RESUMO
The mast cell-rupturing component present in the lysosomes of rabbit exudate PMN neutrophil leukocytes has been identified and some of its physical and chemical properties have been described. The active agent is a low molecular weight (1200 to 2400) polypeptide containing a relatively large proportion of the basic amino acid, arginine. It is thermostable and dialyzable, and does not cause contraction of the isolated guinea pig ileum. The mast cell-rupturing activity of the agent is destroyed by trypsin. A second permeability factor with a larger molecular weight is present in crude extracts of PMN granules. Although this substance does not lyse mast cells, it is capable of evoking delayed permeability responses in rabbit skin.
Assuntos
Inflamação , Leucócitos , Lisossomos , Mastócitos , Neutrófilos , Peptídeos , Animais , CoelhosRESUMO
The present study demonstrates that a granule fraction derived from human polymorphonuclear leukocytes possesses elastinolytic activity, and that the latter can be separated from the collagenase present in these cells. Properties of the human leukocyte elastase differ sufficiently from those of pancreatic elastases of different species as to suggest that the former enzyme is a distinct and separate entity. Thus, soybean trypsin inhibitor and salivary kallikrein inhibitor (Trasylol) fail to inhibit elastolysis by the pancreatic enzyme, but do inhibit the leukocyte elastinolytic agent. Elastolysis by human leukocyte granule extract does not show significant salt inhibition, whereas that catalyzed by pancreatic elastase is markedly reduced when ionic strength is increased to physiological levels. The leukocyte granule extract is at least 10 times more resistant to serum elastase inhibitor than is the purified pancreatic enzyme. Both enzymes show optimal elastolysis above pH 8.5, but the leukocyte factor still retains 50% of its maximal elastolytic activity at pH 6-7; whereas the activity of the pancreatic enzyme falls to 10% or less of its maximal value under the same conditions. The foregoing characteristics of the human leukocyte elastase suggest that it, rather than pancreatic (serum) elastase, may mediate pathological elastolysis during acute arteritis in man. In keeping with this suggestion, the present experiments also show that elastica staining of human arterial vessels is reduced by incubation of tissues with human leukocyte granule extracts in vitro.
Assuntos
Inflamação/enzimologia , Leucócitos/enzimologia , Lisossomos/enzimologia , Elastase Pancreática/metabolismo , Adulto , Aprotinina/farmacologia , Sangue , Colágeno , Tecido Elástico/efeitos dos fármacos , Hemoglobinas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Leucócitos/análise , Masculino , Pâncreas/enzimologia , Elastase Pancreática/análise , Elastase Pancreática/antagonistas & inibidores , Peptídeo Hidrolases/farmacologia , Proteínas/análise , Cloreto de Sódio/farmacologia , Extratos de TecidosRESUMO
Frozen and thawed granules of human, peripheral-blood leukocytes rapidly produce hemorrhage when injected into animal tissues. The effect is blocked by inhibitors of proteolysis. The granule extract can digest vascular basement membrane in vitro at neutral pH. In addition, basement membranes of blood vessels damaged in vivo by the leukocyte fraction are found to be attenuated when examined by electron microscopy. The proteases of human leukocyte granules differ in several important respects from known lysosomal cathepsins and trypsin-like esterases. Polymorphonuclear neutrophils are a major source of the neutral proteases present in circulating white cells, and release these enzymes during phagocytosis of immune complexes.
Assuntos
Membrana Basal/efeitos dos fármacos , Vasos Sanguíneos/efeitos dos fármacos , Hemorragia/induzido quimicamente , Leucócitos/enzimologia , Peptídeo Hidrolases/toxicidade , Animais , Membrana Basal/análise , Vasos Sanguíneos/patologia , Grânulos Citoplasmáticos , Eletroforese , Esterases , Humanos , Intestinos/irrigação sanguínea , Rim/irrigação sanguínea , Masculino , Camundongos , Neutrófilos/enzimologia , Fagocitose , Coelhos , TripsinaRESUMO
Gas-phase, water-soluble components of cigarette smoke cause delayed fibrin self-assembly and prevent fibrin cross-linking by inactivation of factor XIIIa (plasma transglutaminase). These anticoagulant properties of smoke are demonstrable in plasma, suggesting they play a role in the pathophysiology of smoking.
