Complement receptor Mac-1 is an adaptor for NB1 (CD177)-mediated PR3-ANCA neutrophil activation

The glycosylphosphatidylinositol (GPI)-anchored neutrophil-specific receptor NB1 (CD177) presents the autoantigen proteinase 3 (PR3) on the membrane of a neutrophil subset. PR3-ANCA-activated neutrophils participate in small-vessel vasculitis. Since NB1 lacks an intracellular domain, we characterized components of the NB1 signaling complex that are pivotal for neutrophil activation. PR3-ANCA resulted in degranulation and superoxide production in the mNB1(pos)/PR3(high) neutrophils, but not in the mNB1(neg)/PR3(low) subset, whereas MPO-ANCA and fMLP caused similar responses. The NB1 signaling complex that was precipitated from plasma membranes contained the transmembrane receptor Mac-1 (CD11b/CD18) as shown by MS/MS analysis and immunoblotting. NB1 co-precipitation was less for CD11a and not detectable for CD11c. NB1 showed direct protein-protein interactions with both CD11b and CD11a by surface plasmon resonance analysis (SPR). However, when these integrins were presented as heterodimeric transmembrane proteins on transfected cells, only CD11b/CD18 (Mac-1)-transfected cells adhered to immobilized NB1 protein. This adhesion was inhibited by mAb against NB1, CD11b, and CD18. NB1, PR3, and Mac-1 were located within lipid rafts. In addition, confocal microscopy showed the strongest NB1 co-localization with CD11b and CD18 on the neutrophil. Stimulation with NB1-activating mAb triggered degranulation and superoxide production in mNB1(pos)/mPR3(high) neutrophils, and this effect was reduced using blocking antibodies to CD11b. CD11b blockade also inhibited PR3-ANCA-induced neutrophil activation, even when β2-integrin ligand-dependent signals were omitted. We establish the pivotal role of the NB1-Mac-1 receptor interaction for PR3-ANCA-mediated neutrophil activation.     

Proteinase 3 contributes to transendothelial migration of NB1-positive neutrophils

Neutrophil transmigration requires the localization of neutrophils to endothelial cell junctions, in which receptor-ligand interactions and the action of serine proteases promote leukocyte diapedesis. NB1 (CD177) is a neutrophil-expressed surface molecule that has been reported to bind proteinase 3 (PR3), a serine protease released from activated neutrophils. PR3 has demonstrated proteolytic activity on a number of substrates, including extracellular matrix proteins, although its role in neutrophil transmigration is unknown. Recently, NB1 has been shown to be a heterophilic binding partner for the endothelial cell junctional protein, PECAM-1. Disrupting the interaction between NB1 and PECAM-1 significantly inhibits neutrophil transendothelial cell migration on endothelial cell monolayers. Because NB1 interacts with endothelial cell PECAM-1 at cell junctions where transmigration occurs, we considered that NB1-PR3 interactions may play a role in aiding neutrophil diapedesis. Blocking Abs targeting the heterophilic binding domain of PECAM-1 significantly inhibited transmigration of NB1-positive neutrophils through IL-1β-stimulated endothelial cell monolayers. PR3 expression and activity were significantly increased on NB1-positive neutrophils following transmigration, whereas neutrophils lacking NB1 demonstrated no increase in PR3. Finally, using selective serine protease inhibitors, we determined that PR3 activity facilitated transmigration of NB1-positive neutrophils under both static and flow conditions. These data demonstrate that PR3 contributes in the selective recruitment of the NB1-positive neutrophil population.     

Stat6-protease but not Stat5-protease is inhibited by an elastase inhibitor ONO-5046

A short isoform of Stat6 (65-kDa Stat6), a product of proteolytic processing by an undefined protease (Stat6-protease) in the nucleus, downregulates Stat6-mediated signaling in mast cells. Similarly, Stat5-mediated signaling is downregulated by Stat5-protease in myeloid progenitors. These proteases share a number of characteristics, including their nuclear localization and susceptibility to protease inhibitors. Here, we further investigated these Stat proteases. Interestingly, the activity of Stat6-protease but not of Stat5-protease was inhibited by ONO-5046, an elastase inhibitor that inhibits the activity of neutrophil elastase (NE) and NE-related protease proteinase 3 (PR3). Although both NE and PR3 were able to cleave Stat6 in vitro, the cleavage sites of Stat6 by NE or PR3 differed from that by Stat6-protease in mast cells. In addition, both NE and PR3 could also cleave Stat5, but they differed from Stat5-protease in myeloid progenitors. These results suggest that Stat6-protease may belong to the elastase family but differs from NE or PR3.     

