Our investigation focus on the novel functions of two enzymes, originally named kininase I and II that cleave bonds of vasoactive peptides but now their importance extends beyond hydrolyzing peptides. Our first two specific aims are concerned with carboxypeptidase N (CPN), a tetramer of two active and two regulatory subunits. CPN cleaves the C-terminal basic amino acids of proteins and peptides. We will determine how each of the subunits is involved in converting creatine kinase to its subforms. We will use both purified and recombinant enzyme, the latter in quantities sufficient for X-ray crystallography. Our plan is to establish the genomic sequence of both subunits, although they are synthesized on different chromosomes. We will also explore the complex interrelationship of human CPN with the plasmin-plasminogen system, which may affect plasminogen activation and blood clotting. We will determine where the subunits of CPN are cleaved by plasmin, establish the consequences of this cleavage for stability and activity, and measure dissociation of the tetramer. Furthermore, we will see how a complex of the plasmin or plasminogen is formed with the active subunit. The third and fourth specific aims of our research are concerned with the mode of action of angiotensin converting enzyme (kininase II, ACE) inhibitors. These drugs are used by millions of patients, and some of their beneficial effects are attributed to potentiation of bradykinin. We will employ cells transfected with cDNA of human ACE and bradykinin B2 receptor and cultured cells constitutively expressing ACE and B2 receptors to investigate augmentation of the release of signal transduction products by B2 receptor ligands. Inhibitors and slowly hydrolyzed substrates can combine with the active center of membrane- bound ACE and thereby enhance the activity of ligands of the 7- transmembrane B2 receptor, even if the agonists are resistant to ACE. Thus, the actions of ACE inhibitor go beyond protecting bradykinin against inactivation, although they do not act directly on its receptor. We will also employ ultrastructural analysis and microscopy to establish morphologic correlates of functional activities. Our ultimate goal is to show how ACE inhibitors exert their beneficial effects on heart, kidney, etc., by enhancing peptide action at the receptor level.
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