Assuntos
Anticoagulantes , Fator XIII/metabolismo , Fibrina/metabolismo , Nicotiana , Plantas Tóxicas , Fumaça/análise , Testes de Coagulação Sanguínea , Reagentes de Ligações Cruzadas/metabolismo , Fibrinogênio/metabolismo , Humanos , Fumar , Tempo de Trombina , TransglutaminasesRESUMO
Three synthetic inhibitors of proteases (tosyl lysine chloromethyl ketone, tosyl phenylalanine chloromethyl ketone, and tosyl arginine methyl ester) inhibit the tumorigenesis initiated in mouse skin by 7,12-dimethylbenz(a)anthracene and promoted by croton oil or its active principle, phorbol ester. These protease inhibitors, when applied directly to mouse skin, inhibit some of the irritant effects of the tumor promoter and are not toxic.
Assuntos
Antineoplásicos/farmacologia , Arginina , Transformação Celular Neoplásica/efeitos dos fármacos , Cetonas/farmacologia , Neoplasias Cutâneas/induzido quimicamente , Inibidores da Tripsina/farmacologia , Animais , Benzo(a)Antracenos/antagonistas & inibidores , Quimotripsina/antagonistas & inibidores , Óleo de Cróton/antagonistas & inibidores , Depressão Química , Ésteres/farmacologia , Lisina , Camundongos , Papaína/antagonistas & inibidores , Fenilalanina , Terpenos/antagonistas & inibidoresRESUMO
Brief inhalation exposure of rats to three or six puffs of cigarette smoke significantly decreases elastase inhibitory capacity per milligram of alpha 1-antitrypsin in lung lavage fluid. This effect is not observed in ozone-tolerant rats and can be reversed by treating the lung lavage fluid from smoke-exposed rats with reducing agents. Samples of human serum obtained immediately after smoking also show decreased elastase inhibitory capacity per milligram of alpha 1-antitrypsin. Again, elastase inhibitory capacity can be restored by treatment with a reducing agent. Cigarette smoking may cause emphysema by inactivating alpha 1-antitrypsin through oxidation.
Assuntos
Pulmão/enzimologia , Fumar/fisiopatologia , alfa 1-Antitripsina/metabolismo , Animais , Tolerância a Medicamentos , Pulmão/efeitos dos fármacos , Masculino , Oxirredução , Ozônio , Elastase Pancreática/metabolismo , Plantas Tóxicas , Ratos , Fumaça , NicotianaRESUMO
The development of artificial surfactants for the treatment of respiratory distress syndrome (RDS) requires lipid systems that can spread rapidly from solution to the air-water interface. Because hydration-repulsion forces stabilize liposomal bilayers and oppose spreading, liposome systems that undergo geometric rearrangement from the bilayer (lamellar) phase to the hexagonal II (HII) phase could hasten lipid transfer to the air-water interface through unstable transition intermediates. A liposome system containing dipalmitoylphosphatidylcholine was designed; the system is stable at 23 degrees C but undergoes transformation to the HII phase as the temperature increases to 37 degrees C. The spreading of lipid from this system to the air-water interface was rapid at 37 degrees C but slow at 23 degrees C. When tested in vivo in a neonatal rabbit model, such systems elicited an onset of action equal to that of native human surfactant. These findings suggest that lipid polymorphic phase behavior may have a crucial role in the effective functioning of pulmonary surfactant.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Colesterol/química , Lipossomos/química , Complacência Pulmonar/efeitos dos fármacos , Fosfatidiletanolaminas/química , Surfactantes Pulmonares/química , 1,2-Dipalmitoilfosfatidilcolina/farmacologia , Animais , Animais Recém-Nascidos , Fenômenos Químicos , Físico-Química , Colesterol/farmacologia , Bicamadas Lipídicas , Lipossomos/farmacologia , Espectroscopia de Ressonância Magnética , Fosfatidiletanolaminas/farmacologia , Surfactantes Pulmonares/farmacologia , Coelhos , Propriedades de Superfície , Tensão Superficial , Temperatura , Difração de Raios XRESUMO
