RESUMO
The functional changes, associated with the sequential transformation of L'4 into L4 pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) were studied. L'4 enzyme from human erythrocytes shows strong hysteretic behaviour: the initial rate of the enzyme preincubated with an unsaturating concentration of phosphoenolpyruvate is much higher than of the enzyme preincubated with ADP, at the same phosphoenolpyruvate concentration, although the "final activity" (the activity of the linear part of the reaction progress curve) was the same in both cases. This phenomenon was observed both in the presence and absence of fructose 1,6-diphosphate. High concentrations of both Mg2+free and MgATP2- diminish the difference in initial rate, between the ADP and phosphoenolpyruvate preincubated enzymes: Mg2+free by stabilizing the phosphoenolpyruvate-induced form; ATPMg2- by stabilizing the ADP-induced form. The magnitude of the difference in initial rates of the ADP-or phosphoenolpyruvate-preincubated enzyme is a function of both substrates. L4 pyruvate kinase (either from human liver or trypsin treated L'4 enzyme) does not, or to a very slight extent, show such behaviour. L'2L2 pyruvate kinase shows behaviour intermediate between L'4 and L4 enzymes. A model is proposed to describe the kinetic behaviour of L'4 and L4 enzymes.
Assuntos
Precursores Enzimáticos/sangue , Eritrócitos/enzimologia , Piruvato Quinase/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Frutosedifosfatos/metabolismo , Humanos , Cinética , Magnésio/farmacologia , Fosfoenolpiruvato/farmacologia , Piruvato Quinase/sangueRESUMO
In human endothelial cell conditioned medium a fast-acting inhibitor of tissue-type plasminogen activator and urokinase has been detected. Moreover, an inactive inhibitor of these plasminogen activators is present, that can be activated by denaturing agents such as sodium dodecyl sulphate (SDS). The mutual relationship between these inhibitors was studied. The fast-acting plasminogen activator inhibitor from human endothelial cell conditioned medium was purified in a complex with tissue-type plasminogen activator by immune adsorption, using an immobilized anti-tissue-type plasminogen activator antibody. With the complex as an antigen, specific antibodies were raised against this inhibitor in rabbits. The antiserum immunoreacted with both the inactive and the fast-acting plasminogen activator inhibitor. Endothelial cell conditioned medium (containing the inactive plasminogen activator inhibitor) was treated with SDS and the inhibitory activity that emerged was purified. The SDS-generated product formed complexes with tissue-type plasminogen activator with the same molecular mass as those formed with the fast-acting inhibitor. Moreover, the inhibitory activity generated by SDS treatment showed the same kinetic behaviour with tissue-type plasminogen activator as did the fast-acting inhibitor. These data show that the fast-acting and the inactive plasminogen activator inhibitor are immunologically and functionally related to each other, and probably represent different molecular forms of the same protein.
Assuntos
Glicoproteínas/análise , Especificidade de Anticorpos , Células Cultivadas , Reações Cruzadas , Meios de Cultura/análise , Endotélio/análise , Humanos , Imunodifusão , Focalização Isoelétrica , Peso Molecular , Inativadores de Plasminogênio , Dodecilsulfato de Sódio/farmacologia , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidoresRESUMO
Rat brain mitochondrial hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was solubilized by treatment of the mitochondria with glucose 6-phosphate and partly purified. The solubilized enzyme was compared with the cytosolic enzyme fraction. The solubilized and cytosolic enzymes were also compared with the enzyme bound to the mitochondrial membrane. The following observations were made. 1. There is no difference in electrophoretic mobility on cellulose-acetate between the cytosolic and the solubilized enzyme. Both fractions are hexokinase isoenzyme I. 2. There is no difference in kinetic parameters between the cytosolic or solubilized enzymes (P less than 0.001). For the cytosolic enzyme Km for glucose was 0.067 mM (S.E. = 0.024, n = 7); Km for MgATP2- was 0.42 mM (S.E. = 0.13, n = 7) and Ki,app for glucose 1,6-diphosphate was 0.084 mM (S.E. = 0.011, n = 5). For the solubilized enzyme Km for glucose was 0.071 mM (S.E. = 0.021, n = 6); Km for MgATP2- was 0.38 mM (S.E. = 0.11, n = 6) and Ki,app for glucose 1,6-diphosphate was 0.074 mM (S.E. = 0.010, n = 5). However when bound to the mitochondrial membrane, the enzyme has higher affinities for its substrates and a lower affinity for the inhibitor glucose 1,6-diphosphate. For the mitochondrial fraction Km for glucose was 0.045 mM (S.E. = 0.013, n = 7); Km for MgATP2- was 0.13 mM (S.E. = 0.02, n = 7) and Ki,app for glucose 1,6-diphosphate was 0.33 mM (S.E. = 0.03, n = 5). 3. The cytosolic and solubilized enzyme could be (re)-bound to depleted mitochondria to the same extent and with the same affinity. Limited proteolysis fully destroyed the enzyme's ability to bind to depleted mitochondria. 4. Our data support the hypothesis that soluble- and solubilizable enzyme from rat brain are one and the same enzyme, and that there is a simple equilibrium between the enzyme in these two pools.