Neutrophil serine proteinases activate human nonepithelial cells to produce inflammatory cytokines through protease-activated receptor 2

Protease-activated receptors (PARs) compose a family of G protein-coupled receptors activated by proteolysis with exposure of their tethered ligand. Recently, we reported that a neutrophil-derived serine proteinase, proteinase 3 (PR3), activated human oral epithelial cells through PAR-2. The present study examined whether other neutrophil serine proteinases, human leukocyte elastase (HLE), and cathepsin G (Cat G) activate nonepithelial cells, human gingival fibroblasts (HGF). HLE and Cat G as well as PR3 activated HGF to produce IL-8 and monocyte chemoattractant protein 1. Human oral epithelial cells but not HGF express mRNA and protein of secretory leukocyte protease inhibitor, an inhibitor of HLE and Cat G, and recombinant secretory leukocyte protease inhibitor clearly inhibited the activation of HGF induced by HLE and Cat G but not by PR3. HGF express PAR-1 and PAR-2 mRNA in the cells and the proteins on the cell surface. HLE and Cat G cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca(2+) mobilization and rendered cells refractory to subsequent stimulation with HLE and Cat G. The production of cytokine induced by HLE and Cat G and the PAR-2 agonist peptide was completely abolished by inhibition of phospholipase C. These findings suggest that neutrophil serine proteinases have equal ability to activate human nonepithelial cells through PAR-2 to produce inflammatory cytokines and may control a number of inflammatory processes such as periodontitis.     

The association of an elastase with amyloid fibrils

The fibrils of all systemic forms of amyloid (primary, AL; secondary, AA; and hereditary, AF) that had been isolated by the water extraction procedure demonstrated elastolytic enzyme activity when examined in a specific assay using tritiated elastin. The source of this fibril-bound enzyme activity was consistent with human neutrophil elastase (HNE), since it was readily extracted by high salt solutions and inhibited by an elastase-specific chloromethyl ketone inhibitor, human alpha-1-protease inhibitor or by an antibody specific for HNE. The presence of an elastase on the amyloid fibril may suggest physiologic mechanisms of amyloid precursor protein degradation.     

Discriminating between the activities of human neutrophil elastase and proteinase 3 using serpin-derived fluorogenic substrates

Human neutrophil elastase (HNE) has long been linked to the pathology of a variety of inflammatory diseases and therefore is a potential target for therapeutic intervention. At least two other serine proteases, proteinase 3 (Pr3) and cathepsin G, are stored within the same neutrophil primary granules as HNE and are released from the cell at the same time at inflammatory sites. HNE and Pr3 are structurally and functionally very similar, and no substrate is currently available that is preferentially cleaved by Pr3 rather than HNE. Discrimination between these two proteases is the first step in elucidating their relative contributions to the development and spread of inflammatory diseases. Therefore, we have prepared new fluorescent peptidyl substrates derived from natural target proteins of the serpin family. This was done because serpins are rapidly cleaved within their reactive site loop whether they act as protease substrates or inhibitors. The hydrolysis of peptide substrates reflects the specificity of the parent serpin including those from alpha-1-protease inhibitor and monocyte neutrophil elastase inhibitor, two potent inhibitors of elastase and Pr3. More specific substrates for these proteases were derived from the reactive site loop of plasminogen activator inhibitor 1, proteinase inhibitors 6 and 9, and from the related viral cytokine response modifier A (CrmA). This improved specificity was obtained by using a cysteinyl residue at P1 for Pr3 and an Ile residue for HNE and because of occupation of protease S' subsites. These substrates enabled us to quantify nanomolar concentrations of HNE and Pr3 that were free in solution or bound at the neutrophil surface. As membrane-bound proteases resist inhibition by endogenous inhibitors, measuring their activity at the surface of neutrophils may be a great help in understanding their role during inflammation.     

Construction and expression of a recombinant DNA gene encoding a polyomavirus middle-size tumor antigen with the carboxyl terminus of the vesicular stomatitis virus glycoprotein G.