Human polymorphonuclear leukocytes, monocytes, or pulmonary alveolar macrophages, stimulated in vitro by phorbol myristate acetate (PMA), released reactive oxygen species able to suppress the elastase inhibitory capacity (EIC) of human serum. Immunoelectrophoresis using antibodies against alpha(1)-proteinase inhibitor (alpha(1)-Pi) and elastase showed that inactivation of alpha(1)-Pi was responsible for the decreased serum EIC. Treatment of phagocyte-inactivated serum with a reducing agent (dithiothreitol) resulted in significant recovery of EIC, suggesting that alpha(1)-Pi had been oxidatively inactivated. Serum EIC was partially protected by superoxide dismutase or catalase. Hydrogen peroxide alone had no effect on serum EIC. Thus, neither H(2)O(2) nor O(2) (-) alone, but a product of the two, may have oxidatively inactivated alpha(1)-Pi. In support of the foregoing, neutrophils or monocytes from a patient with chronic granulomatous disease failed to produce detectable levels of O(2) (-) after incubation with PMA. These cells also failed to suppress serum EIC. In the case of PMA-stimulated polymorphonuclear leukocytes or monocytes, extracellular myeloperoxidase may have also played a role in alpha(1)-Pi inactivation since serum EIC was partly protected by azide, cyanide, or the depletion of extracellular chloride. Indeed, in a cell-free system consisting of purified myeloperoxidase, a glucose oxidase-H(2)O(2)-generating system, and Cl(-), the EIC of human serum or purified alpha(1)-Pi could also be suppressed. Omission of any single reactant prevented this effect, as did NaN(3) or catalase, suggesting that enzymatically active myeloperoxidase and H(2)O(2) were necessary. Immunoelectrophoresis of myeloperoxidase-inactivated serum showed that, as before, inactivation of alpha(1)-Pi was responsible for the decreased EIC. Treating myeloperoxidase-inactivated serum with dithiothreitol led to significant recovery of EIC, again suggesting that oxidative inactivation of alpha(1)-Pi had occurred. Oxidative inactivation of alpha(1)-Pi in the microenvironment of inflammatory cells, at sites of acute or chronic inflammation, may allow proteases released from these cells to damage adjacent connective tissue components more readily.
Assuntos
Macrófagos/metabolismo , Monócitos/metabolismo , Neutrófilos/metabolismo , Elastase Pancreática/antagonistas & inibidores , alfa 1-Antitripsina/metabolismo , Glucose Oxidase/farmacologia , Doença Granulomatosa Crônica/sangue , Humanos , Imunoeletroforese , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Oxirredução , Peroxidase/farmacologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Human polymorphonuclear leukocytes (PMN) phagocytosing opsonized antigen-antibody complexes, produce dialyzable species of activated oxygen which are capable of partially suppressing the elastase-inhibiting capacity (EIC) of whole human serum or purified human alpha1-proteinase inhibitor. Serum EIC was partially protially protected by superoxide dismutase, catalase, or mannitol, suggesting that hydroxyl radical, formed by interaction of superoxide radical and hydrogen peroxide, might be responsible for this effect. NaN3 also partly protected EIC, implicating myeloperoxidase-mediated reactions as well. An artificial superoxide rradical-generating system, involving xanthine and xanthine-oxidase, could be substituted for phagocytosing PMN with resultant EIC suppression. These results are consistent with previous demonstrations of the release of potent oxidants by stimulated PMN, as well as earlier studies from our laboratory showing sensitivity of alpha1-proteinase inhibitor to inactivation by oxidants. Oxidative inactivation of proteinase inhibitors in the microenvironment of PMN accumulating at sites of inflammation may allow proteases released from these cells to more readily damage adjacent connective tissue structures.
