Latent alpha 1-Antichymotrypsin
A MOLECULAR EXPLANATION FOR THE INACTIVATION OF alpha 1-ANTICHYMOTRYPSIN IN CHRONIC BRONCHITIS AND EMPHYSEMA*

Wun-Shaing W. Chang and David A. LomasDagger

From the Departments of Medicine and Haematology, University of Cambridge, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH, United Kingdom

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

alpha 1-Antichymotrypsin is an acute phase protein that protects the tissues from damage by proteolytic enzymes, but previous studies have shown that alpha 1-antichymotrypsin within the lungs of patients with chronic bronchitis and emphysema is intact but inactive as an inhibitor. Ammonium sulfate fractionation followed by blue Sepharose and DNA-Sepharose chromatography was used to isolate small amounts of intact, monomeric but inactive alpha 1-antichymotrypsin from the plasma of 30 healthy blood donors. This species had a higher DNA binding affinity with more anodal electrophoretic mobility than native alpha 1-antichymotrypsin and was conformationally stable against thermal denaturation, 8 M urea, and 7 M guanidinium chloride. The protein was unable to accept synthetic reactive loop peptides, and the reactive loop was resistant to proteolytic cleavage at the P5-P4 bond but could be cleaved between P1' and P3'. These data suggest that this new alpha 1-antichymotrypsin species was in a conformation similar to those of the crystallographically determined latent serpins, plasminogen activator inhibitor-1 and antithrombin. alpha 1-Antichymotrypsin from lung lavage migrated with the same electrophoretic mobility as the putative latent alpha 1-antichymotrypsin, suggesting that this is the inactive conformation described previously in the lungs of patients with chronic bronchitis and emphysema. This conformational transition of alpha 1-antichymotrypsin, from an active to an inactive state, within the lung may play an important role in the pathogenesis of chronic lung disease.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Neutrophils migrate into the lungs by chemotaxis in response to an inflammatory stimulus. They then degranulate and release destructive proteolytic enzymes, which, if uncontrolled, cause tissue destruction and lung damage (1). In healthy individuals, the lung is protected by proteinase inhibitors of which the most important are members of the serpin1 superfamily typified by alpha 1-antitrypsin and alpha 1-antichymotrypsin (2-4). alpha 1-Antichymotrypsin is an acute phase, DNA-binding glycoprotein whose plasma concentration can increase rapidly within hours of tissue damage (5). It functions by forming stable equimolar complexes with several proteinases including cathepsin G, chymotrypsin and mast cell chymases (6-8).

Like other inhibitory serpins, the crystal structure of active recombinant alpha 1-antichymotrypsin (9) revealed a dominant A beta -sheet together with B and C beta -sheets, eight well defined alpha -helices, and a mobile solvent-exposed reactive loop2 containing the scissile P1-P1' reactive bond, which interacts with and inhibits the cognate proteinase. The mobility of the reactive loop of alpha 1-antichymotrypsin and other inhibitory serpins is illustrated by proteolytically modified structures in which cleavage at the reactive site results in a marked and irreversible conformational change, with the N-terminal portion of the loop being completely inserted into the middle of the A-sheet as strand 4A. The once connected P1 and P1' residues in this conformation are widely separated by approximately 70 Å (11, 12). In some other serpins, the intact reactive loop can also be stably incorporated into the A-sheet in the absence of proteolytic cleavage to form a latent conformation that shares high thermostability and immunological epitopes with the cleaved species (13). This conformation is the structural basis for the latency of plasminogen activator inhibitor-1 (14), and we have recently demonstrated the existence of a similar conformation in antithrombin and alpha 1-antitrypsin (15-17). Initially, the term "latency" was used, since the inhibitory activity of these intact inactive serpins could be regained through a cycle of denaturation and renaturation (17-21). However, it is now broadly used to represent intact serpin species that are structurally similar to those of latent plasminogen activator inhibitor-1 (14) and antithrombin (15). The biological significance of the latent species is unclear, but the loss of activity due to the deformation of their reactive loop would obviously damage the physiological balance between the inhibitors and their target proteinases.

Over a decade ago, studies on bronchoalveolar lavage fluid from patients with chronic obstructive bronchitis and emphysema suggested that the majority of alpha 1-antichymotrypsin in the human lung is intact but inactive as an inhibitor of chymotrypsin-like enzymes (22). The cause for this has remained unclear. We report here the isolation of this unusual, inactive alpha 1-antichymotrypsin species as a minor component of healthy human plasma. The novel alpha 1-antichymotrypsin has high DNA binding affinity and physical properties similar to the latent form of other inhibitory serpins. This species had an electrophoretic mobility similar to that of a conformation of alpha 1-antichymotrypsin in bronchoalveolar lavage, and it provides a molecular explanation for the inactivation of alpha 1-antichymotrypsin in chronic bronchitis and emphysema.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Rabbit anti-human alpha 1-antichymotrypsin antibody and alkaline phosphatase-conjugated goat anti-rabbit immunoglobulins were from Dako A/S (Glostrup, Denmark). Sepharose CL-4B, blue Sepharose CL-6B, chromatography columns, and fraction collectors were from Pharmacia Biotech (St. Albans, UK). Amicon ultrafiltration YM-30 membranes, stirred cells, and Centricon-30 were from Millipore Ltd. (Watford, UK). The synthetic Ac-Ser-Glu-Ala-Ala-Ala-Ser-Thr-Ala-Val-Val-Ile-Ala-OH peptide was prepared by the Protein and Nucleic Acid Chemistry Facility, Department of Biochemistry, University of Cambridge on an Applied Biosystems peptide synthesizer (Warrington, UK). The modular mini-PROTEAN II electrophoresis system was from Bio-Rad Laboratories Ltd. (Hemel Hempstead, UK), the luminescence spectrometer was from Perkin-Elmer (Beaconsfield, UK), and all other reagents were from either Sigma Chemical Co. (Poole, UK) or BDH Chemicals Ltd. (Bristol, UK).

