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1.
Oncogene ; 33(26): 3392-400, 2014 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-23955077

RESUMEN

Lysosomal cysteine cathepsins contribute to proteolytic events promoting tumor growth and metastasis. Their enzymatic activity, however, is tightly regulated by endogenous inhibitors. To investigate the role of cathepsin inhibitor stefin B (Stfb) in mammary cancer, Stfb null mice were crossed with transgenic polyoma virus middle T oncogene (PyMT) breast cancer mice. We show that ablation of Stfb resulted in reduced size of mammary tumors but did not affect their rate of metastasis. Importantly, decrease in tumor growth was correlated with an increased incidence of dead cell islands detected in tumors of Stfb-deficient mice. Ex vivo analysis of primary PyMT tumor cells revealed no significant effects of ablation of Stfb expression on proliferation, angiogenesis, migration and spontaneous cell death as compared with control cells. However, upon treatment with the lysosomotropic agent Leu-Leu-OMe, cancer cells lacking Stfb exhibited a significantly higher sensitivity to apoptosis. Moreover, Stfb-ablated tumor cells were significantly more prone to cell death under increased oxidative stress. These results indicate an in vivo role for Stfb in protecting cancer cells by promoting their resistance to oxidative stress and to apoptosis induced through the lysosomal pathway.


Asunto(s)
Apoptosis/genética , Neoplasias de la Mama/patología , Cistatina B/genética , Neoplasias Mamarias Experimentales/patología , Estrés Oxidativo/genética , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Catepsinas/antagonistas & inhibidores , Movimiento Celular/genética , Proliferación Celular , Inhibidores de Cisteína Proteinasa/genética , Dipéptidos/farmacología , Progresión de la Enfermedad , Femenino , Inmunosupresores/farmacología , Lisosomas/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Noqueados , Metástasis de la Neoplasia/genética , Neovascularización Patológica/genética
2.
Cell Death Differ ; 9(8): 807-17, 2002 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-12107824

RESUMEN

Several receptors that mediate apoptosis have been identified, such as Fas and tumor necrosis factor receptor I. Studies of the signal transduction pathways utilized by these receptors have played an important role in the understanding of apoptosis. Here we report the first ligand-receptor pair-the neuropeptide substance P and its receptor, neurokinin-1 receptor (NK(1)R)-that mediates an alternative, non-apoptotic form of programmed cell death. This pair is widely distributed in the central and peripheral nervous systems, and has been implicated in pain mediation and depression, among other effects. Here we demonstrate that substance P induces a non-apoptotic form of programmed cell death in hippocampal, striatal, and cortical neurons. This cell death requires gene expression, displays a non-apoptotic morphology, and is independent of caspase activation. The same form of cell death is induced by substance P in NK(1)R-transfected human embryonic kidney cells. These results argue that NK(1)R activates a death pathway different than apoptosis, and provide a signal transduction system by which to study an alternative, non-apoptotic cell death program.


Asunto(s)
Apoptosis/fisiología , Células Epiteliales/metabolismo , Riñón/metabolismo , Neuronas/metabolismo , Prosencéfalo/metabolismo , Receptores de Neuroquinina-1/metabolismo , Sustancia P/metabolismo , Triptófano/análogos & derivados , Animales , Anexina A5/metabolismo , Inhibidores de Caspasas , Caspasas/genética , Caspasas/metabolismo , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/ultraestructura , Feto , Humanos , Inmunohistoquímica , Riñón/ultraestructura , Microscopía Electrónica , Antagonistas del Receptor de Neuroquinina-1 , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Piperidinas/farmacología , Prosencéfalo/ultraestructura , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Ratas Sprague-Dawley , Sustancia P/farmacología , Triptófano/farmacología
3.
Curr Pharm Des ; 7(12): 1143-56, 2001 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-11472258

