ELECTRON-MICROSCOPY STUDIES OF ALPHA-2-MACROGLOBULIN CONFORMATIONAL INTERMEDIATES OBTAINED BY DERIVATIZATION WITH CIS-DICHLORODIAMMINEPLATINUM (II)

Abstract

The structure of human .alpha.2-macroglobulin (.alpha.2M) after reaction with cis-dichlorodiammineplatinum (II) (cis-DDP) was studied by electron microscopy. The cis-DDP stabilized a novel conformation of the native inhibitor resembling a doughnut surrounded by two, three, of four well defined spherules. When only two spherules were present, these structures were usually oriented on opposite sides of the doughnut. The protein region joining a spherule to the central structure did not include sufficient mass to exclude stain and was, therefore, invisible. Other images showed spherules that were partially superimposed on the doughnut. A comparison of many molecules suggested great flexibility of the peripheral spherules relative to the central structure. The cis-DDP prevented complete conformational change when the .alpha.2M was reacted with trypsin. The products of this reaction included apparent conformational intermediates. These intermediates most closely resembled either native .alpha.2M or the well established "H" structure of .alpha.2M-proteinase, depending on the initial conditions used to modify the .alpha.2M with cis-DDP. When cis-DDP-treated .alpha.2M was reacted with trypsin, purified by chromatography and subsequently treated with diethyldithiocarbamate, complete conformational change was observed. Based on an analysis of the .alpha.2M structural intermediates obtained using the chemical modification procedures described here, a new model of .alpha.2M conformational change was developed. We postulate that conformational change initially involves contraction of the peripheral spherules towards the central doughnut. These spherules then unfold and elongate in the perpendicular direction to form the lateral walls of the proteinase transformed .alpha.2M H structure.