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PubMed1998

The induction of a prolonged increase in microvascular permeability by human mast cell chymase.

Chymase is a major constituent of the secretory granules of human mast cells, but little is known of the contribution of this serine proteinase in acute allergic reactions. We have purified chymase from human skin tissue, and have investigated its potential to induce microvascular leakage in vivo. Injection of chymase into the skin of guinea pigs provoked an increase in microvascular leakage within 20 min. Although skin reactions were smaller than those elicited with similar quantities of histamine at this time point, they were much longer-lived, and were still apparent 120 min following injection. Chymase induced microvascular leakage was reduced in the presence of soybean trypsin inhibitor, and abolished by heat inactivating the enzyme, indicating dependence on an intact catalytic site. Little evidence was found for synergistic interactions between chymase and either histamine or tryptase. Antihistamine pretreatment of animals did not reduce the magnitude of skin reactions to chymase suggesting that they were not mediated by histamine release. Chymase could contribute to increases in microvascular permeability following mast cell degranulation in allergic disease.

Animals↗
PubMed2002

Beneficial effects of cardiac chymase inhibition during the acute phase of myocardial infarction.

Recently, the presence of the chymase-dependent angiotensin (Ang) II-generating system in hamsters, dogs, monkeys, as well as human cardiovascular tissues has been identified. We have reported that the activation of cardiac chymase was more prominent than that of angiotensin converting enzyme (ACE) and that AT1 receptor antagonist treatment rather than ACE inhibitor treatment alone provided significant beneficial effects on cardiac function and survival after MI in hamsters. The aim of the present study was to determine whether this different effects between AT1 receptor antagonist and ACE inhibitor were due to the activation of cardiac chymase after MI in hamsters by using 4-[1-[[bis-(4-methyl-pheny)-methyl]-carbamoyl]-3-(2-ethoxy-benzyl)-4-oxo-azetidine-2-yloxy]-benzoic acid (BCEAB), a novel, orally active and specific chymase inhibitor. The ACE and chymase activities in the infarcted left ventricle were significantly increased 3 days after MI. BCEAB (100 mg/kg/day, p.o.) treatment starting 3 days before MI significantly suppressed the cardiac chymase activity, while it did not affect the plasma and cardiac ACE activities 3 days after MI. A significant improvement in hemodynamics (maximal negative and positive rates of pressure development; left ventricular systolic pressure) was observed for the treatment with BCEAB 3 days after MI. BCEAB (100 mg/kg/day, p.o.) treatment starting 3 days before MI significantly reduced the mortality rate during 14 days of observation following MI (vehicle, 61.1%, n = 18; BCEAB, 27.8%, n = 18; P < 0.05). These findings demonstrated for the first time that cardiac chymase participates directly in the pathophysiologic state after MI in hamsters.

Acute Disease↗
PubMed2003

Human chymase stimulates Ca2+ signaling in human polymorphonuclear cells.

Human chymase is known to function as a chemoattractant for human leukocytes. To investigate the mechanism of the chymase-induced cell migration, change in intracellular calcium concentration ([Ca(2+)]i) was examined in human polymorphonuclear (PMN) cells using Fluo-3 as a fluorescent Ca(2+) indicator. Treatment of PMN cells with human chymase caused [Ca(2+)]i elevation in a concentration-dependent manner. Depletion of extracellular Ca(2+) from the medium partially attenuated the chymase-induced [Ca(2+)]i increase, showing that both Ca(2+) influx and Ca(2+) release from internal stores might be involved in the [Ca(2+)]i response. Pretreatment of the cells with pertussis toxin completely blocked the chymase-induced [Ca(2+)]i signal, suggesting an involvement of G protein in the chymase-mediated [Ca(2+)]i elevation. The data in the present study raise the possibility that the chymase-induced cell migration is mediated by the [Ca(2+)]i elevation, which might be caused by stimulation of a G-protein-coupled receptor such as protease-activated receptors (PARs).

Calcium↗
PubMed2002

Lys40 but not Arg143 influences selectivity of angiotensin conversion by human alpha-chymase.

