Effect of plant neutrophil elastase inhibitor on leucocyte migration, adhesion and cytokine release in inflammatory conditions

The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (Kiapp 5.3 nM) and cathepsin G (Kiapp 160.0 nM), and the cysteine peptidase cathepsin L (Kiapp 0.2 nM). These three peptidases play important roles in the inflammatory process. We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyte–endothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by elisa.Pretreatment of the animals with BbCI (2.5 mg·kg−1), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1â or tumour necrosis factor-á, however, did not change.Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles.

Structure of cruzipain/cruzain inhibitors isolated from Bauhinia bauhinioides seeds.

The saline extract of Bauhinia bauhinioides dry seeds was shown to inhibit cruzipain, a cysteine proteinase from Trypanosoma cruzi. The inhibitory activity was assigned to a protein with 164 amino acid residues and molecular mass of 18 034 Da that was purified by chromatography on DEAE-Sephadex, trypsin-Sepharose (removal of trypsin inhibitors), Mono Q and a reversed-phase C4 column. The primary structure is homologous to other plant Kunitz-type inhibitors, but it lacks cysteine residues and therefore the disulfide bridges. No methionine residue was identified by amino acid sequencing. The inhibition of cruzipain fits into a slow-tight binding mechanism with a low dissociation constant (Ki 1.2 nM). The studied Bauhinia protein also inhibits cruzain (Ki 0.3 nM), a C-terminally truncated recombinant species of cruzipain. Cathepsin L, a cysteine proteinase with high homology to cruzipain, is also inhibited (Ki 0.22 nM), but not cathepsin B, papain, bromelain or ficin.

Primary sequence determination of a Kunitz inhibitor isolated from Delonix regia seeds.

A serine proteinase inhibitor was purified from Delonix regia seeds a Leguminosae tree of the Caesalpinioideae subfamily. The inhibitor named DrTI, inactivated trypsin and human plasma kallikrein with K(i )values 2.19x10(-8) M and 5.25 nM, respectively. Its analysis by SDS-PAGE 10-20% showed that the inhibitor is a protein with a single polypeptide chain of M(r) 22 h Da. The primary sequence of the inhibitor was determined by Edman degradation, thus indicating that it contained 185 amino acids and showed that it belongs to the Kunitz type family; however, its reactive site did not contain Arg or Lys at the putative reactive site (position 63, SbTI numbering) or it was displaced when compared to other Kunitz-type inhibitors.

Composition and antimicrobial activity of the essential oil of Baccharis notosergila.

The essential oil of the aerial parts of Baccharis notosergila was examined by GC and GC-MS. Thirty-one constituents were identified representing 96.4% and alpha-pinene, limonene, beta-caryophyllene, and spathulenol were found to be the major components. Furthermore, the oil was tested against eight gram-positive and -negative bacteria and it was found that they exhibited moderate antibacterial activity.

Human plasma kallikrein and tissue kallikrein binding to a substrate based on the reactive site of a factor Xa inhibitor isolated from Bauhinia ungulata seeds.

Kunitz type Bauhinia ungulata factor Xa inhibitor (BuXI) was purified from B. ungulata seeds. BuXI inactivates factor Xa and human plasma kallikrein (HuPK) with Ki values of 18.4 and 6.9 nM, respectively. However, Bauhinia variegata trypsin inhibitor (BvTI) which is 70% homologous to BuXI does not inhibit factor Xa and is less efficient on HuPK (Ki = 80 nM). The comparison between BuXI and BvTI reactive site structure indicates differences at Met59, Thr66 and Met67 residues. The hydrolysis rate of quenched fluorescence peptide substrates based on BuXI reactive site sequence, Abz-VMIAALPRTMFIQ-EDDnp (leading peptide), by HuPK and porcine pancreatic kallikrein (PoPK) is low, but hydrolysis is enhanced with Abz-VMIAALPRTMQ-EDDnp, derived from the leading peptide shortened by removing the dipeptide Phe-Ileu from the C-terminal portion, for HuPK (Km = 0.68 microM, k(cat)/Km = 1.3 x 10(6) M(-1) s(-1)), and the shorter substrate Abz-LPRTMQ-EDDnp is better for PoPK (Km = 0.66 microM, k(cat)/Km = 2.2 x 10(3) M(-1) s(-1)). The contribution of substrate methionine residues to HuPK and PoPK hydrolysis differs from that observed with factor Xa. The determined Km and k(cat) values suggest that the substrates interact with kallikreins the same as an enzyme and inhibitor interacts to form complexes.

