Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph.
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.
Bania
Jacek
J
Department of Food Hygiene and Consumer Protection, Faculty of Veterinary Medicine, Agricultural University of Wroclaw, Wroclaw, Poland. bania@ozi.ar.wroc.pl
Samborski
Jaroslaw
J
Bogus
Mieczyslawa
M
Polanowski
Antoni
A
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D020092:Conidiobolus / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006458:Hemolymph / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
D007328:Insulin; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases