesized in myeloid precursor cells and stored in azurophil granules, were investigated by biosynthetic labeling with 14C-leucine of the monoblastic cell line U-937. The proteases were precipitated with specific antibodies and the immunoprecipitates were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by fluorography. The transfer to lysosomes of newly synthesized proteases was demonstrated in pulse-chase labeling experiments followed by centrifugation of cell homogenates in a Percoll gradient. The presence of a closely spaced polypeptide band-doublet at intermediate gradient density suggested cleavage of the specific aminoterminal pro dipeptide extension before storage in lysosomes. The molecular heterogeneity observed for cathepsin G and neutrophil elastase seemed to be due to modifications occurring after sorting into lysosomes, most likely because of C-terminal processing. Modifications of the secreted enzymes were not detectable by SDS-PAGE. In contrast to other lysosomal enzymes, no phosphorylation was demonstrated. Newly synthesized cathepsin G and neutrophil elastase rapidly became resistant to endoglycosidase H, indicating transport through the medial and trans cisternae of the Golgi complex and conversion to "complex" oligosaccharide side chains. This conversion was inhibited by an agent swainsonine, but translocation from the Golgi complex and secretion were unaffected. The processing described may play a role in activation of the proteases.
Asunto(s)
Catepsinas/biosíntesis , Leucemia Mieloide Aguda/enzimología , Elastasa Pancreática/biosíntesis , Acetilglucosaminidasa/metabolismo , Alcaloides/farmacología , Catepsina G , Catepsinas/metabolismo , Fraccionamiento Celular , Centrifugación por Gradiente de Densidad , Electroforesis en Gel de Poliacrilamida , Glicosilación , Aparato de Golgi/enzimología , Técnicas de Inmunoadsorción , Leucemia Mieloide Aguda/patología , Elastasa de Leucocito , Lisosomas/enzimología , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidasa , Peso Molecular , Oligosacáridos/metabolismo , Elastasa Pancreática/metabolismo , Fosforilación , Serina Endopeptidasas , Swainsonina , Células Tumorales CultivadasRESUMEN
The processing and intracellular transport of lactoferrin of the neutrophil specific granules was investigated by biosynthetic labeling with (14C)leucine of bone marrow cells from healthy individuals and patients with chronic myeloid leukemia. Lactoferrin was precipitated with antilactoferrin serum and the immunoprecipitates were analyzed by sodium dodecyl sulfate (SDS), polyacrylamide gel electrophoresis (PAGE) followed by fluorography. In contrast to myeloperoxidase of azurophil granules, lactoferrin was not synthesized as a larger precursor, and it was not found to be phosphorylated. The transfer to granules of newly synthesized lactoferrin was demonstrated in pulse-chase labeling experiments followed by centrifugation of cell homogenate in a Percoll gradient. Monensin, which exchanges protons for Na+ and NH4+ cation, blocked the transfer completely, indicating a need for acidification mechanisms. Unlike myeloperoxidase, newly synthesized lactoferrin rapidly became resistant to endoglycosidase H, indicating a transport through the medial and transcisternae of the Golgi apparatus with conversion of "high mannose" to "complex" oligosaccharide side chains. Intracellular transfer of some major neutrophil azurophil and specific granule constituents is obviously regulated differently. Lactoferrin seems to be processed like proteins destined for secretion, while myeloperoxidase is processed more or less like lysosomal enzymes.
