CONVERSION OF PROENDOTHELIN-1 INTO ENDOTHELIN-1 BY ASPARTYLPROTEASES

The ability of cathepsin D, chymosin, pepsin and renin to produce endo thelin-I (ET-1) from proendothelin-I (proET-1) was compared. No signif icant conversion was observed when proET-1 was incubated with up to 1 U of renin for 15 min at 37-degrees-C. Cathepsin D generated, as well as degraded, ET-1 rapidly. Net production of ET-1 reached a maximum wh en 0.003 U of cathepsin D was used, and about 16% of the initial proET -1 was detected as ET-1 by HPLC. Pepsin up to 1 U converted proET-1 in to ET-1 dose-dependently with a maximum of 71% conversion. A further i ncrease of the amount of pepsin in the reaction mixture produced nonsp ecific cleavage of ET-1. Less than 10% of ET-1 remained in the presenc e of 15 U of pepsin. Chymosin also generated ET-1 dose-dependently, an d a complete conversion was obtained at 1 U of enzyme. Greater than 1 U of chymosin only slightly degraded ET-1; at least 80% of ET-1 was st ill present when 15 U of chymosin was included in the assay. Other pro perties associated with the conversion of proET-1 into ET-1 by chymosi n were investigated. Similar to authentic ET-1, the product of chymosi n treatment caused contraction of isolated rabbit aortic rings, and pr e-incubation of chymosin with pepstatin A abolished this contractile r esponse. Using fast atom bombardment mass spectrometry, the molecular weights of the two peptide fragments generated from proET-1 by chymosi n were shown to be identical to those of authentic ET-1 and the C-term inal fragment of proET-1. The optimal pH of the enzyme reaction was be tween 3.5 and 4.5, and the K(m) and V(max) values for the enzyme were 140 mum and 102 nmol/(mg min), respectively. These results indicate th at chymosin specifically and rapidly converts proET-1 into ET-1 in vit ro. (C) Munksgaard 1993.