Study on interaction between duodenase protease with dual specificity and inhibitors of bowman-birk family

The interaction between duodenase and inhibitors of Bowman-Birk type from soybeans (BBI) and lima beans (LBI) was investigated. Duodenase was shown to interact only with antichymotrypsin site of these inhibitors. The inhibition constants of duodenase by BBI, LBI, BBI-trypsin and LBI trypsin complexes were 4, 23, 400, 600 (n)M respectively.     

Yeast survival following gamma irradiation: delayed colony formation and determination of the proportion of undamaged cells

The experimental method is proposed to determine both a relative number of cells without damages and the effect of the delayed appearance of colonies of haploid and diploid yeast after exposure to ionizing radiation.     

Interaction between duodenase, a proteinase with dual specificity, and soybean inhibitors of Bowman-Birk and Kunitz type

The interaction between duodenase, which belongs to a group of Janus-faced proteinases, and classical Bowman--Birk (BBI) and Kunitz (STI) type inhibitors from soybean was investigated. Duodenase was shown to interact only with the antichymotrypsin site (Leu-Ser) of BBI, whereas the antitrypsin site (Lys-Ser) of the inhibitor appeared to be vacant and capable of interaction with trypsin. The inhibition constants of duodenase by BBI, the BBI--trypsin complex, and STI were 4, 400, and 40 nM, respectively.     

Activation of recombinant proenteropeptidase by duodenase

Duodenase, a serine proteinase from bovine Brunner's (duodenal) glands that was predicted to be a natural activator of enteropeptidase zymogen, cleaves and activates recombinant single-chain bovine proenteropeptidase (kcat/Km = 2700 M(-1) s(-1)). The measured rate of proenteropeptidase cleavage by duodenase was about 70-fold lower compared with the rate of trypsin-mediated cleavage of the zymogen. The role of duodenase is supposed to be the primary activator of proenteropeptidase maintaining a certain level of active enteropeptidase in the duodenum. A new scheme of proteolytic activation cascade of digestive proteases is discussed.     

Effect of reactive center loop structure of antichymotrypsin on inhibition of duodenase activity

Interaction between duodenase (a granase family member) from bovine duodenal mucosa and recombinant antichymotrypsin (rACT) and its P1 variants has been studied. Association rate constants (k _a) were 11, 6.8, and 17 mM^?1·sec^?1 for rACT, ACT L358M, and ACT L358R, respectively. Natural antitrypsin (AT) compared to ACT was a 20 times more effective duodenase inhibitor (in terms of k _a). Duodenase interacted with P1 variants of ACT via a suicide mechanism with stoichiometry of the process SI = 1.2. The nature of the P1 residue of the inhibitor did not influence the interaction if other residues did not meet conformational requirements of the duodenase substrate-binding pocket. Also, interaction of duodenase with ACT variants containing residues from AT reaction center loop (rACT P2-P3′, rACT P3-P4′, rACT P4-P3′, and rACT P6-P4′) was studied. The inhibition type ([E]₀ = 1·10^?7 M, 25°C) was revealed to be reversible-like, and efficacy of inhibition decreased with increase in the substituted part of the reactive center loop. Constants of inhibition (K _i) were measured. Efficacy of interaction between the enzyme (duodenase) and inhibitor depends on topochemical correspondence between a substrate-binding pocket of the enzyme and substrate structure.     

Serine proteases in immune protection of the small intestine

The gastrointestinal tract is subject to a huge antigenic load, which is especially significant in the intestinal lumen. Being the connecting link between the organism and the external environment, the small intestine fulfils not only digestive and transport functions, but also protective ones and acts as a selective barrier for the flow of nutrients. This review considers proteases of the protective system of small intestine cells, their biochemical properties and activation mechanisms, and involvement in biochemical processes responsible for normal functioning and defense reactions of the intestine. Serine proteases of intestinal immunity are multifunctional enzymes making proteolytic attack aimed to immediately exterminate aggressive elements of the intestinal contents (allergens, toxins), to activate (inactivate) zymogens, receptors, and peptide hormones, and to hydrolyze protein precursors and other biologically active factors. Proteases of intestinal immunity control the inflammatory response, proliferation of B-lymphocytes, apoptosis, and secretory and contractive activity of the intestine; they release neurogenic factors, inactivate biologically active substances, and are involved in degradation of the intercellular matrix and in tissue remodeling.     