Assuntos
Encéfalo/enzimologia , Hexoquinase/metabolismo , Mitocôndrias/enzimologia , Animais , Citosol/enzimologia , Eletroforese em Acetato de Celulose , Hexoquinase/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Masculino , Ratos , Ratos EndogâmicosRESUMO
Pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from human liver and red cells has been purified to homogeneity; its subunit structure and some of its kinetic characteristics have been studied. The influence of a partial proteolysis by trypsin on the subunit structure, the isozymic pattern and the kinetic characteristics of red cell and liver enzyme have been investigated. From the results of this study we may conclude that: 1. Liver (L-type) pyruvate kinase is composed of 4 identical L subunits while the major form of erythrocyte enzyme (PK-R2) is a heterotetramer designated as L2L2', the molecular weight of L' being slightly higher than that of L subunits (63 000 and 58 000 respectively). Pyruvate kinase PK-R1, predominant in the erythroblasts and the young red cells, is composed of four identical L' subunits. 2. A mild tryptic attack is able to transform PK-R1 into PK-R2, then PK-R2 into pyruvate kinase L (PK-L). The same proteolytic treatment transforms the L' subunits into L ones. 3. Consequently L-type pyruvate kinase seems to be initially synthesized in the erythroid precursors as an L4' enzyme secondarily partially proteolysed into L2L2'. In liver a very active proteolytic system would be responsible for the total transformation into L4 pyruvate kinase. 4. L4' enzyme exhibits Michaelis-Menten kinetic behaviour with an apparent Michaelis constant of 3.8 mM whereas L4 enzyme shows both positive and negative homotropic interactions towards phosphoenolpyruvate and has [S] 0.5 of 1.2 mM. The characteristics of L2L2' are roughly intermediate between those of L4' and of L4. Fructose 1,6-biphosphate decreases [S]0.5 for these three pyruvate kinase forms without suppressing the differences in the apparent affinity for phosphoenolpyruvate of these enzymes. 5. L4 pyruvate kinase is more inhibited by Mg-ATP than L4', with L2L2' in the intermediate range. 6. Tryptic treatment of each enzyme form studied transforms its kinetic behaviour into that observed for L4.
Assuntos
Eritrócitos/enzimologia , Isoenzimas/genética , Fígado/enzimologia , Piruvato Quinase/genética , Frutosedifosfatos/farmacologia , Humanos , Imunoeletroforese , Cinética , Substâncias Macromoleculares , Peso Molecular , Especificidade de ÓrgãosRESUMO
The phospholipases A2, C and D have been used to investigate the localization of phosphatidylcholine in the phosphatidylcholine exchange protein from beef liver. The rate of enzymatic hydrolysis of the protein-bound phosphatidylcholine was found to be very low. Addition of deoxycholate, isobutanol or dioxane to the native protein, under conditions where delipidation did not occur, greatly enhanced the hydrolytic action of the phospholipases. From these results it is concluded that phosphatidylcholine may be buried in the protein molecule.