RESUMEN

Trypanosoma cruzi, the causative agent of the American Trypanosomiasis, Chagas disease, contains a major cysteine proteinase (CP), cruzipain (also known as cruzain, or GP57/51). The enzyme is a member of the papain C1 family of CPs, with a specificity intermediate between those of cathepsin L and cathepsin B. The enzyme, which is expressed at different levels by different parasite stages, is encoded by a high number of genes (up to 130 in the Tul2 strain), which code for a pre-pro-enzyme. Mature cruzipain consists of a catalytic moiety with high homology to cathepsins S and L, and a C-terminal domain, characteristic of Type I CPs of Trypanosomatids, and absent in all other C1 family CPs described so far. Irreversible inhibitors of cruzipain (peptidyl diazomethylketones, peptidyl fluoromethylketones, peptidyl vinyl sulphones) are able to block the differentiation steps in the parasite's life cycle, and effectively kill the organism. Recently, a vinyl sulphone derivative (N-piperazine-Phe-hPhe-vinyl sulphone phenyl) which is an efficient inhibitor of cruzipain and kills T. cruzi by inducing an accumulation of unprocessed cruzipain in the Golgi cisternae, interfering with the secretory pathway, has been tested in vivo in a mice model (J.H. McKerrow et al.). The curative effects observed, as well as the good bioavailability of the inhibitor and its apparent lack of undesirable side effects, make it a promising lead compound for the development of new drugs for the chemotherapy of Chagas disease.


Asunto(s)
Antiprotozoarios/farmacología , Enfermedad de Chagas/tratamiento farmacológico , Proteínas Protozoarias/antagonistas & inhibidores , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Diseño de Fármacos , Humanos , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo
4.
J Biol Chem ; 276(5): 3149-57, 2001 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-11073962

RESUMEN

We investigated the mechanism of lysosome-mediated cell death using purified recombinant pro-apoptotic proteins, and cell-free extracts from the human neuronal progenitor cell line NT2. Potential effectors were either isolated lysosomes or purified lysosomal proteases. Purified lysosomal cathepsins B, H, K, L, S, and X or an extract of mouse lysosomes did not directly activate either recombinant caspase zymogens or caspase zymogens present in an NT2 cytosolic extract to any significant extent. In contrast, a cathepsin L-related protease from the protozoan parasite Trypanosoma cruzi, cruzipain, showed a measurable caspase activation rate. This demonstrated that members of the papain family can directly activate caspases but that mammalian lysosomal members of this family may have been negatively selected for caspase activation to prevent inappropriate induction of apoptosis. Given the lack of evidence for a direct role in caspase activation by lysosomal proteases, we hypothesized that an indirect mode of caspase activation may involve the Bcl-2 family member Bid. In support of this, Bid was cleaved in the presence of lysosomal extracts, at a site six residues downstream from that seen for pathways involving capase 8. Incubation of mitochondria with Bid that had been cleaved by lysosomal extracts resulted in cytochrome c release. Thus, cleavage of Bid may represent a mechanism by which proteases that have leaked from the lysosomes can precipitate cytochrome c release and subsequent caspase activation. This is supported by the finding that cytosolic extracts from mice ablated in the bid gene are impaired in the ability to release cytochrome c in response to lysosome extracts. Together these data suggest that Bid represents a sensor that allows cells to initiate apoptosis in response to widespread adventitious proteolysis.


Asunto(s)
Apoptosis/fisiología , Endopeptidasas/fisiología , Lisosomas/enzimología , Animales , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Caspasa 3 , Caspasa 7 , Caspasas/metabolismo , Citosol/metabolismo , Humanos , Ratones , Modelos Moleculares , Ratas , Células Tumorales Cultivadas
5.
FEBS Lett ; 469(1): 29-32, 2000 Mar 03.
Artículo en Inglés | MEDLINE | ID: mdl-10708750

RESUMEN

Unlike mammalian lysosomal cysteine proteases, the trypanosomal cysteine protease cruzipain contains a 130-amino acid residue C-terminal domain, in addition to the catalytic domain, and it is stable at neutral pH. The endogenous (with C-terminal domain) and recombinant (without C-terminal domain) cruzipains exhibit similar stabilities at both acid (k(inac)=3.1x10(-3) s(-1) and 4.4x10(-3) s(-1) at pH 2.75 for endogenous and recombinant cruzipain, respectively) and alkaline pH (k(inac)=3.0x10(-3) s(-1) and 3. 7x10(-3) s(-1) at pH 9.15 for endogenous and recombinant cruzipain, respectively). The pH-induced inactivation, which is a highly pH dependent first order process, is irreversible and accompanied by significant changes of secondary and tertiary structure as revealed by circular dichroism measurements. The different stability of cruzipain as compared to related proteases, is therefore due mainly to the different number, nature and distribution of charged residues within the catalytic domain and not due to addition of the C-terminal domain.


