RESUMO
AIM: Duchenne muscular dystrophy (DMD) is an X-linked myopathy affecting one in 3600-6000 live male births. The aim of this study was to gain further insight into how parents experience caring for boys with DMD. METHODS: Using a qualitative design, parents in 12 families with boys with DMD, whereof two pairs of siblings, were interviewed in 2014 and 2015. Participants were parents of boys followed at Oslo University Hospital or other hospitals in the south-eastern health region of Norway. Mean age of the boys was 13 years, range 7-17. RESULTS: Parents expressed the importance of obtaining good information about the diagnosis, supplied with sensitivity for them as clients, and the parents must be involved in timing of interventions. Meeting with others in the same situation was highly appreciated. Many of the parents expressed their own need for support to cope with the difficult situation. Continuity of support in the boys' transition to adulthood was pointed out as important, as well as the need for professional help to talk to the boys about their diagnosis. CONCLUSION: Our study suggests that professional teams should expand the parental perspective and emphasise a holistic approach in their work with patients with DMD.
Assuntos
Distrofia Muscular de Duchenne , Pais/psicologia , Adolescente , Adulto , Criança , Comunicação , Humanos , Masculino , Relações Pais-Filho , Pesquisa Qualitativa , Apoio Social , Fatores de TempoRESUMO
BACKGROUND: The importance of balanced sedation and pain treatment in intensive care units (ICUs) is evident, but regimes and use of medication differ widely. Previous surveys have focused on the use of various medications and regimes. What has not been explored is the process by which nurses and physicians assess patients' needs and work together toward a defined level of sedation and pain for the ICU patient. The purpose of the study was to determine the use of protocols and medications for sedation and analgesia in Norwegian ICUs and the degree of cooperation between nurses and physicians in using them. METHODS: A national survey was conducted in autumn 2007, using postal self-administered questionnaires. RESULTS: Written pain treatment and sedation protocols were not routinely used in Norwegian ICUs; however, half of the departments titrated sedation according to a scoring system, most commonly the Motor Activity Assessment Score. The most commonly used sedatives were propofol and midazolam, while fentanyl and morphine were the most used analgesics. The majority of respondents were concerned about the side effects of sedation and analgesics, leading to circulatory instability and delayed awakening. Nurses and physicians agreed upon the main indications for sedation: patient tolerance for ventilation, tolerance for medical and nursing interventions, and patient symptoms. CONCLUSIONS: Potential factors which may improve sedation and pain management of mechanically ventilated patients in Norwegian ICUs are more systematic assessments of pain and sedation, and the use of written protocols. Strategies which reduce side effects should be addressed.
Assuntos
Analgesia/métodos , Sedação Consciente/métodos , Respiração Artificial/métodos , Analgesia/efeitos adversos , Analgésicos Opioides/efeitos adversos , Analgésicos Opioides/uso terapêutico , Sedação Consciente/efeitos adversos , Coleta de Dados , Interpretação Estatística de Dados , Escala de Coma de Glasgow , Pesquisas sobre Atenção à Saúde , Hipnóticos e Sedativos/efeitos adversos , Hipnóticos e Sedativos/uso terapêutico , Atividade Motora/efeitos dos fármacos , Noruega , Enfermeiras e Enfermeiros , Dor/tratamento farmacológico , Dor/etiologia , Medição da Dor , Médicos , Respiração Artificial/efeitos adversos , Sono/efeitos dos fármacos , Síndrome de Abstinência a Substâncias/prevenção & controle , Inquéritos e Questionários , TraqueotomiaRESUMO
Antithrombin is a plasma protein of the serpin superfamily that may occur as several conformational variants. The native form of antithrombin is a major regulator of blood clotting. In the present study, we have identified the mechanism underlying the antiangiogenic action of a heat-denatured form, denoted latent antithrombin. Fibroblast growth factor (FGF)-induced angiogenesis in the chick embryo and angiogenesis in mouse fibrosarcoma tumors were inhibited by treatment with latent antithrombin at 1 mg/kg/day. Thermolysin-cleaved and native antithrombin were less efficient in these respects. Treatment with latent antithrombin induced apoptosis of cultured endothelial cells and inhibited cell migration toward FGF-2. Under these conditions, FGF-2-stimulated FGF receptor kinase activity was unaffected. However, actin reorganization, activation of focal adhesion kinase, and focal adhesion formation were disturbed by latent antithrombin treatment of FGF-2-stimulated endothelial cells. These data indicate that latent antithrombin induces apoptosis of endothelial cells by disrupting cell-matrix interactions through uncoupling of focal adhesion kinase.