We constructed a molecular clone encoding the N-terminal 379 amino acids of the polyomavirus middle-size tumor antigen, followed by the C-terminal 60 amino acids of the vesicular stomatitis virus glycoprotein G. This hybrid gene contained the coding region for the C-terminal hydrophobic membrane-spanning domain of the G protein in place of the C-terminal hydrophobic domain of the middle-size tumor antigen. The hybrid gene was expressed in COS-1 cells under the control of the simian virus 40 late promoter. The hybrid protein was located in cell membranes and was associated with a tyrosine-specific protein kinase activity, as was the middle-size tumor antigen. Plasmids encoding the hybrid protein failed to transform mouse NIH 3T3 or rat F2408 cells.     

Computational prediction of the binding site of proteinase 3 to the plasma membrane

Proteinase 3 (PR3) is a neutrophil-derived serine proteinase localized within cytoplasmic granules which can be released upon activation. PR3 is exposed at the neutrophil plasma membrane where it can mediate proinflammatory effects. Moreover, PR3 membrane expression is of special relevance in patients with Wegener's granulomatosis, a systemic vasculitis presenting anticytoplasmic neutrophil autoantibodies (ANCA) against PR3, which can bind to PR3 expressed at the surface of neutrophils and amplify their activation state. Therefore, it is of special relevance to unravel the molecular mechanisms governing its association with the membrane to be able to modulate it. To this end, we performed molecular dynamics (MD) simulations of PR3 with the implicit membrane model IMM1-GC to identify its interfacial binding site (IBS). Both the energies and structures resulting from the MD suggest that PR3 associates strongly with anionic membranes. We observe a unique IBS consisting of five basic (R177, R186A, R186B, K187, R222) and six hydrophobic (F165, F166, F224, L223, F184, W218) amino acids. The basic residues provide the driving force to orient PR3 at the membrane surface, so that the hydrophobic residues can anchor into the hydrocarbon region. Energy decomposition and in silico mutations show that only a few residues account for the membrane association. Similar calculations with HNE suggest a different membrane-binding mechanism. Our results agree with previous experimental observations and this work predicts, for the first time, the structural determinants of the binding of PR3 to membranes.     

Rapid inactivation of alpha-1-proteinase inhibitor by neutrophil specific leukolysin/membrane-type matrix metalloproteinase 6

Leukolysin/MT6-MMP is a GPI-anchored matrix metalloproteinase (MMP) primarily expressed by neutrophils. It is stored in intracellular granules at resting state, but rapidly discharged upon stimulations into the extracellular milieu, presumably to promote tissue remodeling or destruction. The proteolytic targets for leukolysin at the inflammatory sites remain unknown. Here, we show that alpha-1-proteinase inhibitor, or alpha1-PI, a known protective shield against destructive serine proteinases, is a physiological target for leukolysin. We show that alpha1-PI failed to accumulate in media conditioned by cells co-expressing alpha1-PI and leukolysin. Purified leukolysin cleaves alpha1-PI efficiently at the Phe376Leu and Pro381Met bonds and the cleaved alpha1-PI lost its anti-proteolytic activity against human neutrophil elastase, cathepsin G (CatG) and proteinase 3 (PR3). In fact, leukolysin preferentially cleaves alpha1-PI when co-incubated with other extracellular molecules such as laminin and gelatin. Kinetically, leukolysin is more active than two known neutrophil MMPs, MMP8 and MMP9, in cleaving and inactivating alpha1-PI. Taken together, these results suggest that neutrophils may mediate tissue destruction by deploying leukolysin to weaken the alpha1-PI protective shield at inflammatory sites.     

Interaction of purified human proteinase 3 (PR3) with reconstituted lipid bilayers.

Proteinase 3 (PR3), the major target autoantigen in Wegener's granulomatosis is a serine proteinase that is normally stored intracellularly in the primary granules of quiescent neutrophils and monocytes. Upon cell activation, a significant portion of this antigen is detected on the cell surface membrane. The nature of the association of PR3 with the membrane and its functional significance are unknown. We investigated the interaction of purified human PR3 with mixtures of zwitterionic (dimyristoyl-L-alpha-phosphatidylcholine, DMPC) and anionic (dimyristoyl-L-alpha-phosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers using differential scanning calorimetry and lipid photolabeling, and measured the affinity of this interaction using spectrophotometry. Two other primary granule constituents, human neutrophil elastase (HNE) and myeloperoxidase (MPO) were investigated for comparison. In calorimetric assays, using lipid vesicles of mixed DMPC/DMPG, increasing PR3 concentrations (protein/lipid molar ratio from 0 to 1 : 110) induced a significant decrease of the main chain transition enthalpy and a shift in chain melting temperatures which is indicative of partial insertion of PR3 into the hydrophobic region of the lipid membranes. This was confirmed by hydrophobic photolabeling using liposomes containing trace amounts of the photoactivable [125I]-labeled phosphatidylcholine analog TID-PC/16. The molar affinity of PR3, HNE, and MPO to lipid vesicles of different DMPC/DMPG ratios was then determined by spectrophotometry. At a DMPC/DMPG ratio of 1 : 1, molar affinities of PR3, Kd = 4.5 +/- 0.3 microm; HNE, 14.5 +/- 1.2 microm; and MPO, 50 +/- 5 microm (n = 3) were estimated. The lipid-associated PR3 exhibited two-fold lower Vmax and Km values, and its enzyme activity was slightly more inhibited (Ki) by the natural alpha1-proteinase inhibitor (alpha1-PI) or an autoantibody to PR3.     

