DIFFERENTIAL-EFFECTS OF HEPARIN AND GLUCOSE ON STRUCTURAL CONFORMATION OF HUMAN ALPHA(1) ANTITRYPSIN - EVIDENCE FOR A HEPARIN-INDUCED CLEAVED FORM OF THE INHIBITOR

alpha(1) Antitrypsin (alpha(1)AT) is the archetypal member of the serp in superfamily. Current knowledge of its inhibitory mechanism does not provide for any heparin-induced enhancement of serine proteinase inhi bition. Since previous results have shown that an apparently altered a lpha(1)AT form may be purified from the plasma of insulin-dependent di abetics by means of heparin-affinity chromatography, in the present wo rk the possibility was tested that heparin at various concentrations m odifies the structural conformation and function of human alpha(1)AT i n the absence and presence of glucose, used at concentrations of 15 mM to mimick mild hyperglycemic conditions. Heparin was observed to bind strongly to alpha(1)AT, causing maximal enhancement of tryptophan flu orescence emission at 50 mu g/ml, mostly in the presence of glucose. C ircular dichroism spectra revealed that heparin with glucose caused th e most relaxed, ordered structure of the inhibitor with increased heat stability. Modification in conformation was accompanied by loss of in hibitory activity, as demonstrated by the inability of alpha(1)AT to b lock bovine trypsin in the specific assay and by alterations of its im munological properties. However, despite inactivation, in the presence of heparin-both with and without glucose-alpha(1)AT was still able to bind trypsin, as revealed by inhibitor-to-proteinase complexes visibl e in both SDS-and nondenaturing electrophoreses. These complexes showe d the same feature regardless of trypsin concentration and differed fr om those formed at a molar excess of the inhibitor in the absence of h eparin, since they underwent rapid, intense fragmentation accompanied by complete loss of the secondary structure of the inhibitor. Even in the absence of trypsin, cleavage of alpha(1)AT was also observed to oc cur at both Val(321)- and Glu(344)- in the primary sequence of the inh ibitor in the presence of 50 mu g/ml heparin, with and without glucose . These results suggest that heparin binding to alpha(1)AT causes prof ound structural changes in the molecule, involving both the expulsion of the reactive site out of the molecule plane and a relaxed, heat-sta ble form of the inhibitor, rendered a substrate for the proteinase. Al though glucose apparently does not affect alpha(1)AT functioning, it d oes enhance the effects of heparin on the alpha(1)AT structure. The po ssibility is discussed that, while heparin and glucose binding occurs at different sites on alpha(1)AT, glucose favors heparin binding by in ducing a partially relaxed form in the inhibitor. Differences in struc ture and charge between the two substances account for both different individual effects on alpha(1)AT and the predominance of the effects o f heparin. (C) 1997 Academic Press.