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Query: UNIPROT:P06889 (Mol)
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A new approach to the molecular modelling of homologous serine proteases is adopted, by including a set of 21 buried waters known to be preserved in enzymes sharing the primary specificity of trypsin, in the homology modelling of rat submaxillary gland kallikrein. Buried waters--water molecules sequestered from bulk solvent within a protein matrix--appear to be integral conserved components of all serine proteases of known structure and should be incorporated into serine protease models built on the basis of sequence/structural homology to this family. The absence of such waters might induce errors in a force field simulation, favouring the formation of nonexistent hydrogen bonds and locally inaccurate structure. The kallikrein model refinement has led to the conclusion that an additional buried water should be added to the original rigid matrix of 21 conserved water molecules. The structurally preserved protein cavities of such waters validate the modelled structure.
J Comput Aided Mol Des 1997 Nov
PMID:Inclusion of conserved buried water molecules in the model structure of rat submaxillary kallikrein. 949 47

A cDNA clone that encodes the 14-kDa bifunctional inhibitor from corn seeds (L. Wen et al., Plant Mol. Biol. 18, 813-814, 1992) has been expressed in Escherichia coli after being incorporated into the pT7 expression vector. This inhibitor protein, referred to as CHFI (for the corn inhibitor of activated Hageman factor) or as the popcorn inhibitor, is an important tool for specific inhibition of human activated Hageman factor (activated forms of coagulation Factor XII) and has been well characterized as isolated from corn seeds. Recombinant CHFI was expressed in E. coli in high levels but was insoluble. We solubilized the expressed protein by sonication in 5 M urea and 1% Triton X-100. Several steps of purification, culminating with reversed-phase HPLC, yielded pure, recombinant corn inhibitor in about 5% yield (about 1 mg per liter of culture). The form with which we have worked most, 7N-CHFI, contains 7 amino acid residues at its N-terminus that are encoded by the expression vector. Physical properties of this recombinant protein indicate it has the expected mass and is properly folded. Functionally, 7N-CHFI is indistinguishable from the inhibitor isolated from corn seeds in its inhibition of porcine trypsin, human beta-Factor XIIa, failure to inhibit human plasma kallikrein, and its inhibition of an insect alpha-amylase. A second recombinant form, (4N-11)-CHFI, which lacks 11 residues from the corn inhibitor's N-terminus, is indistinguishable from 7N-CHFI in its pattern of inhibition of the three test proteinases but is inactive against the insect alpha-amylase. This suggests that the N-terminal region of 7N-CHFI forms at least part of the protein's site of interaction with alpha-amylase.
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PMID:The corn inhibitor of activated Hageman factor: purification and properties of two recombinant forms of the protein. 967 55

Ovarian hyperstimulation syndrome (OHSS) is a severe complication arising from controlled stimulation treatment. Vascular endothelial growth factor (VEGF) has recently emerged as an important factor which may be responsible for the hyperpermeability seen in OHSS. The purpose of the present study was to investigate and compare the mechanisms by which ascites in patients with OHSS and ovarian carcinoma induce increases in vascular permeability in an in vitro assay and an in vivo animal experiment. We found 8-fold lower VEGF levels in ascites from patients with OHSS than in those from patients with ovarian carcinoma. Although VEGF is produced by the ovaries, it is not necessarily the factor responsible for hyperpermeability. We also demonstrated that the vascular hyperpermeability produced by OHSS ascites was not abolished by specific neutralizing anti-VEGF antibodies, and that not all of the VEGF found in the ascites fluid is biologically active. Moreover, our results strongly suggest that the vascular permeability produced by OHSS ascites may depend on activation of the kallikrein-kinin system. Possible evidence for this phenomenon was obtained by demonstrating that the hyperpermeability caused by the ascites could be blocked by Trasylol (known to inhibit bradykinin synthesis) and potentiated by captopril (a kininase II inhibitor). Taken together, the results suggest that, although VEGF is found in ascites fluid from patients with OHSS, it is unlikely that the cause of OHSS involves VEGF production by the ovaries. The kallikrein-kinin system may be more important in the hyperpermeability seen in OHSS.
J Mol Endocrinol 1998 Jun
PMID:The kallikrein-kinin system, but not vascular endothelial growth factor, plays a role in the increased vascular permeability associated with ovarian hyperstimulation syndrome. 968 59