Human alpha-chymase is an efficient angiotensin (AT) converting enzyme, selectively hydrolyzing AT I at Phe8 to generate bioactive AT II, which can promote cardiac hypertrophy, vascular stenosis, and hypertension. Some related enzymes, such as rat beta-chymase 1, are much less selective, destroying AT by cleaving at Tyr4. Comparisons of chymase structure and activity led to speculation that interaction between AT and the side chain of Lys40 or Arg143 accounts for the human enzyme's marked preference for Phe8 over Tyr4. To test these hypotheses, we compared AT hydrolysis by wild-type chymase with that by mutants changing Lys40 or Arg143 to neutral residues. Lys40 was exchanged for alanine, the residue found in canine alpha- and rat beta-chymase 1, the latter being dramatically less selective for hydrolysis at Phe8. Arg143 was exchanged for glutamine found in rat beta-chymase 1. The Lys40Ala mutant is a dog-like enzyme retaining strong preference for Phe8 but with Tyr4 hydrolytic rates enhanced 16-fold compared to wild-type human enzyme. Thus, of 40 residues mismatched between dog and human enzymes, a single residue accounts for most of the difference in specificity between them. The Arg143Gln mutant, contrary to prediction, remains highly Phe8-selective. Therefore, Lys40, but not Arg143, contributes to human chymase's remarkable preference for AT II generation over destruction.

Angiotensins↗
PubMed1997

Crystal structure of phenylmethanesulfonyl fluoride-treated human chymase at 1.9 A.

The X-ray crystal structure of human chymase has been determined to 1.9 A resolution using molecular replacement methods. This first structure of human chymase is present as the Ser 195 ester of alpha-toluenesulfonic acid. The refined structure (Rcryst = 0.183) shows that the inhibitor phenyl moiety lies at the top of the major specificity pocket, S1, while the sulfur is covalently linked to Ser 195-O gamma. The sulfonyl oxygens interact with the oxyanion hole and with His 57-N delta 1. The presence of the inhibitor disturbs the usual gauche position of His 57 and forces it to the trans conformer. Though the primary binding pockets are similarly specific in chymase and chymotrypsin, examination of the extended substrate binding sites reveals the structural basis for chymase's greater discrimination in choosing substrates. The larger 30s loop and its proximity to the active site indicates that it contacts substrate residues C-terminal to the scissile bond. Modeling of substrate at the chymase active site suggests that binding energy may be gained by three main-chain hydrogen bonds provided by substrate residues P2' and P4' and that discriminating interactions with substrate side chains are also likely. The presence of Lys 40 in S1' of human chymase explains its preference for Asp/Glu at P1'. Moreover, the cationic nature of S1' provides a structural basis for human chymase's poor catalytic efficiency when angiotensin II is the substrate.

Binding Sites↗
PubMed2002

Rodent alpha-chymases are elastase-like proteases.

Although the alpha-chymases of primates and dogs are known as chymotrypsin-like proteases, the enzymatic properties of rodent alpha-chymases (rat mast cell protease 5/rMCP-5 and mouse mast cell protease 5/mMCP-5) have not been fully understood. We report that recombinant rMCP-5 and mMCP-5 are elastase-like proteases, not chymotrypsin-like proteases. An enzyme assay using chromogenic peptidyl substrates showed that mast cell protease-5s (MCP-5s) have a clear preference for small aliphatic amino acids (e.g. alanine, isoleucine, valine) in the P1 site of substrates. We used site-directed mutagenesis and computer modeling approaches to define the determinant residue for the substrate specificity of mMCP-5, and found that the mutant possessing a Gly substitution of the Val at position 216 (V216G) lost elastase-like activity but acquired chymase activity, suggesting that the Val216 dominantly restricts the substrate specificity of mMCP-5. Structural models of mMCP-5 and the V216G mutant based on the crystal structures of serine proteases (rMCP-2, human cathepsin G, and human chymase) revealed the active site differences that can account for the marked differences in substrate specificity of the two enzymes between elastase and chymase. These findings suggest that rodent alpha-chymases have unique biological activity different from the chymases of other species.

Amino Acid Sequence↗
PubMed2001

Isoform-selective upregulation of mast cell chymase in the development of skin fibrosis in scleroderma model mice.

The involvement of connective-tissue-type mast cells and chymase, a protease unique to their secretory granules, has been implicated in fibrotic diseases. To elucidate the role of chymase in fibroproliferative inflammation, in this study we examined the enzymatic activity and mRNA expression of chymase in the sclerotic skin of tight-skin mice; syngeneic Pallid mice served as the control. Dorsal skin specimens from mice aged 5, 10, and 20 wk were evaluated by morphometric and biochemical analyses. At ages 10 and 20 wk, the hydroxyproline concentration in tight-skin dermis was higher than that in Pallid. At any age, the subcutaneous fibrous layer was thicker in tight-skin than in Pallid. In accordance with these fibrous changes, both connective-tissue-type mast cell counts and chymase activity were higher in tight-skin skin than in Pallid skin up to 20 wk of age. Age-matched (10-wk-old) tight-skin and Pallid were quantified for their mRNA of connective-tissue-type mast-cell-specific chymase, mouse mast cell protease-4, by the competitive reverse transcriptase polymerase chain reaction technique, which revealed its higher level in tight-skin than Pallid. In contrast, the mRNA level of mouse mast cell protease-5, the chymase isoform of undifferentiated mast cells, in tight-skin skin was only a tenth that of mouse mast cell protease-4 and no different from the mouse mast cell protease-5 mRNA level of Pallid mice. An in situ hybridization study confirmed the higher expression of mouse mast cell protease-4 by connective-tissue-type mast cells in tight-skin skin than Pallid skin. These results strongly support the contention that the connective-tissue-type mast cell chymase plays a crucial role in fibroproliferative remodeling of the skin.

Animals↗
PubMedDate not supplied

Enzyme-histochemical detection of a chymase-like proteinase within bovine mucosal and connective tissue mast cells.

The presence of chymase-like proteinase in bovine mast cells was investigated by an enzyme-histochemical technique (naphthol-AS-D-chloroacetate as substrate) in normal skin, primary bronchus, lung and duodenum. The counts and distribution of chymase-positive and toluidine blue-positive mast cells were compared by means of successive staining. Mast cells with chymase-like activity were detected in all areas, but their proportion was greater in connective than mucosal tissues, with the exception of the skin. These results contrast with those obtained in rodents, in which chymase-like proteinases are detected in all tissues and also in all mast cells. Bovine mast cells are closer to those of human beings, in which chymase-containing mast cells predominate in connective tissues, including skin. The results suggest that more than one chymase subset is present, at least in duodenum. The possible occurrence of dual-specific chymase mast cells, as in other ruminants, is discussed.

Animals↗
PubMed1997

Dog mast cell alpha-chymase activates progelatinase B by cleaving the Phe88-Gln89 and Phe91-Glu92 bonds of the catalytic domain.

In prior work we showed that a metallogelatinase is secreted from dog mastocytoma cells and directly activated by exocytosed mast cell alpha-chymase. The current work identifies the protease as a canine homologue of progelatinase B (92-kDa gelatinase, MMP-9), determines the sites cleaved by alpha-chymase, and explores the regulation of gelatinase expression in mastocytoma cells. To obtain a cDNA encoding the complete sequence of mastocytoma gelatinase B, a 2. 3-kilobase clone encoding progelatinase was isolated from a BR mastocytoma library. The sequenced cDNA predicts a 704-amino acid protein 80% identical to human progelatinase B. Regions thought to be critical for active site latency, such as the Cys-containing propeptide sequence, PRCGVPD, and the catalytic domain sequence, HEFGHALGLDHSS, are entirely conserved. Cleavage of progelatinase B by purified dog alpha-chymase yielded an approximately 84-kDa product that contained two NH2-terminal amino acid sequences, QTFEGDLKXH and EGDLKXHHND, which correspond to residues 89-98 and 92-101 of the cDNA predicted sequence, respectively. Thus, alpha-chymase cleaves the catalytic domain of gelatinase B at the Phe88-Gln89 and Phe91-Glu92 bonds. Like BR cells, the C2 line of dog mastocytoma cells constitutively secrete progelatinase B which is activated by alpha-chymase. By contrast, non-chymase-producing C1 cells secrete a gelatinase B (which remains in its proform) only in response to 12-O-tetradecanoylphorbol-13-acetate. Whereas 12-O-tetradecanoylphorbol-13-acetate stimulation of BR cells produced a approximately 15-fold increase in gelatinase B mRNA expression, dexamethasone down-regulated its expression by approximately 5-fold. Thus, extracellular stimuli may regulate the amount of mast cell progelatinase B expressed by mast cells. These data further support a role for mast cell alpha-chymase in tissue remodeling involving gelatinase B-mediated degradation of matrix proteins.