Preparation of the DNA-Sepharose Chromatography Column-- DNA-Sepharose resin was prepared by dissolving 0.3 g of highly polymerized DNA from calf thymus in 150 ml of distilled H2O and incubating overnight on a rotating wheel with 200 ml of packed volume of ice-cold CNBr-activated Sepharose CL-4B in 10 mM potassium phosphate, 500 mM KCl, pH 8, at 4 °C. Fifteen ml of ethanolamine hydrochloride was then added to DNA-Sepharose resin, and the mixture was incubated on a rotating wheel for at least 4 h at 4 °C. The resin was then transferred to a Hartley funnel and washed with 1 liter of 10 mM potassium phosphate, pH 8, followed by 1 liter of 1 M potassium phosphate, pH 8, 1 liter of 1 M KCl, 1 liter of distilled H2O, and finally 1 liter of 10 mM potassium phosphate, 50 mM KCl, pH 6.8, before packing into a Pharmacia XK16/40 chromatography column. The DNA-Sepharose column (70 ml of resin) was equilibrated with 300 ml of 10 mM potassium phosphate, 50 mM KCl, 5 mM EDTA, pH 6.8, at a flow rate of 1 ml/min.

Isolation of alpha 1-Antichymotrypsin from Plasma-- The preparation of human alpha 1-antichymotrypsin described here is a modification of the original method described by Travis et al. (23, 24) with all purification steps being performed at 4 °C. Two hundred-ml samples of plasma from 30 healthy blood donors were each mixed with 200 ml of saturated ammonium sulfate for 30 min and then centrifuged at 1500 × g for 30 min. The supernatant was pooled, and 160 g of solid ammonium sulfate per liter of supernatant was added, adjusted to pH 6.0 with concentrated H2SO4, and left to stir for 30 min. The solution was centrifuged at 1500 × g for 50 min, the supernatant was discarded, and the pellet was resuspended in a minimum volume of 30 mM sodium phosphate, pH 6.8, and dialyzed against 5 mM EDTA, pH 6.8 (3 × 5 liters) followed by 30 mM sodium phosphate, pH 6.8 (3 × 5 liters). The dialyzed solution was then applied to a blue Sepharose chromatography column (Pharmacia XK50/30 column, 900 ml of resin) at a flow rate of 3 ml/min. The column was washed with 30 mM sodium phosphate, pH 6.8, and alpha 1-antichymotrypsin was eluted with 30 mM sodium phosphate, 0.1 M NaCl, pH 6.8, and confirmed by rocket immunoelectrophoresis (25). The fractions containing alpha 1-antichymotrypsin were dialyzed against 10 mM potassium phosphate, 50 mM KCl, pH 6.8, and concentrated to approximately 10 ml before loading onto the DNA-Sepharose column at 1 ml/min. alpha 1-Antichymotrypsin was then eluted with a 600-ml linear gradient from 50-500 mM KCl. The fractions containing alpha 1-antichymotrypsin were again identified by rocket immunoelectrophoresis and dialyzed against 10 mM potassium phosphate, 50 mM KCl, 5 mM of EDTA, pH 6.8. alpha 1-Antichymotrypsin concentration was determined using the Lowry protein assay, and samples were snap frozen in liquid nitrogen and stored at -80 °C until use.

Polyacrylamide Gel Electrophoresis (PAGE)-- SDS-PAGE was performed using either 10% (w/v) or 7.5-15% (w/v) linear gradient acrylamide gels in 190 mM glycine, 25 mM Tris, pH 8.8, buffer with 0.1% (w/v) SDS (26). All samples were heated in boiling water for 5 min prior to electrophoresis. Nondenaturing PAGE was a modification of the method of Goldenberg (27) and was carried out on 7.5-15% (w/v) linear gradient gels using 53 mM Tris, 68 mM glycine, pH 8.9, as the cathode buffer and 100 mM Tris-HCl, pH 7.8, as the anode buffer.

Measurement of Conformational Stability-- The 20 mM Tris-HCl, pH 7.4, and 20 mM Tris-HCl, 7 M guanidinium chloride (GdmCl), pH 7.4, solutions were filtered through a 0.2-µm membrane prior to fluorescence assays. Each protein sample was incubated at 23 °C for 2 h at a final concentration of 10 µg/ml in 0.5 ml of 20 mM Tris-HCl, pH 7.4, with a range of denaturant concentrations between 0 and 7 M GdmCl. Denaturation curves were then determined by luminescence spectrometry at a wave length of 300-360 nm with slit widths of 10 nm. The unfolded fractions were analyzed as described by Pace et al. (28). Transverse urea gradient gels with a 0-8 M urea gradient were prepared according to the method of Goldenberg (27) and Mast et al. (29). Forty µg of each protein sample was loaded in a single well, and electrophoresis was performed at 15 mA until the dye front reached the base of the gel. The proteins were visualized by staining with Coomassie Blue.

Measurement of Thermal Stability-- The thermostability of both active and inactive alpha 1-antichymotrypsin were determined by incubating the protein at 0.5 mg/ml in 10 mM potassium phosphate, 50 mM KCl, pH 6.8, at constant temperatures between 37 and 100 °C for 2 h followed by brief centrifugation and careful deceleration without braking (17). The solutions were then rapidly cooled on ice, and aliquots were removed for analysis by nondenaturing PAGE.

Immunoblotting-- Purified plasma alpha 1-antichymotrypsin species or lung lavage fluid were separated by SDS-PAGE or nondenaturing PAGE and then electroblotted onto nitrocellulose membrane. alpha 1-Antichymotrypsin was detected using a rabbit anti-human alpha 1-antichymotrypsin antibody and a second alkaline phosphatase-conjugated goat anti-rabbit antibody. The immunoblots were developed using the bromochloroindolyl phosphate/nitro blue tetrazolium substrate according to the method of Harlow and Lane (30).

Active Site Titration and Association Kinetics of alpha 1-Antichymotrypsin-- The activity of alpha 1-antichymotrypsin was determined against bovine alpha -chymotrypsin of known active site, and kinetic parameters for the interaction with bovine alpha -chymotrypsin and cathepsin G were determined at 37 °C as detailed previously (31, 32). All buffers for the cathepsin G kinetics contained 0.1% (v/v) Triton X-100 and the substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide was present at a concentration of 400 µM and 6 mM for bovine alpha -chymotrypsin and cathepsin G, respectively. The Km values for bovine alpha -chymotrypsin and cathepsin G with succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were 0.087 ± 0.007 (31) and 1.9 ± 0.23 mM, respectively (32).

