Mapping the pro-region of carboxypeptidase B by protein engineering. Cloning, overexpression, and mutagenesis of the porcine proenzyme. (1/124)

The proteolytic processing of pancreatic procarboxypeptidase B to a mature and functional enzyme is much faster than that of procarboxypeptidase A1. This different behavior has been proposed to depend on specific conformational features at the region that connects the globular domain of the pro-segment to the enzyme and at the contacting surfaces on both moieties. A cDNA coding for porcine procarboxypeptidase B was cloned, sequenced, and expressed at high yield (250 mg/liter) in the methylotrophic yeast Pichia pastoris. To test the previous hypothesis, different mutants of the pro-segment at the putative tryptic targets in its connecting region and at some of the residues contacting the active enzyme were obtained. Moreover, the complete connecting region was replaced by the homologous sequence in procarboxypeptidase A1. The detailed study of the tryptic processing of the mutants shows that limited proteolysis of procarboxypeptidase B is a very specific process, as Arg-95 is the only residue accessible to tryptic attack in the proenzyme. A fast destabilization of the connecting region after the first tryptic cut allows subsequent proteolytic processing and the expression of carboxypeptidase B activity. Although all pancreatic procarboxypeptidases have a preformed active site, only the A forms show intrinsic activity. Mutational substitution of Asp-41 in the globular activation domain, located at the interface with the enzyme moiety, as well as removal of the adjacent 310 helix allow the appearance of residual activity in the mutated procarboxypeptidase B, indicating that the interaction of both structural elements with the enzyme moiety prevents the binding of substrates and promotes enzyme inhibition. In addition, the poor heterologous expression of such mutants indicates that the mutated region is important for the folding of the whole proenzyme.  (+info)

An integrated study of fibrinogen during blood coagulation. (2/124)

The rate of conversion of fibrinogen (Fg) to the insoluble product fibrin (Fn) is a key factor in hemostasis. We have developed methods to quantitate fibrinopeptides (FPs) and soluble and insoluble Fg/Fn products during the tissue factor induced clotting of whole blood. Significant FPA generation (>50%) occurs prior to visible clotting (4 +/- 0.2 min) coincident with factor XIII activation. At this time Fg is mostly in solution along with high molecular weight cross-linked products. Cross-linking of gamma-chains is virtually complete (5 min) prior to the release of FPB, a process that does not occur until after clot formation. FPB is detected still attached to the beta-chain throughout the time course demonstrating release of only low levels of FPB from the clot. After release of FPB a carboxypeptidase-B-like enzyme removes the carboxyl-terminal arginine resulting exclusively in des-Arg FPB by the 20-min time point. This process is inhibited by epsilon-aminocaproic acid. These results demonstrate that transglutaminase and carboxypeptidase enzymes are activated simultaneously with Fn formation. The initial clot is a composite of Fn I and Fg already displaying gamma-gamma cross-linking prior to the formation of Fn II with Bbeta-chain remaining mostly intact followed by the selective degradation of FPB to des-Arg FPB.  (+info)

Enzymic characterization of a novel member of the regulatory B-like carboxypeptidase with transcriptional repression function: stimulation of enzymic activity by its target DNA. (3/124)

The adipocyte-enhancer binding protein (AEBP) 1 is a novel transcriptional repressor with carboxypeptidase (CP) activity. AEBP1 binds to a regulatory sequence (termed adipocyte enhancer 1, AE-1) located in the proximal promoter region of the adipose P2 (aP2) gene, which encodes the adipocyte fatty-acid binding protein. Sequence comparisons and kinetic studies using known carboxypeptidase substrates, activators and inhibitors have characterized AEBP1 as a member of the regulatory B-like CP family. Significantly, the inherent CP activity of AEBP1 is stimulated by the AE-1 sequence. Our results indicate that AEBP1 is activated by a novel mechanism, wherby the direct binding of DNA enhances its protease activity. These results represent the first demonstration of DNA-mediated regulation of CP activity.  (+info)

Characterization of plasmin-mediated activation of plasma procarboxypeptidase B. Modulation by glycosaminoglycans. (4/124)

Plasma carboxypeptidase B (PCB) is an exopeptidase that exerts an antifibrinolytic effect by releasing C-terminal Lys and Arg residues from partially degraded fibrin. PCB is produced in plasma via limited proteolysis of the zymogen, pro-PCB. In this report, we show that the K(m) (55 nM) for plasmin-catalyzed activation of pro-PCB is similar to the plasma concentration of pro-PCB (50-70 nM), whereas the K(m) for the thrombin- or thrombin:thrombomodulin-catalyzed reaction is 10-40-fold higher than the pro-PCB level in plasma. Additionally, tissue-type plasminogen activator triggers activation of pro-PCB in blood plasma in a reaction that is stimulated by a neutralizing antibody versus alpha(2)-antiplasmin. Together, these results show that plasmin-mediated activation of pro-PCB can occur in blood plasma. Heparin (UH) and other anionic glycosaminoglycans stimulate pro-PCB activation by plasmin but not by thrombin or thrombin:thrombomodulin. Pro-PCB is a more favorable substrate for plasmin in the presence of UH (16-fold increase in k(cat)/K(m)). UH also stabilizes PCB against spontaneous inactivation. The presence of UH in clots prepared with prothrombin-deficient plasma delays tissue-type plasminogen activator-triggered lysis; this effect of UH on clot lysis is blocked by a PCB inhibitor from potato tubers. These results show that UH accelerates plasmin-catalyzed activation of pro-PCB in plasma and PCB, in turn, stabilizes fibrin against fibrinolysis. We propose that glycosaminoglycans in the subendothelial extracellular matrix serve to augment the levels of PCB activity thereby stabilizing blood clots at sites where there is a breach in the integrity of the vasculature.  (+info)

Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries. (5/124)

BACKGROUND: The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites. RESULTS: We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range. CONCLUSIONS: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery.  (+info)

Effect of the reaction field electrostatic term on the molecular dynamics simulation of the activation domain of procarboxypeptidase B. (6/124)

Molecular dynamics simulations of the activation domain of porcine procarboxypeptidase B (ADBp) were performed in order to examine the effects of the inclusion of a reaction field (RF) term into the calculation of electrostatics forces for highly charged proteins. Two simulations were performed with the GROMOS96 package, studying the influence of counterions on the final results. Comparison with previous results without the inclusion of the RF term (Marti-Renom, M.A., Mas,J.M., Oliva,B., Querol,E. and Aviles,F.X., Protein Engng, 1998, 11, 101-110) shows that the structure is well maintained when the RF term is included. Moreover, the analysis of the trajectories shows that simulations of solvated highly-charged proteins are sensitive to the presence of counterions, the secondary structures being more stable when their charges are neutralized.  (+info)

Acute, nontoxic cadmium exposure inhibits pancreatic protease activities in the mouse. (7/124)

Toxic effects of cadmium on liver, kidney, lung, and testes have been well established in experimental animals and in cell model systems. However, little is known about the effect of cadmium on pancreas, though the pancreas has been reported to accumulate high concentrations of cadmium. Therefore, in this study we examined the effects of cadmium on the pancreas of mice. A single sc injection of 1 mg Cd/kg to mice had no obvious toxic effects on the liver, kidney, and pancreas at both 1 and 5 days after cadmium treatment. Within the pancreas, however, the activities of trypsin, chymotrypsin, and carboxypeptidase A were significantly decreased at 1 day after cadmium treatment, whereas the activity of carboxypeptidase B was not changed. All pancreatic enzyme activities returned to the control levels by 5 days after cadmium treatment. The concentrations of cadmium in pancreas were very similar at 1 and 5 days after cadmium treatment, indicating a stable deposition of the metal. The concentration of zinc in pancreas was markedly increased at 5 days after cadmium treatment. In order to more fully examine the inhibitory effects of cadmium on these protease activities in pancreas, the direct effects of cadmium on purified proteases were studied in vitro. Contrary to the results in vivo, cadmium increased the activity of purified trypsin in a concentration-dependent manner. Consistent with the in vivo results, the activity of purified carboxypeptidase A was decreased by cadmium treatment in a concentration-dependent fashion in vitro. The activities of chymotrypsin and carboxypeptidase B did not change by the cadmium exposure in vitro. The enhanced activity of trypsin by cadmium was returned to the control levels by subsequent treatment with EDTA, indicating that enhancement was reversible. In addition, the zinc normally contained in purified carboxypeptidase A and carboxypeptidase B was released by the cadmium treatment. These results indicate that cadmium inhibits protease activities within the pancreas in vivo at doses that do not induce overt hepatic, renal, or pancreatic toxicity. Based on in vitro study, the decreases seen in trypsin and chymotrypsin activities might be based on indirect effects of cadmium, whereas the decreases in carboxypeptidase A are probably due to the direct inhibition by the metal.  (+info)

Thrombin-activable fibrinolysis inhibitor attenuates (DD)E-mediated stimulation of plasminogen activation by reducing the affinity of (DD)E for tissue plasminogen activator. A potential mechanism for enhancing the fibrin specificity of tissue plasminogen activator. (8/124)

A complex of d-dimer noncovalently associated with fragment E ((DD)E), a degradation product of cross-linked fibrin that binds tissue plasminogen activator (t-PA) and plasminogen (Pg) with affinities similar to those of fibrin, compromises the fibrin specificity of t-PA by stimulating systemic Pg activation. In this study, we examined the effect of thrombin-activable fibrinolysis inhibitor (TAFI), a latent carboxypeptidase B (CPB)-like enzyme, on the stimulatory activity of (DD)E. Incubation of (DD)E with activated TAFI (TAFIa) or CPB (a) produces a 96% reduction in the capacity of (DD)E to stimulate t-PA-mediated activation of Glu- or Lys-Pg by reducing k(cat) and increasing K(m) for the reaction; (b) induces the release of 8 mol of lysine/mol of (DD)E, although most of the stimulatory activity is lost after release of only 4 mol of lysine/mol (DD)E; and (c) reduces the affinity of (DD)E for Glu-Pg, Lys-Pg, and t-PA by 2-, 4-, and 160-fold, respectively. Because TAFIa- or CPB-exposed (DD)E produces little stimulation of Glu-Pg activation by t-PA, (DD)E is not degraded into fragment E and d-dimer, the latter of which has been reported to impair fibrin polymerization. These data suggest a novel role for TAFIa. By attenuating systemic Pg activation by (DD)E, TAFIa renders t-PA more fibrin-specific.  (+info)

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