Zymogen Activation (zymogen + activation)

Distribution by Scientific Domains

Selected Abstracts

Zymogen activation in the streptokinase,plasminogen complex

FEBS JOURNAL, Issue 13 2000
Ile1 is required for the formation of a functional active site
Plasminogen (Plgn) is usually activated by proteolysis of the Arg561,Val562 bond. The amino group of Val562 forms a salt-bridge with Asp740, which triggers a conformational change producing the active protease plasmin (Pm). In contrast, streptokinase (SK) binds to Plgn to produce an initial inactive complex (SK感lgn) which subsequently rearranges to an active complex (SK感lgn*) although the Arg561,Val562 bond remains intact. Therefore another residue must substitute for the amino group of Val562 and provide a counterion for Asp740 in this active complex. Two candidates for this counterion have been suggested: Ile1 of streptokinase and Lys698 of Plgn. We have investigated the reaction of SK mutants and variants of the protease domain of microplasminogen (牌lgn) in order to determine if either of these residues is the counterion. The mutation of Ile1 of SK decreases the activity of SK感lgn* by 100-fold (Ile1Val) to ,,104 -fold (Ile1,Ala, Gly, Trp or Lys). None of these mutations perturb the binding affinity of SK, which suggests that Ile1 is not required for formation of SK感lgn but is necessary for SK感lgn*. The substitution of Lys698 of 牌lgn decreases the activity of SK感lgn* by only 10,60-fold. In contrast with the Ile1 substitutions, the Lys698 mutations also decreased the dissociation constant of the SK complex by 15,50-fold. These observations suggest that Lys698 is involved in formation of the initial SK感lgn complex. These results support the hypothesis that Ile1 provides the counterion for Asp740. [source]

Kinetics of intra- and intermolecular zymogen activation with formation of an enzyme,zymogen complex

FEBS JOURNAL, Issue 1 2005
Matilde Esther Fuentes
A mathematical description was made of an autocatalytic zymogen activation mechanism involving both intra- and intermolecular routes. The reversible formation of an active intermediary enzyme,zymogen complex was included in the intermolecular activation route, thus allowing a Michaelis,Menten constant to be defined for the activation of the zymogen towards the active enzyme. Time,concentration equations describing the evolution of the species involved in the system were obtained. In addition, we have derived the corresponding kinetic equations for particular cases of the general model studied. Experimental design and kinetic data analysis procedures to evaluate the kinetic parameters, based on the derived kinetic equations, are suggested. The validity of the results obtained were checked by using simulated progress curves of the species involved. The model is generally good enough to be applied to the experimental kinetic study of the activation of different zymogens of physiological interest. The system is illustrated by following the transformation kinetics of pepsinogen into pepsin. [source]

cDNA sequence, mRNA expression and genomic DNA of trypsinogen from the Indianmeal moth, Plodia interpunctella

Y. C. Zhu
Abstract Trypsin-like enzymes are major insect gut enzymes that digest dietary proteins and proteolytically activate insecticidal proteins produced by the bacterium Bacillus thuringiensis (Bt). Resistance to Bt in a strain of the Indianmeal moth, Plodia interpunctella, was linked to the absence of a major trypsin-like proteinase (Oppert et al., 1997). In this study, trypsin-like proteinases, cDNA sequences, mRNA expression levels and genomic DNAs from Bt-susceptible and -resistant strains of the Indianmeal moth were compared. Proteinase activity blots of gut extracts indicated that the susceptible strain had two major trypsin-like proteinases, whereas the resistant strain had only one. Several trypsinogen-like cDNA clones were isolated and sequenced from cDNA libraries of both strains using a probe deduced from a conserved sequence for a serine proteinase active site. cDNAs of 852 nucleotides from the susceptible strain and 848 nucleotides from the resistant strain contained an open reading frame of 783 nucleotides which encoded a 261-amino acid trypsinogen-like protein. There was a single silent nucleotide difference between the two cDNAs in the open reading frame and the predicted amino acid sequence from the cDNA clones was most similar to sequences of trypsin-like proteinases from the spruce budworm, Choristoneura fumiferana, and the tobacco hornworm, Manduca sexta. The encoded protein included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Northern blotting analysis showed no major difference between the two strains in mRNA expression in fourth-instar larvae, indicating that transcription was similar in the strains. Southern blotting analysis revealed that the restriction sites for the trypsinogen genes from the susceptible and resistant strains were different. Based on an enzyme size comparison, the cDNA isolated in this study corresponded to the gene for the smaller of two trypsin-like proteinases, which is found in both the Bt-susceptible and -resistant strains of the Indianmeal moth. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF064525 for the RC688 strain and AF064526 for HD198). [source]

Characterization of the trypsin-like protease (Ha-TLP2) constitutively expressed in the integument of the cotton bollworm, Helicoverpa armigera,

Yang Liu
Abstract Trypsins belong to the serine endoproteases. They are the most important proteases in insects because of their key roles in food digestion and zymogens activation. But there has been little study of the trypsins in the integuments of insects. In this work, we cloned a trypsin-like protease gene from Helicoverpa armigera and named it trypsin-like protease 2 (Ha-TLP2). Semi-quantitative reverse transcription PCR analysis showed that Ha-TLP2 is constitutively expressed in the integument and can be down-regulated by 20-hydroxyecdysone (20E) and up-regulated by the juvenile hormone (JH) analog methoprene. Immunohistochemistry showed that Ha-TLP2 is located not only in the epidermis, but also in new and old cuticles. Immunoblotting and gelatin-SDS-PAGE revealed that Ha-TLP2 is constitutively expressed with activity in the integument during larval feeding, molting, and metamorphosis. This evidence suggests that Ha-TLP2 is involved in the remodeling of the integument. 2009 Wiley Periodicals, Inc. [source]