Expression of duodenase-like protein in epitheliocytes of Brunner’s glands in human duodenal mucosa

A duodenase, a protease structurally related to human cathepsin G, was found earlier in bovine duodenal mucosa. It was demonstrated that under the influence of duodenase an enteropeptidase zymogen is activated in vitro showing the possible participation of duodenase in the cascade of activation of digestive enzymes. To identify a duodenase functional analog in human duodenum, an immunofluorescence study of duodenal mucosa was conducted by confocal microscopy using antibodies to human cathepsin G and to bovine duodenase. The previously unknown place of synthesis and secretion of cathepsin G — Paneth cells located at the bottom of Lieberkuhn crypts — was revealed. Binding of cathepsin G-specific antibodies in a rough endoplasmic reticulum zone and in the cryptal duct was observed. Duodenase-specific immunofluorescence but not that of cathepsin G was found in the epitheliocytes and secretory ducts of Brunner’s glands, which are characteristic sites of duodenase biosynthesis in cattle. Binding of CD14-specific antibodies in the Brunner’s glands, where the antibodies co-localized with the antibodies to duodenase, was also demonstrated. These data indicate the presence of a protein immunologically similar to duodenase in the human duodenal mucosa. Our study demonstrated the absence of its colocalization with cathepsin G in Brunner’s glands.     

The role of PAR1 in protective action of activated protein C during nonimmune activation of mast cells

Activated protein C (APC) regulates the functional activity of mast cells by reducing release of β-hexosaminidase, the marker of mast cell degranulation. APC modulated not only spontaneous secretion from mast cells, but also secretion induced by the degranulators, proteinase-activated receptor agonist peptide (PAR1-AP) and compound 48/80. PAR1 desensitization by thrombin abolished the decrease of β-hexosaminidase secretion induced by low APC concentrations (≤1.5 nM). APC inactivated by phenylmethylsulfonyl fluoride (PMSF), did non mimic the enzyme action on mast cells. Duodenase (the duodenal proteinase) activated peritoneal mast cell via PAR1. APC abolished the proinflammatory effect of duodenase and PAR1-AP by reducing release of mast cell mediators. The effect of APC could be attributed to nitric oxide generation by mast cells because in the presence of L-NAME the secretory function restored. These data suggest involvement of mast cell PAR1 into regulatory mechanism responsible for the anti-inflammatory effect of APC.     

Proteolytic action of duodenase is required to induce DNA synthesis in pulmonary artery fibroblasts.

Duodenase is a 29-kDa serine endopeptidase that displays selective trypsin- and chymotrypsin-like substrate specificity. This enzyme has been localized to epitheliocytes of Brunner's glands, and as described here, to mast cells within the intestinal mucosa and lungworm-infected lung, implying an important additional role in inflammation and tissue remodelling. In primary cultures of pulmonary artery fibroblasts, duodenase induced a concentration-dependent increase in [3H]thymidine incorporation with a maximal effect observed at 30 nm. Pretreating duodenase with soybean trypsin inhibitor abolished DNA synthesis, confirming that proteolytic activity was an essential requirement for this response. PAR1, PAR2 and PAR4 activating peptides were unable to induce [3H]thymidine incorporation in pulmonary artery fibroblasts. Likewise, pretreatment of fibroblasts with TNFalpha, known to up-regulate PAR2 expression in other systems, and IL-1beta, did not enhance the potential of duodenase to induce DNA synthesis. Furthermore, duodenase increased GTPgammaS binding to fibroblast membranes indicating that a G-protein-coupled receptor may mediate the effects of duodenase. Duodenase-induced DNA synthesis and GTPgammaS binding were both found to be inhibited by pertussis toxin, implying a role for Gi/o. Selective inhibitors of MEK1 (PD98059) and protein kinase C (GF109203X) only partially inhibited duodenase-induced DNA synthesis, but both wortmannin (100 nm) and LY294002 (10 microm) inhibited this response completely, indicating a key role for PtdIns 3-kinase. Furthermore, duodenase induced a 2.3 plus minus 0.1-fold increase in PtdIns 3-kinase activity in p85 immunoprecipitates, which was sensitive to inhibition by wortmannin. These results suggest that duodenase can induce pulmonary artery fibroblast DNA synthesis in a PtdIns 3-kinase-dependent manner via a G-protein-coupled receptor which is activated by a proteolytic mechanism.