Asunto(s)
Médula Ósea/metabolismo , Gránulos Citoplasmáticos/metabolismo , Lactoferrina/biosíntesis , Lactoglobulinas/biosíntesis , Lisosomas/metabolismo , Cloruro de Amonio/farmacología , Transporte Biológico/efectos de los fármacos , Compartimento Celular/efectos de los fármacos , Cloroquina/farmacología , Glicosilación , Hexosaminidasas , Humanos , Técnicas In Vitro , Lactoferrina/metabolismo , Peso Molecular , Monensina/farmacología , Peroxidasa/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
The eosinophil cationic protein (ECP), which has been shown to be secreted both in vitro and in vivo, is a cytotoxic unique constituent of eosinophil granules. To increase the understanding of the mechanisms behind the role of the eosinophil as a cytotoxic effector in disease, a detailed biochemical characterization of ECP was performed. A considerable molecular heterogeneity was revealed when purified ECP was eluted isocratically from a high-resolution cation exchange resin; the separation, reproducibly achieved, of five components was probably due to hydrophobic interaction with the resin. These polypeptides, which reacted quantitatively with anti-ECP antiserum, showed molecular weights (mol wt) of 19,500 and 16,700 and showed almost identical amino acid compositions. The amino-terminal sequence for one of the polypeptides was (in the standard one-letter code) (R-P-X-Q-F-T-R-A-Q-W-F-A-I-Q-H-I-S-L-N-P-R-R-C-T-I-A-M-R-A-I-N-N-Y-). The biosynthesis of ECP was demonstrated in marrow cells from patients with eosinophilia using labeling with (14C)-leucine, followed by immunoprecipitation with anti-ECP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography for visualization of labeled ECP. Biosynthesis was demonstrated of mol wt 22,000 ECP, which may represent precursor ECP, since with time some of it was processed into ECP with a mol wt of 18,000 to 19,000. Monensin, a proton ionophore, blocked the processing of mol wt 22,000 ECP. This study shows that ECP consists of a family of similar polypeptides. These may, however, have different biological activities.
Asunto(s)
Proteínas Sanguíneas/biosíntesis , Médula Ósea/metabolismo , Eosinofilia/metabolismo , Ribonucleasas , Secuencia de Aminoácidos , Aminoácidos/análisis , Proteínas Sanguíneas/aislamiento & purificación , Médula Ósea/patología , Células Cultivadas , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Proteínas en los Gránulos del Eosinófilo , Humanos , Peso MolecularRESUMEN
Myeloperoxidase, stored in azurophil granules of neutrophils, is synthesized in promyelocytes as a larger molecular weight precursor, which is processed to yield a transient Mr 82 000 intermediate and mature polypeptides with molecular weights of 62 000 and 12 000. We have tried to define subcellular sites for processing using metabolic labelling of the promyelocytic leukemia cell line HL-60 in combination with subcellular fractionation on a Percoll gradient. A reasonable separation was achieved between azurophil granules, Golgi elements and endoplasmic reticulum. The finding of almost exclusively fully processed myeloperoxidase in granules and a mixture of unprocessed and processed polypeptide in fractions enriched in Golgi elements suggests that processing occurred mainly in pregranular structures. Monensin, which exchanges protons for Na+, and the base chloroquine blocked processing probably by inhibition of transport through the Golgi apparatus. However, the lysosomotropic NH4+ cation did not inhibit processing or transport indicating that processing is not necessarily influenced by pH-dependent mechanisms. Results from digestion with endoglycosidase H, incubation with tunicamycin and metabolic labelling with [3H]mannose indicated that myeloperoxidase contained high mannose oligosaccharide side chains. Also [32P]phosphate incorporated into Mr 90 000 and Mr 62 000 myeloperoxidase was susceptible to endoglycosidase H indicating that oligosaccharide side chains are modified by phosphorylation as in lysosomal enzymes. Thus, even if myeloperoxidase contained mannose 6-phosphate residues, these may not necessarily be involved in directing transport to the azurophil granules.
Asunto(s)
Líquidos Corporales/enzimología , Furanos/farmacología , Líquido Intracelular/enzimología , Lisosomas/efectos de los fármacos , Monensina/farmacología , Peroxidasa/metabolismo , Cloruro de Amonio/farmacología , Transporte Biológico Activo/efectos de los fármacos , Línea Celular , Cloroquina/farmacología , Humanos , Peso Molecular , Oligosacáridos/metabolismo , Peroxidasa/análisis , FosforilaciónRESUMEN
Human eosinophil peroxidase (EPO) was purified from leukocytes obtained from a patient with hypereosinophilia. EPO was extracted from the granule fraction using 0.2 mol/L sodium acetate pH 4.0, and the extract was subjected to gel chromatography on Sephadex G-75 and ion exchange chromatography on Biorex 70. The mol wt calculated from gel chromatography was approximately 50,000. However, under reducing and denaturing conditions, polyacrylamide gel electrophoresis revealed two subunits with mol wt of 50,000 and 15,000. The biosynthesis of EPO was studied in marrow cells from patients with eosinophilia using labeling with (14C)-leucine, followed by immunoprecipitation with anti-EPO, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography for visualization of labeled EPO. Biosynthesis of an Mr 53,000 subunit was demonstrated. Biosynthetic labeling of the Mr 15,000 subunit was not demonstrated. A labeled Mr 25,000 chain was detected and may represent a degradation product or a chain that, after further modification, produces the Mr 15,000 subunit. Labeling was also detected in two polypeptides with mol wt of 78,000 and 72,000. These forms of EPO seem to represent precursor polypeptides subjected to proteolytic processing in a similar manner as has been reported for myeloperoxidase (MPO). However, Monensin, a proton ionophore, which blocks the processing of MPO, did not inhibit processing of EPO, indicating separate mechanisms by which MPO and EPO are directed to granules.