Amino Acid Sequence↗
PubMed2003

Degradation of phospholipid transfer protein (PLTP) and PLTP-generated pre-beta-high density lipoprotein by mast cell chymase impairs high affinity efflux of cholesterol from macrophage foam cells.

Human atherosclerotic lesions contain mast cells filled with the neutral protease chymase. Here we studied the effect of human chymase on (i) phospholipid transfer protein (PLTP)-mediated phospholipid (PL) transfer activity, and (ii) the ability of PLTP to generate pre-beta-high density lipoprotein (HDL). Immunoblot analysis of PLTP after incubation with chymase for 6 h revealed, in addition to the original 80-kDa band, four specific proteolytic fragments of PLTP with approximate molecular masses of 70, 52, 48, and 31 kDa. This specific pattern of PLTP degradation remained stable for at least 24 h of incubation with chymase. Such proteolyzed PLTP had reduced ability (i) to transfer PL from liposome donor particles to acceptor HDL(3) particles, and (ii) to facilitate the formation of pre-beta-HDL. However, when PLTP was incubated with chymase in the presence of HDL(3), only one major cleavage product of PLTP (48 kDa) was generated, and PL transfer activity was almost fully preserved. Moreover, chymase effectively depleted the pre-beta-HDL particles generated from HDL(3) by PLTP and significantly inhibited the high affinity component of cholesterol efflux from macrophage foam cells. These results suggest that the mast cells in human atherosclerotic lesions, by secreting chymase, may prevent PLTP-dependent formation of pre-beta-HDL particles from HDL(3) and so impair the anti-atherogenic function of PLTP.

Biological Transport↗
PubMed1999

Mast cells in developing subepidermal bullous diseases: emphasis on tryptase, chymase and protease inhibitors.

The possible involvement of mast cell tryptase and chymase in subepidermal bullous diseases was studied enzyme-histochemically in specimens from erythematous and vesicular skin and from non-involved skin of patients with dermatitis herpetiformis, bullous pemphigoid, erythema multiforme, infective bullous eruption and linear IgA dermatosis. Patients with pemphigus were biopsied for comparison. The immunoreactivity of chymase inhibitors, alpha1-proteinase inhibitor (alpha1-PI) and alpha1-antichymotrypsin (alpha1-AC), in mast cells was demonstrated using the sequential double staining method. Tryptase-positive mast cells were unchanged or only slightly increased in number in erythematous lesions and slightly decreased in blistering skin compared with healthy-looking skin. Only occasionally were mast cells seen in apparent contact with the basement membrane zone. Chymase-positive mast cells and the chymase/tryptase ratio steadily decreased during the development of the lesions in each subepidermal bullous disease. The percentage of alpha1-PI+ and/or alpha1-AC+ mast cells increased simultaneously, which could explain the disappearance of chymase activity. Similar results were obtained regardless of the bullous disease. The results were also similar in pemphigus, which is an intraepidermal bullous disease. In conclusion, these results show significant alterations in mast cell chymase and protease inhibitors in a range of different bullous diseases, suggesting mast cell involvement. The apparent inactivation of chymase could be due to the action of chymase inhibitors detected in numerous mast cells. However, these alterations probably reflect general inflammation rather than a specific reaction in a certain bullous disease.

Adolescent↗
PubMed2003

ACE-versus chymase-dependent angiotensin II generation in human coronary arteries: a matter of efficiency?