Assuntos
Inibidores da Angiogênese/farmacologia , Antitrombinas/farmacologia , Apoptose/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Fibrossarcoma/irrigação sanguínea , Neovascularização Patológica/tratamento farmacológico , Alantoide/irrigação sanguínea , Alantoide/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Comunicação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Inibição de Migração Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Embrião de Galinha , Córion/irrigação sanguínea , Córion/efeitos dos fármacos , Fatores de Crescimento Endotelial/antagonistas & inibidores , Endotélio Vascular/citologia , Matriz Extracelular/efeitos dos fármacos , Feminino , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Fibrossarcoma/tratamento farmacológico , Fibrossarcoma/patologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Linfocinas/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/patologia , Proteínas Tirosina Quinases , Receptores Proteína Tirosina Quinases , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Binding constants for the binding of high-affinity heparin to antithrombin at different ionic strengths were determined by fluorescence titrations and were also estimated from dissociation curves of the heparin-antithrombin complex. These curves were monitored by near-ultraviolet circular dichroism or fluorescence. The dependence of the binding constant on the activity of NaCl suggested that maximally 5-6 charged groups are directly involved in the interaction between the two macromolecules. Major pH-dependent changes of the interaction, as evident by changes of the spectroscopic properties of the complex between the molecules, were found to occur below pH 5.5 and above pH 8.5. The acid change, which was irreversible, was most likely caused by an irreversible conformational change of antithrombin. At alkaline pH, however, the gross conformation of antithrombin was stable up to pH 12, while the at pH 8.5. The dissociation curve, which was reversible, had a midpoint around pH 9.5. This is compatible with the loss of affinity being caused by either a local conformational change, by ionization of tyrosine or by titration of one or more amino groups.
Assuntos
Antitrombinas , Heparina , Animais , Bovinos , Fenômenos Químicos , Química , Concentração de Íons de Hidrogênio , Concentração Osmolar , Ovinos , Espectrometria de Fluorescência , Espectrofotometria UltravioletaRESUMO
The exposure to solvent of the aromatic amino acid residues of free bovine antithrombin was probed by three different methods, which gave comparable results. Solvent perturbation suggested that about 35% of the tyrosyl residues and about 40% of the tryptophanyl residues of the protein are exposed to solvent. Fluorescence quenching experiments indicated that 60% of the tryptophanyl fluorescence of antithrombin arose from exposed residues. These values are similar to those previously reported for the human protein. In spectrophotometric titrations, two out of a total of ten tyrosyl residues titrated with a normal pKa and therefore, presumably are accessible to solvent. The binding of low-affinity of high-affinity heparin to antithrombin had neglibible effects on the solvent perturbation spectra and on the spectrophotometric titration curves. Iodide quenching experiments indicated an altered quenching pattern of the fluorescence of the protein in the presence of either form of heparin, but this effect could not be interpreted as a change of the exposure of trytophanyl residues. The binding of heparin to antithrombin therefore apparently leads to, at most, minimal changes of the exposure of the aromatic amino acids of the protein to the surrounding solvent.
Assuntos
Antitrombina III , Heparina , Triptofano/análise , Tirosina/análise , Animais , Antitrombina III/análise , Bovinos , Etilenoglicóis , Concentração de Íons de Hidrogênio , Iodetos , Conformação Proteica , Espectrometria de Fluorescência , Análise EspectralRESUMO
Recent studies have shown that the bovine cysteine proteinase inhibitor, cystatin C, is synthesized as a preprotein containing a 118-residue mature protein. However, the forms of the inhibitor isolated previously from bovine tissues had shorter N-terminal regions than expected from these results, and also lower affinity for proteinases than human cystatin C. In this work, we report the properties of recombinant, full-length bovine cystatin C having a complete N-terminal region. The general characteristics of this form of the inhibitor, as reflected by the isoelectric point, the far-ultraviolet circular dichroism spectrum, the thermal stability and the changes of tryptophan fluorescence on interaction with papain, resembled those of human cystatin C. The affinity and kinetics of inhibition of papain and cathepsins B, H and L by the bovine inhibitor were also comparable with those of the human inhibitor, although certain differences were apparent. Notably, the affinity of bovine cystatin C for cathepsin H was somewhat weaker than that of human cystatin C, and bovine cystatin C bound to cathepsin L with about a four-fold higher association rate constant than the human inhibitor. This rate constant is comparable with the highest values reported previously for cystatin-cysteine proteinase reactions. The full-length, recombinant bovine cystatin C bound appreciably more tightly to proteinases than the shorter form characterized previously. Digestion of the recombinant inhibitor with neutrophil elastase resulted in forms with truncated N-terminal regions and appreciably decreased affinity for papain, consistent with the forms of bovine cystatin C isolated previously having arisen by proteolytic cleavage of a mature, full-length inhibitor.
