The pulmonary vasculature contains enzymes that are resonsible for alterations in components of the pulmonary circulation, thereby changing the composition of the systemic and bronchial blood flow. Angiotensin-1-converting enzymes is an endothelial cell surface enzyme with which we have had considerable experience and which we wish to continue to study in the present proposal. A two-step immunoabsorbent and chromatography system will be used to purify ACE from human plasma, lung, testes and kidney. Physical (e.g., molecular weight and isoelectric point) and chemical (e.g., peptide, carbohydrate and amino acid anylyses) properties of ACE from these different human sources will be compared. ACE from endothelial cells, macrophages, fibroblasts and monocytes and associated culture media will be assessed by electrophoretic and immunoblotting techniques with which isoenzymes of ACE can be discerned. These studies will allow comparisons between cellular, organ and circulating ACE. The turnover of protein and carbohydrate components of ACE in cell culture and in vivo will be studied by immunoprecipitation techniques, utilizing antibody prepared against purified ACE. Factors regulating the synthesis, degradation and release of ACE will be evaluated in cell culture with initial emphasis placed on hormonal regulation, the role of the sugar moiety and possible feedback inhibition by product formation. Similar assessments of regulation will be done in vivo with a rat model. Since it is recognized that ACE activity is elevated in sera of patients with sarcoidosis, possible unique properties of ACE in sera of patients with sarcoidosis will be evaluated by isoelectric focusing and immunoblotting, and the ELISA method we have developed will be used to compare absolute quantity of enzyme with serum enzymatic activity in these patients. Measurement and characterization of ACE and factors regulating its synthesis and release will be studied in peripheral blood monocytes and alveolar macrophages of patients with sarcoidosis. With these studies we hope to gain a broader understanding of structural properties of human ACE and, in particular, of that from the lung; to evaluate the regulation of the synthesis, release and general metabolism of ACE; and to determine the utility of new approaches to studies of this enzyme in sarcoidosis.
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