By introduction of stepwise DEAE Sephadex A-50 and copper-Chelating Sepharose 6B column chromatographies, about 18.5 mg of high molecular weight kininogen (HK) composed of a single polypeptide chain was obtained from 500 ml of porcine plasma. Molecular weights of reduced or non-reduced preparation were estimated to be 110 kDa and 116 kDa, respectively, by SDS-PAGE. Using the preparation, cleavage of HK by porcine plasma kallikrein (KK) was investigated. A single polypeptide HK was cleaved into two chains cross-linked by disulfide bond(s), accompanying the release of kinin. Further degradation was not observed. Molecular weights of heavy-chain (H-chain) and light-chain (L-chain) were estimated to be 61 kDa and 56 kDa, respectively, by SDS-PAGE. The amino- (N-) terminal sequences of intact HK, reduced and carboxymethylated- (RCM-) H-chain, RCM-L-chain and the peptide around the kinin moiety obtained by BrCN digestion were determined. Their sequences were highly homologous with those of bovine or human HK. These results indicate that plasma KK first cleaved the Arg-Ser bond of HK, and formed nicked HK. The second cleavage yielded bradykinin (BK) and kinin-free protein, which was apparently of equal size to the nicked HK. The structure of HK was from the N-terminus to the carboxy- (C-) terminus, H-chain-BK-L-chain.
Comp Biochem Physiol B Biochem Mol Biol 1998 Aug
PMID:Studies on porcine high molecular weight kininogen. An improved method for the purification of porcine high molecular weight kininogen and cleavage of the kininogen by the action of porcine plasma kallikrein. 985 12

This study was designed to investigate the alterations in the levels of various proteases such as angiotensin converting enzyme (ACE), kallikrein, aminopeptidases, urokinase and plasmin in serum-heart and kidney and to find out whether the changes in the levels of these enzymes could explain the pathogeneses of hypertension induced by Dexamethasone (Dex). Dex was administered to Male Wistar rats (180-200 g body weight) at a dosage of 2.5 mg/kg/week subcutaneously on alternate days for 2 weeks. One more week was included in this investigation to oversee the recovery process. Mean Arterial Pressure (MAP) showed significant elevation during administration and after withdrawal of Dex. The levels of enzymes such as angiotensin converting enzyme, carboxypeptidase-N and leucine aminopeptidase were found to be elevated in serum as well as in tissues. The level of kallikrein was observed to decrease in serum and tissues and that of thrombin, plasmin and urokinase exhibited variations. Thus, treatment with Dex altered the levels of these proteases which might have a role in the pathogenesis of hypertension and in altered blood coagulation.
Mol Cell Biochem 1999 Jul
PMID:Dexamethasone induced alterations in the levels of proteases involved in blood pressure homeostasis and blood coagulation in rats. 1048 40

