RESUMO
Human transforming growth factor-alpha (h-TGF alpha), a 50-amino acid residue peptide, was incubated with some purified cell-surface peptidases and with renal microvillar membranes prepared from pig and rat. Hydrolysis was monitored by h.p.l.c. and activity by a biological assay. Prolonged incubation with relatively large amounts of endopeptidase-24.11, aminopeptidase N and peptidyl dipeptidase A (angiotensin-converting enzyme) caused no observable hydrolysis and no detectable loss of biological activity. Incubation with pig renal microvilli also failed to degrade the peptide. In contrast, rat renal microvilli readily degraded h-TGF alpha, as did endopeptidase-2, which is located in rat renal and intestinal brush borders, but is absent from pig kidneys. This enzyme degraded about 30 nmol of h-TGF alpha/h per mg of protein. The physiological significance of these results is discussed.
Assuntos
Membrana Celular/enzimologia , Endopeptidases/metabolismo , Microvilosidades/enzimologia , Fator de Crescimento Transformador alfa/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Dissulfetos/análise , Humanos , Hidrólise , Rim/enzimologia , Cinética , Dados de Sequência Molecular , Conformação Proteica , Ratos , Proteínas Recombinantes/metabolismo , SuínosRESUMO
The tissue distribution and route of clearance of human recombinant interleukin 1 alpha (IL 1 alpha) injected intravenously in rats was studied. The plasma half-life was approximately 2.5 min, and this was increased after nephrectomy, the kidney being the major organ through which the IL 1 alpha was excreted. Two iodinated fragments of IL 1 alpha, of approximately 5 and 9 kDa, were excreted by the kidneys whereas only intact, 17-kDa IL 1 alpha was detected in plasma, suggesting that the protein was being degraded after uptake by the kidney. The results of in vivo experiments in which surface endopeptidase-24.11 was inhibited with phosphoramidon and in vitro experiments in which rat kidney homogenates were incubated with radiolabeled IL 1 alpha suggest that the cytokine was endocytosed and then hydrolysed by lysosomal proteinases.
Assuntos
Interleucina-1/metabolismo , Rim/metabolismo , Animais , Autorradiografia , Meia-Vida , Humanos , Interleucina-1/farmacocinética , Radioisótopos do Iodo , Masculino , Peso Molecular , Nefrectomia , Fragmentos de Peptídeos/isolamento & purificação , Ratos , Ratos Endogâmicos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Fatores de Tempo , Distribuição TecidualRESUMO
A novel two-step enzyme-linked assay for aminopeptidase W is described and validated by comparison with other assays. L-alpha-Glutamyl-L-tryptophan (Glu-Trp) is a favoured substrate for this enzyme. With the use of glutamate dehydrogenase (EC 1.4.1.2) in a second step, the assay measured the release of free glutamate from L-alpha-glutamyl-L-tryptophan by the increase in NADH fluorescence. In the presence of 5 mM-1,10-phenanthroline and 50 microM-cilastatin the contribution of other membrane peptidases, in particular aminopeptidases N and A and microsomal dipeptidase in kidney, was very small. Residual cytosolic activities hydrolysing Glu-Trp were sensitive to inhibition by 2.5 mM-N-ethylmaleimide. The activity of aminopeptidase W was unaffected by these inhibitors. There was good correlation between the fluorimetric assay and those in which the free tryptophan released by kidney membrane fractions was determined by h.p.l.c. or the aminopeptidase W was measured immunoradiometrically with a monoclonal antibody.