Functional significance of Asn-linked glycosylation of proteinase 3 for enzymatic activity, processing, targeting, and recognition by anti-neutrophil cytoplasmic antibodies

Proteinase 3 (PR3) is a neutral serine protease stored in neutrophil granules. It has substantial sequence homology with elastase, cathepsin G and azurocidin. PR3 is the target antigen for autoantibodies (ANCA) in Wegener's granulomatosis, a necrotizing vasculitis syndrome. ANCA have been implicated in the pathogenesis of this disease. PR3 has two potential Asn-linked glycosylation sites. This study was designed to determine the occupancy of these glycosylation sites, and to evaluate their effect on enzymatic function, intracellular processing, targeting to granules and recognition by ANCA. We found that glycosylation occurs at both sites in native neutrophil PR3 and in wild type recombinant PR3 (rPR3) expressed in HMC-1 cells. Using glycosylation deficient rPR3 mutants we found that glycosylation at Asn-147, but not at Asn-102, is critical for thermal stability, and for optimal hydrolytic activity of PR3. Efficient amino-terminal proteolytic processing of rPR3 is dependent on glycosylation at Asn-102. Targeting to granules is not dependent on glycosylation, but unglycosylated rPR3 gets secreted preferentially into media supernatants. Finally, a capture ELISA for ANCA detection, using rPR3 glycosylation variants as target antigens, reveals that in about 20% of patients, epitope recognition by ANCA is affected by the glycosylation status of PR3.     

Comparative properties of human alpha-1-proteinase inhibitor glycosylation variants

Variant forms of human alpha-1-proteinase inhibitor (alpha-1-PI), obtained by the treatment of human Hep G2 cells with specific inhibitors of glycosylation were tested for both inhibitory activity and heat stability. All were found to have the same second-order association rate with human neutrophil elastase, indicating a lack of importance of the carbohydrate moiety. In contrast, incompletely glycosylated forms of alpha-1-PI were found to be heat sensitive relative to the mature protein, suggesting a role for carbohydrate in protein stabilization.     

Proteinase 3, the autoantigen in granulomatosis with polyangiitis, associates with calreticulin on apoptotic neutrophils, impairs macrophage phagocytosis, and promotes inflammation

Proteinase 3 (PR3) is the target of anti-neutrophil cytoplasm Abs in granulomatosis with polyangiitis, a form of systemic vasculitis. Upon neutrophil apoptosis, PR3 is coexternalized with phosphatidylserine and impaired macrophage phagocytosis. Calreticulin (CRT), a protein involved in apoptotic cell recognition, was found to be a new PR3 partner coexpressed with PR3 on the neutrophil plasma membrane during apoptosis, but not after degranulation. The association between PR3 and CRT was demonstrated in neutrophils by confocal microscopy and coimmunoprecipitation. Evidence for a direct interaction between PR3 and the globular domain of CRT, but not with its P domain, was provided by surface plasmon resonance spectroscopy. Phagocytosis of apoptotic neutrophils from healthy donors was decreased after blocking lipoprotein receptor-related protein (LRP), a CRT receptor on macrophages. In contrast, neutrophils from patients with granulomatosis with polyangiitis expressing high membrane PR3 levels showed a lower rate of phagocytosis than those from healthy controls not affected by anti-LRP, suggesting that the LRP-CRT pathway was disturbed by PR3-CRT association. Moreover, phagocytosis of apoptotic PR3-expressing cells potentiated proinflammatory cytokine in vitro by human monocyte-derived macrophages and in vivo by resident murine peritoneal macrophages, and diverted the anti-inflammatory response triggered by the phagocytosis of apoptotic cells after LPS challenge in thioglycolate-elicited murine macrophages. Therefore, membrane PR3 expressed on apoptotic neutrophils might amplify inflammation and promote autoimmunity by affecting the anti-inflammatory "reprogramming" of macrophages.     

