The α-macroglobulins are proteinase inhibitors that form part of a superfamily along with components of the complement system. Internal β-cysteinyl–γ-glutamyl thiol ester bonds are an important structural feature of most α-macroglobulins and several complement components. We have studied the reversibility of thiol ester cleavage caused by NH3 or CH3NH2 in tetrameric human α2-macroglobulin (α2M) and monomeric rat α1-inhibitor-3 (α1 I3). When employing NH3 as the nucleophile, the thiol ester in α1I3 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, α2M once inactivated did not regain proteinase-inhibitory capacity at room temperature. At elevated temperatures, however, the reaction between α2M 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 α2M to function as an inhibitor. As demonstrated by non-denaturing PAGE, the conformation of native α2M is restored when the proteinase-inhibitory capacity is recovered.
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Research Article|
September 01 1996
Structural and functional analysis of the spontaneous re-formation of the thiol ester bond in human α2-macroglobulin, rat α1-inhibitor-3 and chemically modified derivatives
Hanne GRØN;
Hanne GRØN
1Department of Pathology, Duke University Medical Center, P.O. Box 3712, Durham, NC 27710, U.S.A.
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Ida B. THØGERSEN;
Ida B. THØGERSEN
1Department of Pathology, Duke University Medical Center, P.O. Box 3712, Durham, NC 27710, U.S.A.
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Jan J. ENGHILD;
Jan J. ENGHILD
1Department of Pathology, Duke University Medical Center, P.O. Box 3712, Durham, NC 27710, U.S.A.
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Salvatore V PIZZO
Salvatore V PIZZO
*
*To whom correspondence should be addressed.
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Publisher: Portland Press Ltd
Received:
December 14 1995
Revision Received:
May 09 1996
Accepted:
May 13 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 318 (2): 539–545.
Article history
Received:
December 14 1995
Revision Received:
May 09 1996
Accepted:
May 13 1996
Citation
Hanne GRØN, Ida B. THØGERSEN, Jan J. ENGHILD, Salvatore V PIZZO; Structural and functional analysis of the spontaneous re-formation of the thiol ester bond in human α2-macroglobulin, rat α1-inhibitor-3 and chemically modified derivatives. Biochem J 1 September 1996; 318 (2): 539–545. doi: https://doi.org/10.1042/bj3180539
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