Assuntos
Cistatinas/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Animais , Catepsina B/antagonistas & inibidores , Catepsina H , Catepsinas/antagonistas & inibidores , Catepsinas/farmacologia , Bovinos , Dicroísmo Circular , Cistatina C , Cistatinas/química , Cistatinas/metabolismo , Cisteína Endopeptidases/farmacologia , Escherichia coli/metabolismo , Cinética , Papaína/antagonistas & inibidores , Proteínas Recombinantes/farmacologia , TemperaturaRESUMO
The N-terminal region of human cystatin C has been shown to be of crucial importance for the interaction of the inhibitor with cysteine proteinases. However, several studies have been unable to identify the corresponding region in bovine cystatin C, indicating that the binding of proteinases to the bovine inhibitor may not be dependent on this region. With the aim to resolve this apparent discrepancy and to elucidate the relation of bovine cystatin C to other cystatins, we have isolated a cDNA clone encoding bovine precystatin C. The sequence of this cDNA was similar to that of the human inhibitor and showed a putative signal peptidase cleavage site consistent with the N-terminal regions of the bovine and human inhibitors being of comparable size. This suggestion was verified by determination of the relative molecular mass of the mature bovine inhibitor isolated from cerebrospinal fluid under conditions minimising proteolysis. The N-terminal of the purified inhibitor was blocked, but the sequence of the N-terminal peptide produced by digestion with endopeptidase LysC could be unequivocally determined by tandem mass spectroscopy. Together, these results show that bovine cystatin C has 118 residues, in contrast with 110-112 residues reported previously, and has an N-terminal region analogous to that of human cystatin C. This region presumably is of similar importance for tight binding of target proteinases as in the human inhibitor.
Assuntos
Cistatinas/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Bovinos , Clonagem Molecular , Cistatina C , Cistatinas/líquido cefalorraquidiano , Cistatinas/genética , Inibidores de Cisteína Proteinase/química , Humanos , Espectrometria de Massas , Metaloendopeptidases , Dados de Sequência Molecular , Ligação Proteica , Precursores de Proteínas/química , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Cystatin B is unique among cysteine proteinase inhibitors of the cystatin superfamily in having a free Cys in the N-terminal segment of the proteinase binding region. The importance of this residue for inhibition of target proteinases was assessed by studies of the affinity and kinetics of interaction of human and bovine wild-type cystatin B and the Cys 3-to-Ser mutants of the inhibitors with papain and cathepsins L, H, and B. The wild-type forms from the two species had about the same affinity for each proteinase, binding tightly to papain and cathepsin L and more weakly to cathepsins H and B. In general, these affinities were appreciably higher than those reported earlier, perhaps because of irreversible oxidation of Cys 3 in previous work. The Cys-to-Ser mutation resulted in weaker binding of cystatin B to all four proteinases examined, the effect varying with both the proteinase and the species variant of the inhibitor. The affinities of the human inhibitor for papain and cathepsin H were decreased by threefold to fourfold and that for cathepsin B by approximately 20-fold, whereas the reductions in the affinities of the bovine inhibitor for papain and cathepsins H and B were approximately 14-fold, approximately 10-fold and approximately 300-fold, respectively. The decreases in affinity for cathepsin L could not be properly quantified but were greater than threefold. Increased dissociation rate constants were responsible for the weaker binding of both mutants to papain. By contrast, the reduced affinities for cathepsins H and B were due to decreased association rate constants. Cys 3 of both human and bovine cystatin B is thus of appreciable importance for inhibition of cysteine proteinases, in particular cathepsin B.
