RESUMO
1. Two chromatographically distinct multicatalytic proteinases (MCP's) were isolated from the cytoplasm of chicken red blood cells and one MCP was purified from the nuclei. 2. The nuclear and the majority (97-99%) of the cytoplasmic multicatalytic proteolytic activity were chromatographically similar and differed from the minor cytoplasmic activity in their elution from hydroxylapatite, number of subunits on 2D-SDS-PAGE, and in their sensitivity to proteinase inhibitors. 3. Dichloroisocoumarin, a serine proteinase inhibitor, inhibited the hydrolysis of fluorogenic peptides but stimulated the degradation of casein by the multicatalytic proteinases suggesting that this enzyme has distinct active sites for protein and peptide hydrolysis.
Assuntos
Núcleo Celular/enzimologia , Cisteína Endopeptidases/metabolismo , Citoplasma/enzimologia , Eritrócitos/enzimologia , Complexos Multienzimáticos/metabolismo , Animais , Galinhas , Cromatografia DEAE-Celulose , Cromatografia em Gel , Cisteína Endopeptidases/efeitos dos fármacos , Cisteína Endopeptidases/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Ponto Isoelétrico , Masculino , Complexos Multienzimáticos/efeitos dos fármacos , Complexos Multienzimáticos/isolamento & purificação , Polilisina/farmacologia , Complexo de Endopeptidases do ProteassomaRESUMO
The contribution of metabolic energy to the degradation of intracellular proteins in skeletal muscle was investigated. Isolated chick skeletal muscles deprived of oxygen and muscles incubated in buffer under nonphysiological conditions containing inhibitors of glycolysis and mitochondrial respiration had lower concentrations or undetectable levels of ATP and faster rates of proteolysis. Both total protein breakdown and the breakdown of myofibrillar proteins were stimulated 35-124% in ATP-depleted tissues. However, ATP-depleted muscles incubated in buffer to which no Ca2+ was added showed slower rates of total protein breakdown and no significant change in myofibrillar proteolysis compared with control muscles. Trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), a compound that inhibits the calpains and the lysosomal cysteine proteases, completely blocked the Ca(2+)-stimulated breakdown of nonmyofibrillar and myofibrillar proteins in ATP-depleted muscles. However, Ca(2+)-stimulated proteolysis was not inhibited in ATP-depleted muscles incubated with weak bases to prevent lysosome function. These data suggest that intracellular proteins can be degraded in skeletal muscle in the absence of metabolic energy and that the calpains play a major role in the enhanced proteolysis in skeletal muscles depleted of ATP.
Assuntos
Trifosfato de Adenosina/deficiência , Cálcio/farmacologia , Proteínas Musculares/metabolismo , Animais , Animais Recém-Nascidos , Calcimicina/farmacologia , Cálcio/fisiologia , Calpaína/farmacologia , Galinhas , Inibidores de Cisteína Proteinase/farmacologia , Metabolismo Energético , Hipóxia/metabolismo , Ionomicina/farmacologia , Masculino , Peptídeo Hidrolases/metabolismoRESUMO
We have purified the multicatalytic proteinase from the nucleus and cytoplasm of chicken red blood cells. Both enzymes have an Mr of 700 kDa and can be separated into 8-10 bands (22-32 kDa) on 1D-SDS-PAGE.
Assuntos
Cisteína Endopeptidases/sangue , Eritrócitos/enzimologia , Complexos Multienzimáticos/sangue , Sequência de Aminoácidos , Animais , Núcleo Celular/enzimologia , Galinhas , Cisteína Endopeptidases/química , Cisteína Endopeptidases/isolamento & purificação , Citoplasma/enzimologia , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Peso Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/isolamento & purificação , Oligopeptídeos/química , Complexo de Endopeptidases do Proteassoma , Especificidade por SubstratoRESUMO
Reticulocytes contain a soluble nonlysosomal proteolytic pathway that requires ATP and ubiquitin. Polyamines at physiological concentrations were found to inhibit rapidly the ATP-dependent proteolytic system in reticulocyte lysates; spermidine and putrescine inhibited this process by 26-72% and spermine by 71-96%. Spermine had little effect on the ATP-independent breakdown of oxidant-treated hemoglobin. By fractionating the ATP-dependent system, we show that polyamines inhibit the ATP-dependent degradation of ubiquitin-protein conjugates.