Assuntos
Fígado/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolipases/farmacologia , Proteínas/metabolismo , Receptores de Droga/efeitos dos fármacos , Animais , Transporte Biológico Ativo , Butanóis/farmacologia , Bovinos , Ácido Desoxicólico/farmacologia , Dioxanos/farmacologia , Cinética , Fígado/efeitos dos fármacos , Ligação ProteicaRESUMO
In human umbilical artery and vein endothelial cell conditioned medium fibrinolytic inhibitors have been detected by two different techniques. A fast-acting inhibitor of tissue-type plasminogen activator (t-PA) and urokinase has been detected and quantified by its capacity to neutralize the above-mentioned plasminogen activators in a kinetic assay. By reverse fibrin autography after SDS-polyacrylamide gel electrophoresis a fibrinolytic inhibitor can be detected with a molecular mass of 52 kDa. The mutual relationship between these two inhibitors was studied. Neutralization of the fast-acting inhibitor by t-PA results in the formation of a complex with a molecular mass of 100 kDa. The t-PA added to endothelial cell conditioned medium in excess of the fast-acting inhibitor is fully stable. However, the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography is not affected by complete neutralization of the fast-acting inhibitor, and removal of the formed complexes by immune adsorption with immobilized anti-t-PA IgG. This suggests that the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography does not react with t-PA. Moreover, endothelial cell conditioned medium that is depleted of the fast-acting inhibitor does not show lysis resistance when directly applied to the reverse fibrin autography indicator gel (without previous electrophoresis), although the inhibitor is still present in the zymogram after SDS-polyacrylamide gel electrophoresis. This suggests that the inhibitor is induced by the SDS treatment. Heating the endothelial cell conditioned medium for 15 min at 70 degrees C fully destroys the fast-acting inhibitory activity, but leaves the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography unaffected. Moreover, at least one additional fibrinolytic inhibitor is detected in the zymogram after SDS-polyacrylamide gel electrophoresis. We conclude that the fast-acting inhibitor is not the same as the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography; the latter inhibitor is not operational in endothelial cell conditioned medium, but is induced by SDS-polyacrylamide gel electrophoresis.
Assuntos
Fibrinólise , Ativadores de Plasminogênio/análise , Artérias Umbilicais/citologia , Veias Umbilicais/citologia , Ativador de Plasminogênio Tipo Uroquinase/análise , Células Cultivadas , Meios de Cultura , Endotélio/citologia , Endotélio/metabolismo , Feminino , Humanos , Cinética , Peso Molecular , Gravidez , TermodinâmicaRESUMO
K-type pyruvate kinase was purified from human kidney by immunoadsorbant chromatography. Monoclonal antibodies secreting hybridomas were made using conventional techniques. Two clones were established which produced antibodies against K-type not cross-reacting with the other pyruvate kinase isoenzymes, named the M, L and R-types. The specificity of the monoclonal antibodies was proven by enzyme-linked immunosorbent assay, immunoprecipitation and immunoblotting experiments. The M- and K-isoenzymes are produced from the same gene probably by alternative splicing, and all differences between both enzymes originate from one exon coding for 45 amino acids (Noguchi et al. J. Biol. Chem. 261, 13807-13812 (1986]. The monoclonal antibodies are specific for K-type under denaturing conditions. Thus, it is likely that these antibodies recognize (a) continuous epitope(s), of which at least some amino acids are coded in the K-specific exon. The monoclonal antibodies could be successfully used in immunohistochemical studies. Neurons and astrocytes in brain, Kupffer cells in liver, connective tissue cells and vascular smooth muscle cells showed immunoreactivity. However, striated muscle cells in skeletal muscle and heart and hepatocytes were not immunoreactive. Other types of glial cells, e.g., oligodendrocytes and microglia, so far studied, showed no reaction either.
Assuntos
Anticorpos Monoclonais/imunologia , Formação de Anticorpos , Isoenzimas/imunologia , Piruvato Quinase/imunologia , Anticorpos Monoclonais/análise , Glioma/metabolismo , Humanos , Imuno-Histoquímica , Piruvato Quinase/metabolismoRESUMO
Inhibition of tissue-type plasminogen activator (t-PA) by pooled plasma could be ascribed for only 60% to the endothelial cell type PA inhibitor. The residual inhibition is ascribed to a so-far undescribed plasma component present at 0.2 nmol/l. This component shows reversible binding to t-PA with an apparent Ki of 10 pmol/l (does not hinder t-PA binding to fibrin); also reacts with urokinase, but not with DIP-t-PA; is stable at 37 degrees C and does not occur in media of endothelial cells, hepatocytes and fibroblasts. This PA binding component in plasma adds to the regulation of plasminogen activator activities.