Assuntos
Neutrófilos/fisiologia , Oxigênio , Elastase Pancreática/antagonistas & inibidores , Fagocitose , Inibidores de Proteases/sangue , Superóxidos , Catalase/farmacologia , Humanos , Manitol/farmacologia , Neutrófilos/efeitos dos fármacos , Superóxido Dismutase/farmacologia , Xantina Oxidase/metabolismo , Xantinas/farmacologiaRESUMO
Human polymorphonuclear leukocyte (PMN) elastase has been implicated in various pathological conditions. However, its physiological role remains undefined. One possible function of this enzyme may be digestion of bacterial proteins after phagocytosis. To test this hypothesis, we prepared Escherichia coli labeled with [3H]arginine and treated these bacteria with a lipid-soluble, active-site-directed chloromethyl ketone inactivator of pancreatic and granulocyte elastases (carbobenzoxy-L-glycyl-L-leucyl-L-alanine chloromethyl ketone, dissolved in methanol). Control bacteria were treated with methanol alone. When E. coli pretreated with the inactivator were incubated with solutions of porcine pancreatic elastase or with PMN granule extract, release of trichloroacetic acid-soluble radioactivity was significantly lower than in the control E. coli. Similar results were obtained when treated and control E. coli were fed to viable human PMN. In contrast, release of trichloroacetic acid-soluble radioactivity from E. coli containing [3H]thymidine was not affected by pretreatment of bacteria with elastase inactivator before feeding them to PMN, suggesting that phagocytosis of E. coli had not been inhibited by the chloromethyl ketone. When treated and control bacteria were fed to PMN, no significant difference was observed in the activity of lysosomal beta-glucuronidase recovered from post-granule supernatant fractions of homogenized leukocytes, suggesting that lysosomal degranulation had not been suppressed by the inactivator. However, elastase activity of the same fractions was depressed if the leukocytes had phagocytized chloromethyl ketone-treated E. coli, suggesting that inhibition of PMN elastase had occurred. We conclude that PMN elastase participates in digestion of E. coli proteins by human PMN.
Assuntos
Proteínas de Bactérias/metabolismo , Lisossomos/enzimologia , Neutrófilos/enzimologia , Elastase Pancreática/fisiologia , Quimotripsina , Escherichia coli , Metanol , FagocitoseRESUMO
Cigarette smoking is associated with significant increases in the number of pulmonary mononuclear phagocytes and neutrophils. A potent chemoattractant for these cells is C5a, a peptide generated during complement (C) activation. We, therefore, investigated the possibility that cigarette smoke could activate the complement system in vitro. Our results show that factor(s) (mol wt less than 1,000) present in an aqueous solution of whole, unfiltered cigarette smoke can deplete the hemolytic capacity of whole human serum in a dose-dependent manner. The particle-free, filtered gas phase of cigarette smoke is inactive. The smoke factor(s) do not activate serum C1, but do deplete serum C4 activity. Treatment of purified human C3 with whole smoke solution modifies the molecule such that its subsequent addition to serum (containing Mg/EGTA to block the classical pathway) results in consumption of hemolytic complement by activation of the alternative pathway. Smoke-modified C3 shows increased anodal migration in agarose electrophoresis, but this is not due to proteolytic cleavage of the molecule as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast to methylamine-treated C3, C3 treated with smoke is only partially susceptible to the action of the complement regulatory proteins Factors H and I. In addition, smoke-modified C3 has diminished binding to Factor H as compared with methylamine-treated C3. Finally, smoke-modified C3 incorporates [14C]methylamine which suggests that the thiolester bond may be intact. These data indicate that aqueous whole cigarette smoke solution can modify C3 and activate the alternative pathway of complement, perhaps by a previously unrecognized mechanism. Should this occur in vivo, complement activation might partly account for the extensive pulmonary leukocyte recruitment observed in smokers.
Assuntos
Ativação do Complemento , Complemento C3/metabolismo , Via Alternativa do Complemento , Fumar , Complemento C1/metabolismo , Complemento C3/imunologia , Proteínas Inativadoras do Complemento C3b/metabolismo , Proteínas Inativadoras do Complemento C3b/farmacologia , Complemento C4/metabolismo , Fator H do Complemento , Fator I do Complemento , Endopeptidases/farmacologia , Hemólise , Humanos , Técnicas In Vitro , Iodoacetamida/metabolismo , Metilaminas/metabolismo , Ligação Proteica , SoluçõesRESUMO
Extracts of human peripheral blood polymorphonuclear leukocyte granules, and two purified proteases derived from such extracts, an elastase and a chymotrypsin-like enzyme, degrade isolated bovine nasal cartilage proteoglycan at neutral pH. Viscosity studies indicate that the leukocyte granule extracts lack hyaluronidase activity and that their degradative effect on proteoglycan at physiological pH is due entirely to proteolytic action. Sepharose 4B gel chromatography and SDS-polyacrylamide gel electrophoresis of proteoglycan fractions treated with leukocyte granule enzymes at pH 7.0 indicate that they degrade one of the proteoglycan link proteins, release a fragment from the hyaluronic acid-binding portion of the proteoglycan subunit core protein, and break down the remainder of the proteoglycan subunit molecule into peptide fragments with varying numbers of chondroitin sulfate chains. Immunodiffusion studies indicate that the antigenic determinants of the proteoglycan subunit core protein and the link proteins survive treatment with granule proteases. Similar degradation of human articular cartilage proteoglycan by granule neutral proteases can be presumed to occur, in view of the similarity of structure of human articular and bovine nasal cartilage proteoglycans. The release of granule enzymes in the course of neutrophil-mediated inflammation can thus result in the degradation of cartilage matrix proteoglycan, leading to cartilage destruction and joint injury.
