RESUMO
The antiserum was raised in rabbits against intracellular inhibitors I-1, I-2 and I-3 isolated from the soluble phase of disrupted pig peripheral leucocytes. It was demonstrated with double immunodiffusion and with immunoelectrophoresis that the isolated inhibitors with different biochemical characteristics are three different, specific and unrelated proteins. With the techniques used, it was clearly confirmed that the inhibitors were isolated in a pure form and that they are located in cytoplasm and nucleus. The suppression of inhibitors by antiinhibitors antibodies was also demonstrated.
Assuntos
Leucócitos/análise , Inibidores de Proteases/imunologia , Animais , Anticorpos/imunologia , Núcleo Celular/análise , Citoplasma/análise , Imunodifusão , Imunoeletroforese , Inibidores de Proteases/isolamento & purificação , SuínosRESUMO
Two monoclonal antibodies against the native ammodytoxin A and four site-directed polyclonal antibodies against synthetic peptides derived from the primary structure of the toxin were prepared in order to estimate the localization of its toxic site. Some of the antibodies neutralized the lethal toxicity of the toxin, thus indicating an approximate position of the toxic or receptor binding site on the molecule that is different from those predicted by comparison with a number of known sequences.
Assuntos
Fosfolipases A/química , Venenos de Víboras/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Sítios de Ligação , Testes Imunológicos de Citotoxicidade , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fosfolipases A/imunologia , Conformação Proteica , Estereoisomerismo , Venenos de Víboras/imunologiaRESUMO
A plasminogen activator inhibitor (PA-I) which inhibits primarily plasminogen activator of the urokinase type (u-PA) was isolated from the cytosol of human peripheral leukocytes. The inhibitor was isolated using ion exchange chromatography, gel filtration and FPLC. This inhibitor has an apparent molecular weight of 45 kDa, determined by SDS-PAGE, and a pI of 5.5-5.7. The inhibitor is a fast reacting inhibitor, is thermally unstable and is inactivated outside the pH range 7-9. Treatment of cytosol to pH 9 for 30 min at 37 degrees C resulted in a large increase in inhibitory activity. Antibodies against human placental UK-I completely quenched the inhibitory activity of human leucocyte UK-I.
Assuntos
Proteínas Sanguíneas , Glicoproteínas/sangue , Leucócitos/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Proteínas Sanguíneas/imunologia , Reações Cruzadas , Feminino , Humanos , Técnicas In Vitro , Ponto Isoelétrico , Peso Molecular , Placenta/metabolismo , Inativadores de Plasminogênio , GravidezRESUMO
Cysteine proteinase inhibitors (CpI) of all three families were found in ascites fluid from patients with ovarian carcinoma. CPIs were isolated by affinity chromatography on carboxymethylated papain Sepharose, followed by gel filtration, anti-stefin-Sepharose and ion exchange chromatography. The highest apparent inhibition against cathepsin B (Cat B) was found in the low molecular mass (LMM) CPI fraction. Immunochemical analysis of this fraction revealed the presence of cystatin C and both stefins A and B while the high molecular mass (HMM) CPI fraction contained kininogens. We demonstrated that CPIs were not completely associated with cysteine proteinases (CPs): about 20% of HMM CPIs and 50% of LMM CPIs were free in native ascites fluid. Affinity chromatography on anti-Cat B-Sepharose revealed that the major LMM CPI, associated with Cat B in native ascites fluid, was the full length form of cystatin C, pI 9.3, and not its truncated form, pI 7.85. The latter was isolated and found to inhibit Cat B in vitro with apparent Ki 0.18 +/- 0.2 nM. Stefin A was isolated from alkaline activated ascites fluid in its two isoforms, pI 4.6 and 4.9. In native ascites, the pI 4.9 isoform was mostly associated with Cat B. Ki for Cat B was 3.55 +/- 1.7 nM, not significantly different from the Ki values measured for stefin A, isolated from other human tissues and biological fluids.
