Two Kunitz-type inhibitors with activity against trypsin and papain from Pithecellobium dumosum seeds: purification, characterization, and activity towards pest insect digestive enzyme.
Two trypsin inhibitors (called PdKI-3.1 and PdKI-3.2) were purified from the seeds of the Pithecellobium dumosum tree. Inhibitors were obtained by TCA precipitation, affinity chromatography on Trypsin-Sepharose and reversed-phase-HPLC. SDS-PAGE analysis with or without reducing agent showed that they are a single polypeptide chain, and MALDI-TOF analysis determined molecular masses of 19696.96 and 19696.36 Da, respectively. The N-terminal sequence of both inhibitors showed strong identity to the Kunitz family trypsin inhibitors. They were stable over a wide pH (2-9) and temperature (37 to 100 degrees C) range. These inhibitors reduced over 84% of trypsin activity with inhibition constant (Ki) of 4.20 x 10(-8) and 2.88 x 10(-8) M, and also moderately inhibited papain activity, a cysteine proteinase. PdKI-3.1 and PdKI-3.2 mainly inhibited digestive enzymes from Plodia interpunctella, Zabrotes subfasciatus and Ceratitis capitata guts. Results show that both inhibitors are members of the Kunitz-inhibitor family and that they affect the digestive enzyme larvae of diverse orders, indicating a potential insect antifeedant.
Oliveira
A S
AS
Laboratório de Química eFunção de Proteínas, Departamento de Bioquímica, Centro de Biociências-UFRN, Natal-RN, Brazil.
Migliolo
L
L
Aquino
R O
RO
Ribeiro
J K C
JK
Macedo
L L P
LL
Bemquerer
M P
MP
Santos
E A
EA
Kiyota
S
S
de Sales
M P
MP
D000595:Amino Acid Sequence; D000818:Animals; D001963:Bromelains / Q000037:antagonists & inhibitors / Q000378:metabolism; D002417:Cattle; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D007887:Fabaceae / Q000737:chemistry*; D007814:Larva / Q000187:drug effects / Q000201:enzymology; D007915:Lepidoptera / Q000187:drug effects* / Q000201:enzymology; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors; D010206:Papain / Q000037:antagonists & inhibitors*; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D010940:Plant Proteins / Q000737:chemistry / Q000494:pharmacology*; D012639:Seeds / Q000737:chemistry; D016415:Sequence Alignment; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism*
C053865:Kunitz-type protease inhibitor, plant; C547255:PdKI-3.1 protein, Pithecellobium dumosum; C547256:PdKI-3.2 protein, Pithecellobium dumosum; D010455:Peptides; D010940:Plant Proteins; D001963:Bromelains; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin; D010206:Papain