DIFFERENTIAL CLEAVAGE OF URODILATIN AND ATRIAL-NATRIURETIC-FACTOR BY THROMBIN AND PROTEASE 3.4.24.11

Human urodilatin (residues 95-126) and atrial natriuretic factor (residues 99-126, based on ANF prohormone sequence) were incubated separately with three proteases, thrombin, angiotensin converting enzyme (ACE), and neutral endopeptidase 3.4.24.11 (NEP). Thrombin cleaved urodilatin on the carboxyl side of arginine(98) to yield ANF but under the same conditions did not cleave h-ANE Neither urodilatin nor ANF was cleaved by ACE. ANF was rapidly degraded by NEP resulting in a major product cleaved between amino acid residues Cys(105) and Phe(106). Urodilatin was also cleaved by NEP and the amino acid sequencing of the cleaved product revealed the site of cleavage to be the same Cys(105)-Phe(106) site as for ANF with a second cleavage site at Gly(118)-Leu(119). However, cleavage of urodilatin by NEP proceeded much more slowly when compared to ANF A comparison of the affinities of ANF and urodilatin for purified NEP from rabbit kidney revealed K-m Values of 11.7 and 3.1 mu M, respectively. The turnover rates (k(cat)/K-m) for urodilatin and h-ANF with NEP were 4.6 and 37.3 min(-1) mu M, respectively. Thus, urodilatin is much less efficiently hydrolyzed by purified NEP than is ANF The four residue extension at the N-terminus of urodilatin may be important for protection against rapid biological inactivation.