Formation of Binary Complex of alpha 1-Antichymotrypsin with a Synthetic Reactive Loop Peptide-- Thirty µg of active or inactive alpha 1-antichymotrypsin was incubated at a final concentration of 0.5 mg/ml in 50 mM Tris, 50 mM NaCl, pH 7.4, with a 100-fold molar excess of the synthetic 12-mer antithrombin reactive loop peptide (Ac-SEAAASTAVVIA) at 37 °C for up to 48 h. Five µg of protein was removed at each time point, snap frozen in liquid nitrogen, and stored at -80 °C until required. The formation of protein-peptide binary complexes was then assessed by separating the proteins on nondenaturing PAGE as described above.

Analysis of the Conformation of the Reactive Loop by Limited Proteolysis-- Cleavage of alpha 1-antichymotrypsin by porcine pancreatic trypsin (33) was performed at a 100:1 (w/w) ratio of serpin to enzyme in 20 mM Tris, 20 mM CaCl2, pH 8.3, for 0, 30, 60, and 90 min at 37 °C. alpha 1-Antichymotrypsin was cleaved by Bitis arietans snake venom (33, 34) by incubation at a 20:1 (w/w) ratio of serpin to enzyme in 20 mM Tris-HCl, pH 7.4, for 0, 30, 60, and 90 min at 37 °C. The final concentration of alpha 1-antichymotrypsin in each reaction was 0.2 mg/ml. At each time point, 4 µg of alpha 1-antichymotrypsin was taken, and the cleavage reaction was terminated by heating at 100 °C for 10 min before separating the proteins on SDS-PAGE. At the end of the cleavage reaction, an aliquot of the cleaved alpha 1-antichymotrypsin was immediately snap frozen in liquid nitrogen and subjected to N-terminal sequencing.

Bronchoalveolar Lavage-- Lavage samples were obtained from 10 consecutive patients undergoing bronchoscopy for investigation of bronchogenic carcinoma. One hundred ml of normal saline was instilled in 20-ml aliquots into the lung opposite of that under investigation. The fluid was aspirated and stored on ice before being filtered through cotton gauze to remove mucus and centrifugation at 1500 × g for 10 min to remove the cell pellet. The supernatant was concentrated and then assayed by SDS-PAGE, nondenaturing PAGE, and immunoblotting to detect alpha 1-antichymotrypsin. This study was approved by the Local Research Ethics Committee, and all patients gave informed consent.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

alpha 1-Antichymotrypsin was isolated from the plasma of healthy blood donors by ammonium sulfate fractionation followed by blue Sepharose and DNA-Sepharose chromatography. Purification using the DNA-Sepharose column yielded less than 2 mg of protein but reproducibly (in 30 separate experiments) fractionated alpha 1-antichymotrypsin into two peaks, denoted peak 1 and peak 2, which eluted at KCl concentrations of approximately 185 and 255 mM, respectively (Fig. 1a). The protein yield from peak 2 was typically only 50-100 µg of protein, and multiple preparations were therefore required to fully characterize this conformation. Peak 1 eluted at a salt concentration similar to that of native alpha 1-antichymotrypsin, and peak 2 eluted at the same point as reactive loop-cleaved alpha 1-antichymotrypsin. The cleaved alpha 1-antichymotrypsin control was prepared by incubating native protein with B. arietans snake venom. HPLC purification and amino-terminal sequencing of the cleavage fragments confirmed that the venom cleaved at P1'-P2' (Ser-Ala) and P2'-P3' (Ala-Leu) of the reactive loop and the Asn8-Leu9 bond of the amino terminus. alpha 1-Antichymotrypsin polymers formed by heating native protein at 0.2 mg/ml at 60 °C for 2 h (Fig. 1b) did not bind to the column, suggesting that polymerization resulted in the obfuscation of some or all of the lysine residues at positions 210-212, 391, and 396 that constitute the DNA binding domain (35). Nondenaturing PAGE showed that both peak 1 and peak 2 alpha 1-antichymotrypsin species were monomeric and that peak 2 migrated more anodally than peak 1 with the same electrophoretic mobility as reactive loop-cleaved alpha 1-antichymotrypsin (Fig. 1b). Such a difference in electrophoretic mobility between peak 1 and peak 2 alpha 1-antichymotrypsin on nondenaturing PAGE did not result from amino-terminal heterogeneity, since both species had mixed N-terminal sequences of His-2-Pro-1-Asn1-Ser2-Pro3 or Asn1-Ser2-Pro3, in keeping with the findings of others (36, 37). The integrity of both peak 1 and peak 2 alpha 1-antichymotrypsin was confirmed for all preparations by SDS-PAGE in comparison with a reactive loop-cleaved control (Fig. 1c). In no case did peak 2 alpha 1-antichymotrypsin migrate with similar electrophoretic mobility to the cleaved protein. Finally, this conformation was not an artifact of plasma storage, since similar results were also obtained with fresh, nonfrozen plasma.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   DNA-Sepharose chromatography showing the separation of peak 1 and peak 2 alpha 1-antichymotrypsin isolated from human plasma. The proteins were loaded onto a DNA-Sepharose column in 10 mM potassium phosphate, 50 mM KCl, pH 6.8, and eluted with a linear gradient of 50-500 mM KCl (dotted line). a, two fractions of alpha 1-antichymotrypsin, denoted peak 1 and peak 2, were isolated from the plasma of healthy subjects. b, 7.5-15% (w/v) nondenaturing PAGE. Lane P, alpha 1-antichymotrypsin polymers prepared by heating native alpha 1-antichymotrypsin at 60 °C for 2 h; lane 1, peak 1 alpha 1-antichymotrypsin; lane 2, peak 2 alpha 1-antichymotrypsin; lane C, reactive loop-cleaved alpha 1-antichymotrypsin control. c, 10% (w/v) SDS-PAGE. Lane 1, peak 1 alpha 1-antichymotrypsin; lane 2, peak 2 alpha 1-antichymotrypsin; lane C, reactive loop-cleaved alpha 1-antichymotrypsin control. The cathode is at the top and the anode is at the bottom of the gels, and each lane contains 5 µg of protein.

