The citrate ion increases the conformational stability of alpha(1)-antitrypsin.

Sodium citrate has previously been shown to convert native alpha(1)-antitrypsin into the inactive latent state and cause alpha(1)-antitrypsin to polymerize via the C-sheet pathway instead of the more common A-sheet pathway. In order to begin to understand these dramatic effects, we have examined the influence of low concentrations of sodium citrate upon the structure, stability and function of alpha(1)-antitrypsin. In 0.5 M citrate, the midpoint of guanidine hydrochloride-induced unfolding was increased by 1.8 M and the rate of heat inactivation was decreased approximately 30-fold compared with Tris or phosphate buffer. alpha(1)-Antitrypsin was fully active in the presence of a range of citrate concentrations (0. 1-0.5 M), forming a stable 1:1 complex with chymotrypsin. The association rate constant between alpha(1)-antitrypsin and chymotrypsin was decreased with increasing citrate concentration. Fluorescence and circular dichroism spectroscopy demonstrated no significant changes in the tertiary structure due to the presence of citrate. However, the insertion rate of exogenous reactive-center loop peptide increased with increasing citrate concentration, indicating some structural changes in the A beta-sheet region. Taken together, these data suggest that in the presence of 0.5 M citrate alpha(1)-antitrypsin adopts a highly stable but active conformation.

Probing the equilibrium denaturation of the serpin alpha(1)-antitrypsin with single tryptophan mutants; evidence for structure in the urea unfolded state.

The native conformation of proteins in the serpin superfamily is metastable. In order to understand why serpins attain the native state instead of more stable conformations we have begun investigations into the equilibrium-unfolding of alpha(1)-antitrypsin. alpha(1)-Antitrypsin contains two tryptophan residues, Trp194 and Trp238, situated on the A and B beta-sheets, respectively. Site-directed mutagenesis was used to construct two single-tryptophan variants. Both variants were fully active and had similar secondary structure and stabilities to alpha(1)-antitrypsin. The denaturation of alpha(1)-antitrypsin and its variants was extremely similar when followed by far-UV CD, indicating the presence of a single intermediate. Fluorescence analysis of the unfolding behavior of each single tryptophan variant indicated that the sole tryptophan residue reported the structural changes within its immediate environment. These data suggest that the A beta-sheet is expanded in the intermediate state whilst no structural change around the B beta-sheet has occurred. In the urea-induced unfolded state, Trp238 does not become fully solvated, suggesting the persistence of structure around this residue. The implications of these data on the folding, misfolding and function of the serpin superfamily are discussed.

Intrinsic fluorescence changes and rapid kinetics of proteinase deformation during serpin inhibition.

The X-ray crystal structure of the serpin-proteinase complex suggested that the serpin deformed the proteinase thereby inactivating the molecule. Using a variant of alpha(1)-antitrypsin in which both tryptophan residues have been replaced by phenylalanine, we have shown that the proteinase becomes partially unfolded during serpin inhibition. The tryptophan free variant, alpha(1)-antitrypsin((FF)), is fully active as an inhibitor of thrombin. Thrombin has a fluorescence emission maximum of 340 nm which blue shifts to 346 nm, concomitant with a 40% increase in intensity, upon formation of the serpin-proteinase complex indicative of substantial conformational change within the proteinase. Stopped-flow analysis of the fluorescence changes within the proteinase indicated a two-step mechanism. A fast bimolecular reaction with a rate constant of 2.8x10(6) M(-1) s(-1) is followed by a slow unimolecular process with a rate of 0.26 s(-1) that is independent of concentration. We propose that the first rate is formation of an initial complex which is then followed by a slower process involving the partial unfolding of the proteinase during its translocation to the opposite pole of the serpin.

Prevention of polymerization of M and Z alpha1-Antitrypsin (alpha1-AT) with trimethylamine N-oxide. Implications for the treatment of alpha1-at deficiency.

alpha1-Antitrypsin (alpha1-AT) is the most abundant circulating proteinase inhibitor. The Z variant results in profound plasma deficiency as the mutant polymerizes within hepatocytes. The retained polymers are associated with cirrhosis, and the lack of circulating protein predisposes to early onset emphysema. We have investigated the role of the naturally occurring solute trimethylamine N-oxide (TMAO) in modulating the polymerization of normal M and disease-associated Z alpha1-AT. TMAO stabilized both M and Z alpha1-AT in an active conformation against heat-induced polymerization. Spectroscopic analysis demonstrated that this was due to inhibition of the conversion of the native state to a polymerogenic intermediate. However, TMAO did not aid the refolding of denatured alpha1-AT to a native conformation; instead, it enhanced polymerization. These data show that TMAO can be used to control the conformational transitions of folded alpha1-AT but that it is ineffective in promoting folding of the polypeptide chain within the secretory pathway.

Thrombin, a survival factor for cultured myoblasts.

Three members of the family of protease-activated receptors (PARs), PARs-1, -3 and -4, have been identified as thrombin receptors. PAR-1 is expressed by primary myoblast cultures, and expression is repressed once myoblasts fuse to form myotubes. The current study was undertaken to investigate the hypothesis that thrombin inhibits myoblast fusion. Primary rodent myoblast cultures were deprived of serum to promote myoblast fusion and then cultured in the presence or absence of thrombin. Thrombin inhibited myoblast fusion, but another notable effect was observed; 50% of control cells were apoptotic within 24 h of serum deprivation, whereas less than 15% of thrombin-treated cells showed signs of apoptosis. Proteolysis was required for the effect of thrombin, but no other serine protease tested mimicked the action of thrombin. Neither a PAR-1- nor a PAR-4-activating peptide inhibited apoptosis or fusion, and myoblast cultures were negative for PAR-3 expression. Myoblasts exposed to thrombin for 1 h and then changed to medium without thrombin accumulated apoptosis inhibitory activity in their medium over the subsequent 20 h. Thus the protective action of thrombin appears to be effected through cleavage of an unidentified thrombin receptor, leading to secretion of a downstream apoptosis inhibitory factor. These results demonstrate that thrombin functions as a survival factor for myoblasts and is likely to play an important role in muscle development and repair.