Assuntos
Glicoproteínas/sangue , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Temperatura Alta , Humanos , Imunoquímica , Cinética , Inativadores de Plasminogênio , Ligação Proteica , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
Current anti-HIV-1 screening tests combine an excellent anti-HIV-1 sensitivity with a sensitivity of only 28-93% for anti-HIV-2 positive plasma or serum samples. The reactivity of anti-HIV-2 sera in anti-HIV-1 screening tests is based mainly on the immunological cross-reactivity of the GAG and POL proteins of HIV-1 and HIV-2. We describe here a sandwich immunoassay, in which HIV-1 viral lysate is combined with an HIV-2 ENV synthetic peptide, corresponding to the immunodominant envelope epitope, as the coating antigens on microELISA plates. This immunoassay has a sensitivity of 100% for anti-HIV-1 (128 sera tested) and 100% for anti-HIV-2 (109 sera tested). Assay specificity with fresh human donor sera was 99.9% (2256 sera tested).
Assuntos
Anticorpos Anti-HIV/análise , HIV-1/imunologia , HIV-2/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Sensibilidade e EspecificidadeRESUMO
A polyclonal antibody raised against plasminogen activator (PA-)inhibitor from endothelial cells fully precipitates the PA-inhibitor in endothelial cell conditioned medium but only a part of the PA-inhibitory activity in blood plasma. This indicates that the PA-inhibitory activity in blood plasma is not due to a single inhibitory component. Performing the assay for PA-inhibitory activity in plasma both in the presence and absence of saturating concentrations of anti-endothelial cell PA-inhibitor antibodies, allows the determination of endothelial cell type PA-inhibitor in plasma. The assay gives a linear dose-response curve of amount of plasma added versus t-PA neutralised. Values for endothelial cell type PA-inhibitor in plasma of a group of 20 healthy individuals are in the range of 0.0-16.8 IU/ml and are not normally distributed (median value 3.0 IU/ml). This method also reveals a second, so far unidentified, PA-inhibitory component in human plasma.
Assuntos
Glicoproteínas/sangue , Testes de Neutralização/métodos , Ativadores de Plasminogênio/antagonistas & inibidores , Inativadores de Plasminogênio , Vasos Sanguíneos/análise , Endotélio/análise , Glicoproteínas/imunologia , Humanos , Ativador de Plasminogênio Tecidual/antagonistas & inibidoresRESUMO
An assay for plasminogen activator inhibitor in human platelets is described. With this assay we find an average value of 6.8 X 10(-8) IU/platelet (S.D. = 3.0 X 10(-8); n = 20) in a healthy population. We characterized the PA-inhibitor from platelets and identified it as endothelial cell type plasminogen activator inhibitor, by its immunologic and functional properties. Besides the plasma pool of plasminogen activator inhibitor with a very high turnover rate, platelets constitute a second pool of plasminogen activator inhibitor in the circulation of the same order of magnitude. The two different pools of plasminogen activator inhibitor might have a different physiologic function.
Assuntos
Plaquetas/metabolismo , Glicoproteínas/sangue , Ativadores de Plasminogênio/antagonistas & inibidores , Inativadores de Plasminogênio , Endotélio/metabolismo , Fibrinólise , HumanosRESUMO
Human foreskin microvascular endothelial cells synthesize and release tissue-type plasminogen activator (t-PA) in similar amounts as do endothelial cells from umbilical cord artery and vein. Human thrombin increases the production of t-PA by these cells, which could be visualized from 8 h after addition of 0.1-5 units/ml thrombin by fibrin autography after SDS polyacrylamide gel electrophoresis of the endothelial cell conditioned media. Thrombin also increased the secretion of t-PA antigen. Together with t-PA, human microvascular cells release urokinase-type plasminogen activator (u-PA) antigen and endothelial cell-type PA inhibitor, PA inhibitor-1, which were both demonstrated by specific immunoprecipitation from radiolabeled endothelial cell conditioned medium. Thrombin increases the release of u-PA antigen, but no u-PA activity could be demonstrated. Thrombin induced a two-fold stimulation of the synthesis and secretion of PA inhibitor-1 antigen. At 0.1 unit/ml thrombin also an increase in PA inhibitor activity was found. At high concentrations of thrombin a decrease of PA inhibitor activity was found, due to the conversion of the active 46 kD PA inhibitor-1 into a 42 kD product without PA inhibitor activity. Our data indicate that interaction of thrombin with microvascular endothelial cells will shift the balance between t-PA, u-PA and PA inhibitor-1, and thus affects the regulation of fibrinolysis.