OBJECTIVE: The objective of this study was to investigate ACE- and chymase-dependent angiotensin I-to-II conversion in human coronary arteries (HCAs). METHODS AND RESULTS: HCA rings were mounted in organ baths, and concentration-response curves to angiotensin II, angiotensin I, and the chymase-specific substrate Pro(11)-D-Ala(12)-angiotensin I (PA-angiotensin I) were constructed. All angiotensins displayed similar efficacy. For a given vasoconstriction, bath (but not interstitial) angiotensin II during angiotensin I and PA-angiotensin I was lower than during angiotensin II, indicating that interstitial (and not bath) angiotensin II determines vasoconstriction. PA-angiotensin I increased interstitial angiotensin II less efficiently than angiotensin I. Separate inhibition of ACE (with captopril) and chymase (with C41 or chymostatin) shifted the angiotensin I concentration-response curve approximately 5-fold to the right, whereas a 10-fold shift occurred during combined ACE and chymase inhibition. Chymostatin, but not captopril and/or C41, reduced bath angiotensin II and abolished PA-Ang I-induced vasoconstriction. Perfused HCA segments, exposed luminally or adventitially to angiotensin I, released angiotensin II into the luminal and adventitial fluid, respectively, and this release was blocked by chymostatin. CONCLUSIONS: Both ACE and chymase contribute to the generation of functionally active angiotensin II in HCAs. However, because angiotensin II loss in the organ bath is chymase-dependent, ACE-mediated conversion occurs more efficiently (ie, closer to AT(1) receptors) than chymase-mediated conversion.

Adolescent↗
PubMed2000

Increased chymase activity in internal thoracic artery of patients with hypercholesterolemia.

Apart from ACE, various angiotensin II (Ang II)-forming serine proteinases (eg, chymase, kallikrein, and cathepsin G) are known to exist in human tissues, but their clinical significance or the regulatory mechanisms that control their activities are not well established. A recent clinical study has shown that chymase activity was significantly increased in human atherosclerotic or aneurysmal aorta. The association between vascular Ang II-forming activities (AIIFAs) in the human internal thoracic artery (ITA) and various clinical parameters was studied with the use of ITAs obtained from 32 patients who underwent coronary artery bypass graft surgery. Total and ACE- and chymase-dependent AIIFAs in homogenates of ITAs were determined. Total AIIFA was 8.67+/-0.86 (nmol Ang II formed. min(-1). mg protein(-1) [U]), and approximately 95% of the activities were due to chymase. Serum total cholesterol level, but no other risk factors, significantly correlated with chymase- (r=0. 60, P<0.001) and ACE- (r=0.35, P<0.05) dependent AIIFAs, respectively. LDL cholesterol level was also correlated with chymase-dependent AIIFAs (r=0.47, P<0.05). Mast cells identified through the use of toluidine blue or immunohistochemical staining appeared in the adventitia but not in the intima or media of ITAs. Our results suggest that an increased plasma LDL cholesterol level may induce increased arterial chymase and ACE activity.

Aged↗
PubMed1988

Chymase and tryptase in dog mastocytoma cells: asynchronous expression as revealed by enzyme cytochemical staining.

Mast cell populations can be distinguished by differences in the content and substrate specificity of their two major cytoplasmic granule proteases, the chymases and the tryptases. To explore the origins of differences in the types of proteases present in mast cells, we used a double cytochemical staining technique to reveal both chymase and tryptase in cells from four lines of dog mast cell tumors containing both enzymes. We expected that if chymase and tryptase were expressed together during cell development the relative staining intensity of chymase compared to tryptase would be constant among different cells of each tumor. Instead, we found substantial variation in the relative intensity of chymase and tryptase staining among cells of a given mastocytoma line, each of which contained cells presumed to be monoclonal in origin but heterogeneous with respect to cell development. The overall staining intensity for chymase or tryptase correlated with the amount of protease activity in extracts of tumor homogenates. Staining specificity was established by use of selective inhibitors and competitive substrates and was tested on various types of dog cells obtained by bronchoalveolar lavage. The results suggest that active chymase and tryptase may be expressed differently during mast cell differentiation and support the possibility of a close developmental relationship between mast cells differing in protease phenotype. Moreover, the success of the staining procedures applied to mastocytoma cells suggests that they may be of general utility in phenotyping of mast cells according to the protease activities present in their granules.

Animals↗
PubMed2002

Time-dependent expression of chymase and angiotensin converting enzyme in the hamster heart under pressure overload.

