The induction of macrophage spreading: role of coagulation factors and the complement system.
(1/1021)
Unstimulated mouse peritoneal macrophages, attached to either glass or plastic substrates, responded to factors generated in serum and plasma by spreading and increasing their apparent surface area up to eightfold. Two distinct and dissociable systems were involved. The first appears related to the distinct and dissociable systems were involved. The first appears related to the contact phase of blood coagulation. It is activated by glass and not plastic surfaces, depleted by kaolin adsorption, and inhibited by soybean trypsin inhibitor. In contrast, a separate complement-dependent system can be generated in kaolin-adsorbed plasma. Activation of the complement system can occur either by the alternate or classical pathways and generates a relatively small effector molecule which is dialyzable. These factors presumably influencing the surface membrane and underlying structures may explain the rapid spreading of activated macrophages observed after both infections and chemical peritoneal inflammatory agents. (+info)
Intracellular proteolytic processing of the heavy chain of rat pre-alpha-inhibitor. The COOH-terminal propeptide is required for coupling to bikunin.
(2/1021)
Pre-alpha-inhibitor is a serum protein consisting of two polypeptides named bikunin and heavy chain 3 (H3). Both polypeptides are synthesized in hepatocytes and while passing through the Golgi complex, bikunin, which carries a chondroitin sulfate chain, becomes covalently linked to the COOH-terminal amino acid residue of H3 via its polysaccharide. Immediately prior to this reaction, a COOH-terminal propeptide of 33 kDa is cleaved off from the heavy chain. Using COS-1 cells transfected with rat H3, we found that in the absence of bikunin, the cleaved propeptide remained bound to the heavy chain and that H3 lacking the propeptide sequence did not become linked to coexpressed bikunin. Sequencing of H3 secreted from COS-1 cells showed that part of the molecules had a 12-amino acid residue long NH2-terminal propeptide. Cleavage of this propeptide, which occurred in the endoplasmic reticulum, was found to require basic amino acid residues at P1, P2, and P6 suggesting that it is mediated by a Golgi enzyme in transit. Deletion of the NH2-terminal propeptide or blocking of its release affected neither transport nor coupling of the heavy chain to bikunin. (+info)
Inhibition of plant-pathogenic fungi by a corn trypsin inhibitor overexpressed in Escherichia coli.
(3/1021)
The cDNA of a 14-kDa trypsin inhibitor (TI) from corn was subcloned into an Escherichia coli overexpression vector. The overexpressed TI was purified based on its insolubility in urea and then refolded into the active form in vitro. This recombinant TI inhibited both conidium germination and hyphal growth of all nine plant pathogenic fungi studied, including Aspergillus flavus, Aspergillus parasiticus, and Fusarium moniliforme. The calculated 50% inhibitory concentration of TI for conidium germination ranged from 70 to more than 300 microgram/ml, and that for fungal growth ranged from 33 to 124 microgram/ml depending on the fungal species. It also inhibited A. flavus and F. moniliforme simultaneously when they were tested together. The results suggest that the corn 14-kDa TI may function in host resistance against a variety of fungal pathogens of crops. (+info)
Reduction of sodium deoxycholic acid-induced scratching behaviour by bradykinin B2 receptor antagonists.
(4/1021)
1. Subcutaneous injection of sodium deoxycholic acid into the anterior of the back of male ddY mice elicited dose-dependent scratching of the injected site with the forepaws and hindpaws. 2. Up to 100 microg of sodium deoxycholic acid induced no significant increase in vascular permeability at the injection site as assessed by a dye leakage method. 3. Bradykinin (BK) B2 receptor antagonists, FR173657 and Hoe140, significantly decreased the frequency of scratching induced by sodium deoxycholic acid. 4. Treatment with aprotinin to inhibit tissue kallikrein reduced the scratching behaviour induced by sodium deoxycholic acid, whereas treatment with soybean trypsin inhibitor to inhibit plasma kallikrein did not. 5. Although injection of kininase II inhibitor, lisinopril together with sodium deoxycholic acid did not alter the scratching behaviour, phosphoramidon, a neutral endopeptidase inhibitor, significantly increased the frequency of scratching. 6. Homogenates of the skin excised from the backs of mice were subjected to gel-filtration column chromatography followed by an assay of kinin release by trypsin from each fraction separated. Less kinin release from the fractions containing kininogen of low molecular weight was observed in the skin injected with sodium deoxycholic acid than in normal skin. 7. The frequency of scratching after the injection of sodium deoxycholic acid in plasma kininogen-deficient Brown Norway Katholiek rats was significantly lower than that in normal rats of the same strain, Brown Norway Kitasato rats. 8. These results indicate that BK released from low-molecular-weight kininogen by tissue kallikrein, but not from high-molecular-weight kininogen by plasma kallikrein, may be involved in the scratching behaviour induced by the injection of sodium deoxycholic acid in the rodent. (+info)
Role of P225 and the C136-C201 disulfide bond in tissue plasminogen activator.
