The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites.

MNEI (monocyte/neutrophil elastase inhibitor) is a 42 kDa serpin superfamily protein characterized initially as a fast-acting inhibitor of neutrophil elastase. Here we show that MNEI has a broader specificity, efficiently inhibiting proteases with elastase- and chymotrypsin-like specificities. Reaction of MNEI with neutrophil proteinase-3, an elastase-like protease, and porcine pancreatic elastase demonstrated rapid inhibition rate constants >10(7) M(-1) s(-1), similar to that observed for neutrophil elastase. Reactions of MNEI with chymotrypsin-like proteases were also rapid: cathepsin G from neutrophils (>10(6) M(-1) s(-1)), mast cell chymase (>10(5) M(-1) s(-1)), chymotrypsin (>10(6) M(-1) s(-1)), and prostate-specific antigen (PSA), which had the slowest rate constant at approximately 10(4) M(-1) s(-1). Inhibition of trypsin-like (plasmin, granzyme A, and thrombin) and caspase-like (granzyme B) serine proteases was not observed or highly inefficient (trypsin), nor was inhibition of proteases from the cysteine (caspase-1 and caspase-3) and metalloprotease (macrophage elastase, MMP-12) families. The stoichiometry of inhibition for all inhibitory reactions was near 1, and inhibitory complexes were resistant to dissociation by SDS, further indicating the specificity of MNEI for elastase- and chymotrypsin-like proteases. Determination of the reactive site of MNEI by N-terminal sequencing and mass analysis of reaction products identified two reactive sites, each with a different specificity. Cys(344), which corresponds to Met(358), the P(1) site of alpha1-antitrypsin, was the inhibitory site for elastase-like proteases and PSA, while the preceding residue, Phe(343), was the inhibitory site for chymotrypsin-like proteases. This study demonstrates that MNEI has two functional reactive sites corresponding to the predicted P(1) and P(2) positions of the reactive center loop. The data suggest that MNEI plays a regulatory role at extravascular sites to limit inflammatory damage due to proteases of cellular origin.

Inhibition of human chymase by 2-amino-3,1-benzoxazin-4-ones.

A series of 2-sec.amino-4H-3,1-benzoxazin-4-ones was evaluated as acyl-enzyme inhibitors of human recombinant chymase. The compounds were also assayed for inhibition of human cathepsin G, bovine chymotrypsin, and human leukocyte elastase. Introduction of an aromatic moiety into the 2-substituent resulted in strong inhibition of chymase, cathepsin G, and chymotrypsin. Extension of the N(Me)CH2Ph substituent by one methylene unit was unfavourable to inhibit these proteases. Towards chymase, 2-(N-benzyl-N-methylamino)-4H-3,1-benzoxazin-4-one (32) and 2-(N-benzyl-N-methylamino)-6-methyl-4H-3,1-benzoxazin-4-one (33) were found to exhibit Ki values of 11 and 17 nM, respectively, and form stable acyl-enzymes with half-lives of 53 and 25 min, respectively. Benzoxazinone 33 also inhibited the human chymase-catalyzed formation of angiotensin 11 from angiotensin I.

Trypsin-induced, neurokinin-mediated contraction of guinea pig bronchus.

Proteases may act as cell signaling molecules via protease-activated receptors (PARs). PAR1, PAR3, and PAR4, but not PAR2, are activated by thrombin, whereas trypsin can activate PAR2 and PAR4. In this study, trypsin (3-100 nM) evoked concentration-dependent contractions of guinea pig isolated bronchus, however, thrombin (3-300 nM) was a weak spasmogen. Neither the PAR2-activating peptide SLIGRL (100 microM) nor mast cell tryptase (100 nM), a trypsin-like protease known to activate PAR2, evoked contraction. A role for neurokinins in trypsin-induced contraction is suggested by our observation that contractions to trypsin were markedly attenuated in the presence of neurokinin receptor antagonists. Depletion of neurokinins in sensory nerves with capsaicin also markedly reduced the ability of trypsin to evoke contraction. In electrophysiological studies, trypsin did not evoke action potentials in C-fiber afferents whose receptive fields were located in the trachea or main bronchi. The results from this study support the hypothesis that trypsin activates a mechanism allowing for local release of sensory neurokinins from afferent C-fibers and that this release occurs independently of the sensory function of these nerves.