Assuntos
Capilares/metabolismo , Glicoproteínas/biossíntese , Ativadores de Plasminogênio/biossíntese , Trombina/farmacologia , Capilares/citologia , Endotélio/citologia , Endotélio/metabolismo , Humanos , Masculino , Pênis , Inativadores de Plasminogênio , Pele/irrigação sanguíneaRESUMO
The plasminogen activator inhibitor (PAI-1) from endothelial cells is a potentially important regulator of plasminogen activator activity. Cultured human endothelial cells increase their PAI-1 production upon stimulation with LPS and TNF, agents that are known to cause an increase in PAI-1 levels in vivo. We isolated a PAI-1 cDNA probe, and by RNA hybridization analysis studied the regulation of PAI-1 mRNA synthesis in human endothelial artery cells. Freshly isolated endothelial cells do not contain detectable amounts of PAI-1 mRNA, but after adherence and incubation for 18 h in growth medium produce considerable amounts of PAI-1 activity and contain PAI-1 mRNA levels comparable to those found in subcultured cells. When subcultured endothelial cells are incubated for 6 h with LPS or TNF, both species of PAI-1 mRNA increase 10 to 20 fold, while PAI-1 activity in the growth medium increases only 1.5 to 2 fold. Stimulation of endothelial cells in the presence of cycloheximide (CHX) results in superinduction of mainly the 3.0 kb PAI-1 mRNA. The 3' end of this mRNA contains a 60 bp AT-rich sequence, that resembles 3' sequences present in a number of other genes superinducible with CHX.
Assuntos
Endotélio Vascular/citologia , Regulação da Expressão Gênica , Glicoproteínas/genética , RNA Mensageiro/genética , Sequência de Bases , Northern Blotting , Células Cultivadas , Clonagem Molecular , Meios de Cultura , Cicloeximida/farmacologia , Sondas de DNA , Eletroforese em Gel de Ágar , Humanos , Lipopolissacarídeos/farmacologia , Hibridização de Ácido Nucleico , Inativadores de Plasminogênio , RNA/isolamento & purificação , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
1. Maleic acid is shown to be able to bind the thiol compound 2-mercaptoethanol. This is fully consistent with the data of Morgan and Friedman (1938). 2. Human erythrocyte pyruvate kinase dissolved and quantitated in Tris-maleate shows a loss of positive homotropic interactions, as compared to the same preparation in Tris-HCl. Hill coefficients (n) of n = 1.0-1.2 and n = 1.6-1.8 are obtained in Tris-maleate and Tris-HCl respectively. Half saturation [S] 0.5 and Vmax remain unchanged. Pyruvate kinase in Tris-maleate is slightly more stable to heating at 60 degrees C than in Tris-HCl. Incubation of the enzyme in Tris-maleate for one h with high concentrations of dithiotreitol restores the positive homotropic interactions. 3. It is proposed, that the abnormalities of the pyruvate kinase of some patients with acquired pyruvate kinase deficiency, obtained from a study in Tris-maleate, may partly be induced by the buffer itself.
Assuntos
Eritrócitos/enzimologia , Maleatos/farmacologia , Piruvato Quinase/sangue , Humanos , Cinética , Piruvato Quinase/deficiênciaRESUMO
1. In isolated erythrocytes the ratio of reduced to oxidized glutathione was modified by the addition of diazinedicarboxylic acid bis-dimethylamide (diamide). Incubation of erythrocytes, with a decreased GSH/GSSG ratio, resulted in an increase in [S]0.5 of pyruvate kinase of phosphoenolpyruvate as measured in haemolysates. We presume this increase to be due to oxidation of the enzyme. 2. The apparent affinity of pyruvate kinase for phosphoenolpyruvate returned to normal when the GSSG formed was reduced to GSH intracellularly. Oxidation of pyruvate kinase could also be reversed by incubation of haemolysates with reducing agents such as 2-mercaptoethanol or dithioerythritol. 3. Intracellular oxidation of pyruvate kinase caused no significant changes in the Hill coefficient (n) or Vmax of the enzyme. However, the heat stability of the oxidized enzyme was lower than normal. Lability increased with increasing oxidation of the enzyme. 4. The possible role of oxidation processes in pyruvate kinase deficiency is discussed. It is concluded that not only 'in vitro' but also in the intact erythrocyte, pyruvate kinase is sensitive to oxidizing agents and intracellular redox state. However, that a decreased GSH/GSSG ratio can be a single cause of acquired pyruvate kinase deficiency seems highly improbable.
