Purification and characterization of a trypsin-papain inhibitor from Pithecelobium dumosum seeds and its in vitro effects towards digestive enzymes from insect pests.
A novel trypsin-papain inhibitor, named PdKI-2, was purified from the seeds of Pithecelobium dumosum seeds by TCA precipitation, Trypsin-Sepharose chromatography and reversed-phase HPLC. PdKI-2 had an M(r) of 18.1 kDa as determined by SDS-PAGE and was composed of a single polypeptide chain. The inhibition on trypsin was stable at pH range 2-10, temperature of 50 degrees C and had a K(i) value of 1.65 x 10(-8)M, with a competitive inhibition mechanism. PdKI-2 was also active to papain, a cysteine proteinase, and showed a noncompetitive inhibition mechanism and K(i) value of 5.1 x 10(-7)M. PdKI-2 was effective against digestive proteinase from bruchids Zabrotes subfasciatus and Callosobruchus maculatus; Dipteran Ceratitis capitata; Lepidopterans Plodia interpunctella and Alabama argillacea, with 74.5%, 70.0%, 70.3%, 48.7%, and 13.6% inhibition, respectively. Results support that PdKI-2 is a member of Kunitz-inhibitor family and its effect on digestive enzyme larvae from diverse orders indicated this protein as a potent insect antifeedant.
Oliveira
Adeliana S
AS
Departamento Bioquímica, Laboratório de Química e Função de Proteínas, Centro de Biociências, Campus UFRN, Av. Salgado Filho s/n, 59072-970 Natal, RN, Brazil.
Migliolo
Ludovico
L
Aquino
Rodrigo O
RO
Ribeiro
Jannison K C
JK
Macedo
Leonardo L P
LL
Andrade
Lucia B S
LB
Bemquerer
Marcelo P
MP
Santos
Elizeu A
EA
Kiyota
Sumika
S
de Sales
Maurício P
MP
D000818:Animals; D004064:Digestive System / Q000201:enzymology*; D004175:Diptera / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D019476:Insect Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D007313:Insecta / Q000201:enzymology; D007700:Kinetics; D007915:Lepidoptera / Q000201:enzymology; D010206:Papain / Q000037:antagonists & inhibitors*; D011480:Protease Inhibitors / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology; D012639:Seeds / Q000378:metabolism*; D013696:Temperature; D014361:Trypsin Inhibitors / Q000302:isolation & purification* / Q000378:metabolism / Q000494:pharmacology
D019476:Insect Proteins; D011480:Protease Inhibitors; D014361:Trypsin Inhibitors; D010206:Papain