Affinity chromatography of alpha-1-protease inhibitor using Sepharose-4B-bound anhydrochymotrypsin

Sepharose 4B-bound bovine anhydrochymotrypsin (AnhCT), a catalytically inactive form of chymotrypsin, was shown to be effective for retaining active alpha-1-protease inhibitor (alpha-1-PI, also alpha-1-antitrypsin) from human plasma, while showing no measurable affinity for oxidized or protease complexed alpha-1-PI, or for most other plasma proteins. alpha-1-PI eluted from this resin with 0.1 M chymostatin retained full activity against trypsin, chymotrypsin, and elastase. In addition to alpha-1-PI, AnhCT-Sepharose binds a limited number of other plasma proteins. Using monospecific antisera to plasma protease inhibitors, one of these proteins was identified as inter-alpha-trypsin inhibitor, and it was recoverable in active form. Therefore, an AnhCT-Sepharose 4B resin has been demonstrated to be of value for isolating active forms of alpha-1-PI from solutions, and may also be useful for the isolation of inter-alpha-trypsin inhibitor.     

Importance of a surface hydrophobic pocket on protein phosphatase-1 catalytic subunit in recognizing cellular regulators

Cellular functions of protein phosphatase-1 (PP1), a major eukaryotic serine/threonine phosphatase, are defined by the association of PP1 catalytic subunits with endogenous protein inhibitors and regulatory subunits. Many PP1 regulators share a consensus RVXF motif, which docks within a hydrophobic pocket on the surface of the PP1 catalytic subunit. Although these regulatory proteins also possess additional PP1-binding sites, mutations of the RVXF sequence established a key role of this PP1-binding sequence in the function of PP1 regulators. WT PP1alpha, the C-terminal truncated PP1alpha-(1-306), a chimeric PP1alpha containing C-terminal sequences from PP2A, another phosphatase, PP1alpha-(1-306) with the RVXF-binding pocket substitutions L289R, M290K, and C291R, and PP2A were analyzed for their regulation by several mammalian proteins. These studies established that modifications of the RVXF-binding pocket had modest effects on the catalytic activity of PP1, as judged by recognition of substrates and sensitivity to toxins. However, the selected modifications impaired the sensitivity of PP1 to the inhibitor proteins, inhibitor-1 and inhibitor-2. In addition, they impaired the ability of PP1 to bind neurabin-I, the neuronal regulatory subunit, and G(M), the skeletal muscle glycogen-targeting subunit. These data suggested that differences in RVXF interactions with the hydrophobic pocket dictate the affinity of PP1 for cellular regulators. Substitution of a distinct RVXF sequence in inhibitor-1 that enhanced its binding and potency as a PP1 inhibitor emphasized the importance of the RVXF sequence in defining the function of this and other PP1 regulators. Our studies suggest that the diversity of RVXF sequences provides for dynamic physiological regulation of PP1 functions in eukaryotic cells.     

Activation of human oral epithelial cells by neutrophil proteinase 3 through protease-activated receptor-2

Proteinase 3 (PR3), a 29-kDa serine proteinase secreted from activated neutrophils, also exists in a membrane-bound form, and is suggested to actively contribute to inflammatory processes. The present study focused on the mechanism by which PR3 activates human oral epithelial cells. PR3 activated the epithelial cells in culture to produce IL-8 and monocyte chemoattractant protein-1 and to express ICAM-1 in a dose- and time-dependent manner. Incubation of the epithelial cells for 24 h with PR3 resulted in a significant increase in the adhesion to neutrophils, which was reduced to baseline levels in the presence of anti-ICAM-1 mAb. Activation of the epithelial cells by PR3 was inhibited by serine proteinase inhibitors and serum. The epithelial cells strongly express protease-activated receptor (PAR)-1 and PAR-2 mRNA and weakly express PAR-3 mRNA. The expression of PAR-2 on the cell surface was promoted by PR3, and inhibited by cytochalasin B, but not by cycloheximide. PR3 cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca(2+) mobilization, and rendered cells refractory to subsequent stimulation with PR3 and vice versa. The production of cytokine induced by PR3 and the PAR-2 agonist peptide was completely abolished by a phospholipase C inhibitor. These findings suggest that neutrophil PR3 activates oral epithelial cells through G protein-coupled PAR-2 and actively participates in the process of inflammation such as periodontitis.     