Assessment of the Conformation of Peak 1 and 2 alpha 1-Antichymotrypsin-- Active site titration showed that peak 1 alpha 1-antichymotrypsin had a specific activity of 57-77% against bovine alpha -chymotrypsin, whereas the peak 2 species consistently showed <1% inhibition. Ultraviolet scans of peak 2 alpha 1-antichymotrypsin were similar to those of peak 1 protein, indicating that the loss of activity did not result from residual DNA binding from the purification protocol. Kinetic analysis of native alpha 1-antichymotrypsin, identical to that of peak 1 but purified on a higher capacity DNA-cellulose (32) rather than a DNA-Sepharose column, revealed an association rate constant (ka) of 5.0 ± 0.02 × 105 M-1 s-1 and a Ki value of 42.8 ± 0.2 pM with bovine alpha -chymotrypsin (n = 4 experiments) and a ka of 1.7 ± 0.2 × 105 M-1 s-1 and a Ki of 809 ± 39 pM against cathepsin G (n = 3 experiments). These results are similar to the values reported previously for recombinant alpha 1-antichymotrypsin (32, 38) with the association rate constant of plasma alpha 1-antichymotrypsin with cathepsin G being over 100-fold less than that determined by Beatty et al. (7). The assessment of the thermal stability of the two alpha 1-antichymotrypsin conformations from the DNA-Sepharose column showed that peak 1 alpha 1-antichymotrypsin formed high molecular mass polymers between 50 and 60 °C, but peak 2 and venom-cleaved alpha 1-antichymotrypsin remained stable and monomeric at temperatures up to 100 °C (Fig. 2). Similarly, peak 1 alpha 1-antichymotrypsin displayed the characteristic unfolding transition of the serpins (29) with increasing concentration of urea (Fig. 3a), whereas peak 2 and venom-cleaved alpha 1-antichymotrypsin were resistant to unfolding in up to 8 M urea (Fig. 3, b and c). The significant difference in stability between the active peak 1 and inactive peak 2 alpha 1-antichymotrypsin was clearly evident when an equal mixture of the two conformations was assessed on the same transverse urea gradient gel (Fig. 3d). This resistance to unfolding in high concentrations of urea, in the absence of reactive-loop cleavage, is characteristic of latent serpins (16, 17). One of the determinants of latency is the restoration of activity following denaturation with and refolding from GdmCl (17, 18), although the latent form of antithrombin appears to be resistant to refolding (16). Refolding of peak 2 alpha 1-antichymotrypsin with 6 M GdmCl did not restore inhibitory activity against bovine alpha -chymotrypsin, and fluorescence analysis was used to determine if this concentration of GdmCl unfolded the peak 2 protein. Peak alpha 1-antichymotrypsin unfolded at approximately 3 M GdmCl, but peak 2 alpha 1-antichymotrypsin was resistant to unfolding in 7 M GdmCl (Fig. 4), which explains the finding that higher concentrations of denaturant could not be used to refold and reactivate this thermostable conformation.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 2.   Assessment of the thermal stability of peak 1, peak 2, and reactive loop-cleaved alpha 1-antichymotrypsin. All samples were heated at 0.5 mg/ml for 2 h at the temperature indicated and then assessed by 7.5-15% (w/v) nondenaturing PAGE. Each lane contains 5 µg of protein except for samples on gel a heated at a temperature higher than 60 °C, where 10 µg of protein was loaded. The cathode is at the top and the anode is at the bottom of the gel. a, peak alpha 1-antichymotrypsin; b, peak 2 alpha 1-antichymotrypsin; c, venom-cleaved alpha 1-antichymotrypsin control.


View larger version (70K):
[in this window]
[in a new window]
 
Fig. 3.   7.5% (w/v) transverse urea gradient PAGE analysis of the unfolding of peak 1, peak 2, and reactive loop-cleaved alpha 1-antichymotrypsin. All panels contain 40 µg of protein unless otherwise stated. a, peak 1 alpha 1-antichymotrypsin; b, peak 2 alpha 1-antichymotrypsin; c, venom-cleaved peak 1 alpha 1-antichymotrypsin; d, mixture of peak 1 (40 µg) and peak 2 (40 µg) alpha 1-antichymotrypsin; e, heat-induced alpha 1-antichymotrypsin polymers. The urea gradient (0-8 M) runs from left to right, and the direction of protein migration is from the top (cathode) to the bottom (anode) of each gel.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 4.   Unfolding fluorescence of peak 1 and peak 2 alpha 1-antichymotrypsin with increasing concentrations of guanidinium chloride. The denaturing curves of peak 1 (bullet ) and peak 2 (triangle ) alpha 1-antichymotrypsin were measured by luminescence spectrometry, and the values of each unfolding transition were calculated using equations as described by Pace et al. (28).

The remarkable stability of peak 2 alpha 1-antichymotrypsin implies that the protein has been stabilized by the insertion of the reactive loop peptide into the A-sheet. This was probed with a synthetic Nalpha -acetyl 12-mer peptide representing residues P14-P3 of antithrombin (Ac-SEAAASTAVVIA), which is capable of forming a heterologous serpin-peptide binary complex by insertion into the A-sheet of inhibitory serpins (39). The acquisition of additional negative charge from the incorporated acetylated peptide results in greater anodal electrophoretic migration on nondenaturing PAGE. Peak 1 alpha 1-antichymotrypsin accepted the exogenous reactive loop peptide to form the characteristic binary complex (Fig. 5), whereas incubation with peak 2 alpha 1-antichymotrypsin resulted in no such electrophoretic shift, indicating that the A-sheet of peak 2 protein was already occupied. The conformation of the reactive loop in peak 2 alpha 1-antichymotrypsin was probed by limited proteolysis with porcine pancreatic trypsin, which cleaves alpha 1-antichymotrypsin at the P5-P4 (Lys-Ile) reactive loop bond (33). This proteinase cleaved peak 1 alpha 1-antichymotrypsin at the same position but failed to cleave peak 2 alpha 1-antichymotrypsin (Fig. 6a), suggesting that the loop is inaccessible in this species. If the loop adopts a latent configuration, it may be accessible to cleavage on the prime side of the reactive loop between the P1 bond and s1C. B. arietans snake venom cleaves alpha 1-antichymotrypsin at the P1'-P2' (Ser-Ala) and P2'-P3' (Ala-Leu) peptide bonds. Both peak 1 and peak 2 alpha 1-antichymotrypsin were cleaved at the P1'-P2' and P2'-P3' peptide bonds in a ratio of 1:2, respectively, following incubation with venom (Fig. 6b).


