Abstract: | AbstractHuman urodilatin (residues 95–126) and atrial natriuretic factor (residues 99–126, based on ANF prohor-mone 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 arginine98 to yield ANF but under the same conditions did not cleave h-ANF. Neither urodilatin nor ANF was cleaved by ACE. ANF was rapidly degraded by NEP resulting in a major product cleaved between amino acid residues Cysl05 and Phe106. Urodilatin was also cleaved by NEP and the amino acid sequencing of the cleaved product revealed the site of cleavage to be the same Cys105-Phe106 site as for ANF with a second cleavage site at Gly118-Leu119. 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 Km values of 11.7 and 3.1 μM, respectively. The turnover rates (kcat/Km) for urodilatin and h-ANF with NEP were 4.6 and 37.3 min?1 μM?1, 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. |