(5/1021)
The protease domain of tissue plasminogen activator (tPA), a key fibrinolytic enzyme, was expressed in Escherichia coli with a yield of 1 mg per liter of media. The recombinant protein was titrated with the Erythrina caraffa trypsin inhibitor (ETI) and characterized in its interaction with plasminogen and the natural inhibitor plasminogen activator inhibitor-1 (PAI-1). Analysis of the catalytic properties of tPA using a library of chromogenic substrates carrying substitutions at P1, P2, and P3 reveals a strong preference for Arg over Lys at P1, unmatched by other serine proteases like thrombin or trypsin. In contrast to these proteases and plasmin, tPA shows little or no preference for Pro over Gly at P2. A specific inhibition of tPA by Cu2+ was discovered. The divalent cation presumably binds to H188 near D189 in the primary specificity pocket and inhibits substrate binding in a competitive manner with a Kd = 19 microM. In an attempt to engineer Na+ binding and enhanced catalytic activity in tPA, P225 was replaced with Tyr, the residue present in Na+-dependent allosteric serine proteases. The P225Y mutation did not result in cation binding, but caused a significant loss of specificity (up to 100-fold) toward chromogenic substrates and plasminogen and considerably reduced the inhibition by PAI-1 and ETI. Interestingly, the P225Y substitution enhanced the ability of Cu2+ to inhibit the enzyme. Elimination of the C136-C201 disulfide bond, that is absent in all Na+-dependent allosteric serine proteases, significantly enhanced the yield (5 mg per liter of media) of expression in E. coli, but caused no changes in the properties of the enzyme whether residue 225 was Pro or Tyr. These findings point out an unanticipated crucial role for residue 225 in controlling the catalytic activity of tPA, and suggest that engineering of a Na+-dependent allosteric enhancement of catalytic activity in this enzyme, must involve substantial changes in the region homologous to the Na+ binding site of allosteric serine proteases. (+info)
Bifunctional inhibitors of the trypsin-like activity of eukaryotic proteasomes.
(6/1021)
BACKGROUND: The 20S proteasome is a multicatalytic protease complex that exhibits trypsin-like, chymotrypsin-like and post-glutamyl-peptide hydrolytic activities associated with the active sites of the beta2, beta5 and beta1 subunits, respectively. Modulation of these activities using inhibitors is essential for a better understanding of the proteasome's mechanism of action. Although there are highly selective inhibitors of the proteasome's chymotryptic activity, inhibitors of similar specificity have not yet been identified for the other activities. RESULTS: The X-ray structure of the yeast proteasome reveals that the sidechain of Cys118 of the beta3 subunit protrudes into the S3 subsite of the beta2 active site. The location of this residue was exploited for the rational design of bidentated inhibitors containing a maleinimide moiety at the P3 position for covalent linkage to the thiol group and a carboxy-terminal aldehyde group for hemiacetal formation with the Thr1 hydroxyl group of the active site. Structure-based modelling was used to determine the optimal spacing of the maleinimide group from the P2-P1 dipeptide aldehydes and the specificity of the S1 subsite was exploited to limit the inhibitory activity to the beta2 active site. X-ray crystallographic analysis of a yeast proteasome-inhibitor adduct confirmed the expected irreversible binding of the inhibitor to the P3 subsite. CONCLUSIONS: Maleoyl-beta-alanyl-valyl-arginal is a new type of inhibitor that is highly selective for the trypsin-like activity of eukaryotic proteasomes. Despite the reactivity of the maleinimide group towards thiols, and therefore the limited use of this inhibitor for in vitro studies, it might represent an interesting new biochemical tool. (+info)
Characterization of low-molecular-mass trypsin isoinhibitors from oil-rape (Brassica napus var. oleifera) seed.
(7/1021)
A new low-molecular-mass (6767.8 Da) serine proteinase isoinhibitor has been isolated from oil-rape (Brassica napus var. oleifera) seed, designated 5-oxoPro1-Gly62-RTI-III. The 5-oxoPro1-Gly62-RTI-III isoinhibitor is longer than the Asp2-Pro61-RTI-III and the Ser3-Pro61-RTI-III forms, all the other amino acid residues being identical. In RTI-III isoinhibitors, the P1-P1' reactive site bond (where residues forming the reactive site have been identified as PnellipsisP1 and P1'ellipsisPn', where P1-P1' is the inhibitor scissile bond) has been identified at position Arg21-Ile22. The inhibitor disulphide bridges pattern has been determined as Cys5-Cys27, Cys18-Cys31, Cys42-Cys52 and Cys54-Cys57. The disulphide bridge arrangement observed in the RTI-III isoinhibitors is reminiscent of that found in a number of toxins (e.g. erabutoxin b). Moreover, the organization of the three disulphide bridges subset Cys5-Cys27, Cys18-Cys31 and Cys42-Cys52 is reminiscent of that found in epidermal growth factor domains. Preliminary 1H-NMR data indicates the presence of alphaalphaNOEs and 3JalphaNH coupling constants, typical of the beta-structure(s). These data suggest that the three-dimensional structure of the RTI-III isoinhibitors may be reminiscent of that of toxins and epidermal growth factor domains, consisting of three-finger shaped loops extending from the crossover region. Values of the apparent association equilibrium constant for RTI-III isoinhibitors binding to bovine beta-trypsin and bovine alpha-chymotrypsin are 3.3 x 109 m-1 and 2.4 x 106 m-1, respectively, at pH 8.0 and 21.0 degrees C. The serine proteinase : inhibitor complex formation is a pH-dependent entropy-driven process. RTI-III isoinhibitors do not show any similarity to other serine proteinase inhibitors except the low molecular mass white mustard trypsin isoinhibitor, isolated from Sinapis alba L. seed (MTI-2). Therefore, RTI-III and MTI-2 isoinhibitors could be members of a new class of plant serine proteinase inhibitors. (+info)
Near-UV circular dichroism of trypsin inhibitor of adzuki beans attributable to disulfide groups.
(8/1021)
The trypsin inhibitor of adzuki (Phaseolus angularis) beans shows a CD spectrum with a negative extremum at 280 nm and a positive shoulder around 245 nm. Since the inhibitor lacks tryptophan and tyrosine, the observed CD spectrum can be attributed to the six disulfide groups in the molecule. The CD features completely disappeared on reduction of the disulfide groups, and converged into a single negative extremum at 270 nm when the groups were modified to form mixed disulfides with glutathione. These observations of the CD properties of the inhibitor strongly suggest the presence of disulfide groups constrained with respect to their dihedral angles. (+info)