Structural and functional analysis of the spontaneous re-formation of the thiol ester bond in human alpha 2-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 structural feature of most alpha-macroglobulins and several complement components. We have studied the reversibility of thiol ester cleavage caused by NH3 or CH3NH2 in tetrameric human alpha 2-macroglobulin (alpha 2M) and monomeric rat alpha 1-inhibitor-3 (alpha 1I3). When employing NH3 as the nucleophile, the thiol ester in alpha 1I3 re-formed spontaneously at room temperature after gel filtration to remove excess 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 2M once inactivated did not regain proteinase-inhibitory capacity at room temperature. At elevated temperatures, however, the reaction between alpha 2M and NH3 or CH3NH2 was reversible and the inhibitory capacity could be recovered. Modification of the cysteinyl groups from the thiol ester prevented its re-formation but did not prevent the heat-induced retrieval of inhibitory capacity, suggesting that conformational features rather than the thiol ester are essential for alpha 2M to function as an inhibitor. As demonstrated by non-denaturing PAGE, the conformation of native alpha 2M is restored when the proteinase-inhibitory capacity is recovered.
Grøn
H
H
Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA.
Thøgersen
I B
IB
Enghild
J J
JJ
Pizzo
S V
SV
D000209:Acute-Phase Proteins / Q000737:chemistry* / Q000494:pharmacology*; D000818:Animals; D003545:Cysteine; D004952:Esters; D018698:Glutamic Acid; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology; D051381:Rats; D013438:Sulfhydryl Compounds; D014357:Trypsin / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000737:chemistry* / Q000494:pharmacology*
D000209:Acute-Phase Proteins; D004952:Esters; D046911:Macromolecular Substances; D011480:Protease Inhibitors; D013438:Sulfhydryl Compounds; C017894:alpha 1-inhibitor 3; D000511:alpha-Macroglobulins; D018698:Glutamic Acid; D014357:Trypsin; D003545:Cysteine