Human chymase inhibitors based on the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold.

A series of compounds that utilize the 1,2,5-thiadiazolidin-3-one 1,1 dioxide scaffold was synthesized and shown to be highly effective inhibitors of recombinant human skin chymase.

Highly efficient inhibition of human chymase by alpha(2)-macroglobulin.

The inhibition of human chymase by the protease inhibitor alpha(2)-macroglobulin (alpha2M) was investigated. Titration of chymase hydrolytic activity with purified alpha2M showed that approximately 1 mol of alpha2M tetramer inhibits 1 mol of chymase. Inhibition was associated with cleavage of the alpha2M bait region and formation of a 200-kDa covalent complex. NH(2)-terminal sequencing of chymase-treated alpha2M revealed cleavage at bonds Phe684-Tyr685 and Tyr685-Glu686 of the bait region. alpha2M pretreated with methylamine, an inactivator of alpha2M, did not inhibit chymase. The apparent second-order rate constant for inhibition (k(ass)) was 5 x 10(6) M(-1) s(-1), making alpha2M the most efficient natural protein protease inhibitor of chymase so far described. The k(ass) value for inhibition was decreased approximately 10-fold by addition of heparin, a glycosaminoglycan produced by mast cells that binds to chymase. Heparin did not change significantly the stoichiometry of inhibition or block covalent complex formation. These results indicate that alpha2M is an important inhibitor to consider in the regulation of human chymase.

The 2.2 A crystal structure of human chymase in complex with succinyl-Ala-Ala-Pro-Phe-chloromethylketone: structural explanation for its dipeptidyl carboxypeptidase specificity.

Human chymase (HC) is a chymotrypsin-like serine proteinase expressed by mast cells. The 2.2 A crystal structure of HC complexed to the peptidyl inhibitor, succinyl-Ala-Ala-Pro-Phe-chloromethylketone (CMK), was solved and refined to a crystallographic R-factor of 18.4 %. The HC structure exhibits the typical folding pattern of a chymotrypsin-like serine proteinase, and shows particularly similarity to rat chymase 2 (rat mast cell proteinase II) and human cathepsin G. The peptidyl-CMK inhibitor is covalently bound to the active-site residues Ser195 and His57; the peptidyl moiety juxtaposes the S1 entrance frame segment 214-217 by forming a short antiparallel beta-sheet. HC is a highly efficient angiotensin-converting enzyme. Modeling of the chymase-angiotensin I interaction guided by the geometry of the bound chloromethylketone inhibitor indicates that the extended substrate binding site contains features that may generate the dipeptidyl carboxypeptidase-like activity needed for efficient cleavage and activation of the hormone. The C-terminal carboxylate group of angiotensin I docked into the active-site cleft, with the last two residues extending beyond the active site, is perfectly localized to make a favorable hydrogen bond and salt bridge with the amide nitrogen of the Lys40-Phe41 peptide bond and with the epsilon-ammonium group of the Lys40 side-chain. This amide positioning is unique to the chymase-related proteinases, and only chymases from primates possess a Lys residue at position 40. Thus, the structure conveniently explains the preferred conversion of angiotensin I to angiotensin II by human chymase.

The reactive site loop of the serpin SCCA1 is essential for cysteine proteinase inhibition.

