Partial purification and characterization of trypsin-like proteinases from insecticide-resistant and -susceptible strains of the maize weevil, Sitophilus zeamais.
Serine proteinases from three strains of Sitophilus zeamais (Coleoptera: Curculionidae), one susceptible and two resistant to insecticides--one exhibiting fitness cost (resistant cost strain) and the other lacking it (resistant no-cost strain), were partially purified using an aprotinin-agarose affinity column providing purification factors ranging from 36.5 to 51.2%, with yields between 10 and 15% and activity between 529 and 875 microM/min/mg protein with the substrate N-alpha-benzoyl-L-Arg-p-nitroanilide (L-BApNA). SDS-PAGE of the purified fraction revealed a 56,000 Da molecular mass band in all strains and a 70,000 Da band more visible in the resistant no-cost strain. The purified proteinases from all strains were inhibited by phenylmethyl sulphonyl fluoride (PMSF), N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), aprotinin, benzamidine and soybean trypsin inhibitor (SBTI) characterizing them as trypsin-like serine proteinases. Trypsin-like proteinases from the resistant strains exhibited higher affinity for L-BApNA. The resistant no-cost strain exhibited V(max)-values 1.5- and 1.7-fold higher than the susceptible and resistance cost strains, respectively. A similar trend was also observed when using N-alpha-p-tosyl-L-Arg methyl ester (L-TAME) as substrate. These results provide support to the hypothesis that the enhanced serine proteinase activity may be playing a role in mitigating physiological costs associated with the maintenance of insecticide resistance mechanisms in some maize weevil strains.
Silva
L B
LB
Departamento de Biologia Animal, Universidade Federal de Viçosa, MG 36571-000, Brazil.
Reis
A P
AP
Pereira
E J G
EJ
Oliveira
M G A
MG
Guedes
R N C
RN
D000818:Animals; D001586:Benzoylarginine Nitroanilide / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D001517:Coleoptera / Q000145:classification / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007305:Insecticide Resistance; D007306:Insecticides / Q000633:toxicity*; D007700:Kinetics; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D057057:Serine Proteases / Q000302:isolation & purification / Q000378:metabolism*; D013045:Species Specificity; D013379:Substrate Specificity; D013696:Temperature; D014357:Trypsin / Q000302:isolation & purification / Q000378:metabolism; D003313:Zea mays / Q000469:parasitology*
D019476:Insect Proteins; D007306:Insecticides; D011480:Protease Inhibitors; D010664:Phenylmethylsulfonyl Fluoride; D001586:Benzoylarginine Nitroanilide; D057057:Serine Proteases; D014357:Trypsin