Detection and partial characterization of a protein in mouse fibroblasts that binds and inhibits trypsin.

The existence of proteins in BALB/c 3T3 cells which interact strongly with trypsin was investigated in order to learn more about how the activities of proteases with trypsin-like specificity might be regulated in mammalian cells. Cell extracts were found to contain 7 to 12 pmol/miUion cells of a protein (TCP) which binds and inhibits trypsin. The trypsin-TCP complex which forms upon incubation of cell extracts with trypsin was demonstrated to have a Stokes' radius of 3.25 nm, a sedimentation coefficient of 4.5 S , and a molecular weight of 60 to 63 X lo3 by gel filtration and sucrose density gradient centrifugation. Gel filtration of the cell extracts prior to reaction with trypsin revealed that 90% of the trypsin-inhibitory activity of the extracts had an elution volume corresponding to that of an M, = 42,500 globular protein standard. This M, = 42,500 trypsin-inhibitory fraction also inhibited thrombin, but it did not inhibit chymotrypsin. After incubation of I z a L trypsin with theM, = 42,500 trypsin-inhibitory fraction, the radiochemical label of the trypsin co-eluted with trypsin-TCP upon gel filtration. This result supports the view that a single protein or group of proteins with a molecular weight of about 42,500 combines with trypsin (Mr = 23,300) to form a M, = 60 to 63 X IO3 eatalytically inactive trypsin -TCP complex. The trypsin TCP complex could be dissociated by incubation with excess lima bean trypsin inhibitor (LBTI). Experiments using trypsin-TCP in which either the trypsin or tbe TCP was radiochemically labeled indicated that the incubation with LBTI resulted in formation of a trypsin. LBTI complex with concomitant release of free TCP.