Cloning of a kinin-tensin enzyme and kinin-destruction enzyme in the dog heart

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We have previously shown that the purified kinin-forming enzyme from the dog heart is similar to tissue kalikrein and is also able to convert angiotensin (Ang) I to Ang II.The aim of the present study is to clarify the structure and tissue localization of this enzyme. Antiserum against the purified enzyme from the heart was raised in rabbits. Western blot analysis indicated the presence of this enzyme not only the heart (atria, ventiricule, coronary artery) but also other tissues such as sorta, kidney, pancreas, small intestine, and skeletal muscle. Its presence was also demonstrated in MDCK cell originated fron canine distal tubules. The kallikrein-like enzyme has been purified from homogenates of the kidney and aorta by a DEAE-Sepharose and aprotinin affinity columnn. Purified enzyme from the kidney and aorta was visualized at the relative molecular weight of 65 kDa on Western blotting which is identical to that of the kallikrein-like enzyme purified from the heart. The porified enzyme from the heart was proceeded to amino acid composition and sequence analysis. It was cleaved by lysyl-endopeptidase at 37ﾟC for 14 hrs and fractionated on a C18 column with a linear (0-90%) acetonitrile gradient in 0.1% trigluoroacetic acid. After fractionation, it was used for sequence analysis. However, the amino acid composition of purified kallikrein-like enzeme from the heart was different from that of dog urinary kallikrein. It has N-terminal bloked amino acid. the present study has shown that kallikrein-like enzyme is distributed not only in the heart, but also in the other tissues such as aorta, kidney, pancreas, small intestine, and skeletal muscle. This enzyme may exert pathophysiological effects through generating kinins and Ang II.The cloning og this enzyme iks now ongoing using antibody against the enzyme.