Assuntos
Cistatinas/química , Cistatinas/metabolismo , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/metabolismo , Cisteína/metabolismo , Animais , Bovinos , Cistatina B , Cistatinas/antagonistas & inibidores , Cistatinas/genética , Cisteína/genética , Inibidores de Cisteína Proteinase/genética , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutação , Papaína/química , Papaína/metabolismo , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , TermodinâmicaRESUMO
The three-dimensional structures of cystatins, and other evidence, suggest that the flexible N-terminal region of these inhibitors may bind to target proteinases independent of the two rigid hairpin loops forming the remainder of the inhibitory surface. In an attempt to demonstrate such two-step binding, which could not be identified in previous kinetics studies, we introduced a cysteine residue before the N-terminus of cystatin A and labeled this residue with fluorescent probes. Binding of AANS- and AEDANS-labeled cystatin A to papain resulted in approximately 4-fold and 1.2-fold increases of probe fluorescence, respectively, reflecting the interaction of the N-terminal region with the enzyme. Observed pseudo-first-order rate constants, measured by the loss of papain activity in the presence of a fluorogenic substrate, for the reaction of the enzyme with excess AANS-cystatin A increased linearly with the concentration of the latter. In contrast, pseudo-first-order rate constants, obtained from measurements of the change of probe fluorescence with either excess enzyme or labeled inhibitor, showed an identical hyperbolic dependence on the concentration of the reactant in excess. This dependence demonstrates that the binding occurs in two steps, and implies that the labeled N-terminal region of cystatin A interacts with the proteinase in the second step, subsequent to the hairpin loops. The comparable affinities and dissociation rate constants for the binding of labeled and unlabeled cystatin A to papain indicate that the label did not appreciably perturb the interaction, and that unlabeled cystatin therefore also binds in a similar two-step manner. Such independent binding of the N-terminal regions of cystatins to target proteinases after the hairpin loops may be characteristic of most cystatin-proteinase reactions.
Assuntos
Cistatinas/química , Papaína/química , Animais , Galinhas , Cistatinas/metabolismo , Cisteína/química , DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes/metabolismo , Humanos , Cinética , Papaína/metabolismo , Ligação Proteica , EspectrofotometriaRESUMO
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.
Assuntos
Cisteína Endopeptidases/metabolismo , Endopeptidases , Cininogênios/metabolismo , Sequência de Aminoácidos , Animais , Catepsina H , Catepsina L , Catepsinas/metabolismo , Bovinos , Galinhas , Humanos , Cinética , Cininogênios/química , Dados de Sequência Molecular , Peso Molecular , Papaína/metabolismo , Espectrometria de Fluorescência , UltracentrifugaçãoRESUMO
The residues contributing to the thioester bonds in bovine alpha 2-macroglobulin were differentially labelled by modification of the Glu moiety with [14C]methylamine and of the Cys moiety with iodo[3H]acetate. The labelled region was identified and analyzed in a tryptic peptide. Two amino acid replacements between human and bovine alpha 2-macroglobulin were found at positions +3 (Val/Ala) and +4 (Leu/Arg) from the Glu moiety of the thioester. Thus, marked differences exist between the human and bovine proteins in side chain size and charge close to the thioester bonds. These differences may explain the greater conformational stability of bovine alpha 2-macroglobulin, compared with that of the human inhibitor, after cleavage of the thioester bonds.
Assuntos
alfa-Macroglobulinas , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Humanos , Conformação Proteica , Especificidade da Espécie , Compostos de Sulfidrila/análise , alfa-Macroglobulinas/antagonistas & inibidoresRESUMO
Far-ultraviolet circular dichroism and tryptophan fluorescence measurements showed that the reversible unfolding of the cysteine proteinase inhibitor, chicken cystatin, by guanidinium chloride is a two-step process with transition midpoints at approximately 3.4 and approximately 5.4 M denaturant. The partially unfolded intermediate had both far- and near-ultraviolet circular dichroism and fluorescence emission spectra comparable to those of the native protein. The largely retained tertiary structure suggests that the intermediate represents a species in which a separate region of lower stability has been unfolded, rather than an intermediate of the 'molten globule' type. Such a structurally independent region is apparent in the three-dimensional structure of the inhibitor.
Assuntos
Cistatinas/química , Guanidinas/farmacologia , Animais , Galinhas , Dicroísmo Circular , Guanidina , Conformação Proteica , Desnaturação Proteica , Espectrometria de FluorescênciaRESUMO
The far-ultraviolet circular dichroism spectra of fibrinogens phosphorylated by protein kinase C or casein kinase II indicated a conformational change corresponding to an increase in ordered secondary structure. The spectra of protein kinase A- or casein kinase I-phosphorylated fibrinogens did not differ substantially from the control. Fluorescence studies indicated changes in the tertiary structure around tryptophan residues for protein kinase A- or C-phosphorylated fibrinogens, but failed to show any such change for fibrinogen phosphorylated by either of the casein kinases. This latter result was also confirmed by circular dichroism measurements in the near-ultraviolet region. The apparent increase in ordered structure was proposed as an explanation for the slower rate of plasmin degradation seen in fibrinogens after phosphorylation by protein kinase C [6], and casein kinase II, especially as both spectral changes and plasmin degradation rate were unaffected by alkaline phosphatase.
