Isolation and characterization of alpha 2-macroglobulin-protease complexes from purified mouse mammary tumor virus and culture supernatants from virus-infected cell lines.

Cleavage of oligopeptide substrates mimicking the maturation sites in the Gag polyproteins of the mouse mammary tumor virus was assayed using lysed virus. Cleavage at the expected P1-P1' positions was detected in four of seven synthetic peptides. However, studies with specific inhibitors of retroviral proteases showed that only two of them could be unequivocally attributed to the viral enzyme. In an attempt to characterize other proteolytic activities that copurify with the virus, we isolated a multicatalytic high molecular mass protease (700 kDa) that copurifies with the virus. This protein has been identified as an alpha 2-macroglobulin-protein complex according to its biochemical properties and ultrastructure. The proteases forming these complexes are mainly serine proteases and can be inhibited by phenylmethylsulfonyl fluoride. However, other compounds such as chymostatin and elastatinal are more effective inhibitors. The relative efficacy of each compound depends on the substrate, since the complexes described herein appear to be multicatalytic. Elastatinal is a very good inhibitor of the cleavages found at Ala-Ala bonds in peptides representing the capsid/nucleocapsid site, while chymostatin inhibits certain cleavages at the carboxyl terminus of bonds involving leucine and valine in three of the substrates used. Therefore, the alpha 2-macroglobulin present in the cell culture medium is able to bind proteases, forming high molecular weight complexes, which are active against peptide substrates, copurify with the virus and are responsible for the nonviral proteolytic activities found in the purified virus. Elastase appears to be the main proteolytic activity which can be detected in the alpha 2-macroglobulin-protease complexes associated with the virus.