RESUMO
Although about 90% of human renin circulates as inactive prorenin, the mechanism of prorenin activation in vivo is not known. We found that human polymorphonuclear leukocytes (PMN) activate prorenin at a neutral pH. Prorenin was partially purified from human amniotic fluid, and its activation was measured by the release of angiotensin I from sheep angiotensinogen. In control experiments, thermolysin was the standard activator. PMN cells were separated from blood and, after N2 cavitation or degranulation by cytochalasin, were fractionated by differential centrifugation. Elastase and cathepsin G activities were determined with synthetic fluorescent substrates. The activators of prorenin concentrated in the azurophil granules were released by Triton; most of the activation was due to elastase. Elastase, purified from human PMN, activated prorenin completely. The activation by the granular fraction was inhibited 77% by a specific elastase inhibitor in the presence of a detergent, but only 22% by a cathepsin G inhibitor. After inhibition of elastase, the residual activity was inhibited by diisopropylfluorophosphate; thus, it was due to a serine protease(s) such as cathepsin G. We suggest that human renin fully activated by elastase may still contain an N-terminal pentapeptide fragment of the propeptide.
Assuntos
Precursores Enzimáticos/fisiologia , Neutrófilos/fisiologia , Elastase Pancreática/fisiologia , Renina/fisiologia , Líquido Amniótico , Ativação Enzimática , Humanos , Renina/biossíntese , Frações Subcelulares/fisiologiaRESUMO
Neutral endopeptidase (EC 3.4.24.11, NEP) is an integral membrane protein of human neutrophils. NEP is identical with the common acute lymphoblastic leukemia antigen (CALLA) of leukemic cells. The expression of NEP on the surface of neutrophils is down-regulated by endocytosis which can be induced by phorbol 12-myristate 13-acetate (PMA) at 37 degrees C. The activity of the enzyme on the surface of intact cells decreases by 76% within 5 min. The activity can be recovered, however, if the cells are lysed within 5 min of the endocytosis. After 30 min, only 32% of the NEP activity is present in the neutrophil lysates. The loss of activity is presumably due to proteolytic inactivation. Diacylglycerol and monoclonal antibody to CALLA/NEP also induce internalization of NEP. PMA induces endocytosis even at 4 degrees C, but NEP is not inactivated at that temperature. The disappearance of NEP activity after adding PMA was inhibited by various agents. Among the most active were the phospholipase inhibitor 4-bromophenacyl bromide and a combination of the serine protease and cathepsin inhibitors, diisopropylfluorophosphate and N-ethylmaleimide. The employment of fluorescent monoclonal antibody confirmed the down-regulation and internalization of NEP antigen on the neutrophils. Since NEP inactivates chemotactic peptides and thereby affects chemotaxis of neutrophils (Painter, R. G., Dukes, R., Sullivan, J., Carter, R., Erdös, E. G., and Johnson, A. R. (1988) J. Biol. Chem. 263, 9456-9461), the down-regulation of NEP activity on the cell membrane may modulate the function of these cells in inflammation.