Assuntos
Fibrinogênio/química , Caseína Quinases , Dicroísmo Circular , Fibrinogênio/metabolismo , Humanos , Fosforilação , Conformação Proteica , Proteínas Quinases/metabolismo , Espectrometria de FluorescênciaRESUMO
The calpain-binding domain 2 of the kininogens, the major plasma inhibitors of cysteine proteinases, was expressed in Escherichia coli. Expression of soluble protein was optimal at 15 degrees C and was augmented by growing the bacteria in sorbitol and betaine. The recombinant domain showed high affinity (Ki 0.3-1 nM) for cathepsin L and papain, and a somewhat lower affinity (Ki approximately 15 nM) for calpain. The binding to cathepsin H was substantially weaker, and no inhibition of actinidin and cathepsin B was detected. The affinity for cathepsin L was comparable to that reported for the domain isolated from plasma L-kininogen, whereas the affinities for papain and calpain were about tenfold lower. The latter difference may be due to the recombinant domain being nonglycosylated.
Assuntos
Catepsinas/metabolismo , Cisteína Endopeptidases/metabolismo , Endopeptidases , Cininogênios/metabolismo , Papaína/metabolismo , Animais , Catepsina L , Bovinos , Escherichia coli , Humanos , Cininogênios/genética , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , OvinosRESUMO
Stopped-flow kinetics showed that the inhibition of the lysosomal cysteine proteinase, cathepsin B, by its endogenous inhibitor, cystatin C, occurs by a two-step mechanism, in which an initial, weak interaction is followed by a conformational change. The initial interaction most likely involves binding of the N-terminal region of the inhibitor to the proteinase. Considerable evidence indicates that the subsequent conformational change is due to the inhibitor displacing the occluding loop of the proteinase that partially obscures the active site. The presence of this loop, which allows the enzyme to function as an exopeptidase, thus complicates the inhibition mechanism, rendering cathepsin B much less susceptible than other cysteine proteinases to inhibition by cystatins.
Assuntos
Catepsina B/antagonistas & inibidores , Cistatinas/farmacologia , Sítios de Ligação , Catepsina B/química , Catepsina B/genética , Cistatina C , Cistatinas/genética , Cistatinas/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Cinética , Lisossomos/enzimologia , Mutagênese Sítio-Dirigida/genética , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
The first EGF-like module of human coagulation factor IX contains a single functionally important calcium ion binding site. We have now shown the dissociation constant for this site to be approximately 160 microM in a recombinant protein fragment consisting of residues 1-85 in human fIX. This represents a approximately 10-fold increase in affinity as compared with the isolated EGF module (residues 46-85). The Gla module (here with Glu instead of Gla) thus increases the affinity of the EGF module calcium ion binding site. Each of two mutations, V46E and Q50E, made to investigate whether the extra negative charge would increase the affinity of the calcium binding site manifested a negligible increase in affinity.
Assuntos
Cálcio/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fator IX/metabolismo , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Mutação , Valina/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Fator de Crescimento Epidérmico/genética , Fator IX/genética , Ácido Glutâmico/genética , Glutamina/genética , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Spodoptera/citologia , Valina/genéticaRESUMO
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.
Assuntos
Cistatinas/farmacologia , Cisteína Endopeptidases/metabolismo , Trypanosoma cruzi/enzimologia , Animais , Galinhas , Cistatina A , Cisteína Endopeptidases/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Humanos , Focalização Isoelétrica , Cinética , Peso Molecular , Proteínas de Protozoários , Proteínas Recombinantes/farmacologiaRESUMO
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).
Assuntos
Cistatinas/isolamento & purificação , Inibidores de Cisteína Proteinase/isolamento & purificação , Sequência de Aminoácidos , Animais , Catepsinas/antagonistas & inibidores , Bovinos , Cistatina A , Cinética , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Pele/enzimologiaRESUMO
Cystatins A and C were both shown to inhibit cathepsin B by a two-step mechanism, involving an initial weak interaction followed by a conformational change. Disruption of the major salt bridge anchoring the occluding loop of cathepsin B to the main body of the enzyme by mutation of His110 to Ala converted the binding to an apparent one-step reaction. The second step of cystatin binding to cathepsin B must therefore be due to the inhibitor having to alter the conformation of the enzyme by displacing the occluding loop to allow a tight complex to be formed. Cystatin A was appreciably less effective in displacing the loop than cystatin C, resulting in a considerably lower overall inhibition rate constant.
Assuntos
Catepsina B/antagonistas & inibidores , Catepsina B/química , Cistatinas/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Histidina , Alanina , Substituição de Aminoácidos , Catepsina B/genética , Cistatina C , Humanos , Cinética , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologiaRESUMO
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.