LIMITED PROTEOLYSIS OF HUMAN KIDNEY ANGIOTENSIN-CONVERTING ENZYME ANDGENERATION OF CATALYTICALLY ACTIVE N-TERMINAL AND C-TERMINAL DOMAINS

The somatic form of angiotensin converting enzyme is a class I ectoenz yme that is bound to the surface of endothelial calls, It consists of two homologous, catalytic domains of approximately 600 residues each; a juxtamembrane ''stalk'' region; a transmembrane, hydrophobic sequenc e; and a 30 residue, C-terminal cytosolic domain, We have used limited proteolysis to probe the structural and functional properties of the enzyme, Endoproteinase Asp-N cleaves both the Thr(615)-Asp(616) and th e Leu(1219)-Asp(1220) peptide bonds to generate the two catalytic doma ins which were isolated by a combination of immunoaffinity and lisinop ril Sepharose affinity chromatography, The enzymatic characteristics o f the N and C fragments were examined with angiotensin I, hippuryl-His -Leu, and luteinizing hormone-releasing hormone and indicate that both fragments contain catalytically active sites that retain their indivi dual functional integrity. (C) 1997 Academic Press.