Asunto(s)
Cisteína Endopeptidasas/química , Trypanosoma cruzi/enzimología , Animales , Sitios de Unión , Dicroismo Circular , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Protozoarias , Proteínas Recombinantes/química , Electricidad Estática
6.
Biol Chem ; 380(5): 589-92, 1999 May.
Artículo en Inglés | MEDLINE | ID: mdl-10384966

RESUMEN

Cathepsin S has been isolated for the first time from human tissue. It has a molecular mass of 24 kDa and an isoelectric point in the range of 8.2 to 8.6. The enzyme is inhibited by equistatin, which belongs to the thyropins, a new family of protein inhibitors, with an inhibition constant of Ki = 0.40 +/- 0.07 nM. Cruzipain, a cathepsin L-like enzyme sharing a 130 amino acid long C-terminal extension, is also strongly inhibited by equistatin (Ki = 0.028 +/- 0.006 nM). Together with previously reported data, these results further indicate that a functional heterogeneity exists among thyropin inhibitors, as demonstrated by their interaction with cathepsin S and cruzipain.


Asunto(s)
Catepsinas/antagonistas & inhibidores , Cisteína Endopeptidasas/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas/farmacología , Anémonas de Mar/química , Animales , Cromatografía por Intercambio Iónico , Inhibidores de Cisteína Proteinasa/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Humanos , Focalización Isoeléctrica , Cinética , Proteínas/aislamiento & purificación , Proteínas Protozoarias
7.
FEBS Lett ; 429(2): 129-33, 1998 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-9650575

RESUMEN

Endogenous and recombinant cruzipain, the major cysteine proteinase from the protozoan parasite Trypanosoma cruzi, exhibit differences in the protein and circular dichroism spectra probably attributed to the absence of the C-terminal domain in the recombinant enzyme. Substrate hydrolysis of both molecules at 25 degrees C and neutral pH obeyed Michaelis-Menten kinetics whereas significant substrate inhibition was observed above neutral pH. The results suggest that substrate inhibition of cruzipain is pH-dependent, and that the C-terminal domain does not play an essential role in this process.


Asunto(s)
Cisteína Endopeptidasas/metabolismo , Trypanosoma cruzi/enzimología , Animales , Sitios de Unión , Dicroismo Circular , Cisteína Endopeptidasas/química , Inhibidores de Cisteína Proteinasa , Relación Dosis-Respuesta a Droga , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Oligopéptidos/metabolismo , Conformación Proteica , Proteínas Protozoarias , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato
8.
Biol Chem ; 378(1): 1-10, 1997 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-9049059

RESUMEN

Trypanosoma cruzi, the parasitic protozoan which causes the American Trypanosomiasis, Chagas disease, contains a major cysteine proteinase (CP), cruzipain. The enzyme belongs to the papain family, but contains, as other CPs from Trypanosomatids, an unusual C-terminal extension. This C-terminal domain contains a number of post-translational modifications and is responsible for the immunodominant antigenic character of cruzipain in natural human infections. In addition, this domain is probably the cause of most of the microheterogeneities found in natural cruzipain. Irreversible inhibitors of CPs are able to block the parasite's life cycle at the differentiation steps, suggesting an essential role for CPs for parasite survival, and opening up possibilities of developing new chemotherapeutic agents against Chagas disease based on specific cruzipain inhibitors.


Asunto(s)
Cisteína Endopeptidasas , Trypanosoma cruzi/enzimología , Secuencia de Aminoácidos , Animales , Catálisis , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/inmunología , Cisteína Endopeptidasas/metabolismo , Humanos , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Protozoarias
9.
FEBS Lett ; 401(2-3): 259-61, 1997 Jan 20.
Artículo en Inglés | MEDLINE | ID: mdl-9013899

RESUMEN

A peptide fragment derived from the p41 form of the invariant chain (Ii) associated with the major histocompatibility complex (MHC) class II molecule has been shown to inhibit the mammalian lysosomal cysteine proteinase, cathepsin L, and to be a novel cysteine proteinase inhibitor, distinct from cystatins. Here we report that this same fragment also binds to and inhibits cruzipain, the cathepsin L-like enzyme from the protozoan parasite Trypanosoma cruzi. The binding of the Ii fragment to cruzipain is fast (k(ass) = 2.4 x 10(7) M(-1) s(-1) and tight (Ki = 5.8 x 10(-11) M). The inhibition is competitive. These results suggest the possibility of using the invariant chain as a model for the specific inhibition of cruzipain in vivo, i.e. as a potential drug to combat Chagas' disease.