The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inactivate cysteine proteinases is unknown. Our previous studies suggest that SCCA1 inhibits papain-like cysteine proteinases in a manner similar to that observed for serpin-serine proteinase interactions. However, we could not preclude the possibility of an inhibitory mechanism that did not require the serpin RSL. To test this possibility, we employed site-directed mutagenesis to alter the different residues within the RSL. Mutations to either the hinge or the variable region of the RSL abolished inhibitory activity. Moreover, RSL swaps between SCCA1 and the nearly identical serpin, SCCA2 (an inhibitor of chymotrypsin-like serine proteinases), reversed their target specificities. Thus, there were no unique motifs within the framework of SCCA1 that independently accounted for cysteine proteinase inhibitory activity. Collectively, these data suggested that the sequence and mobility of the RSL of SCCA1 are essential for cysteine proteinase inhibition and that serpins are likely to utilize a common RSL-dependent mechanism to inhibit both serine and cysteine proteinases.

Spontaneous inactivation of human tryptase involves conformational changes consistent with conversion of the active site to a zymogen-like structure.

The conformational changes accompanying spontaneous inactivation and dextran sulfate (DS) mediated reactivation of the serine protease human tryptase were investigated by analysis of (i) intrinsic fluorescence, (ii) inhibitor binding, and (iii) catalytic efficiency. Spontaneous inactivation produced a marked decrease in fluorescence emission intensity that was reversed by the addition of DS. Fluorescence decreases at high (4.0 microM) and low (0.1 microM) tryptase concentrations were similar at early times and coincided with loss of enzymatic activity but deviated significantly from activity loss at later times by showing a difference in the extent of change. The fluorescence losses were best described by a two-step kinetic model in which the major decrease correlated to activity loss (t1/2 of 4.3 min in 0.2 M NaCl, pH 6.8, 30 degrees C) and was followed by a further decrease (t1/2 approximately 60 min) whose extent differed with tryptase concentration. The ability to bind the competitive inhibitor p-aminobenzamidine was reversibly lost upon spontaneous inactivation, providing evidence for conformational changes affecting the major substrate binding site (S1-pocket). Estimation of catalytic efficiency using an active site titrant showed that the specific activity of tryptase remained unchanged upon inactivation and reactivation. Return of enzymatic activity, intrinsic fluorescence, and the S1 pocket appeared to occur in the same time frame (t1/2 approximately 3 min). These studies indicate that spontaneous inactivation involves reversible changes which convert the active site to a nonfunctional state. The association of activity loss with an intrinsic fluorescence decrease and loss of the S1-pocket is consistent with the disruption of a critical ionic bond at the active site. Formation of this ionic bond is the basis of zymogen activation for the chymotrypsin family of serine proteases.

Reaction of mast cell proteases tryptase and chymase with protease activated receptors (PARs) on keratinocytes and fibroblasts.

Protease activated receptors (PARs) compose a family of G protein signal transduction receptors activated by proteolysis. In this study, the susceptibility of PARs expressed on human keratinocytes and dermal fibroblasts to the human mast cell proteases tryptase and chymase was evaluated. PAR activation was measured by monitoring cytosolic [Ca2+] in cells loaded with the fluorescent Ca2+ probe Fura-2. Tryptase produced transient cytosolic Ca2+ mobilization in keratinocytes, but not in fibroblasts. Ca2+ mobilization in keratinocytes required enzymatically active tryptase, demonstrated desensitization, and was blocked by pretreatment of cells with the PAR-2 peptide agonist SLIGKV, trypsin, or the phospholipase inhibitor U73122. Heparin, a GAG that binds to tryptase, stabilizing its functional form, also inhibited tryptase-induced Ca2+ mobilization. The maximal response elicited by tryptase was smaller than that observed upon treatment of keratinocytes with trypsin, a known activator of PAR-2, and keratinocytes made refractory to tryptase by pretreatment with the protease remained responsive to trypsin. Pretreatment of keratinocytes with thrombin, an activator of PAR-1 and -3 (thrombin receptors), had no detectable effect on the tryptase or trypsin responses. These data suggest that in keratinocytes tryptase may be activating a subpopulation of PAR-2 receptors. Treatment of keratinocytes or fibroblasts with human chymase did not produce Ca2+ mobilization, nor did it affect Ca2+ mobilization produced by trypsin. However, chymase pretreatment of fibroblasts rapidly inhibited the ability of these cells to respond to thrombin. Inhibition was dependent on chymase enzymatic activity and was not significantly affected by the presence of heparin. This finding is consistent with studies indicating that PAR-1 may be susceptible to proteases with chymotrypsin-like specificity. These results suggest that the proteases tryptase and chymase secreted from mast cells in skin may affect the behavior of surrounding cells by the hydrolysis of PARs expressed by these cells.