The role of a dual angiotensin (Ang) II-forming pathway from the local renin angiotensin system (RAS) of the cardiac tissue was determined in a hamster model of cardiac hypertrophy. Time-dependent expressions of chymase and angiotensin converting enzyme (ACE) genes and their enzymes activities, and Ang II levels were measured in the hamster heart at 3 days, and at 4 and 8 weeks after pressure overload. Cardiac hypertrophy was induced by an operation to constrict the abdominal aorta. Compared to the sham-operated group, the cardiomyocyte diameters of hamster hearts at 3 days after overload underwent no obvious changes, while those at 4 and 8 weeks after overload increased markedly (p<0.01), and both transcriptional expressions of chymase and ACE genes gradually increased in the hamster hearts at 3 days, and at 4 and 8 weeks after overload, but the transcriptional expressions of angiotensin II type 1 receptor (AT1R) gene gradually decreased. Chymase and ACE activities (U/mg) (0.441+/-0.040 vs. 0.175+/-0.014, 0.446+/-0.036 vs. 0.160+/-0.016 and 0.522+/-0.014 vs. 0.148+/-0.038) (p<0.01) and (0.142+/-0.023 vs. 0.056+/-0.038, 0.317+/-0.017 vs. 0.079+/-0.016 and 0.466+/-0.010 vs. 0.098+/-0.003) (p<0.01), respectively and Ang II levels (pg/g) (98.7+/-4.5 vs. 71.2+/-4.9, 134.4+/-7.8 vs. 71.9+/-12.8 and 151.6+/-10.1 vs. 80.7+/-3.0) gradually increased in the hamster hearts, vs. sham treatment, respectively, at 3 days, and at 4 and 8 weeks after overload. However, the increases in chymase and ACE activities were much higher than those in their respective mRNA levels, and the levels of chymase activities were also higher than those of ACE activities during the development of cardiac hypertrophy. The results suggested that the increase in Ang II levels via the dual pathway of Ang II formation by chymase and ACE plays an important role in the cardiac hypertrophy of hamsters caused by the overloaded state. Importantly, in the non-hypertrophied hamster heart in the early stage after overload (at 3 days), chymase could be activated by mechanical stress in advance of an increase in its mRNA, and the Ang II level increased significantly.

Angiotensin II↗
PubMed2000

Identification of domains in apoA-I susceptible to proteolysis by mast cell chymase. Implications for HDL function.

When stimulated, rat serosal mast cells degranulate and secrete a cytoplasmic neutral protease, chymase. We studied the fragmentation of apolipoprotein (apo) A-I during proteolysis of HDL(3) by chymase, and examined how chymase-dependent proteolysis interfered with the binding of eight murine monoclonal antibodies (Mabs) against functional domains of apoA-I. Size exclusion chromatography of HDL(3) revealed that proteolysis for up to 24 h did not alter the integrity of the alpha-migrating HDL, whereas a minor peak containing particles of smaller size with prebeta mobility disappeared after as little as 15 min of incubation. At the same time, generation of a large (26 kDa) polypeptide containing the N-terminus of apoA-I was detected. This large fragment and other medium-sized fragments of apoA-I produced after prolonged treatment with chymase were found to be associated with the alphaHDL; meanwhile, small lipid-free peptides were rapidly produced. Incubation of HDL(3) with chymase inhibited binding of Mab A-I-9 (specific for prebeta(1)HDL) most rapidly (within 15 min) of the eight studied Mabs. This rapid loss of binding was paralleled by a similar reduction in the ability of HDL(3) to induce high-affinity efflux of cholesterol from macrophage foam cells, indicating that proteolysis had destroyed an epitope that is critical for this function. In sharp contrast, prolonged degradation of HDL(3) by chymase failed to reduce the ability of HDL(3) to activate LCAT, even though it led to modification of three epitopes in the central region of apoA-I that are involved in lecithin cholesterol acyltransferase (LCAT) activation. This differential sensitivity of the two key functions of HDL(3) to the proteolytic action of mast cell chymase is compatible with the notion that, in reverse cholesterol transport, intactness of apoA-I is essential for prebeta(1)HDL to promote the high-affinity efflux of cellular cholesterol, but not for the alpha-migrating HDL particles to activate LCAT.

Animals↗
PubMed2004

[Correlations between chymase activity, levels of tryptase, and eotaxin in the sputum of patients with chronic obstructive pulmonary disease and their clinical implications].