Asunto(s)
Antígenos de Diferenciación de Linfocitos B/farmacología , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Antígenos de Histocompatibilidad Clase II/farmacología , Trypanosoma cruzi/enzimología , Empalme Alternativo , Animales , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Histocompatibilidad Clase II/genética , Cinética , Fragmentos de Péptidos/farmacología , Proteínas Protozoarias
10.
FEBS Lett ; 391(1-2): 109-12, 1996 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-8706894

RESUMEN

Fluorescence titrations showed that high-molecular-weight kininogen binds two molecules of papain, cruzipain and cathepsin S with high affinity. The 2:1 binding stoichiometry was confirmed by stopped-flow kinetic measurements of papain binding, which also revealed that the two sites bind the enzyme with different association rate constants (kass,1 = 23.0 x 10(6) M-1 s-1 and kass,2 = 3.4 x 10(6) M-1 s-1). As for low-molecular-weight kininogen, comparison of these kinetic constants with previous data for intact low- and high-molecular-weight kininogen and the separated domains indicated that the faster-binding site is also the tighter-binding site and is that of domain 3, whereas the slower-binding, lower-affinity site is on domain 2. The results further demonstrate that there is no appreciable steric interference between the two domains or by the kininogen light chain in the binding of proteinases. Similarly, the binding of kininogen via its light chain to a surface, as indicated by the binding to the model surface, heparin, did not affect the inhibitory properties of kininogen. The M(r) of high-molecular-weight kininogen was determined to be 83,500 by sedimentation equilibrium measurements, in agreement with the value calculated from amino acid sequence and carbohydrate analysis.


Asunto(s)
Cisteína Endopeptidasas/metabolismo , Quininógenos/sangre , Animales , Sitios de Unión , Catepsinas/metabolismo , Bovinos , Pollos , Humanos , Cinética , Quininógenos/aislamiento & purificación , Papaína/metabolismo , Unión Proteica , Proteínas Protozoarias , Espectrometría de Fluorescencia
11.
J Exp Med ; 183(4): 1331-8, 1996 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-8666891

RESUMEN

The invariant chain (Ii) is associated with major histocompatibility complex class II molecules during early stages of their intracellular transport. In an acidic endosomal/lysosomal compartment, it is proteolytically cleaved and removed from class II heterodimers. Participation of aspartic and cysteine proteases has been observed in in vitro degradation of Ii, but the specific enzymes responsible for its in vivo processing are as yet undefined. We have previously isolated a noncovalent complex of the lysosomal cysteine protease cathepsin L with a peptide fragment derived from the p41 form of Ii from human kidney. Here we show that this Ii fragment, which is identical to the alternatively spliced segment of p41, is a very potent competitive inhibitor of cathepsin L (equilibrium inhibition constant Ki = 1.7 X 10(-12) M). It inhibits two other cysteine proteases, cathepsin H and papain, but to much lesser extent. Cysteine proteases cathepsins B, C, and S, as well as representatives of serine, aspartic, and metalloproteases, are not inhibited at all. These findings suggest a novel role for p41 in the regulation of various proteolytic activities during antigen processing and presentation. The Ii inhibitory fragment shows no sequence homology with the known cysteine protease inhibitors, and may, therefore, represent a new class.


Asunto(s)
Antígenos de Diferenciación de Linfocitos B/farmacología , Catepsinas/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/farmacología , Endopeptidasas , Antígenos de Histocompatibilidad Clase II/farmacología , Riñón/química , Lisosomas/enzimología , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Antígenos de Diferenciación de Linfocitos B/aislamiento & purificación , Antígenos de Diferenciación de Linfocitos B/metabolismo , Catepsina L , Catepsinas/metabolismo , Cisteína Endopeptidasas , Inhibidores de Cisteína Proteinasa/metabolismo , Antígenos de Histocompatibilidad Clase II/aislamiento & purificación , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Unión Proteica
12.
Protein Sci ; 4(9): 1874-80, 1995 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-8528085

