RESUMEN
Overactivation of calcium-activated neutral protease (calpain) has been implicated in the pathophysiology of several degenerative conditions, including stroke, myocardial ischemia, neuromuscular degeneration, and cataract formation. Alpha-mercaptoacrylate derivatives (exemplified by PD150606), with potent and selective inhibitory actions against calpain, have been identified. PD150606 exhibits the following characteristics: (i) Ki values for mu- and m-calpains of 0.21 microM and 0.37 microM, respectively, (ii) high specificity for calpains relative to other proteases, (iii) uncompetitive inhibition with respect to substrate, and (iv) it does not shield calpain against inactivation by the active-site inhibitor trans-(epoxysuccinyl)-L-leucyl-amido-3-methylbutane, suggesting a nonactive site action for PD150606. The recombinant calcium-binding domain from each of the large or small subunits of mu-calpain was found to interact with PD150606. In low micromolar range, PD15O6O6 inhibited calpain activity in two intact cell systems. The neuroprotective effects of this class of compound were also demonstrated by the ability of PD150606 to attenuate hypoxic/hypoglycemic injury to cerebrocortical neurons in culture and excitotoxic injury to Purkinje cells in cerebellar slices.
Asunto(s)
Acrilatos/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Glicoproteínas/farmacología , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Hipoxia de la Célula , Línea Celular , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Hipoglucemia/fisiopatología , Técnicas In Vitro , Datos de Secuencia Molecular , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores , Células de Purkinje/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/toxicidadAsunto(s)
Acrilatos/farmacología , Calpaína/antagonistas & inhibidores , Inhibidores de Cisteína Proteinasa/farmacología , Acrilatos/farmacocinética , Animales , Permeabilidad de la Membrana Celular , Inhibidores de Cisteína Proteinasa/farmacocinética , Humanos , Fármacos Neuroprotectores/farmacocinética , Fármacos Neuroprotectores/farmacologíaRESUMEN
Human mu-calpain is activated maximally by 100-200 microM Ca2+. Both the 80 kDa and 29 kDa subunits of mu-calpain have a EF-hand type calcium-binding domain. It is known that trivalent terbium ion (Tb3+) mimics Ca2+ in many biological systems. We found that Tb3+ alone transiently activated calpain. However, in the presence of Ca2+, Tb3+ inhibited mu-calpain with an IC50 of about 100 microM. As high as 10 mM Ca2+ did not significantly shift the IC50 of Tb3+. Preincubating mu-calpain by Ca2+ (before Tb3+ and substrate were added) did not diminish the inhibition by Tb3+. On the other hand, pretreating mu-calpain with Tb3+ produced that Tb3+ has a slow dissociation rate for the calcium-binding sites when compared to Ca2+. Electrophoretic analysis revealed that terbium ion transiently activated mu-calpain followed by the aggregation of the proteinase.
Asunto(s)
Calpaína/metabolismo , Terbio/farmacología , Sitios de Unión , Calcio/farmacología , Calpaína/antagonistas & inhibidores , Calpaína/química , Caseínas/metabolismo , Diálisis , Ácido Egtácico/farmacología , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Eritrocitos/química , Humanos , Immunoblotting , Unión Proteica , Terbio/metabolismoRESUMEN
The activity of cytosolic calcium-dependent neutral protease (calpain) is commonly measured using casein as a substrate. A novel, modified caseinolysis assay is now developed. It involves incubation of calpain with substrate casein, followed by removal of an aliquot to which Coomassie brilliant blue G-250 dye reagent is added. The assay is based on the observation that the dye interacts only with protein but not the proteolytic products (small peptides and amino acids). Unlike the existing caseinolysis assay, this novel assay does not require separation of substrate from products and measurement is done in the visible range (595 nm). It is also more sensitive than existing colorimetric assays which use dye-conjugated protein substrates. The assay can also be used to measure the activity of other proteases such as trypsin and papain.