Crystallization and characterization of a new protease in mitochondria of bone marrow cells.

A new protease was found in mitochondria of bone marrow cells. The protease was purified from human bone marrow cells by using the following methods: disruption of cells by sonication; buffer extraction; column chromatography using DEAE-cellulose, Sephadex G-75, and CM-cellulose. It was then crystallized in the presence of polyethylene glycol. The crystallized enzyme is homogeneous as judged by polyacrylamide disc gel electrophoresis. The molecular weight is 31,800 and optimum pH 8.5. Diisopropyl fluorophosphate (10 PM) inhibits the protease activity completely, whilep-chloromercuribenzoate (1 mM) has no effect. The enzyme is similar to elastase in that it hydrolyzes ester substrates for elastase and is inhibited by Elastatinal, an inhibitor of elastase. However, it is not identical with elastase, because it possesses no elastolytic activity and no ability to hydrolyze amide substrates for elastase. The protease inactivates the apoform of certain pyridoxal enzymes. Both granulocytes and erythroblasts contain the protease, but it was not detected in lymphocytes or thrombocytes. Amino acid analysis of the protease revealed that it contains a large proportion of arginine and a small proportion of lysine. Histidine, serine, and one carboxyl group were proved to be essential to the protease activity. Equimolar amounts of cY,-antitrypsin inhibit the protease. The protease was shown to be located on the inner membrane of mitochondria from bone marrow cells. An inverse relationship was obtained between &aminolevulinic acid synthetase activity and the protease activity in erythroblasts. Moreover, the rate of inactivation of &aminolevulinic acid synthetase activity in erythroblasts was diminished by the addition of Elastatinal.