Characterization of a tissue kallikrein inhibitor isolated from Bauhinia bauhinioides seeds: inhibition of the hydrolysis of kininogen related substrates.

Trypsin inhibitors were purified from a saline extract of Bauhinia bauhinioides seeds by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q ion-exchange chromatography or, alternatively, by affinity chromatography on trypsin-Sepharose. Both B. bauhinioides isolated inhibitors, BbTI-I and BbTI-II, inhibit trypsin being the dissociation constant 0.6 and 0.36 nM, respectively. BbTI-II only inhibits porcine pancreatic kallikrein hydrolysis of H-Pro-Phe-Arg-AMC (Ki 2.0 nM); the bradykinin-containing sequence LGMISLMKRPPGFSPFRSSRI-NH2 and the two kininogen related flanking quenched substrates Abz-MISLMKRP-EDDnp (Ki 2.0 nM) and Abz-FRSSRQ-EDDnp (Ki 2.5 nM).

The complete amino acid sequence of a trypsin inhibitor from Bauhinia variegata var. candida seeds.

Trypsin inhibitors of two varieties of Bauhinia variegata seeds have been isolated and characterized. Bauhinia variegata candida trypsin inhibitor (BvcTI) and B. variegata lilac trypsin inhibitor (BvlTI) are proteins with Mr of about 20,000 without free sulfhydryl groups. Amino acid analysis shows a high content of aspartic acid, glutamic acid, serine, and glycine, and a low content of histidine, tyrosine, methionine, and lysine in both inhibitors. Isoelectric focusing for both varieties detected three isoforms (pI 4.85, 5.00, and 5.15), which were resolved by HPLC procedure. The trypsin inhibitors show Ki values of 6.9 and 1.2 nM for BvcTI and BvlTI, respectively. The N-terminal sequences of the three trypsin inhibitor isoforms from both varieties of Bauhinia variegata and the complete amino acid sequence of B. variegata var. candida L. trypsin inhibitor isoform 3 (BvcTI-3) are presented. The sequences have been determined by automated Edman degradation of the reduced and carboxymethylated proteins of the peptides resulting from Staphylococcus aureus protease and trypsin digestion. BvcTI-3 is composed of 167 residues and has a calculated molecular mass of 18,529. Homology studies with other trypsin inhibitors show that BvcTI-3 belongs to the Kunitz family. The putative active site encompasses Arg (63)-Ile (64).

Purification and primary structure determination of a Bowman-Birk trypsin inhibitor from Torresea cearensis seeds.

A Bowman-Birk-type trypsin inhibitor (TcTI) was purified from seeds of Torresea cearensis, a Brazilian native tree of the Papilionoideae sub-family of Leguminosae. Three forms of the inhibitor were separated by anion exchange chromatography. The major form with 63 amino acids was entirely sequenced; it shows a high structural similarity to the Bowman-Birk inhibitors from other Leguminosae. The putative reactive sites of the inhibitor are a lysine residue at position 15 and a histidine at position 42 as identified by alignment to related inhibitors, direct chemical modification and specific enzymatic degradation. Immunoprecipitation with antibodies raised in rats is reduced significantly if TcTI is complexed with chymotrypsin and, to a lesser degree, if complexed with trypsin. TcTI forms a ternary complex with trypsin and chymotrypsin. The binary complexes with trypsin or chymotrypsin were isolated by gel filtration. Dissociation constants of the complexes with trypsin, plasmin, chymotrypsin, and factor XIIa are 1, 36, 50, 1450 nM, respectively; human plasma kallikrein, human factor Xa, porcine pancreatic kallikrein and bovine thrombin are not inhibited. TcTI prolongs blood clotting time of the contact phase activation pathway by inhibition of FXIIa.

