Independent analysis of bait region cleavage dependent and thiolester bond cleavage dependent conformational changes by cross-linking of alpha 2-macroglobulin with cis-dichlorodiammineplatinum(II) and dithiobis(succinimidyl propionate).
Treatment of the human plasma proteinase inhibitor alpha 2-macroglobulin (alpha 2M) with proteinase results in conformational changes in the inhibitor and subsequent activation and cleavage of the internal thiolester bonds of alpha 2M. Previous studies from this laboratory have shown that cross-linking the alpha 2M subunits with cis-dichlorodiammineplatinum(II) (cis-DDP) prevents the proteinase-induced conformational changes which lead to the activation and cleavage of the internal thiolester bonds of alpha 2M. In addition, cis-DDP treatment prevents the proteinase- or CH3NH2-induced conformational changes in alpha 2M which lead to a "slow" to "fast" change in nondenaturing polyacrylamide gel electrophoresis. In this paper, we demonstrate that treatment of alpha 2M with dithiobis(succinimidyl propionate) (DSP) also results in cross-linking of the subunits of alpha 2M with concomitant loss of proteinase inhibitory activity. Although proteinase is not inhibited by DSP-treated alpha 2M, bait region specific proteolysis of the alpha 2M subunits still occurs. Unlike cis-DDP-treated alpha 2M, however, incubation of DSP-treated alpha 2M with proteinase does not prevent the bait region cleavage dependent conformational changes which lead to activation and cleavage of the internal thiolester bonds in alpha 2M. On the other hand, cross-linking of alpha 2M with DSP does prevent the conformational changes which trigger receptor recognition site exposure following cleavage of the alpha 2M thiolester bonds by CH3NH2. These conformational changes, however, occur following incubation of the CH3NH2-treated protein with proteinase.(ABSTRACT TRUNCATED AT 250 WORDS)
Roche
P A
PA
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710.
Moncino
M D
MD
Pizzo
S V
SV
D002945:Cisplatin / Q000493:pharmacokinetics*; D003432:Cross-Linking Reagents / Q000493:pharmacokinetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D011480:Protease Inhibitors / Q000494:pharmacology; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013388:Succinimides / Q000493:pharmacokinetics*; D000511:alpha-Macroglobulins / Q000032:analysis*
D003432:Cross-Linking Reagents; D011480:Protease Inhibitors; D013388:Succinimides; D000511:alpha-Macroglobulins; C011240:dithiobis(succinimidylpropionate); D002945:Cisplatin