Assuntos
Líquido Ascítico/química , Cistatinas/isolamento & purificação , Neoplasias Ovarianas/química , Adulto , Idoso , Líquido Ascítico/enzimologia , Catepsina B/antagonistas & inibidores , Catepsina B/metabolismo , Cromatografia de Afinidade , Cistatina B , Cistatinas/metabolismo , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Cinética , Pessoa de Meia-Idade , Peso Molecular , Neoplasias Ovarianas/enzimologiaRESUMO
Cellular and molecular events during the development of inflammatory disease are accompanied by the release of host lysosomal cysteine proteinases (CPs) affecting not only degradation of matrix proteins but possibly also antigen processing and chemotaxis of neutrophils. Activity measurements of Cat B and Cat L could not be used as an accurate indicator of disease activity in individual patients, although average values were higher in patients with more advanced periodontal inflammation. In contrast, simultaneous decrease of cystatin C and alpha 2-macroglobulin (alpha 2-M) in inflamed gingiva and gingival fluid, respectively, might be useful diagnostic/prognostic factors. While the total and the free form of alpha 2-M in gingival fluid decreased with the progression of the disease, the complexed alpha 2-M form was hardly detectable. This indicates an increased consumption of this inhibitor by various proteinases and clearance of protease: alpha 2-M complexes by macrophages. Elevated serum levels of alpha 2-M were found in patients with more pronounced disease, suggesting a systemic host response. In addition, high levels of stefin A and moderate levels of kininogen were observed in gingival tissue homogenates. Stefin A was also found to play a role in the inhibition of neutrophil chemotaxis. In addition, other proteinases which are released at inflammatory sites from neutrophils, macrophages, lymphocytes, and/or bacteria may degrade the cystatins, thereby further increasing CP activities. Increased CP activity may inactivate serine protease inhibitors, leading to the so-called "proteolytic burst."
Assuntos
Cisteína Endopeptidases/fisiologia , Inibidores de Cisteína Proteinase/fisiologia , Gengivite/enzimologia , Periodontite/enzimologia , Animais , HumanosRESUMO
The amount of the low molecular-weight inhibitor, cystatin C, was determined by the enzyme-linked immunosorbent assay. Gingival tissue samples were obtained during periodontal surgery from 22 patients with different degrees of inflammatory periodontal disease, as indicated by gingival index and probing depth (PD). The concentration of cystatin C was in the range from 0.21 to 3.82 micrograms/g tissue and was significantly decreased (p less than 0.01) in samples taken from sites with increased PD.
Assuntos
Cistatinas/análise , Inibidores de Cisteína Proteinase , Gengivite/enzimologia , Adulto , Cistatina C , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Pig leucocytes contain inhibitors of neutral and thiol proteinases. These proteins could be isolated from post-granule supernatant fraction as well as from nuclear extract using ion exchange chromatography, gel chromatography and affinity chromatography. Inhibitors differ in molecular weight, isoelectric point, immunologically and their inhibition ability against tested enzymes.
Assuntos
Leucócitos/enzimologia , Inibidores de Proteases/sangue , Animais , Eletroforese em Gel de Poliacrilamida , Humanos , Leucócitos/ultraestrutura , Inibidores de Proteases/isolamento & purificação , Frações Subcelulares/enzimologia , Especificidade por SubstratoAssuntos
Catepsinas/imunologia , Grânulos Citoplasmáticos/imunologia , Leucócitos , Inibidores de Proteases/imunologia , Animais , Bovinos , Reações Cruzadas , Concentração de Íons de Hidrogênio , Soros Imunes , Imunodifusão , Imunoeletroforese , Peso Molecular , Precipitinas , Coelhos , alfa 1-Antitripsina/imunologiaRESUMO
Extra- and intracellular proteinases from various Claviceps purpurea strains grown in submerged culture have been studied. A maximum level of intracellular proteinases was observed on the 6th day of culture growth, whereas extracellular activity continued to increase throughout the culture growth. Proteinases were purified and characterized. The ergotamine strain secreted one aspartic and two serine proteinases, whereas from the disrupted mycelium only the aspartic proteinase could be isolated. The ergocornine strain secreted the aspartic proteinase in two forms and the ergocristine strain produced an aspartic and a serine proteinase.
