Alpha-Macroglobulin (alpha2M), a general proteinase inhibitor ubiquitous in the plasma of vertebrates, is believed to serve as a general proteinase scavenger thereby protecting blood and tissue proteins from degradation. As a proteinase inhibitor, it is involved in the regulation of proteinase activity in fibrinolysis, coagulation and complement activation. It interacts with cytokines and growth factors and it may be of physiological importance in the degradation of thrombi. A better understanding of the structure-function relationships of alpha2M will result from the determination of the structure of its various complexes by stain and cryo-electron microscopy, and image processing. The importance of the thioester site which is cleaved during the transformation of alpha2M to the proteolyzed (activated) form will be evaluated by determining the structure in which this site has been eliminated by site directed mutagenesis. Plasmin (Mr=80,000) forms a binary complex with alpha2M whereas smaller proteinases such as alpha- chymotrypsin (Mr=25,000) form a ternary complex. The determination of the 3-D structure of the binary complex and its comparison with our 3-D structure of the ternary alpha2M-chymotrypsin complex should reveal the location of thrombin in the structure and help to assess the significance of the larger size of plasmin on the manner in which it is bound to alpha2M. Thrombin (Mr=33,500) also forms a binary complex, possibly because its reaction with alpha2M is considerably slower than those proteinases that form a ternary complex. A genetically engineered alpha2M in which the bait region more closely matches the specificity of thrombin will be prepared in order to determine whether increasing the rate constant for the reaction results in the formation of ternary complex. The 3-D structure of the engineered alpha2M and its complex with thrombin will complement these studies. Further understanding of the complex transformations that are involved in the activation of alpha2M will result from the determination of (1) the 3_D structure of intermediate forms and (2) the disposition of N- and C-terminal Fabs bound to the native, intermediate, and activated structures. Immunoelectron microscopy will also be utilized to determine the position of the bait and receptor binding sites and a site-specific gold label will determine the position of the thiols released from the thiol ester sites in the intermediate and activated structures. A better understanding of the manner in which proteinases are bound to alpha2M should result from determining the orientation of the active site of papain labelled with a gold cluster in the complex. The subunit organization of the two dimers that comprise alpha2M will be assessed by determining the shape of monomeric rat alpha1I3 which has extensive sequence identity with rat alpha2M.