The serine proteinase plasmin is, together with tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), involved in the dissolution of blood clots in a fibrin-dependent manner. Moreover, plasmin plays a key role in a variety of other activation cascades such as the activation of metalloproteinases, and has also been implicated in wound healing, pathogen invasion, cancer invasion and metastasis. The leech-derived (Hirudo medicinalis) antistasin-type inhibitor bdellastasin represents a specific inhibitor of trypsin and plasmin and thus offers a unique opportunity to evaluate the concept of plasmin inhibition. The complexes formed between bdellastasin and bovine as well as porcine beta-trypsin have been crystallised in a monoclinic and a tetragonal crystal form, containing six molecules and one molecule per asymmetric unit, respectively. Both structures have been solved and refined to 3.3 A and 2.8 A resolution. Bdellastasin turns out to have an antistasin-like fold exhibiting a bis-domainal structure like the tissue kallikrein inhibitor hirustasin. The interaction between bdellastasin and trypsin is restricted to the C-terminal subdomain of bdellastasin, particularly to its primary binding loop, comprising residues Asp30-Glu38. The reactive site of bdellastasin differs from other antistasin-type inhibitors of trypsin-like proteinases, exhibiting a lysine residue instead of an arginine residue at P1. A model of the bdellastasin-microplasmin complex has been created based on the X-ray structures. Our modelling studies indicate that both trypsin and microplasmin recognise bdellastasin by interactions which are characteristic for canonically binding proteinase inhibitors. On the basis of our three-dimensional structures, and in comparison with the tissue-kallikrein-bound and free hirustasin and the antistasin structures, we postulate that the binding of the inhibitors toward trypsin and plasmin is accompanied by a switch of the primary binding loop segment P5-P3. Moreover, in the factor Xa inhibitor antistasin, the core of the molecule would prevent an equivalent rotation of the P3 residue, making exosite interactions of antistasin with factor Xa imperative. Furthermore, Arg32 of antistasin would clash with Arg175 of plasmin, thus impairing a favourable antistasin-plasmin interaction and explaining its specificity.
J Mol Biol 1999 Oct 15
PMID:Structure of the complex of the antistasin-type inhibitor bdellastasin with trypsin and modelling of the bdellastasin-microplasmin system. 1051 18

Organophosphorus pesticide toxicology is normally evaluated in relation to inhibition of cholinesterases (acetyl and butyryl), neuropathy target esterase, and carboxylesterases, with less attention given to other physiologically important hydrolases. This study considers the relative organophosphate sensitivities of the aforementioned serine hydrolases compared with purified blood-clotting factors (thrombin, plasmin, and kallikrein) and digestive enzymes (alpha-chymotrypsin, trypsin, and elastase), assayed under similar conditions. Inhibitors that we examined are organophosphorus insecticides or their activated metabolites (paraoxon, chlorpyrifos oxon, and profenofos) and other toxicants (phenyl saligenin cyclic phosphonate and tribufos) for comparison with values that are found in the literature for the fluorophosphonates (isoflurophate and sarin). Thrombin is the most sensitive blood-clotting factor with IC-50 values of 19 to 160 microM for tribufos, the cyclic phosphonate, isoflurophate, and profenofos; plasmin and kallikrein are less affected (IC-50 >100 microM). Alpha-Chymotrypsin, trypsin, and elastase are most sensitive to the cyclic phosphonate (IC-50 1.3-15 microM) and less so to isoflurophate, sarin, and profenofos (IC-50 values from 3.6 to greater than 100 microM). The cholinesterases, carboxylesterase, and neuropathy target esterase are the most sensitive to inhibition with IC-50 values for the insecticides of less than 0.001 to 0.6, 0.002 to 0.009, and 0.15 to 100 microM, respectively. The generally low potency of these organophosphates for blood-clotting factors and digestive enzymes suggests that associated toxic effects are unlikely at sublethal doses.
J Biochem Mol Toxicol 2000
PMID:Sensitivity of blood-clotting factors and digestive enzymes to inhibition by organophosphorus pesticides. 1056 Oct 82