Isolation and partial characterization of an endopeptidase from Enterolobium contortisiliquum seeds.

1. Aqueous extracts of Enterolobium contortisiliquum seeds contain an endopeptidase of M(r) 60,000 with specificity for basic amino acid residues. The enzyme was purified by chromatography on DEAE Sephadex, followed by gel filtration on Sephadex G-75 and affinity chromatography on Zinc-Sepharose. The overall purification was 300-fold and the yield about 46%. 2. The endopeptidase hydrolyzes benzoyl-arginine-p-nitroanilide (Bz-Arg-pNan) and acetyl-phenylalanine-arginine-p-nitroanilide (Ac-Phe-Arg-pNan) with Km 14.4 mM and 0.062 mM, respectively. Succinyl-phenylalanine-p-nitroanilide (Suc-Phe-pNan) and tosyl-arginine methyl ester (TAME) were not hydrolyzed. E. contortisiliquum endopeptidase also cleaves a seed protein of low molecular weight from the same E. contortisiliquum seeds, and converts Met-Lys-bradykinin into bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). 3. Metals (1.0 mM) such as Cr3+, Fe3+ and Zn2+ ions inactivate the enzyme when Bz-Arg-pNan was the substrate. Enzyme activity is abolished by EDTA but is partially restored by Cu2+, Al3+, Ba2+, Mn2+, Mg2+, Fe2+, Ca2+ and Co2+ ions. The endopeptidase is not inhibited by the previously purified E. contortisiliquum inhibitors of trypsin and cysteine proteinases, or by soybean trypsin inhibitor (Oliva et al. (1987). Brazilian Journal of Medical and Biological Research, 20:767-770).

Action of plant proteinase inhibitors on enzymes of the kallikrein kinin system.

Serine proteinase inhibitors, in the seeds of several Leguminosae from the Pantanal region (West Brazil), were studied using bovine trypsin, Factor XIIa and human plasma kallikrein. The inhibitors were purified from Enterolobium contortisiliquum (Mr = 23,000), Torresea cearensis (Mr = 13,000), Bauhinia bauhinioides (Mr = 20,000), Bauhinia mollis (Mr = 20,000) and Bauhinia pentandra (Mr = 20,000). E. contortisiliquum inhibitor inactivates all three enzymes, whereas the T. cearensis inhibitor inactivates trypsin and Factor XIIa, but does not affect plasma kallikrein. B. bauhinioides and B. pentrandra inhibitors, on the other hand, inactivate trypsin and plasma kallikrein but only the B. pentandra inhibitor affects Factor XIIa, and B. mollis inhibitor causes trypsin inactivation only. Calculated Ki values were between 10(-7) and 10(-9) M. Chymotrypsin, like trypsin, is also inhibited, but with lower affinity. The trypsin inhibitors, isolated from E. contortisiliquum, B. pentandra, B. bauhinioides and B. mollis seem to be of the Kunitz type; the inhibitor purified from T. cearensis is of the Bowman-Birk type.

Proteinase inhibitors in Brazilian Leguminosae.

Serine proteinase inhibitors, in the seeds of several Leguminosae from the Pantanal region (West Brazil), were studied using bovine trypsin, a digestive enzyme, Factor XIIa and human plasma kallikrein, two blood clotting factors. The inhibitors were purified from Enterolobium contortisiliquum (M(r) = 23,000), Torresea cearensis (M(r) = 13,000), Bauhinia pentandra (M(r) = 20,000) and Bauhinia bauhinioides (M(r) = 20,000). E. contortisiliquum inhibitor inactivates all three enzymes, whereas the T. cearensis inhibitor inactivates trypsin and Factor XIIa, but does not affect plasma kallikrein; both Bauhinia inhibitors, on the other hand, inactivate trypsin and plasma kallikrein but only the B. pentandra inhibitor affects Factor XIIa. Ki values were calculated between 10(-7) and 10(-8) M.

A Bothrops jararaca plasma cysteine-proteinase inhibitor related to mammalian kininogen.