Recombinant expression of human mast cell proteases chymase and tryptase.

Expression of recombinant human chymase and tryptase was achieved in a baculovirus-insect cell system using a fusion protein construct. Recombinant baculovirus was produced with DNA coding for a NH2-ubiquitin-chymase-COOH or NH2-ubiquitin-tryptase-COOH fusion protein inserted immediately downstream of the signal sequence for the secreted envelope protein, glycoprotein 67. In each construct, the natural prepropeptide sequence of the protease was replaced by the amino acid sequence for the enterokinase cleavage site of trypsinogen. High Five insect cells infected with either of the modified baculovirus produced mg quantities of each fusion protein per liter of culture. Treatment of the chymase-fusion protein with enterokinase or the tryptase-fusion protein with enterokinase in the presence of a highly charged polysaccharide (dextran sulfate or heparin) produced enzymatically active proteases with properties of the native enzymes. A procedure for the purification of mg quantities of recombinant chymase from infected-cell medium is presented.

Role of the P6-P3' region of the serpin reactive loop in the formation and breakdown of the inhibitory complex.

Serpins have a large external peptide loop known as the reactive loop. Part of the reactive loop functions as the primary recognition site for target proteases; however, the complete role of the reactive loop in determining serpin specificity is unclear. In the current study, we investigated the reactive loop region that could potentially interact with the extended binding site of target proteases; the P6-P3' region. We utilized a reactive loop switching strategy to determine the extent to which the inhibitory activity of alpha-1-protease inhibitor (PI) against human neutrophil elastase (HNE) could be transferred to alpha-1-antichymotrypsin (ACT), a serpin that does not inhibit HNE. A series of ACT-PI chimeras were constructed in which segments of increasing length taken from the P6-P3' region of PI replaced the corresponding residues of ACT. The effectiveness of each chimera as an inhibitor of HNE was assessed by measuring (1) the rate of inhibitory complex formation and (2) the rate of complex breakdown (complex stability). Although all the ACT-PI chimeras were fully functional against chymotrypsin-like proteases, the series of chimeras showed no consistent progress toward the production of an inhibitor with the inhibitory properties of PI. The most rapid complex formation and most stable complexes were observed for chimeras with the P3-P1 residues of PI, whereas extending the replacement region to the P6 residue resulted in a considerable decrease in both inhibitory parameters. In order to study two additional features of the PI reactive loop that may play a role in the presentation of the P6-P3' region to HNE, we constructed variants that contained a P4' proline and deleted the P6'-P9' residues. Changes on the prime side appeared to have little effect on rates of inhibition or complex stability. Overall, even the most effective chimeras demonstrated an inhibition rate constant at least 60-fold less than that observed for PI inhibition of HNE and the most long lived chimera-HNE complexes broke down more rapidly than PI-HNE complexes. These results indicate that residues in the reactive loop region predicted to contact a specific target protease cannot fully transfer inhibitory activity from one serpin to another, suggesting that specific reactive loop-serpin body and serpin body-protease body interactions play a significant role in determining serpin inhibitory activity against target proteases.

Diverse effects of pH on the inhibition of human chymase by serpins.