RESUMEN

Human low-molecular-weight kininogen (LK) was shown by fluorescence titration to bind two molecules of cathepsins L and S and papain with high affinity. By contrast, binding of a second molecule of cathepsin H was much weaker. The 2:1 binding stoichiometry was confirmed by titration monitored by loss of enzyme activity and by sedimentation velocity experiments. The kinetics of binding of cathepsins L and S and papain showed the two proteinase binding sites to have association rate constants kass,1 = 10.7-24.5 x 10(6) M-1 s-1 and kass,2 = 0.83-1.4 x 10(6) M-1 s-1. Comparison of these kinetic constants with previous data for intact LK and its separated domains indicate that the faster-binding site is also the tighter-binding site and is present on domain 3, whereas the slower-binding, lower-affinity site is on domain 2. These results also indicate that there is no appreciable steric hindrance for the binding of proteinases between the two binding sites or from the kininogen light chain.


Asunto(s)
Cisteína Endopeptidasas/metabolismo , Endopeptidasas , Quininógenos/metabolismo , Secuencia de Aminoácidos , Animales , Catepsina H , Catepsina L , Catepsinas/metabolismo , Bovinos , Pollos , Humanos , Cinética , Quininógenos/química , Datos de Secuencia Molecular , Peso Molecular , Papaína/metabolismo , Espectrometría de Fluorescencia , Ultracentrifugación
13.
FEBS Lett ; 370(1-2): 101-4, 1995 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-7649285

RESUMEN

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi epimastigotes, purified to a sequentially pure form, exists in multiple forms with pI values between 3.7 and 5.1, and an apparent molecular mass of 41 kDa. The enzyme is stable between pH 4.5-9.5. Cruzipain was found to be rapidly and tightly inhibited by various protein inhibitors of the cystatin superfamily (kass = 1.7-79 x 10(6) M-1s-1, Kd = 1.4-72 pM). These results suggest a possible defensive role for the host's cystatins after parasite infection, and may be of use for the design of new therapeutic drugs.


Asunto(s)
Cistatinas/farmacología , Cisteína Endopeptidasas/metabolismo , Trypanosoma cruzi/enzimología , Animales , Pollos , Cistatina A , Cisteína Endopeptidasas/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Humanos , Focalización Isoeléctrica , Cinética , Peso Molecular , Proteínas Protozoarias , Proteínas Recombinantes/farmacología
14.
Biol Chem Hoppe Seyler ; 376(4): 225-30, 1995 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-7626231

RESUMEN

The kinetics of pH-induced inactivation of human cathepsins B and L was studied by conventional and stopped-flow methods. The inactivation of both enzymes was found to be an irreversible, first-order process. The inactivation rate constants increased exponentially with pH for both enzymes. From log kinac vs pH plots, 3.0 and 1.7 protons were calculated to be desorbed for pH-induced inactivation of cathepsins L and B. Cathepsin B was thus substantially more stable than cathepsin L (approximately 15-fold at pH 7.0 and 37 degrees C). Cathepsin B was efficiently inhibited by cystatin C at pH 7.4, whereas the inhibition by stefin B and high molecular weight kininogen was only moderate. In contrast, cathepsin L was efficiently inhibited by both chicken cystatin and stefin B at this pH kass approximately 3.3 x 10(7) m-1 s-1).


Asunto(s)
Cistatinas/fisiología , Cisteína Endopeptidasas/metabolismo , Endopeptidasas , Lisosomas/enzimología , Animales , Catepsina B/antagonistas & inhibidores , Catepsina L , Catepsinas/antagonistas & inhibidores , Pollos , Humanos , Concentración de Iones de Hidrógeno , Cinética
15.
FEBS Lett ; 360(2): 101-5, 1995 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-7875311

RESUMEN

For the first time, three different stefins, A, B and C, have been isolated from a single species. The complete amino acid sequence of bovine stefin A was determined. The inhibitor, with a calculated M(r) of 11,123, consists of 98 amino acid residues. Although it exhibits considerable similarity to human and rat stefin A, some significant differences in inhibition kinetics were found. Bovine stefin A bound tightly and rapidly to cathepsin L (kass = 9.6 x 10(6) M-1.s-1, Ki = 29 pM). The binding to cathepsin H was also rapid (kass = 2.1 x 10(6) M-1.s-1), but weaker (Ki = 0.4 nM) due to a higher dissociation rate constant. In contrast, the binding to cathepsin B was much slower (kass = 1.4 x 10(5) M-1.s-1), but still tight (Ki = 1.9 nM).