View larger version (87K):
[in this window]
[in a new window]
 
Fig. 5.   Interaction of peak 1 and peak 2 alpha 1-antichymotrypsin with an exogenous reactive loop peptide. A synthetic 12-mer antithrombin reactive loop peptide (Ac-SEAAASTAVVIA) was incubated with either peak 1 or peak 2 alpha 1-antichymotrypsin at a peptide:serpin molar ratio of 100:1 at 37 °C. The more anodal (lower) migration of the binary complex (BC) in this 7.5-15% (w/v) nondenaturing PAGE is due to the acquisition of additional negative charge from the incorporated acetylated peptide. Lanes 1-5 represent aliquots of the incubation of peak 1 alpha 1-antichymotrypsin with the peptide taken at 0, 6, 12, 24, and 48 h, respectively. Lanes 6-10 represent aliquots of the incubation of peak 2 alpha 1-antichymotrypsin with the peptide taken at 0, 6, 12, 24, and 48 h, respectively. Each lane contains 5 µg of protein.


View larger version (50K):
[in this window]
[in a new window]
 
Fig. 6.   Limited proteolysis of alpha 1-antichymotrypsin by porcine pancreatic trypsin and by snake venom. The proteins were examined on 7.5-15% (w/v) SDS-PAGE. a, cleavage of alpha 1-antichymotrypsin by porcine pancreatic trypsin at a serpin:enzyme ratio of 100:1 (w/w). Lanes 1-4, samples removed from the cleavage reaction of peak 1 alpha 1-antichymotrypsin at 0, 30, 60, and 90 min, respectively; lanes 5-8, samples removed from the cleavage reaction of peak alpha 1-antichymotrypsin at 0, 30, 60, and 90 min, respectively. b, cleavage of alpha 1-antichymotrypsin by B. arietans venom at a serpin:enzyme ratio of 20:1 (w/w). Lane 1, molecular weight markers; lanes 2-5, samples removed from the cleavage reaction of peak 1 alpha 1-antichymotrypsin at 0, 30, 60, and 90 min, respectively; lanes 6-9, samples removed from the cleavage reaction of peak 2 alpha 1-antichymotrypsin at 0, 30, 60, and 90 min, respectively. Lane 10, control peak 2 alpha 1-antichymotrypsin. Each lane contains 4 µg of protein.

Peak 2 alpha 1-antichymotrypsin has many of the properties of a latent serpin. Both alpha 1-antitrypsin and antithrombin can be induced to adopt a latent conformation following heating at 60-68 °C with stabilizing concentrations of sodium citrate (16-17). Peak 1 alpha 1-antichymotrypsin was therefore incubated with 0-0.8 M sodium citrate and heated at 65 °C for 12 h. The protein was dialyzed against 20 mM Tris-HCl, pH 7.4, and then assessed by nondenaturing PAGE. Prolonged incubation produced polymeric alpha 1-antichymotrypsin and a small amount of a new monomeric alpha 1-antichymotrypsin, which migrated more anodally on nondenaturing PAGE in a similar position to peak 2. This band was present in all lanes (even the control without citrate) and in retrospect was seen after the assessment of peak 1 alpha 1-antichymotrypsin in thermal stability assays (Fig. 2a). Assessment of the protein by SDS-PAGE showed no evidence of reactive loop cleavage. Repeated attempts to produce peak 2 alpha 1-antichymotrypsin by the prolonged incubation of peak 1 protein under more physiological conditions resulted in reactive loop cleavage.

Bronchoalveolar Lavage-- There were similarities between the characteristics of peak 2 alpha 1-antichymotrypsin and the observation by Berman et al. (22) that alpha 1-antichymotrypsin in lung lavage was monomeric and inactive. Bronchoalveolar lavage was obtained from 10 consecutive patients undergoing fiberoptic bronchoscopy for the investigation of bronchogenic carcinoma. The lavage was concentrated in an Amicon cell, and the conformation of alpha 1-antichymotrypsin was assessed by SDS- and nondenaturing PAGE followed by Western blot analysis (Fig. 7). Immunoblotting of SDS-PAGE for alpha 1-antichymotrypsin showed that alpha 1-antichymotrypsin from bronchoalveolar lavage was not cleaved at the reactive loop (Fig. 7b). Analysis of alpha 1-antichymotrypsin by nondenaturing PAGE revealed a band that migrated with the same electrophoretic mobility as peak 2 alpha 1-antichymotrypsin (Fig. 7a), making it likely that the conformation of alpha 1-antichymotrypsin in peak 2 is the same as the inactive species described previously (22) in the lungs of patients with chronic bronchitis and emphysema. The concentration of alpha 1-antichymotrypsin in lavage was too low to allow the isolation of peak 2 alpha 1-antichymotrypsin for further analysis.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 7.   Immunoblotting of bronchoalveolar lavage fluid. A representative bronchoalveolar lavage sample from a patient undergoing investigation for bronchogenic carcinoma is shown. The fluid was separated by 7.5-15% (w/v) nondenaturing PAGE and 10% (w/v) SDS-PAGE followed by transfer onto nitrocellulose membrane for immunoblotting. Fig. 7a, lanes 1 and 2, mixture of plasma peak 1 and peak 2 alpha 1-antichymotrypsin; lane 3, plasma peak 2 alpha 1-antichymotrypsin; lane 4, bronchoalveolar lavage sample in which the two different alpha 1-antichymotrypsin species were clearly defined. The upper band migrated with the same electrophoretic mobility as peak 1 alpha 1-antichymotrypsin and the lower band migrated with peak 2 alpha 1-antichymotrypsin. b, lane 1, plasma peak 1 alpha 1-antichymotrypsin; lanes 2 and 3, bronchoalveolar lavage samples showing the integrity of alpha 1-antichymotrypsin from lung lavage on SDS-PAGE. The cathode is at the top and the anode is at the bottom of the gels.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

alpha 1-Antichymotrypsin was first isolated from human serum in 1965, but the purification procedure was lengthy and complicated (40). Since then, several groups have attempted to simplify the isolation procedure using a variety of chromatographic techniques (6, 23, 24, 37, 38, 41, 42). The yield and quality of alpha 1-antichymotrypsin was diverse, and two groups reported unexplained additional alpha 1-antichymotrypsin bands. Laine and Hayem (42) consistently observed two bands of pure alpha 1-antichymotrypsin on analytical PAGE, especially if the protein had been lyophilized. Travis et al. (23) detected two well separated lines when they examined whole human plasma using an antiserum against alpha 1-antichymotrypsin. There was no explanation for these observations, but the authors speculated that a form of desialylated alpha 1-antichymotrypsin might exist in whole plasma. Berman et al. (22) found that alpha 1-antichymotrypsin in lung lavage from patients with chronic bronchitis and emphysema was intact but inactive against cognate proteinases. The mechanism of this inactivation was unclear. It was these studies and our recent description of latent alpha 1-antitrypsin and antithrombin (16, 17) that raised the possibility that alpha 1-antichymotrypsin may adopt a latent conformation in vivo.