Assuntos
Cartilagem Articular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Modelos Animais de Doenças , Glicosaminoglicanos/metabolismo , Artropatias/metabolismo , Leucócitos/ultraestrutura , Proteoglicanas/metabolismo , Animais , Artrite/etiologia , Cartilagem Articular/efeitos dos fármacos , Bovinos , Fenômenos Químicos , Química , Sulfatos de Condroitina/metabolismo , Cromatografia em Gel , Quimotripsina/farmacologia , Eletroforese em Gel de Poliacrilamida , Humanos , Imunodifusão , Septo Nasal , Elastase Pancreática/farmacologia , Proteoglicanas/análise , Dodecilsulfato de Sódio , ViscosidadeRESUMO
The present work was undertaken to explore the effect of two purified neutral proteases derived from human peripheral blood polymorphonuclear leukocytes (PMN) on articular cartilage as a model of joint injury. Human leukocyte elastase and chymotrypsin-like enzyme, purified by affinity chromatography, released 32SO4 from labeled rabbit articular cartilage slices in vitro. Release of isotope was initially delayed, suggesting that either a lag in enzyme penetration occurs or that size of degradation fragments is a limiting factor in diffusion of label out of the tissue. The release of 35SO4 was inhibited by preincubation of elastase and chymotrypsin-like enzyme with human alpha 1-anti-trypsin, or with their specific chloromethyl ketone inactivators, and the action of elastase was also inhibited by a monospecific antiserum to PMN elastase, freed of major serum proteinase inhibitors. Immunohistochemical staining procedures revealed the presence of PMN elastase inside the matrix of cartilage slices after a 20-min exposure of tissue to either the pure enzyme or crude PMN granule extract. Serum alpha 1-antitrypsin failed to penetrate into the cartilage slices under identical in vitro conditions. In association with the results reported in the accompanying paper, these findings suggest a model of cartilage matrix degradation by PMN neutral proteases in which local protease-antiprotease imbalance, coupled with different rates of penetration of protease and antiprotease into target tissue, plays a key role in accounting for matrix damage.
Assuntos
Cartilagem Articular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Modelos Animais de Doenças , Glicosaminoglicanos/metabolismo , Artropatias/metabolismo , Leucócitos/ultraestrutura , Peptídeo Hidrolases/metabolismo , Proteoglicanas/metabolismo , Animais , Cartilagem Articular/efeitos dos fármacos , Quimotripsina/isolamento & purificação , Quimotripsina/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Soros Imunes , Cinética , Leucócitos/enzimologia , Oligopeptídeos/farmacologia , Elastase Pancreática/isolamento & purificação , Elastase Pancreática/farmacologia , Fenilalanina/análogos & derivados , Coelhos , Radioisótopos de Enxofre , alfa 1-Antitripsina/metabolismo , alfa 1-Antitripsina/farmacologiaRESUMO
Concentrations of 0.25 and 0.5 mug/ml calcium chromate (CaCrO4-2H2O)dissolved in Dulbecco's medium were found to alter the growth behavior of BHK21 cells in culture. Treated cells grew as shortened fibroblasts and in random orientation. The changes detected during the first two weeks of culture in the presence of the metal became more pronounced as the number of growth passages increased. In addition to the alterations noted above, chromate-treated cells grew into large clusters in Methocel (an alternative technique to the agar suspension system), while untreated cells underwent, at most, only one or two divisions in Methocel. These alterations in growth properties were irreversible and persisted after removal of the treated cells from chromate-containing medium, suggesting that a heritable change had occurred as opposed to a transient, chromate-dependent alteration of cell growth. This experimental observation suggests that chromate salts and perhaps salts of other metals can transform BHK21 cells in vitro or can select for spontaneously transformed cells.