Cloning, expression, purification, and biological activity of recombinant native and variant human alpha 1-antichymotrypsins

Human alpha 1-antichymotrypsin has been cloned, sequenced and expressed in Escherichia coli and recombinant protein as well as point-specific mutants have been purified and characterized. The corrected gene-deduced amino acid sequence has 45% overall identity with alpha 1-protease inhibitor, which is higher than the 42% previously reported (Chandra, T., Stackhouse, R., Kidd, V. J., Robson, J. H., and Woo, S. L. C. (1983) Biochemistry 22, 5055-5060). Recombinant antichymotrypsin (rACT) is similar to natural antichymotrypsin with respect to the specificity of its interactions with proteases. Its second-order rate constant for association with bovine chymotrypsin is 6-8 x 10(5) M-1 s-1, which is identical to that of the serum-derived inhibitor. Site-specific mutagenesis has been used to produce two variants of rACT in which the P1 position has been changed from leucine to either methionine (L358M-rACT) or arginine (L358R-rACT). L358M-rACT has a specificity of inhibitory activity toward serine proteases closely similar to that of native rACT. By contrast, the specificity of L358R-rACT is quite different from that of native rACT, most notably in efficiently inhibiting trypsin and human thrombin while showing a decreased ability to inhibit chymotrypsin.     

Evaluation of assays for detecting alpha-1-protease inhibitor during purification from rat serum

We purified the R1 alpha-1-protease inhibitor from rat serum and developed a convenient assay for its detection during purification procedures. Purification was accomplished by desalting, DEAE-Sephacel, zinc chelate, and reactive green-agarose columns. The resultant antiprotease had a molecular weight of 54,000 and inhibited elastase, chymotrypsin, and trypsin. By isoelectric focusing, five bands were produced with pI values from 4.3 to 4.7. Functional assays utilizing protease substrates imbedded in agarose plates were evaluated for the ability to distinguish the R1 alpha-1-protease inhibitor from the other serum antiproteases eluted in column chromatography fractions. This technique of screening for anti-protease activity was compared to conventional spectrophotometric methods and was found to correlate well when quantifying inhibition of elastase and chymotrypsin, but not trypsin. The presence of alpha-1-protease inhibitor was most reliably detected by testing for anti-elastase activity. Technician time and expense were saved by employing protease substrate plates to test chromatogrpahy fractions. This technique may facilitate purification of other protease inhibitors.     

Isolation of tri-antennary enriched and tri-antennary depleted alpha-1-protease inhibitor

Alpha-1-protease inhibitor, (alpha-1-PI), the major inhibitor of serine proteases in human plasma, has three asparagine-linked carbohydrate chains located at positions 46, 83 and 247. The protein has a microheterogeneity which is seen on isoelectric focusing and which is a result of whether the various carbohydrate chains are in bi- or tri-antennary forms. Tri-antennary enriched forms of alpha-1-PI are associated with inflammation. By using a combination of three methods, reductive salting out, Sepharose-bound Concanavalin A affinity chromatography, and Sepharose-bound anhydrochymotrypsin, biologically active alpha-1-PI was obtained in tri-antennary enriched and tri-antennary depleted forms. These preparations should be useful for studies on the physiological role of the carbohydrate moiety in alpha-1-PI.     

Polymorphic serum prealbumin (Pa) of pig, identified as an a!-protease inhibitor

Pig serum proteins were analysed by horizontal polyacrylamide gel electrophoresis, with a discontinuous buffer system (pH 9.0). A 12 % acrylamide concentration in the separation gel was used. Each of the two prealbumin (Pa) alleles gave rise to two closely migrating fractions. The polymorhic Pa was identified as an a,-protease inhibitor as the Pa fractions inhibited the esterolytic activity of both bovine trypsin and chymotrypsin. Therefore, it has been proposed that the locus symbol for this prealbumin be changed to Pi-1. The protease inhibitory spectra and electrophoretic mobility of the Pa (Pi-1) fractions suggested that this protein was probably the same as the pig serum a,-protease inhibitor described in some earlier studies and that it corresponds to human serum a,-protease inhibitor (Pi).