Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis.
Trypsin-like proteases from the midgut of Anticarsia gemmatalis Hubner (Lepidoptera: Noctuidae) were purified on an aprotinin-agarose column equilibrated with 0.01 M Tris-HCl containing 5 mM CaCl2 (pH 7.5). The yield was 66.7% with a purification factor of 107 and a final specific activity of 6.88 mM/min/mg protein with the substrate N-alpha-benzoyl-L-Arg-p-nitroanilide (L-BApNA). The purified fraction showed three bands with proteolytic activity and molecular weights of 66,000, 71,000 and 91,000 (sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE)). Enzyme specificity assays were carried out using seven synthetic peptides containing 13 amino acid residues, but differing only on the 5th residue (K, R, Y, L, W or P). Peptide cleavage takes place only with amino acids K or R at the 5th position, which is typical of trypsin. The partially purified enzymes hydrolyzed casein and the synthetic trypsin substrates L-BApNA and N-alpha-p-tosyl-L-Arg methyl ester (L-TAME). Higher activity was observed at pH 8.5 and 35 degrees C when using L-BApNA as substrate and at pH 8.0 and 30 degrees C when using L-TAME. Maximum enzyme activity against L-BApNA was obtained with 20 mM CaCl2 in the reaction mixture. The partially purified enzymes showing trypsin activity were sensitive to inhibition by ethylenediaminetetraacetic acid (EDTA), phenylmethyl sulphonyl fluoride (PMSF), N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), benzamidine and aprotinin. Highest inhibition was obtained with TLCK and benzamidine. KM values obtained were 0.32 mM for L-BApNA and 52.5 microM for L-TAME.
Oliveira
M G A
MG
Departamento de Bioquímica e Biologia Molecular, Instituto de Biotecnologia Aplicada a Agropecuária (BIOAGRO), Universidade Federal de Viçosa, Viçosa, MG 36571-000, Brazil. malmeida@ufv.br
De Simone
S G
SG
Xavier
L P
LP
Guedes
R N C
RN
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D007915:Lepidoptera / Q000201:enzymology*; D010455:Peptides / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D014106:Tosylarginine Methyl Ester / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
D002614:Chelating Agents; D010455:Peptides; D011480:Protease Inhibitors; D015842:Serine Proteinase Inhibitors; D014106:Tosylarginine Methyl Ester; D004492:Edetic Acid; D012697:Serine Endopeptidases; D014357:Trypsin; D002118:Calcium