Inhibition of human chymase by the serpins alpha1-antichymotrypsin (ACT) and alpha1-proteinase inhibitor (PI) at pH 8.0 produces a complex stable to dissociation by SDS/dithiothreitol and a second product, hydrolyzed/inactivated serpin. The first product is the presumed trapped acyl-enzyme complex typical of serpin inhibition, and the second is the result of a concurrent substrate-like reaction. As a result of the hydrolytic reaction, stoichiometries of inhibition (SI) appear greater than 1; values of 4 and 6.0 are observed for the chymase-ACT and -PI reactions. In this study the effect of pH on the inhibition rate constant (kinh) and the SI of each reaction were evaluated to better define the rate-limiting steps of the inhibitory and hydrolytic reaction pathways associated with chymase inhibition. Reactions were evaluated over a pH range to correlate kinh and SI with the ionizations (pK values of 7 and 9) that typically regulate serine protease catalytic activity. The results show that the effects of pH on SI and kinh differ for each inhibitor. On reducing the pH from 8.0 to 5.5, the chymase-ACT reaction exhibited a decrease in SI (to about 1) and little change in kinh, whereas the chymase-PI reaction revealed an increase in SI and a marked decrease in kinh. On increasing the pH from 8.0 to 10.0, the chymase-ACT reaction exhibited little change in SI and a marked decrease in kinh, whereas the chymase-PI reaction revealed a decrease in SI and a marked increase in kinh. Chymase catalytic properties determined for a peptide substrate were atypical over the high pH range exhibiting increases for kcat/Km and kcat and decreases for Km. This behavior suggests the presence of a high pH enzyme form with enhanced hydrolytic activity. From these results and others involving analyses of ACT/PI reactive loop chimeras and ACT point variants exhibiting a range of SI values, we suggest that the diverse pH effects on kinh and SI are caused largely by a difference in the abilities of ACT and PI to interact with low (catalytically inactive) and high (catalytically enhanced) pH forms of chymase. The constancy of kinh for the chymase-ACT reaction over the low pH range suggests that the rate-limiting step for inhibition is pH insensitive and not reflective of diminished chymase hydrolytic activity. Low pH did not appear to affect the rate of SDS-stable complex formation as complex accumulation, assessed qualitatively by SDS-PAGE, correlated with the loss of chymase enzymatic activity.

Chymase cleavage of stem cell factor yields a bioactive, soluble product.

Stem cell factor (SCF) is produced by stromal cells as a membrane-bound molecule, which may be proteolytically cleaved at a site close to the membrane to produce a soluble bioactive form. The proteases producing this cleavage are unknown. In this study, we demonstrate that human mast cell chymase, a chymotrypsin-like protease, cleaves SCF at a novel site. Cleavage is at the peptide bond between Phe-158 and Met-159, which are encoded by exon 6 of the SCF gene. This cleavage results in a soluble bioactive product that is 7 amino acids shorter at the C terminus than previously identified soluble SCF. This research shows the identification of a physiologically relevant enzyme that specifically cleaves SCF. Because mast cells express the KIT protein, the receptor for SCF, and respond to SCF by proliferation and degranulation, this observation identifies a possible feedback loop in which chymase released from mast cell secretory granules may solubilize SCF bound to the membrane of surrounding stromal cells. The liberated soluble SCF may in turn stimulate mast cell proliferation and differentiated functions; this loop could contribute to abnormal accumulations of mast cells in the skin and hyperpigmentation at sites of chronic cutaneous inflammation.

Soybean Bowman-Birk protease inhibitor is a highly effective inhibitor of human mast cell chymase.

Soybean Bowman-Birk protease inhibitor (BBI) is an inhibitor of serine proteases with two functional inhibitory domains of different specificities: one is specific for chymotrypsin-like proteases, the other for trypsin-like proteases. Chymase and tryptase are serine proteases which are stored in mast cell granules and released upon degranulation. This work investigated the inhibition of human chymase and tryptase by BBI. Active-site titration of human skin chymase by BBI demonstrated that BBI was a highly effective inhibitor of human chymase. Virtually stoichiometric inhibition of chymase by BBI was observed at 10 nM chymase. Kinetic studies of the inhibition reaction yielded an association rate constant of 4.0 x 10(5) M(-1) s(-1) and a dissociation rate constant of 1.7 x 10(-5) s(-1). From these two constants we estimate a K(i) of 50 pM. Chymase/BBI complexes did not dissociate in SDS-PAGE analyses under nonreducing conditions, consistent with the formation of a very tight complex with little tendency to dissociate. In contrast to chymase, human tryptase was not inhibited by BBI. These studies demonstrate that BBI is a good inhibitor of human chymase, exhibiting reaction properties better than physiological inhibitors described to date.