Assuntos
Eritrócitos/enzimologia , Glutationa/sangue , Piruvato Quinase/sangue , Diamida/farmacologia , Estabilidade de Medicamentos , Humanos , Cinética , Oxirredução , TemperaturaRESUMO
A new wet-state membrane characterization method, thermoporometry, was used to study the effect on membrane structure of commonly used sterilization methods for artificial kidney membranes. The porosity and pore size distribution of differently sterilized hollow fiber Hemophan hemodialysis membranes were determined. Also the effect of a glycerol treatment (before sterilization) on porosity and pore size distribution after sterilization was studied. Hemophan was found to have a pore size distribution of pores with radii between 1.5 and 12 nm. Most of the samples had a maximum pore volume at a pore radius of 2.5 nm, only the steam sterilized and non glycerol treated sample had a maximum pore volume at 1.5 nm. The porosity was found to vary between 14 and 31% and was dependent on the applied treatment.
Assuntos
Materiais Biocompatíveis , Celulose/análogos & derivados , Rins Artificiais , Membranas Artificiais , Esterilização/métodos , Calorimetria , Óxido de Etileno , Glicerol , Humanos , Porosidade , VaporRESUMO
The aim of this paper is to provide an outline of the current developments in drug discovery. However, in doing so, it is important to put these developments into an historical perspective and to acknowledge the impact that the previous research effort has had on research in progress.
Assuntos
Bloqueadores Neuromusculares/química , Química Farmacêutica , Desenho de Fármacos , Avaliação de Medicamentos , Bloqueadores Neuromusculares/farmacologia , Receptores Nicotínicos/fisiologiaRESUMO
In conditioned medium (CM) from cultured human endothelial cells, two forms of plasminogen-activator inhibitor (PA-inhibitor) can be demonstrated: a fast-acting active form and an immunologically related, inactive form. Evidence is presented that endothelial cells produce active PA-inhibitor which is rapidly inactivated upon secretion into the medium. This inactivation can, at least partly, be prevented by culturing cells with excess of tissue-type plasminogen activator (t-PA). This results in the formation of large amounts of t-PA-PA-inhibitor complex at the cost of accumulation of inactive PA-inhibitor. No complex was detectable when inactive PA-inhibitor preparations were incubated with t-PA either in the absence or in the presence of cells. Furthermore, in cell extracts, predominantly functionally active PA-inhibitor was present. PA-inhibitor derived from the t-PA-PA-inhibitor complex showed an Mr approx. 4000 lower by polyacrylamide-gel electrophoresis than that of the inactive form. The rapid inactivation seems to be confined to newly synthesized molecules, since PA-inhibitor molecules in CM are inactivated much more slowly (even with cells or cell homogenates) than necessary to explain the excessive production of inactivated PA-inhibitor by cells. It could not be prevented by inhibitors of oxidative processes, like butylated hydroxytoluene, dithiothreitol, superoxide dismutase and catalase.
Assuntos
Endotélio/metabolismo , Glicoproteínas/antagonistas & inibidores , Ativadores de Plasminogênio/antagonistas & inibidores , Inativadores de Plasminogênio , Células Cultivadas , Precipitação Química , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/imunologia , Humanos , CinéticaRESUMO
Primary cultures of human hepatocytes and the human hepatocellular cell line Hep G2 are shown to produce fast-acting inhibitors of tissue-type plasminogen activator (tPA) and urokinase. The tPA inhibitory activities in conditioned medium of these liver cell types are very similar to those present in human endothelial cell conditioned medium. They are stable at pH 2.5, have similar dissociation constants with tPA (1.5 to 5 pmol/L), and are similar in thermostability. Addition of tPA to conditioned medium of Hep G2 and endothelial cells that has been depleted of tPA and urokinase reveals a 100 kilodalton tPA-inhibitor complex. The fast-acting tPA inhibitory activity in human plasma has comparable properties, and may originate from the liver or the vascular endothelium or both. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis of conditioned medium from hepatocytes, Hep G2, and endothelial cells, additional fibrinolytic inhibition at 52 kilodaltons was visualized. This was not found with human plasma.