Asunto(s)
Cistatinas/aislamiento & purificación , Inhibidores de Cisteína Proteinasa/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Catepsinas/antagonistas & inhibidores , Bovinos , Cistatina A , Cinética , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Piel/enzimología
16.
FEBS Lett ; 339(1-2): 155-9, 1994 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-8313966

RESUMEN

The kinetics of the complex formation between bovine cathepsin S and bovine stefin B was studied by conventional and stopped-flow techniques. The inhibition at low inhibitor concentrations was tight and reversible (kass = 5.8 x 10(7) M-1.s-1, kdiss = 4.9 x 10(-4) s-1 at pH 6.0 and 25 degrees C), whereas at higher inhibitor concentrations it was pseudo-irreversible (kass = 6.14 x 10(7) M-1.s-1). The complex was formed directly lacking the fast pre-equilibrium step with the dissociation equilibrium constant of approximately 8 pM. The competitive nature of inhibition was confirmed. The kass was found to be pH-independent between pH 6.0 and 7.5 and decreased at lower or higher pH values in a way that strongly suggests involvement of two ionizable groups in the interaction (pKi = 5.2, pK2 = 8.3). The enzyme-substrate interaction seems to be influenced by different ionizable groups (pKi = 4.4, pK2 = 7.8).


Asunto(s)
Catepsinas/antagonistas & inhibidores , Cistatinas/farmacología , Animales , Unión Competitiva , Catepsinas/metabolismo , Bovinos , Cistatina B , Cistatinas/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Espectrometría de Fluorescencia , Temperatura
17.
FEBS Lett ; 336(2): 289-92, 1993 Dec 27.
Artículo en Inglés | MEDLINE | ID: mdl-8262248

RESUMEN

A new stefin type low-M(r) cysteine proteinase inhibitor (PLCPI) was isolated from pig polymorphonuclear leukocytes as a contaminant of the cathelin sample. The inhibitor consists of 103 amino acids, and its M(r) was calculated to be 11,768. The inhibitor exhibits considerable sequence identity with inhibitors from the stefin family, particularly with human stefin A. The PLCPI is a fast acting inhibitor of papain and cathepsins L and S (k(ass) > or = 1 x 10(6) M-1 x s-1) and forms very tight complexes with these enzymes (Ki < or = 190 pM). The affinity for cathepsins B and H (Ki > or = 125 nM) was lower. These results also show that the inhibitory activity previously ascribed to cathelin was due to the presence of PLCPI.


Asunto(s)
Cistatinas/clasificación , Inhibidores de Cisteína Proteinasa/aislamiento & purificación , Leucocitos Mononucleares/química , Proteínas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Catepsinas/antagonistas & inhibidores , Cistatina B , Inhibidores de Cisteína Proteinasa/clasificación , Inhibidores de Cisteína Proteinasa/farmacología , Humanos , Cinética , Datos de Secuencia Molecular , Papaína/antagonistas & inhibidores , Proteínas/clasificación , Proteínas/farmacología , Homología de Secuencia de Aminoácido , Porcinos
18.
FEBS Lett ; 334(3): 340-2, 1993 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-8243643

RESUMEN

Since peptidyl diazomethyl ketones are useful irreversible inhibitors for inactivating cysteinyl proteinases in vitro and in vivo and in order to reveal their role, we set out to obtain selective and effective reagents for cathepsin S. A number of such derivatives with hydrophobic amino acid residues, such as valine, leucine and tryptophane in positions adjacent to the primary specificity site were synthesized and these provided inhibitors rapidly acting at high dilution. For example, 1 nM Z-Leu-Leu-Nle-CHN2 inactivates cathepsin S with k2nd = 4.6 x 10(6) M-1 x s-1 at pH 6.5, 25 degrees C. Similarities to the specificities of cathepsin L and calpain were evident. However, Z-Val-Val-NleCHN2 is over 300 times more effective in inactivating S than L. On the other hand, Z-Phe-Tyr(t-Bu)CHN2 is about 10(4) more effective against L than S. Reagents are thus now available for a clear discrimination between these proteases.


Asunto(s)
Calpaína/antagonistas & inhibidores , Catepsinas/antagonistas & inhibidores , Diazometano/análogos & derivados , Endopeptidasas , Cetonas/farmacología , Secuencia de Aminoácidos , Catepsina L , Cisteína Endopeptidasas , Diazometano/farmacología , Datos de Secuencia Molecular
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