Assuntos
Carcinógenos , Transformação Celular Neoplásica , Cromatos/toxicidade , Animais , Cálcio , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Cricetinae , Meios de Cultura , Rim , Metilcelulose , Neoplasias Experimentais/induzido quimicamenteRESUMO
The ether-lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3) has anticancer activity, but systemic toxicity has restricted its therapeutic use. In this report "free" ET-18-OCH3 and a stable, well-characterized, liposome-based formulation of ET-18-OCH3 (ELL-12) were compared for in vivo toxicity in normal mice and for therapeutic efficacy in three mouse tumor model systems. The entrapment of ET-18-OCH3 in liposomes decreased the acute toxicity of ET-18-OCH3 after i.v. administration. The maximum tolerated dose for a single i.v. dose of free ET-18-OCH3 was found to be approximately 25 mg/kg, whereas the maximum tolerated dose for ELL-12 was approximately 200 mg/kg. ELL-12 was much less hemolytic in vivo than ET-18-OCH3. The therapeutic efficacy of free ET-18-OCH3 and ELL-12 was investigated against i.p. P388 leukemia, Lewis lung cancer lung metastases, and B16/F10 melanoma (lung tumor nodules) in mice. Although ET-18-OCH3 had some anticancer activity, it was found that ELL-12 was more effective than ET-18-OCH3 in all three tumor models at lower and nontoxic dose schedules. These results suggest that association of ET-18-OCH3 in stable, well-characterized liposomes transforms it into an effective antitumor agent.
Assuntos
Antineoplásicos/administração & dosagem , Éteres Fosfolipídicos/administração & dosagem , Animais , Antineoplásicos/toxicidade , Líquido Ascítico/patologia , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Hemólise , Leucemia P388/tratamento farmacológico , Leucemia P388/patologia , Lipossomos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Necrose , Éteres Fosfolipídicos/toxicidadeRESUMO
A chymotrypsin-like enzyme (EC 3.4.21.-) was purified from granules of human neutrophiles (polymorphonuclear leucocytes). The isolation procedure included differential salt extractions of the granules followed by affinity chromatography on 4-phenylbutylamine-Affi-Gel. This rapid purification method resulted in obtaining pure enzyme in relatively high yield in short time. The purified granulocyte chymotrypsin-like enzyme has a minimum Mr of 22 378, calculated from its amino acid composition. The Mr value obtained by sodium dodecyl sulphate gel electrophoresis was 20 000-23 000. The enzyme did not react with antibodies which are monospecific to granulocyte elastase. The granulocyte chymotrypsin-like enzyme was inactivated by Dip-F and by the chloromethyl ketone derivatives Z-PheCH2Cl and Z-(Gly)2-PheCH2Cl but not by Tos-PheCH2Cl. It therefore appears that the enzyme has serine and histidine side chains in its active site, like pancreatic chymotrypsin. The granulocyte enzyme substrate specificity is similar to that of pac-Tyr-Nan and Ac-Phe-1-ONap. It also has an intrinsic weak hydrolytic activity towards some classical elastase substrates such as Boc-Ala-ONp and Ac-DL-Ala-1-ONap. The granulocyte enzyme is inhibited by human serum and by human alpha1-antitrypsin. Its affinity for alpha1-antitrypsin is weaker than that of granulocyte elastase for the same inhibitor. The enzyme is stable at neutral pH at 37 degrees C, but unstable at pH 3.5 and at elevated temperature.