Cleavage of type I procollagen by human mast cell chymase initiates collagen fibril formation and generates a unique carboxyl-terminal propeptide.

The ability of human mast cell chymase and tryptase to process procollagen was examined. Purified human intestinal smooth muscle cell procollagen was incubated with human mast cell tryptase or human mast cell chymase. Purified chymase, but not tryptase, exhibited procollagen proteinase activity in the presence of EDTA. Addition of purified porcine heparin over a range of 0.1-100 microg/ml did not affect either the rate or the products of procollagen chymase cleavage. The cleavage site of chymase on the pro-alpha1(I) collagen carboxyl terminus was found to be in the propeptide region at Leu-1248-Ser-1249. Cleavage at this site suggested that the collagen products would form fibrils and confirmed the production of a unique carboxyl-terminal propeptide. Turbidometric fibril formation assay demonstrated de novo formation of chymase-generated collagen fibrils with characteristic lag, growth, and plateau phases. When observed by dark field microscopy, these fibrils were similar to fibrils formed by the action of procollagen proteinases. Thus, mast cell chymase, but not tryptase, exhibits procollagen peptidase-like activity as evidenced by its ability to process procollagen to fibril-forming collagen with concurrent formation of a unique carboxyl-terminal propeptide. These data demonstrate that mast cell chymase has a potential role in the regulation of collagen biosynthesis and in the pathogenesis of fibrosis.

Squamous cell carcinoma antigen 2 is a novel serpin that inhibits the chymotrypsin-like proteinases cathepsin G and mast cell chymase.

The squamous cell carcinoma antigen (SCCA) serves as a serological marker for more advanced squamous cell tumors. Molecular cloning of the SCCA genomic region revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2. Analysis of the primary amino acid sequences shows that both genes are members of the high molecular weight serpin superfamily of serine proteinase inhibitors. Although SCCA1 and SCCA2 are nearly identical in primary structure, the reactive site loop of each inhibitor suggests that they may differ in their specificity for target proteinases. SCCA1 has been shown to be effective against papain-like cysteine proteinases. The purpose of this study was to determine whether SCCA2 inhibited a different family of proteolytic enzymes. Using recombinant DNA techniques, we prepared a fusion protein of glutathione S-transferase and full-length SCCA2 . The recombinant SCCA2 was most effective against two chymotrypsin-like proteinases from inflammatory cells, but was ineffective against papain-like cysteine proteinases. Serpin-like inhibition was observed for both human neutrophil cathepsin G and human mast cell chymase. The second order rate constants for these associations were on the order of approximately 1 x 10(5) M-1 s-1 and approximately 3 x 10(4) M-1 s-1 for cathepsin G and mast cell chymase, respectively. Moreover, SCCA2 formed SDS-stable complexes with these proteinases at a stoichiometry of near 1:1. These data showed that SCCA2 is a novel inhibitor of two physiologically important chymotrypsin-like serine proteinases.

Distortion of the active site of chymotrypsin complexed with a serpin.

There is no complete understanding of how serine protease inhibitors of the serpin family inhibit their target enzymes. Structural and biochemical studies have suggested that serpins utilize a mechanism that is distinct from the standard mechanism of inhibition proposed for most small protein protease inhibitors. Proton nuclear magnetic resonance spectroscopy was used in the present study to demonstrate a fundamental difference in the atomic environment of the catalytic triad of enzyme in complex with serpins when compared to uncomplexed enzyme and enzyme in complex with standard mechanism inhibitors. This work demonstrates that the active site of chymotrypsin is distorted when complexed to a serpin and makes tenable a mechanism of inhibition in which the serpin induces a conformational change in the enzyme that dramatically reduces or completely abrogates the catalytic activity of the protease.

