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Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph.
Arch Insect Biochem Physiol. 2006 Aug; 62(4):186-96.AI

Abstract

The relatively little-investigated entomopathogen Conidiobolus coronatus secretes several proteinases into culture broth. Using a combination of ion-exchange and size-exclusion chromatography, we purified to homogeneity a serine proteinase of Mr 30,000-32,000, as ascertained by SDS-PAGE. The purified enzyme showed subtilisin-like activity. It very effectively hydrolyzed N-Suc-Ala(2)-Pro-Phe-pNa with a Km-1.36 x 10(-4) M and Kcat-24 s(-1), and N-Suc-Ala(2)-Pro-Leu-pNa with Km-6.65 x 10(-4) M and Kcat-11 s(-1). The specificity index k(cat)/K(m) for the tested substrates was calculated to be 176,340 s(-1) M(-1) and 17,030 s(-1) M(-1), respectively. Using oxidized insulin B chain as a substrate, the purified proteinase exhibited specificity to aromatic and hydrophobic amino-acid residues, such as Phe, Leu, and Gly at the P1 position, splitting primarily the peptide bonds: Phe(1)-Val(2), Leu(15)-Tyr(16), and Gly(23)-Phe(24). The proteinase appeared to be sensitive to the specific synthetic inhibitors of the serine proteinases DFP (diisopropyl flourophosphate) and PMSF (phenyl-methylsulfonyl fluoride) as well as to some naturally occurring protein inhibitors of chymotrypsin. It is worth noting that the enzyme exhibited the highest sensitivity to inhibition by AMCI-1 (with an association constant of 3 x 10(10) M(-1)), an inhibitor of cathepsin G/chymotrypsin from the larval hemolymph of Apis mellifera, reinforcing the possibility of involvement of inhibitors from hemolymph in insect innate immunity. The substrate specificity and proteinase inhibitor effects indicate that the purified proteinase from the fermentation broth of Conidiobolus coronatus is a subtilisin-like serine proteinase.

Authors+Show Affiliations

Bania J
Department of Food Hygiene and Consumer Protection, Faculty of Veterinary Medicine, Agricultural University of Wroclaw, Wroclaw, Poland. bania@ozi.ar.wroc.pl
Samborski J
No affiliation info available
Bogus M
No affiliation info available
Polanowski A
No affiliation info available

MeSH

AnimalsChromatography, GelChromatography, Ion ExchangeConidiobolusElectrophoresis, Polyacrylamide GelHemolymphHydrogen-Ion ConcentrationInsulinKineticsSerine EndopeptidasesSerine Proteinase InhibitorsSubstrate Specificity

Pub Type(s)

Comparative Study
Journal Article

Language

eng

PubMed ID

16933280

Citation

Bania, Jacek, et al. "Specificity of an Extracellular Proteinase From Conidiobolus Coronatus and Its Inhibition By an Inhibitor From Insect Hemolymph." Archives of Insect Biochemistry and Physiology, vol. 62, no. 4, 2006, pp. 186-96.
Bania J, Samborski J, Bogus M, et al. Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph. Arch Insect Biochem Physiol. 2006;62(4):186-96.
Bania, J., Samborski, J., Bogus, M., & Polanowski, A. (2006). Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph. Archives of Insect Biochemistry and Physiology, 62(4), 186-96.
Bania J, et al. Specificity of an Extracellular Proteinase From Conidiobolus Coronatus and Its Inhibition By an Inhibitor From Insect Hemolymph. Arch Insect Biochem Physiol. 2006;62(4):186-96. PubMed PMID: 16933280.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph. AU - Bania,Jacek, AU - Samborski,Jaroslaw, AU - Bogus,Mieczyslawa, AU - Polanowski,Antoni, PY - 2006/8/26/pubmed PY - 2006/10/25/medline PY - 2006/8/26/entrez SP - 186 EP - 96 JF - Archives of insect biochemistry and physiology JO - Arch Insect Biochem Physiol VL - 62 IS - 4 N2 - The relatively little-investigated entomopathogen Conidiobolus coronatus secretes several proteinases into culture broth. Using a combination of ion-exchange and size-exclusion chromatography, we purified to homogeneity a serine proteinase of Mr 30,000-32,000, as ascertained by SDS-PAGE. The purified enzyme showed subtilisin-like activity. It very effectively hydrolyzed N-Suc-Ala(2)-Pro-Phe-pNa with a Km-1.36 x 10(-4) M and Kcat-24 s(-1), and N-Suc-Ala(2)-Pro-Leu-pNa with Km-6.65 x 10(-4) M and Kcat-11 s(-1). The specificity index k(cat)/K(m) for the tested substrates was calculated to be 176,340 s(-1) M(-1) and 17,030 s(-1) M(-1), respectively. Using oxidized insulin B chain as a substrate, the purified proteinase exhibited specificity to aromatic and hydrophobic amino-acid residues, such as Phe, Leu, and Gly at the P1 position, splitting primarily the peptide bonds: Phe(1)-Val(2), Leu(15)-Tyr(16), and Gly(23)-Phe(24). The proteinase appeared to be sensitive to the specific synthetic inhibitors of the serine proteinases DFP (diisopropyl flourophosphate) and PMSF (phenyl-methylsulfonyl fluoride) as well as to some naturally occurring protein inhibitors of chymotrypsin. It is worth noting that the enzyme exhibited the highest sensitivity to inhibition by AMCI-1 (with an association constant of 3 x 10(10) M(-1)), an inhibitor of cathepsin G/chymotrypsin from the larval hemolymph of Apis mellifera, reinforcing the possibility of involvement of inhibitors from hemolymph in insect innate immunity. The substrate specificity and proteinase inhibitor effects indicate that the purified proteinase from the fermentation broth of Conidiobolus coronatus is a subtilisin-like serine proteinase. SN - 0739-4462 UR - https://www.unboundmedicine.com/medline/citation/16933280/Specificity_of_an_extracellular_proteinase_from_Conidiobolus_coronatus_and_its_inhibition_by_an_inhibitor_from_insect_hemolymph_ DB - PRIME DP - Unbound Medicine ER -