Assuntos
Quimotripsina/sangue , Granulócitos/enzimologia , Leucócitos/enzimologia , Peptídeo Hidrolases/sangue , Aminoácidos/análise , Animais , Sangue , Bovinos , Quimotripsina/isolamento & purificação , Grânulos Citoplasmáticos/enzimologia , Estabilidade de Medicamentos , Humanos , Cetonas/farmacologia , Cinética , Especificidade de Órgãos , Pâncreas/enzimologia , Elastase Pancreática/metabolismo , Peptídeo Hidrolases/isolamento & purificação , Especificidade da Espécie , Temperatura , alfa 1-Antitripsina/farmacologiaRESUMO
Human granulocyte elastase (EC 3.4.21.-) was isolated and purified (yield = 62%, purity = 91-100%) by a new short procedure using affinity chromatography using phenylbutylamine covalently linked to Affi-Gel. The granulocyte elastase was found to have a molecular weight of 34 400 by sodium dodecyl sulphate gel electrophoresis and the molecular weight obtained from the amino acid composition was 34 970. The composition of elastase purified from normal leucocytes showed some significant differences from that of enzyme purified by others from leukemic leucocytes. The granulocyte elastase hydrolysed typical pancreatic elastase substrates like Boc-Ala-ONp and Ac-(Ala)3-Nan. The enzyme was also found to have a weak enzymatic activity in hydrolysing acetyl-L-phenylalanine-alpha-naphthyl ester, a typical chymotrypsin substrate. A monospecific antiserum raised against the purified enzyme gave a single precipitin line with the pure enzyme and also with crude granular extract, both lines being identical.
Assuntos
Granulócitos/enzimologia , Leucócitos/enzimologia , Elastase Pancreática/sangue , Aminoácidos/análise , Cromatografia de Afinidade/métodos , Eletroforese Descontínua , Humanos , Imunodifusão , Peso Molecular , Elastase Pancreática/imunologia , Elastase Pancreática/isolamento & purificaçãoRESUMO
Purified cytoplasmic and outer membranes isolated from cells of wild-type Escherichia coli grown at different temperatures were labelled with 1,6-diphenyl-1,3,5-hexatriene and analyzed using fluorescence polarization techniques. Lipids extracted from the membranes were similarly analyzed using fluorescence polarization. The thermotropic structural transition in outer membranes changed as a function of growth temperature. The structural transition in cytoplasmic membranes and lipids extracted from either cytoplasmic or outer membranes did not change with growth temperature. These data suggest that adaptive changes which occur in the outer membrane determine the temperature range of growth of E. coli. These changes apparently require alterations in outer membrane components other than phospholipids.
Assuntos
Membrana Celular/ultraestrutura , Escherichia coli/crescimento & desenvolvimento , Lipídeos de Membrana/fisiologia , Fosfolipídeos/fisiologia , Difenilexatrieno , Escherichia coli/ultraestrutura , Cinética , Espectrometria de Fluorescência , TemperaturaRESUMO
Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43 degrees C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20 degrees C, but in a liquid crystalline state when cells were grown at 37 and 43 degrees C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.
Assuntos
Membrana Celular/ultraestrutura , Escherichia coli/ultraestrutura , Lipídeos de Membrana/análise , Espectroscopia de Ressonância de Spin Eletrônica , Escherichia coli/crescimento & desenvolvimento , Temperatura , ViscosidadeRESUMO
The ability of elastatinal and chymostatin, protease inhibitors of microbial origin, to inhibit human leucocyte proteases (EC 3.4.-) was studied. Elastatinal and chymostatin are capable of inhibiting the pancreatic enzymes elastase and chymotrypsin, respectively. It was found in these studies, with synthetic substrates, that elastatinal is a much weaker inhibitor of human leucocyte elastase than it is of porcine pancreatic elastase. Elastatinal caused no inhibition of the activity of human leucocyte chymotrypsin-like protease. Chymostatin was found to be a powerful inhibitor of human leucocyte chymotrypsin-like protease. Its affinity to the leucocyte protease was higher than its affinity to bovine pancreatic alpha-chymotrypsin. Chymsotatin had a weak inhibitory effect on the activity of human leucocyte elastase. Studies were also carried out on the ability of chymostatin to inhibit the release of 35SO2-4 from rabbit articular cartilage by human leucocyte chymotrypsin-like protease. Preincubation of the chymostatin with the protease before the latter was added to the 35SO2-4 -labeled cartilage caused inhibition of proteolysis as measured by 35SO2-4 release. Preincubation of chymostatin with 35SO2-4 -labeled cartilage prior to addition of the human chymotrypsin-like protease to the tissue also inhibited 35SO2-4 release. However, in the case of preincubation of cartilage with alpha1 -antitrypsin there was no such inhibition. It therefore appeared that chymostatin, unlike alpha1 -antitrypsin, was capable of penetrating the cartilage matrix and exerting its inhibitory effect upon the human leucocyte chymotrypsin-like protease that was subsequently added to the tissue.