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

Abstract

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.

Proteolytic inactivation of α1‐antitrypsin and α1‐antichymotrypsin by neutrophils in arthritic joints

“…Although the absolute concentrations of ia,AT and ia,ACT in normal plasma were comparable, for example, in pooled normal plasma 310 nM and 285 nM, respectively, the levels expressed as a percentage of the total level of the inhibitor differed considerably, i.e., 0.8% for a,AT versus 5% for a,ACT. Mast et al (50) have shown that ia,AT is cleared more rapidly from the circulation than is native a,AT, whereas ia,ACT and native a, ACT are removed at a similar rate. It is tempting to speculate that this difference in clearance of ia,AT and ia,ACT explains their different percentage levels in normal individuals.…”

SERPINB11 Is a New Noninhibitory Intracellular Serpin

“…4C). This finding suggested that the R-form of Serpinb11 was not as stable as those of Serpinb3b and other serpins, which typically retain their solubility within this temperature range (14,(37)(38)(39). Uncleaved SERPINB11d showed a thermal denaturation profile similar to Serpinb11 (Fig.…”

“…To further assess the conformations of SERPINB11d and the revertants, we next examined their ability to undergo the stressed (S) to relaxed (R) transition typical of serpins with mobile RSLs, a requisite function for inhibitory activity (14,(37)(38)(39). Serpins in the S-conformation (RSL noncleaved and exposed) are metastable and in most cases precipitate upon heating to ϳ55-65°C.…”

39 Structure function relationships of plasminogen activator inhibitor type-2 (PAI-2)

“…In contrast, inhibitory serpins R state have unfolding temperatures above 100°C (Gettins & Harten 1988;Bruch et al 1988). Furthermore, Mast et al (1991) showed that serpins in the R state are very resistant to urea induced denaturation, with unfolding undetectable even at urea concentrations of 8M. Using this approach, they estimated the conformational stabilit (AG[H 2 OJ) of inhibitory serpins in the S state to be 5 -15 kJ.mol" 1 , significantly lo than that of most globular proteins.…”

“…The hepatic serpin clearance receptor provides a common pathway for the elimination of a number of different serpins, complexed with their target proteases, from the circulation (eg. oc,-PI, a r ACT, antithrombin III) (Mast et al 1991). The exact mechanism of internalisation of uPA:PAI-2 complexes is not yet clear.…”