Ammonium sulfate fractionation followed by blue Sepharose and DNA-Sepharose chromatography was used to purify two species of alpha 1-antichymotrypsin from human plasma. Sequence analysis revealed that both alpha 1-antichymotrypsin species contained the His-2-Pro-1-Asn1-Ser2-Pro3 and Asn1-Ser2-Pro3 isoforms that correspond to the amino-terminal microheterogeneity that has been described previously (36, 37). The physiological importance of these isoforms is unknown, but they are generally believed to be the result of signal peptidase cleavage or post-translational proteolysis (37). There are no differences in apparent molecular mass, charge microheterogeneity, or inhibitory activity between the two isoforms (37), and since they were found in both peaks from DNA-Sepharose chromatography, it is unlikely that they account for any of the differences between the two species of alpha 1-antichymotrypsin. Both of the alpha 1-antichymotrypsin species from DNA-Sepharose chromatography were intact and monomeric (Fig. 1), but whereas alpha 1-antichymotrypsin in the first peak was functional as an inhibitor of proteinases, the alpha 1-antichymotrypsin in peak 2 had no inhibitory activity. The relatively modest association rate constant for the interaction of alpha 1-antichymotrypsin with cathepsin G and the high Ki raises the possibility that cathepsin G may not be the primary target of this proteinase inhibitor.

Peak 1 alpha 1-antichymotrypsin readily formed high molecular mass polymers on heating at 50-60 °C (Fig. 2) and showed unfolding profiles in urea (Fig. 3) and GdmCl (Fig. 4) that are characteristic of inhibitory members of the serpin superfamily. Similarly, this protein accepted an exogenous reactive loop peptide, inferentially as s4A, to form a binary complex with a distinctive band shift on nondenaturing PAGE (Fig. 5). alpha 1-Antichymotrypsin from peak 2, like latent antithrombin and alpha 1-antitrypsin (16, 17), was resistant to conformational transitions induced by heating to 100 °C, 8 M urea, and 7 M GdmCl and failed to accept exogenous reactive loop peptides (Figs. 2-5). These results strongly suggest that peak 2 alpha 1-antichymotrypsin has been stabilized by the insertion of an extra strand into the A-sheet as s4A. This is recognized in the serpin superfamily following reactive loop cleavage (43), the formation of latent protein (15-17), and the annealing of reactive loop peptides in the formation of high molecular mass loop-sheet polymers (17, 44) or serpin-reactive loop peptide binary complexes (39, 43). Peak 2 alpha 1-antichymotrypsin was not polymeric as assessed by nondenaturing PAGE and was not cleaved at the reactive loop as assessed by SDS-PAGE (Fig. 1c). The protein migrated on the nondenaturing PAGE with the electrophoretic mobility of cleaved protein, making it unlikely that it contained an exogenous reactive loop peptide that had annealed to the A-sheet to form a binary complex. Thus, the small quantity of alpha 1-antichymotrypsin that was reproducibly isolated from the plasma of healthy blood donors was likely to be in the latent conformation.

The crystallographic structure of latent antithrombin (15) showed the N-terminal portion of the reactive loop (P14-P3) to be stably incorporated into the A-sheet as s4A and the C-terminal portion of the loop (P1-P12') to be externalized (Fig. 8). Porcine pancreatic trypsin cleaves native alpha 1-antichymotrypsin at the P5-P4 residues (33). This enzyme cleaved peak 1 alpha 1-antichymotrypsin at the P5-P4 bond, but no equivalent cleavage was found in the peak 2 species (Fig. 6a), consistent with the hypothesis that the N-terminal portion of its reactive loop is buried in the A-sheet. Snake venom was able to cleave peak 1 and peak 2 alpha 1-antichymotrypsin at both the P1'-P2' and P2'-P3' positions (Fig. 6b), in keeping with the model presented in Fig. 8 showing that the C-terminal region of the loop in latent alpha 1-antichymotrypsin is located on the outer aspect of the molecule and therefore accessible to proteolytic attack. This provides further evidence that the conformation of peak 2 alpha 1-antichymotrypsin differs significantly from the native species but closely resembles that of latent antithrombin and latent plasminogen activator inhibitor-1 (14, 15, 45).


View larger version (46K):
[in this window]
[in a new window]
 
Fig. 8.   Comparison of the x-ray structures of active native (left) and inactive latent (right) antithrombin. Two structures are shown from residues Arg47-Lys432; residues Asn396-Ala404 of latent antithrombin were modeled, since they were not visible in the original structure (15). The reactive loop (black) is fully inserted into the A beta -sheet in the latent conformation, rendering it inactive as an inhibitor, thermostable, and unreceptive to exogenous peptides. The P5-P4 bond is clearly accessible in the native structure but is buried within the bottom of the A-sheet in the latent species, making it inaccessible to limited proteolysis by porcine pancreatic trypsin. The P1'-P3' positions are still located on the outer aspect of both molecules, making them accessible to B. arietans venom cleavage. The transition to latency also perturbs the "gate" region (s3C and s4C and the turn between the two beta -strands) shown here by an arrow in the native conformation.