Activation of the kallikrein-kinin system in lung injury has long been recognized. However, the effects of bradykinin (BK) on human lung fibroblasts (HLF) remain to be elucidated. We determined whether BK stimulates HLF to release chemotactic activity for neutrophils and monocytes (NCA and MCA, respectively). We evaluated HLF supernatant fluids for chemotactic activity through a blind-well chamber technique. HLF released NCA and MCA in a dose- and time-dependent manner in response to BK. The release of chemotactic activity was inhibited by lipoxygenase inhibitors and cycloheximide. Molecular sieve column chromatography revealed that both NCA and MCA had multiple chemotactic peaks. NCA was inhibited by a leukotriene (LT) B(4) receptor antagonist and by antibodies to interleukin (IL)-8 and granulocyte colony-stimulating factor (G-CSF). MCA was attenuated by the LTB(4) receptor antagonist and by antibodies to monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and transforming growth factor (TGF)-beta. Both the LTB(4) receptor antagonist and these antibodies inhibited chemotactic activity of the molecular weights corresponding to MCP-1, GM-CSF, and TGF-beta, separated by column chromatography. The concentrations of IL-8, G-CSF, MCP-1, GM-CSF, and TGF-beta in supernatant fluids increased significantly in a time-dependent manner in response to BK. The receptors responsible for the release of NCA, MCA, and individual chemokines included both BKB(1) and BKB(2) receptors. These data suggest that BK may stimulate lung fibroblasts to release inflammatory cytokines, which may modulate lung inflammation.
Am J Respir Cell Mol Biol 2000 Jan
PMID:Bradykinin stimulates lung fibroblasts to release neutrophil and monocyte chemotactic activity. 1061 68

Microneme organelles are present in all apicomplexan protozoa and contain proteins that are critical for parasite motility and host cell invasion. One apicomplexan-wide family of microneme proteins has been identified with members that are characterised by the possession of thrombospondin type I repeats, conserved adhesive motifs which are implicated in binding to glycosaminoglycan chains. In this paper we describe a micronemal glycoprotein, EtMIC 5, from Eimeria tenella which contains eleven cysteine-rich motifs that have striking similarity to the adhesive Apple (A-) domains of blood coagulation factor XI and plasma pre-kallikrein. EtMIC 5 is confined to an intracellular location in resting sporozoites but is translocated to the parasite surface and secreted into the culture supernatant during parasite infection of MDBK cells. During intracellular replication, the protein is switched off in early schizogony and is then re-expressed within the apical tips of newly formed merozoites. A-domain sequences were also found in microneme proteins from Sarcocystis muris and Toxoplasma gondii and in a protein of unknown localisation from Eimeria acervulina. These studies suggest that A-domain containing proteins may comprise a novel apicomplexan-wide family of microneme adhesins.
Mol Biochem Parasitol 2000 Mar 15
PMID:A microneme protein from Eimeria tenella with homology to the Apple domains of coagulation factor XI and plasma pre-kallikrein. 1071 5

To determine the identity of porcine follipsin, a plasma kallikrein cDNA clone was isolated from a porcine liver cDNA library. The clone encoded a protein of 643 amino acids, exhibiting identities 79.7, 72. 9, and 74.4% homologous to human, rat, and mouse plasma prekallikrein, respectively. The amino acid sequences of four internal peptides isolated from the tryptic digest of follipsin were all found in the deduced sequence. Authentic plasma kallikrein was purified from porcine plasma and compared directly with follipsin. Actions on synthetic substrates and behaviors with proteinase inhibitors were indistinguishable between these two enzymes. The cDNA was expressed in COS-7 cells and the recombinant protein was prepared from the culture medium of these cells. No active enzyme could be obtained, but the expressed protein was reacted with anti-porcine plasma kallikrein antibody. The mRNA was detected only in the liver in northern blot analysis. RT-PCR analysis of RNAs revealed that porcine testis, in addition to the liver, expressed the corresponding mRNA. In the ovary, plasma kallikrein was detected as a main band of the active form (Mr = 85,000) and the band of the minor inactive precursor form (Mr = 80,000), respectively. In contrast, the liver extract contained only the precursor form. Incubation of high molecular weight kininogen with follicular fluid plasma kallikrein resulted in an increased production of bradykinin. Further, the fresh fluid of large-sized follicles of porcine ovaries was found to contain this peptide hormone at a detectable level. These results indicate that porcine follipsin is plasma kallikrein, and that the enzyme may be involved in the production of bradykinin within ovarian follicles.
Mol Reprod Dev 2000 Sep
PMID:Identification of porcine follipsin as plasma kallikrein, and its possible involvement in the production of bradykinin within the follicles of porcine ovaries. 1095 59


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