The trypsin-inhibitory efficiency of human alpha(2)-macroglobulin in the presence of alpha(1)-proteinase inhibitor: Evidence for the formation of an alpha(2)-macroglobulin-alpha(1)-proteinase inhibitor complex

The inhibition of bovine pancreatic trypsin was studied at pH 7, 25 degrees C, using mixtures of purified human alpha(2)-macroglobulin (alpha(2)M) and alpha(1)-proteinase inhibitor (alpha(1)PI). The partitioning of the enzyme between the two inhibitors was determined by comparing control esterase ac tivity, assayed with N-benzoyl-L-arginine ethyl ester as substrate, with th at remaining after incubation with inhibitory mixtures. (At [I](0) > [E](0) , remaining esteratic activity reflects the concentration of alpha(2)M-asso ciated enzyme (alpha(2)M-E*) and the concentration of alpha(1)PI-associated , inactive enzyme (alpha(1)PI-E*) is given by the difference, [E](0) - [alp ha(2)M-E*].) The pattern of product distribution was found to be incompatib le with an inhibitory model involving parallel, second-order reactions of E with alpha(2)M and alpha(1)PI. The data pointed to complex formation betwe en the two inhibitors, limiting the level of alpha(2)M readily available fo r reaction with E. Analysis based on the binding equilibrium, alpha(2)M (di meric unit) + alpha(1)PI reversible arrow alpha(2)M - alpha(1)PI, yielded K -d = 2.1 +/- 0.3 mu M. Complex formation between alpha(2)M and alpha(1)PI w as verified by gel permeation experiments. alpha(2)M was found to restrict the volume of distribution of alpha(1)PI in Sephadex G200 beds. K-d, deduce d from gel permeation behaviour, was 0.8 +/- 0.32 mu M. Preliminary kinetic experiments with dialyzed plasma suggested that the alpha(2)M-alpha(1)PI i nteraction is effective also in vivo. Given Kd and the mean plasma levels o f the two inhibitors ([alpha(2)M] = 2 mu M; [alpha(1)PI] = 36 mu M), it was estimated that > 90% of alpha(2)M in human circulation must be complexed t o alpha(1)PI and lack immediate antiproteinase activity.