The formation of a latent serpin may be described as a two-step process: an initial rapid transition, including partial incorporation of the N-terminal portion of the reactive loop into the A-sheet with the concomitant release of strand 1C from the C-sheet (16), and a second, much slower conformational change allowing full insertion of the reactive loop into the A-sheet concurrent with sufficient position exchange between the reactive loop and s3C/s4C to stabilize the conformation (21, 46). This conformational transition is likely to be facilitated in alpha 1-antichymotrypsin, since it contains four extra residues on the P' side of the reactive loop (47). The crystal structure of latent and native antithrombin obtained from a loop-sheet dimer (15, 45) and shown in Fig. 8 allows a direct comparison of the structural perturbation that results from the conformational transition to latency. In addition to the striking changes in the conformation of the reactive loop, there are changes in the "gate" region comprising the residues of s3C and s4C and the turn between the two beta -strands (16, 46). The positive charges of Lys210-Lys211-Lys212 located on the s3C/s4C turn contribute to the DNA binding domain of alpha 1-antichymotrypsin (35), and it is plausible that destabilizing this region in latent alpha 1-antichymotrypsin would allow increased lysine contact with DNA and hence the increased binding affinity seen on elution from the DNA cellulose column. Similarly, the formation of high molecular mass polymers must obscure this region, abolishing the binding of this conformation to DNA. The function of the DNA binding domain of alpha 1-antichymotrypsin in vivo is unclear, but any biological role may be modulated by transition from the native to the latent or polymeric conformation.

Despite evidence that alpha 1-antichymotrypsin can inhibit several proteinases in vitro and that it is selectively concentrated and synthesized in the lung (48), its physiological role remains unclear. The removal of alpha 1-antichymotrypsin from bronchoalveolar lavage had little effect on the total inhibitory capacity, suggesting that the majority of lung alpha 1-antichymotrypsin is inactive as a proteinase inhibitor (22). Unlike alpha 1-antitrypsin, which has a P1 methionine residue (49), the P1 leucine of alpha 1-antichymotrypsin is resistant to oxidation and inactivation. Moreover, our data demonstrate that this inactive alpha 1-antichymotrypsin is intact and cannot be the product of indiscriminate proteolytic attack by nontarget enzymes or dissociation of proteinase-inhibitor complexes. Similarly the results are unlikely to be explained by aberrant glycosylation (48), since peak 1 and peak 2 alpha 1-antichymotrypsins from plasma and lavage have the same molecular mass on SDS-PAGE. The co-migration of bronchoalveolar lavage alpha 1-antichymotrypsin with latent peak 2 protein on nondenaturing PAGE (Fig. 7a) provides strong support for our hypothesis that lung alpha 1-antichymotrypsin may be inactivated by transition to the latent conformation. The identification of this form in the lungs of patients with chronic bronchitis and emphysema (22) implies that it may contribute to the pathogenesis of this disease.

    ACKNOWLEDGEMENTS

We are grateful to Prof. Robin Carrell (Department of Hematology, University of Cambridge) for helpful comments, Peter Elliott (Department of Hematology, University of Cambridge) for preparing Fig. 8, Dr. Stephen Bottomley (Department of Hematology, University of Cambridge) for advice on the fluorescence measurements, Dr. Len Packman (Department of Biochemistry, University of Cambridge) for peptide synthesis and N-terminal sequence analysis, and Dr. Clare LaRoche (Chest Medical Unit, Papworth NHS Trust) for providing the bronchoalveolar lavage.

    FOOTNOTES

* This work was supported by the Wellcome Trust (United Kingdom).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Haematology, University of Cambridge, MRC Centre, Hills Road, Cambridge CB2 2QH, UK. Tel.: 44 1223 336825; Fax: 44 1223 336827; E-mail: dal16{at}cam.ac.uk.

2 Residue numbers used for alpha 1-antichymotrypsin in this report are counted from asparagine. Thus, the normal amino-terminal sequence for alpha 1-antichymotrypsin is either His-2-Pro-1-Asn1-Ser2-Pro3 or Asn1-Ser2-Pro3. The reactive loop amino acids are numbered from the Leu358--Ser359 P1-P1' sessile bond, in keeping with the convention of Schechter and Berger (10). Residues to the amino terminus of this bond are labeled P2, P3, P4, etc., while those to the carboxyl terminus are termed P2', P3', P4', etc.