A kininogen-like protein was purified from Bothrops jararaca plasma by DEAE-Sephadex ion-exchange and carboxy-methyl-papain-Sepharose affinity chromatography. The molecular weight, estimated by SDS-gel electrophoresis, is about 100,000 and a species of about 75,000 is formed after incubation with horse urinary kallikrein. After incubation with trypsin, only traces of biological activity were detected in tests on guinea pig ileum. The purified protein inhibits papain and bromelain, does not correct the clotting time of a kininogen-depleted human plasma, and does not affect the clotting time of plasma from Waglerophis merremii, a nonpoisonous snake; the same type of inhibitor was found in this nonpoisonous snake. The dissociation constant (Ki) for the papain-inhibitor complex is approximately 1.6 nM.

Purification and preliminary characterization of Torresea cearensis trypsin inhibitor.

An inhibitor against serine proteinases was purified from Torresea cearensis by affinity chromatography on trypsin-Sepharose. The protein is a single polypeptide of molecular weight 13,600 after reduction and has a high content of cysteine residues. Both trypsin (Ki = 0.34 nM) and chymotrypsin (Ki = 0.15 microM) are inhibited by Torresea cearensis inhibitor. Blood clotting factor XII is also inhibited (Ki = 0.24 microM), but not plasma kallikrein, tissue kallikrein or thrombin. The stoichiometry of the inhibitor-proteinase complex with trypsin is 1:1.

A Bothrops jararaca plasma cysteine proteinase inhibitor related to Mammalian kininogen

A kininogen-like protein was purified from Bothrops jararaca plasma by DEAE-Sephadex ion-exchange and carboxy-methul-papain-Sepharose affinity chromatography. The molecular weight, estimated by SDS-gel electrophoresis, is about 100,000 and a species of about 75,000 is formed after incubation with hosrse urinary kallikrein. After incubation with rrypsin, only traces of biological activity were detected in tests on guinea pig ileum. The purified protein inhibits papain and bromelain, does not correct the clotting time of a kininogen-depleted human plasma, and does not affect the clotting time ogf plasma from Waglerophis merremii, a nonpoisonous snake; the same type of inhibitor was foind in this nonpoisonous snake. The dissociation cosntant (Ki) for the papain-inhibitor complex is approximately 1.6 nM

Purification and partial characterization of a thiol proteinase inhibitor from Enterolobium contortisiliquum beans.

A thiol proteinase inhibitor was purified from Enterolobium contortisiliquum beans by affinity chromatography on carboxy-methylated-papain-Sepharose. The inhibitor represents a single polypeptide chain with a molecular mass of 60 kDa and inactivates papain (Ki = 0.58 x 10(-9) M) and bromelain. The inhibitor shows activity in the pH range 2 to 10 and at temperatures up to 60 degrees C.

Serine- and SH-proteinase inhibitors from Enterolobium contortisiliquum beans. Purification and preliminary characterization.

Two types of proteinase inhibitors were purified from Enterolobium contortisiliquum beans. The inhibitor of serine-proteinases inhibited trypsin (Ki = 5 nM), chymotrypsin (Ki = 10 nM) and plasma kallikrein, but not tissue kallikreins. The molecular weight is approximately 23 kDal and two polypeptide chains are detected after reduction. The second inhibitor with activity directed against SH-proteinases was isolated by CM-papain-Sepharose. The molecular weight is approximately 60 kDal and only one polypeptide chain was detected after reduction. Papain (Ki = 0.6 nM) and bromelain are inhibited.

Serine and SH-proteinase inhibitors from Enterolobium contortisiliquum beans: purification and preliminary characterization

Two types inhibitors were prufied from Enterolobium contortisiliquum beans. The inhibitor of serine-proteinases inhibited trypasin (Ki = 5 nM) chymostrypsin (Ki = 10 nM) and plasma kallikrein, but not tissue kallikreins. The molecular weight is approximately 23 KDal and two polypeptide chains detected after reduction. The second inhibitor with activity directed against SH-proteinase was isolated by CM-papain-Sepharose. The molecular weight is approximately 60 KDal and only one polupeptide chain was detected after reduction. Papain (Ki = 0.6 nM) and bromelain are inhibited