OBJECTIVE: To investigate the correlations between chymase activity, levels of tryptase, interleukin-8 (IL-8), eotaxin, neutrophil and eosinophil numbers in the sputum of elderly patients with chronic obstructive pulmonary disease (COPD) and their clinical implications. METHODS: A total of 73 elderly patients with COPD (21 severe, 21 moderate and 31 mild) were recruited into the study. IL-8 and eotaxin levels were detected with Sandwich enzyme-linked immunosorbent assay (ELISA) procedures and tryptase levels were measured with antibody against tryptase with Uni CAP system. Chymase activity was determined spectrophotometrically (410 nm) by the rate of hydrolysis of 0.7 mmol/L N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (SAAPP) in NaCl 1.5 mol/L and Tris 0.3 mol/L The specific activity of chymase was expressed as mU/ml. Actions of protease inhibitors on chymase activities were also examined with enzyme assay. RESULTS: (1) At acute attack stage, the sputum levels of tryptase of patients with severe, moderate and mild COPD were 284.0, 215.0 and 59.5 ng/L, respectively. The sputum levels of tryptase in the severe and moderate patients were greater than that in mild patients (P <0.01). The tryptase levels in the sputum of all patients were significantly decreased following treatment( 151.0, 92.0, 3.3 ng/L respectively; P <0. 01). At acute attack stage, the sputum levels of IL-8 and eotaxin were 1 299. 8,454. 9,78.7 ng/L; 22.7, 15.1, 7.4 ng/L, respectively. Severe and moderate-patients were greater than that of patients at mild stages. At convalescent stage,the sputum levels of IL-8 and eotaxin were 1 037. 5,326.6, 67.9 ng/L; 7.9, 6.3, 6.8 ng/L, respectively. The IL-8 and eotaxin levels in the sputum of severe and moderate patients were significantly decreased following treatment (P < 0.01). (2) The specific chymase activities of severe and moderate patients were higher than mild patients. Native protease inhibitors alpha1-antitrypsin (alpha1-AT) and soy bean trypsin inhibitor (SBTI) inhibited 83% and 89% enzymatic chymase activity, respectively. (3) The numbers of neutrophils and eosinophils in sputum were increased in patients with COPD at the acute stage compared with the mild convalescent stage. At all remission stages, the elevated numbers of cells were significantly reduced. (4) At acute attack stage of patients with COPD, there were correlations between the sputum levels of tryptase, IL-8, eotaxin, neutrophils and eosinophils; and there were correlations between the sputum levels of tryptase, chymase and neutrophils. CONCLUSION: COPD is not merely a neutrophil associated disease, but mast cells, eosinophils and their released mediators also actively contribute to the pathogenesis of the disease.

Aged↗
PubMed1991

Multiple determinants for the high substrate specificity of an angiotensin II-forming chymase from the human heart.

Human heart chymase, a chymotrypsin-like serine proteinase that hydrolyzes the Phe8-His9 bond in angiotensin I (Ang I) to yield the octapeptide hormone angiotensin II (Ang II) and His-Leu, is the most specific, efficient Ang II-forming enzyme described. Other mammalian chymases display a much broader substrate specificity. To better define its substrate specificity, we have mapped the extended substrate-binding site of human heart chymase using Ang I analogs. The enzyme has a preference for aromatic amino acids phenylalanine, tyrosine, and tryptophan at the P1 site. At the S2 subsite there is a significant preference for proline over hydrophobic or hydrophilic amino acids. There is no clear preference for hydrophobic or hydrophilic amino acids at the S'1 and S'2 subsites, but an Ang I analog containing a P'1 proline is not hydrolyzed and one with a P'2 proline is hydrolyzed poorly. An increasing reduction in reactivity occurs when the P position amino acids in Ang I are deleted sequentially from the N terminus. An increase or decrease in the length of the His-Leu leaving group also produces a marked decrease in reactivity. No single determinant in Ang I is preeminently required for efficient catalysis, but several factors acting synergistically appear to be important. Thus, we propose that ideal substrates for human heart chymase should contain the structure nXaa-Pro-[Phe, Tyr, or Trp]-Yaa-Yaa, where n greater than or equal to 6; Xaa = any amino acid; Yaa = any amino acid except proline. This structure exists in Ang I and neurotensin, both of which are good substrates for human heart chymase. These findings indicate that the selection of the scissile bond by the extended substrate-binding site of human heart chymase is more restricted than that in other chymases.

Amino Acid Sequence↗