Purification, properties and substrate specificity of a digestive trypsin from Periplaneta americana (Dictyoptera) adults.
A digestive trypsin from the American cockroach (Periplaneta americana, Dictyoptera) males was purified by a combination of anionic chromatographies in low and high pressure systems. The yield was 70% with a final specific activity of 2,000 units per mg protein (substrate: benzoyl-Arg-p-nitroanilide, BRpNA). Chemical modification with TLCK (k(obs)=3.3 M(-1) s(-1); stoichiometry 1:1) and PMSF (k(obs)=0.18 M(-1) s(-1); stoichiometry 1:1) confirmed that this peptidase is a trypsin. This enzyme has a molecular weight of 29 kDa (SDS-PAGE), a pI of 6.0 and a pH optimum of 8.9. Kinetic parameters using different colorimetric, fluorimetric and internally-quenched substrates indicated that P. americana trypsin prefers to hydrolyze synthetic substrates containing more than one amino acid residue and with an arginine residue at P1 position and a hydrophobic residue at P2. This enzyme presented a Km of 120 microM for BRpNA and is competitively inhibited by benzamidine (Ki=0.25 microM). Soybean trypsin inhibitor is a tight-binding inhibitor presenting a K(D) of 0.4 nM. Differences in substrate specificity and in the reactivity of the trypsin active site groups can be related to adaptation of insects to different hosts. P. americana trypsin is an excellent model for comparison as a basal group on evolutionary studies of insect trypsins.
Lopes
A R
AR
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, C.P. 26077, 05513-970 São Paulo, Brazil.
Terra
W R
WR
D000595:Amino Acid Sequence; D000818:Animals; D004064:Digestive System / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D010526:Periplaneta / Q000201:enzymology*; D013379:Substrate Specificity; D014357:Trypsin / Q000302:isolation & purification / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000494:pharmacology
D014361:Trypsin Inhibitors; D014357:Trypsin