Assuntos
Claviceps/enzimologia , Peptídeo Hidrolases/metabolismo , Ergolinas/metabolismo , Ergotamina/metabolismo , Fermentação , Concentração de Íons de Hidrogênio , Fatores de TempoRESUMO
Leucocytes contain an urokinase inhibitor, that can be inactivated by cathepsin D. In this work biochemical and immunological studies on the inactivation of this inhibitor by cathepsin D are presented. Examinations by polyacrylamide gel electrophoresis and SDS electrophoresis indicate that cathepsin D inactivates urokinase inhibitor by hydrolysis of the inhibitor molecule and that the degradation needed for total inactivation is different from that for formation of the precipitin line with antibodies. The conversion of active inhibitor into inactive protein proceeds catalytically.
Assuntos
Proteínas Sanguíneas/antagonistas & inibidores , Catepsinas/farmacologia , Leucócitos/enzimologia , Animais , Catepsina D , Bovinos , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Imunodifusão , Imunoeletroforese , Cinética , Peso Molecular , Fragmentos de Peptídeos , SuínosRESUMO
In the present work we demonstrate the presence of cysteine proteinase inhibitors of all three classes: kininogens, stefin A, and cystatin C, in inflamed human gingiva. Using cystatin C, in inflamed human gingiva. Using immunochemical methods we found that stefin A is the major inhibitor of cysteine proteinases, followed by kininogen and cystatin C. The values for stefin A and cystatin C ranged from 7.0--400 micrograms/g and 1.5--6.1 micrograms/g tissue, respectively, as determined by enzyme-linked immunosorbent assay in inflamed gingival homogenates from patients with different degrees of periodontal disease.
Assuntos
Cistatinas , Gengiva/enzimologia , Gengivite/enzimologia , Inibidores de Proteases/isolamento & purificação , Formação de Anticorpos , Cromatografia de Afinidade , Cistatina B , Inibidores de Cisteína Proteinase , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoquímica , Imunodifusão , Imunoeletroforese , Focalização Isoelétrica , Inibidores de Proteases/imunologia , Inibidores de Proteases/farmacologia , Proteínas/isolamento & purificaçãoRESUMO
The concentration of total, free and bound (complexed forms) of alpha 2-macroglobulin was measured by crossimmunoelectrophoresis in patients with different degrees of periodontal disease as indicated by the gingival index (GI) and the proportion of alveolar bone loss (ABL). The concentration of total alpha 2-M (bound and free forms) is lower in gingival fluid taken from sites with more inflamed gingivae. Its concentration decreases with an increase in the pocket depth and the alveolar bone loss at the sites of fluid collection. The concentration of alpha 2-M bound form, i.e., the presence of alpha 2-M: protease complexes, in gingival fluid is low or absent at the sites with more pronounced bone loss (above 15%). Our results support the hypothesis that unbalanced protease activity damages the periodontal tissue. Not only the proteolytic but also inhibitory activities are altered and correspond to the severity of the disease.
Assuntos
Processo Alveolar/metabolismo , Reabsorção Óssea/metabolismo , Líquido do Sulco Gengival/metabolismo , Gengivite/metabolismo , Doenças Periodontais/metabolismo , alfa-Macroglobulinas/análise , Adulto , Feminino , Humanos , Masculino , Bolsa Periodontal/metabolismo , Bolsa Periodontal/patologiaRESUMO
A new intracellular inhibitor of plasmin and trypsin was isolated from porcine leukocytes by ion exchange chromatography and affinity chromatography. In dodecyl sulphate gel electrophoresis a single protein band with an apparent molecular mass of 15 kDa was found under reducing conditions. On isoelectric focusing three protein bands with isoelectric points between pH 4.0 and 4.5 were found. The association rate constants and the inhibition constants were determined for porcine plasmin and bovine trypsin. The inhibitor shows no immunologic cross-reactivity with any of the tested leukocyte inhibitors. On the basis of its N-terminal amino-acid sequence a great degree of similarity to Kunitz-type inhibitors was observed.