Asunto(s)
Eosinofilia/enzimología , Eosinófilos/enzimología , Peroxidasas/biosíntesis , Anticuerpos Monoclonales/inmunología , Humanos , Peroxidasa/aislamiento & purificación , Peroxidasas/aislamiento & purificaciónRESUMEN
The processing and intracellular transport of myeloperoxidase were studied in the human promyelocytic leukaemia cell line HL-60 and in normal marrow cells labelled with [35S]methionine or [14C]leucine. Myeloperoxidase was precipitated with antimyeloperoxidase serum; the immunoprecipitates were subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and radiolabelled myeloperoxidase visualized by fluorography. During a 1 h pulse, myeloperoxidase was labelled in a chain of apparent Mr 90 000. With a subsequent chase, the Mr 90 000 polypeptide disappeared and was replaced by chains of Mr 62 000 and 12 400 corresponding roughly to the size of neutrophil myeloperoxidase subunits. The identification of the radioactive polypeptides as different forms of myeloperoxidase was established also by the similarity in patterns generated by partial proteolysis with V8 proteinase from Staphylococcus aureus. Processing of myeloperoxidase in HL-60 was slow; mature polypeptides were significantly increased only after 6 h. Another myeloperoxidase chain of apparent Mr 82 000 was an intermediate precursor or degradation form. Pulse-chase experiments in combination with sucrose-density-gradient separations of homogenates showed that the Mr 90 000 precursor was located in light density organelles only and not in granule fractions, whereas the Mr 82 000 precursor was located only in intermediate density organelles, suggesting that the latter is a product of the former. Processed mature myeloperoxidase was concentrated in the granule fraction, but some occurred in lower density organelles, which may indicate processing during intracellular transport. Only the Mr 90 000 polypeptide was secreted into the culture medium; this was also the only form found in the cytosol fraction.
Asunto(s)
Médula Ósea/enzimología , Leucemia Mieloide Aguda/enzimología , Peroxidasa/biosíntesis , Peroxidasas/biosíntesis , Línea Celular , Electroforesis en Gel de Poliacrilamida , Hexosaminidasas/metabolismo , Humanos , Manosidasas/metabolismo , Metionina/metabolismo , Fragmentos de Péptidos/análisis , Peroxidasa/metabolismo , Fracciones Subcelulares/enzimología , alfa-Manosidasa , beta-N-AcetilhexosaminidasasRESUMEN
A comparison was made between eosinophils from normal persons and patients with eosinophilia. Highly purified eosinophils were obtained by centrifugation in a Percoll density gradient. Studies were carried out on density distribution, oxygen consumption upon adherence to serum-coated Sephadex and expression of cell surface receptors for IgG and complement. In eosinophil leukaemia the density of eosinophils was abnormally low. Abnormal light density fractions of blood eosinophils were also detected in the hypereosinophilic syndrome (HES). Light density eosinophils of HES showed morphological signs of degranulation consonant with the finding of a low content of granular eosinophil cationic protein (ECP) suggesting degranulation in the circulation or abnormal granule formation in the marrow. In addition, such cells exhibited a higher oxygen consumption than eosinophils with normal density upon adherence to serum-coated Sephadex. Low density eosinophils showed a greater number of cells with Fc-IgG and complement receptors than high density cells. Likewise exudate eosinophils displayed an abnormally low density with higher than normal oxygen consumption indicating that eosinophils may be activated in the tissues. In one patient with HES, a febrile episode resulted in a disappearance of eosinophils with a normal density while abnormal low density eosinophils increased. Our findings suggest that eosinophils from some patients with eosinophilia may be 'activated' in the circulation.