1 The abbreviations used are: serpin(s), serine proteinase inhibitor(s); PAGE, polyacrylamide gel electrophoresis; GdmCl, guanidinium chloride; HPLC, high performance liquid chromatography.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Calverley, P., and Pride, N. (1995) Chronic Obstructive Pulmonary Disease, Chapman & Hall, London
  2. Laurell, C. B., and Eriksson, S. G. (1963) Scand. J. Clin. Lab. Invest. 15, 132-140
  3. Stockley, R. A., and Burnett, D. (1992) in Biochemistry of Pulmonary Emphysema (Grassi, C., Travis, J., Casali, L., and Luisetti, M., eds), pp. 47-69, Springer-Verlag, London
  4. Travis, J., and Salvesen, G. S. (1983) Annu. Rev. Biochem. 52, 655-709[CrossRef][Medline] [Order article via Infotrieve]
  5. Aronsen, K. F., Ekelund, G., Kindmark, C. O., and Laurell, C. B. (1972) Scand. J. Clin. Lab. Invest. 29, (suppl.) 127-136
  6. Travis, J., Bowen, J., and Baugh, R. (1978) Biochemistry 17, 5651-5656[Medline] [Order article via Infotrieve]
  7. Beatty, K., Bieth, J., and Travis, J. (1980) J. Biol. Chem. 255, 3931-3934[Abstract/Free Full Text]
  8. Gaffar, S. A., Princler, G. L., McIntire, K. R., Braatz, J. A. (1980) J. Biol. Chem. 255, 8334-8339[Free Full Text]
  9. Wei, A., Rubin, H., Cooperman, B. S., Christianson, D. W. (1994) Nature Struct. Biol. 1, 251-258[Medline] [Order article via Infotrieve]
  10. Schechter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162[Medline] [Order article via Infotrieve]
  11. Loebermann, H., Tokuoka, R., Deisenhofer, J., and Huber, R. (1984) J. Mol. Biol. 177, 531-556[Medline] [Order article via Infotrieve]
  12. Baumann, U., Huber, R., Bode, W., Grosse, D., Lesjak, M., and Laurell, C. B. (1991) J. Mol. Biol. 218, 595-606[Medline] [Order article via Infotrieve]
  13. Munch, M., Heegaard, C., Jensen, P. H., Andreasen, P. A. (1991) FEBS Letts. 295, 102-106[CrossRef][Medline] [Order article via Infotrieve]
  14. Mottonen, J., Strand, A., Symersky, J., Sweet, R. M., Danley, D. E., Geoghegan, K. F., Gerard, R. D., Goldsmith, E. J. (1992) Nature 355, 270-273[CrossRef][Medline] [Order article via Infotrieve]
  15. Carrell, R. W., Stein, P. E., Fermi, G., and Wardell, M. R. (1994) Structure 2, 257-270[Abstract]
  16. Wardell, M. R., Chang, W. S. W., Bruce, D., Skinner, R., Lesk, A. M., Carrell, R. W. (1997) Biochemistry 36, 13133-13142[CrossRef][Medline] [Order article via Infotrieve]
  17. Lomas, D. A., Elliott, P. R., Chang, W.-S. W., Wardell, M. R., Carrell, R. W. (1995) J. Biol. Chem. 270, 5282-5288[Abstract/Free Full Text]
  18. Hekman, C. M., and Loskutoff, D. J. (1985) J. Biol. Chem. 260, 11581-11587[Abstract/Free Full Text]
  19. Levin, E. G., and Santell, L. (1987) Blood 70, 1090-1098[Abstract]
  20. Lawrence, D., Strandberg, L., Grundstrom, T., and Ny, T. (1989) Eur. J. Biochem. 186, 523-533[Abstract]
  21. Lawrence, D. A., Strandberg, L., Ericson, J., and Ny, T. (1990) J. Biol. Chem. 265, 20293-20301[Abstract/Free Full Text]
  22. Berman, G., Afford, S. C., Burnett, D., and Stockley, R. A. (1986) J. Biol. Chem. 261, 14095-14099[Abstract/Free Full Text]
  23. Travis, J., Garner, D., and Bowen, J. (1978) Biochemistry 17, 5647-5651[Medline] [Order article via Infotrieve]
  24. Travis, J., and Morii, M. (1981) Methods Enzymol. 80, 765-771
  25. Weeke, B. (1973) Scand. J. Immunol. 2, Suppl. 1, 37-46
  26. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  27. Goldenberg, D. P. (1989) in Protein Structure: A Practical Approach (Creighton, T. E., ed), pp. 225-250, IRL Press, Oxford
  28. Pace, C. N., Shirley, B. A., and Thomson, J. A. (1989) in Protein Structure: A Practical Approach (Creighton, T. E., ed), pp. 311-330, IRL Press, Oxford
  29. Mast, A. E., Enghild, J. J., Pizzo, S. V., Salvesen, G. (1991) Biochemistry 30, 1723-1730[Medline] [Order article via Infotrieve]
  30. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, pp. 471-510, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
  31. Lomas, D. A., Evans, D. L., Stone, S. R., Chang, W. S. W., Carrell, R. W. (1993) Biochemistry 32, 500-508[Medline] [Order article via Infotrieve]
  32. Lomas, D. A., Stone, S. R., Llewellyn-Jones, C., Keogan, M.-T., Wang, Z., Rubin, H., Carrell, R. W., Stockley, R. A. (1995) J. Biol. Chem. 270, 23437-23443[Abstract/Free Full Text]
  33. Potempa, J., Fedak, D., Dubin, A., Mast, A., and Travis, J. (1991) J. Biol. Chem. 266, 21482-21487[Abstract/Free Full Text]
  34. Kress, L. F., and Hufnagel, M. E. (1984) Comp. Biochem. Physiol. 77B, 431-436
  35. Naidoo, N., Cooperman, B. S., Wang, Z., Liu, X., and Rubin, H. (1995) J. Biol. Chem. 270, 14548-14555[Abstract/Free Full Text]
  36. Morii, M., and Travis, J. (1983) Biochem. Biophys. Res. Commun. 111, 438-443[Medline] [Order article via Infotrieve]
  37. Lindmark, B., Lilja, H., Alm, R., and Eriksson, S. (1989) Biochim. Biophys. Acta 997, 90-95[Medline] [Order article via Infotrieve]
  38. Rubin, H., Wang, Z., Nickbarg, E. B., Mclarney, S., Naidoo, N., Schoenberger, O. L., Johnson, J. L., Cooperman, B. S. (1990) J. Biol. Chem. 265, 1199-1207[Abstract/Free Full Text]
  39. Chang, W. S. W., Wardell, M. R., Lomas, D. A., Carrell, R. W. (1996) Biochem. J. 314, 647-653[Medline] [Order article via Infotrieve]
  40. Heimburger, N., and Haupt, H. (1965) Clin. Chim. Acta 12, 116-118[Medline] [Order article via Infotrieve]
  41. Kilpatrick, L., Johnson, J. L., Nickbarg, E. B., Wang, Z. M., Clifford, T. F., Banach, M., Cooperman, B. S., Douglas, S. D., Rubin, H. (1991) J. Immunol. 146, 2388-2393[Abstract/Free Full Text]
  42. Laine, A., and Hayem, A. (1981) Biochim. Biophys. Acta 668, 429-438[Medline] [Order article via Infotrieve]
  43. Mast, A. E., Enghild, J. J., and Salvesen, G. (1992) Biochemistry 31, 2720-2728[Medline] [Order article via Infotrieve]
  44. Lomas, D. A., Elliott, P. R., Sidhar, S. K., Foreman, R. C., Finch, J. T., Cox, D. W., Whisstock, J. C., Carrell, R. W. (1995) J. Biol. Chem. 270, 16864-16870[Abstract/Free Full Text]
  45. Skinner, R., Abrahams, J. P., Whisstock, J. C., Lesk, A. M., Carrell, R. W., Wardell, M. R. (1997) J. Mol. Biol. 266, 601-609[CrossRef][Medline] [Order article via Infotrieve]
  46. Tucker, H. M., Mottonen, J., Goldsmith, E. J., Gerard, R. D. (1995) Nat. Struct. Biol. 2, 442-445[Medline] [Order article via Infotrieve]
  47. Huber, R., and Carrell, R. W. (1989) Biochemistry 28, 8951-8966[Medline] [Order article via Infotrieve]
  48. Cichy, J., Potempa, J., Chawla, R. K., Travis, J. (1995) J. Clin. Invest. 95, 2729-2733[Medline] [Order article via Infotrieve]
  49. Johnson, D., and Travis, J. (1979) J. Biol. Chem. 254, 4022-4026[Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.