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Proteases in egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine proteases from adult.
Comp Biochem Physiol B Biochem Mol Biol. 2005 Oct; 142(2):192-200.CB

Abstract

Proteolytic activity of 0-12 day old eggs, miracidium and adult worm of Fasciola gigantica was assessed and proteases were partially purified by DEAE-Sepharose and CM-cellulose columns. Four forms of protease were separated, PIa, PIb, PIc and PII. Purifications were completed for PIc and PII using Sephacryl S-200 chromatography. A number of natural and synthetic proteins were tested as substrates for F. gigantica PIc and PII. The two proteases had moderate activity levels toward azoalbumin and casein compared to azocasein, while gelatin, hemoglobin, albumin and fibrin had very low affinity toward the two enzymes. Amidolytic substrates are more specific to protease activity. PIc had higher affinity toward BAPNA-HCl (N-benzoyl-arginine-p-nitroanilide-HCl) and BTPNA-HCl (N-benzoyl-tyrosine-p-nitroanilide-HCl) at pH 8.0 indicating that the enzyme was a serine protease. However, PII had higher affinity toward BAPNA at pH 6.5 in the presence of sulfhydryl groups (beta-mercaptoethanol) indicating that the enzyme was a cysteine protease. The effect of specific protease inhibitors on these enzymes was studied. The results confirmed that proteases PIc and PII could be serine and cysteine proteases, respectively. The molecular weights of F. gigantica PIc and PII were 60,000 and 25,000, respectively. F. gigantica PIc and PII had pH optima at 7.5 and 5.5 and K(M) of 2 and 5 mg azocasein/mL, respectively. For amidolytic substrates, PIc had K(M) of 0.3 mM BAPNA/mL and 0.5 mM BTPNA/mL at pH 8.0 and PII had K(M) of 0.6 mM BAPNA/mL at pH 6.5 with reducing agent. F. gigantica PIc and PII had the same optimum temperature at 50 degrees C and were stable up to 40 degrees C. All examined metal cations tested had inhibitory effects toward the two enzymes. From substrate specificity and protease inhibitor studies, PIc and PII could be designated as serine PIc and cysteine PII, respectively.

Authors+Show Affiliations

Mohamed SA
Molecular Biology Department, National Research Centre, Cairo, Egypt. saleh38@hotmail.com
Fahmy AS
No affiliation info available
Mohamed TM
No affiliation info available
Hamdy SM
No affiliation info available

MeSH

AnimalsCysteine EndopeptidasesFasciolaHelminth ProteinsLarvaLife Cycle StagesMolecular WeightOvumSerine Endopeptidases

Pub Type(s)

Journal Article

Language

eng

PubMed ID

16102991

Citation

Mohamed, Saleh A., et al. "Proteases in Egg, Miracidium and Adult of Fasciola Gigantica. Characterization of Serine and Cysteine Proteases From Adult." Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, vol. 142, no. 2, 2005, pp. 192-200.
Mohamed SA, Fahmy AS, Mohamed TM, et al. Proteases in egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine proteases from adult. Comp Biochem Physiol B Biochem Mol Biol. 2005;142(2):192-200.
Mohamed, S. A., Fahmy, A. S., Mohamed, T. M., & Hamdy, S. M. (2005). Proteases in egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine proteases from adult. Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, 142(2), 192-200.
Mohamed SA, et al. Proteases in Egg, Miracidium and Adult of Fasciola Gigantica. Characterization of Serine and Cysteine Proteases From Adult. Comp Biochem Physiol B Biochem Mol Biol. 2005;142(2):192-200. PubMed PMID: 16102991.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Proteases in egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine proteases from adult. AU - Mohamed,Saleh A, AU - Fahmy,Afaf S, AU - Mohamed,Tarek M, AU - Hamdy,Soha M, PY - 2005/04/07/received PY - 2005/06/26/revised PY - 2005/07/05/accepted PY - 2005/8/17/pubmed PY - 2005/12/13/medline PY - 2005/8/17/entrez SP - 192 EP - 200 JF - Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology JO - Comp Biochem Physiol B Biochem Mol Biol VL - 142 IS - 2 N2 - Proteolytic activity of 0-12 day old eggs, miracidium and adult worm of Fasciola gigantica was assessed and proteases were partially purified by DEAE-Sepharose and CM-cellulose columns. Four forms of protease were separated, PIa, PIb, PIc and PII. Purifications were completed for PIc and PII using Sephacryl S-200 chromatography. A number of natural and synthetic proteins were tested as substrates for F. gigantica PIc and PII. The two proteases had moderate activity levels toward azoalbumin and casein compared to azocasein, while gelatin, hemoglobin, albumin and fibrin had very low affinity toward the two enzymes. Amidolytic substrates are more specific to protease activity. PIc had higher affinity toward BAPNA-HCl (N-benzoyl-arginine-p-nitroanilide-HCl) and BTPNA-HCl (N-benzoyl-tyrosine-p-nitroanilide-HCl) at pH 8.0 indicating that the enzyme was a serine protease. However, PII had higher affinity toward BAPNA at pH 6.5 in the presence of sulfhydryl groups (beta-mercaptoethanol) indicating that the enzyme was a cysteine protease. The effect of specific protease inhibitors on these enzymes was studied. The results confirmed that proteases PIc and PII could be serine and cysteine proteases, respectively. The molecular weights of F. gigantica PIc and PII were 60,000 and 25,000, respectively. F. gigantica PIc and PII had pH optima at 7.5 and 5.5 and K(M) of 2 and 5 mg azocasein/mL, respectively. For amidolytic substrates, PIc had K(M) of 0.3 mM BAPNA/mL and 0.5 mM BTPNA/mL at pH 8.0 and PII had K(M) of 0.6 mM BAPNA/mL at pH 6.5 with reducing agent. F. gigantica PIc and PII had the same optimum temperature at 50 degrees C and were stable up to 40 degrees C. All examined metal cations tested had inhibitory effects toward the two enzymes. From substrate specificity and protease inhibitor studies, PIc and PII could be designated as serine PIc and cysteine PII, respectively. SN - 1096-4959 UR - https://www.unboundmedicine.com/medline/citation/16102991/Proteases_in_egg_miracidium_and_adult_of_Fasciola_gigantica__Characterization_of_serine_and_cysteine_proteases_from_adult_ DB - PRIME DP - Unbound Medicine ER -