Inhibition of human mast cell chymase by secretory leukocyte proteinase inhibitor: enhancement of the interaction by heparin.

The inhibition of human chymase, a chymotrypsin-like proteinase stored in mast cell granules, by secretory leukocyte proteinase inhibitor (SLPI) is investigated in this study. SLPI is a serine proteinase inhibitor present in human mucus secretions and tissues. It binds heparin, a highly sulfated glycosaminoglycan also found in mast cell secretary granules, and the interaction increases its effectiveness as an inhibitor of neutrophil elastase. Analysis of the chymase-SL interaction by equilibrium and kinetic methods indicates that the inhibition of chymase results from the reversible formation of a stable 1:1 enzyme-inhibitor complex. The dissociation equilibrium constant (determined in reactions containing 0.18 M or 1.0M NaCl (pH 8.0, 25 degrees C) was 5 X 10(-8) and 2 x 10(-8) M, respectively. Addition of heparin to the low-salt reaction decreased the Ki approximately 10-fold to a value of 3 x 10(-9) M, making SLPI a more effective inhibitor of human chymase. The decrease was due primarily to an approximately 10-fold increase in the association rate constant (kass) from 2 X 10(4) to 3 X 10(5) M-1 s-1. The magnitudes of the rate and dissociation equilibrium constants indicate that SLPI has the potential to be a good chymase inhibitor in vivo, especially if chymase and heparin are released from mast cell granules simultaneously. The enhanced interaction in the presence of heparin supports the importance of this glycosaminoglycan to the inhibitory function of SLPI.

Demonstration of tryptase in bovine cutaneous and tumor mast cells.

We examined three tissue samples from each of four cows with non-lesional skin, tissue samples from a cow with multiple cutaneous mast cell tumors, and samples from another cow in which mast cells were infiltrating multiple lymphosarcomas of the skin, for the presence of tryptase and chymase by enzyme cytochemical and immunohistological methods. The enzyme activities of tryptase and chymase were tested using N-carbobenzoxy-glycilglycil-L-arginine-2-naphthylamide (Z-Gly-Gly-Arg-NA) and naphthol-AS-D-chloroacetate (N-AS-D-CA) as substrates, respectively. Tryptase reactivity could be demonstrated in frozen and Carnoy-fixed paraffin sections. Chymase reactivity was seen in neither frozen nor paraffin sections of formalin- or Carnoy-fixed skin tissues. Antibody linkage with a polyclonal rabbit anti-human skin tryptase antibody was highly specific in bovine normal cutaneous, infiltrating, and tumor mast cells. More than 90% of the tumor mast cells were distinctly tryptase-positive. With alcian blue, only slightly more than 10% of the mast cells stained clearly positive and with methylene blue hardly any staining of mast cell granules could be demonstrated. No antibody labeling of mast cell granules in any of the tissue sections was detected by the use of rabbit anti-dog chymase antiserum. These results indicate that there is a striking antigenic similarity of bovine tryptase to its canine and human equivalents. The demonstration of tryptase is an important tool in confirming the diagnosis of undifferentiated mast cell tumors. In contrast to other species, chymase appears to be completely absent in bovine skin mast cells.

Production of active recombinant human chymase from a construct containing the enterokinase cleavage site of trypsinogen in place of the native propeptide sequence.

Human chymase, a chymotrypsin-like proteinase found in mast cells, was produced in an enzymatically active recombinant form. The protein was expressed in Escherichia coli as part of an insoluble fusion protein which was solubilized and renatured. The structure of the fusion protein was NH2-ubiquitin-enterokinase cleavage site-chymase-COOH. The enterokinase cleavage site of trypsinogen replaced the native propeptide sequence of chymase, allowing for activation by a readily available proteinase (enterokinase) of known specificity. Characterization of refolded-activated recombinant chymase with substrates and inhibitors demonstrated properties identical to that of the native proteinase isolated from skin.