Assuntos
Fibrinolisina/antagonistas & inibidores , Leucócitos/enzimologia , Inibidores da Tripsina/isolamento & purificação , Sequência de Aminoácidos , Animais , Aprotinina , Bovinos , Dados de Sequência Molecular , Suínos , Inibidores da Tripsina/análiseRESUMO
The increased expression of proteolytic systems is one of the characteristics of transformed and malignant cells and their evaluations in whole tumor homogenates were considered as possible diagnostic and/or prognostic factors. Abnormal intracellular distribution, increased activities and secretion of cysteine proteinases (CPs) cathepsin B (Cat B) and L (Cat L), were associated with tumor progression. In the present study of matched pairs of breast carcinoma and normal breast tissue, the activities of Cat B and Cat L in breast carcinoma homogenates were found to be 20 and 50 fold higher, respectively, than in normal tissues. In contrast, a decrease in total inhibitory activity of cysteine proteinase inhibitors (CPIs) was observed but an average ratio between tumor and normal tissues was only 0.75. One of the CPIs, stefin A, was also determined immunochemically. The activities of CPs and CPIs were compared to the increased levels of cathepsin D (Cat D) activities in individual patients, but no statistically significant correlations were found. We correlated CPs and CPIs with morphological and receptor data as well as the axillary lymph node metastases. There was no statistical correlation of CP and CPIs with the number of lymph node metastases. However, highly elevated levels of Cat B and Cat L and lowered CPI activities in tumor cytosols were often associated with poorly differentiated carcinomas and those with negative ER and PR values. We conclude that cysteine-dependent proteolysis may play an important role in breast tumors.
Assuntos
Neoplasias da Mama/enzimologia , Catepsinas/metabolismo , Cistatinas/metabolismo , Endopeptidases , Sequência de Aminoácidos , Catepsina B/metabolismo , Catepsina L , Cistatina A , Cisteína Endopeptidases , Inibidores de Cisteína Proteinase/farmacologia , Citosol/enzimologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Dados de Sequência Molecular , Metástase Neoplásica , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismoRESUMO
Leucocytes and spleen contain four different types of protein proteinase inhibitors. Two of them can be inactivated by cathepsin D. In this work biochemical and immunological studies of the inactivation of I-2 by cathepsin D are presented. Polyacrylamide gel electrophoretic examinations indicate that cathepsin D inactivates I-2 by hydrolysis of the inhibitor molecule. The conversion of the active inhibitor into inactive protein proceeds catalytically. The studies on the inhibitor mechanism of the isoinhibitors of I-1 type explain the unusual inhibitor property of this type of inhibitor to inhibit two different types of proteinases, cysteine and serine. The evidence suggests that the inhibitory mechanism is based on an active sulfhydryl group of the inhibitor which may interact with the disulfide bridge of the inhibited proteinase.
Assuntos
Leucócitos/enzimologia , Inibidores de Proteases/metabolismo , Baço/enzimologia , Animais , Catepsina B , Catepsina D , Catepsinas/antagonistas & inibidores , Catepsinas/farmacologia , Cisteína/farmacologia , Cisteína Endopeptidases , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/patologia , Papaína/antagonistas & inibidores , Inibidores de Proteases/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Serina Endopeptidases , SuínosRESUMO
In the study of 50 matched pairs of breast carcinoma and normal breast tissue, the activities of cysteine proteinases (CPs), cathepsin (Cat) B and Cat L in tumors were increased on average by 18.5-fold and 52.5-fold respectively. The differences in activity of cysteine proteinase inhibitors (CPIs) between tumor and control breast tissues was also observed: in approximately two thirds of carcinomas, lowered CPI activity was measured (group-I patients), while similar or higher tumor CPI activity was measured in the remaining samples (group-II patients). Relative increases in specific activity of Cat B and Cat L in group I were significantly higher than in group II. In group I more patients with histopathological tumor grade III and negative estrogen (ER) and progesterone receptor (PR) levels were found, but the metastatic involvement of regional lymph nodes was similar in both groups. A 2-year follow-up study showed a significant inverse correlation between disease-free survival and increased Cat L activity, but the differences in group I and group II patients were not significant in this short time interval. In 20 matched pairs of breast carcinoma and normal breast tissue, the mean activity of Cat D was 5.8-fold higher in tumors compared with controls. The hypothesis that elevated Cat D activity increased CP activity and/or lowered tumor CPI activity due to post-translational proteolytic modification appeared less likely, since no correlations between corresponding activities were observed. We suggested that lowered CPI might rather reflect changes in transcription of intracellular CPIs, the stefins. Immunoassay and Northern blot analysis showed that the average value of stefin A protein and mRNA content respectively in the majority of investigated breast carcinoma samples were lowered, suggesting the possible value of stefin A in diagnosis and/or prognosis of the disease.