A membrane-associated cysteine protease inhibitor from murine hepatoma

A cysteine protease inhibitor was purified from total membrane fractions of an invasive murine hepatoma, Hepa cl 9. On gel filtration under non-reducing conditions the purified inhibitor was eluted in a single peak of M(r) 10-15 kDa, but resolved as two bands at 14 and 70 kDa on SDS-PAGE under reducing conditions. By isoelectric focusing, the inhibitor ran at an isoelectric point of 4.75. Immunoblotting studies using the enhanced chemiluminescence technique indicated no crossreactivity with monoclonal antibodies to stefin B and cystatin C or with a polyclonal antibody to low M(r) kininogen. In contrast, the 14 kDa and 70 kDa bands both crossreacted with a polyclonal antibody to stefin A, suggesting that the cysteine protease inhibitor associated with Hepa cl 9 membranes may be a modified form of stefin A.