Analysis of the plasma elimination kinetics and conformational stabilities of native, proteinase-complexed, and reactive site cleaved serpins: comparison of alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, antithrombin III, alpha 2-antiplasmin, angiotensinogen, and ovalbumin.
Proteinase inhibitors of the serpin superfamily may exist in one of three distinct conformations: the native form, a fully active protein with the reactive site loop intact; the proteolytically modified form in which inhibitory capacity is abolished; and the proteinase-complexed form, a stable equimolar complex between the inhibitor and a target proteinase. Here, the specificity and kinetics of the plasma elimination of different serpin conformations are compared. Proteinase-complexed serpins were rapidly cleared from the circulation. However, the native and modified forms were not cleared rapidly, indicating that the receptor-mediated pathways which recognize the complexes fail to recognize the native and modified forms. This result suggests that significant structural differences exist between modified and proteinase-complexed serpins. The structural differences were probed by using transverse urea gradient gel electrophoresis, a technique that allows comparisons of the conformational stabilities of proteins. With the exception of the noninhibitory serpins ovalbumin and angiotensinogen, the modified and proteinase-complexed serpins were both stabilized thermodynamically compared to the native forms. In addition, the proteinase component of the serpin-proteinase complex was usually thermodynamically stabilized. These data are used to compare the conformations of serpin-proteinase complexes with those of native and modified serpins; they are discussed in terms of a model whereby serpins inhibit proteinases in a manner similar to that described for other types of protein inhibitors of serine proteinases.
Mast
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AE
Pathology Department, Duke University Medical Center, Durham, North Carolina 27710.
Enghild
J J
JJ
Pizzo
S V
SV
Salvesen
G
G
D000595:Amino Acid Sequence; D000808:Angiotensinogen / Q000378:metabolism*; D000818:Animals; D000990:Antithrombin III / Q000378:metabolism*; D001665:Binding Sites; D004355:Drug Stability; D005341:Fibrinolysin / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000378:metabolism*; D015843:Serpins / Q000737:chemistry / Q000378:metabolism*; D000514:alpha 1-Antichymotrypsin / Q000378:metabolism*; D000515:alpha 1-Antitrypsin / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000378:metabolism*
D015843:Serpins; D000514:alpha 1-Antichymotrypsin; D000515:alpha 1-Antitrypsin; D000511:alpha-Macroglobulins; C024668:plasmin-alpha(2)-macroglobulin complex; D000808:Angiotensinogen; D000990:Antithrombin III; D010047:Ovalbumin; D012697:Serine Endopeptidases; D005341:Fibrinolysin