STRUCTURAL AND FUNCTIONAL-ANALYSIS OF THE SPONTANEOUS RE-FORMATION OFTHE THIOL ESTER BOND IN HUMAN ALPHA-MACROGLOBULIN, RAT ALPHA(1)-INHIBITOR-3 AND CHEMICALLY-MODIFIED DERIVATIVES

The alpha-macroglobulins are proteinase inhibitors that form part of a superfamily along with components of the complement system. Internal beta-cysteinyl-gamma-glutamyl thiol ester bonds are an important struc tural feature of most alpha-macroglobulins and several complement comp onents. We have studied the reversibility of thiol ester cleavage caus ed by NH3 or CH3NH2 in tetrameric human alpha(2)-macroglobulin (alpha( 2)M) and monomeric rat alpha(1)-inhibitor-3 (alpha(1)I(3)). When emplo ying NH3 as the nucleophile, the thiol ester in alpha(1)I(3) re-formed spontaneously at room temperature after gel filtration to remove exce ss nucleophile, and an active proteinase inhibitor was regained. When CH3NH2 was employed as the nucleophile, thiol ester reversibility was more energy-demanding. With either nucleophile, alpha(2)M once inactiv ated did not regain proteinase-inhibitory capacity at room temperature . At elevated temperatures, however, the reaction between alpha(2)M an d NH3 or CH3NH2 was reversible and the inhibitory capacity could be re covered. Modification of the cysteinyl groups from the thiol ester pre vented its re-formation but did not prevent the heat-induced retrieval of inhibitory capacity, suggesting that conformational features rathe r than the thiol ester are essential for alpha(2)M to function as an i nhibitor. As demonstrated by nondenaturing PAGE, the conformation of n ative alpha(2)M is restored when the proteinase-inhibitory capacity is recovered.