Leucaena leucocephala serine proteinase inhibitor: primary structure and action on blood coagulation, kinin release and rat paw edema.
A serine proteinase inhibitor isolated from Leucaena leucocephala seeds (LlTI) was purified to homogeneity by acetone fractionation, ion exchange chromatography, gel filtration and reverse phase chromatography (HPLC). SDS-PAGE indicated a protein with M(r) 20000 and two polypeptide chains (alpha-chain, M(r) 15000, and beta-chain, M(r) 5000), the sequence being determined by automatic Edman degradation and by mass spectroscopy. LlTI is a 174 amino acid residue protein which shows high homology to plant Kunitz inhibitors, especially those double chain proteins purified from the Mimosoideae subfamily. LlTI inhibits plasmin (K(i) 3.2 x 10(-10) M), human plasma kallikrein (K(i) 6.3 x 10(-9) M), trypsin (K(i) 2.5 x 10(-8) M) and chymotrypsin (K(i) 1.4 x 10(-8) M). Factor XIIa activity is inhibited but K(i) was not determined, and factor Xa, tissue kallikrein and thrombin are not inhibited by LlTI. The action of LlTI on enzymes that participate in the blood clotting extrinsic pathway is confirmed by the prolongation of activated partial thromboplastin time, used as clotting time assay. The inhibition of the fibrinolytic activity of plasmin was confirmed on the hydrolysis of fibrin plates. LlTI inhibits kinin release from high molecular weight kininogen by human plasma kallikrein in vitro and, administered intravenously, causes a decrease in paw edema induced by carrageenin or heat in male Wistar rats. In addition, lower concentrations of bradykinin were found in limb perfusion fluids of LlTI-treated rats.
Oliva
M L
ML
Departamento de Bioquímica, Universidade Federal de São Paulo, Rua Três de Maio 100, 04044-020, São Paulo, Brazil. maysa.bioq@epm.br
Souza-Pinto
J C
JC
Batista
I F
IF
Araujo
M S
MS
Silveira
V F
VF
Auerswald
E A
EA
Mentele
R
R
Eckerskorn
C
C
Sampaio
M U
MU
Sampaio
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D001777:Blood Coagulation / Q000187:drug effects*; D001920:Bradykinin / Q000378:metabolism*; D004487:Edema / Q000188:drug therapy / Q000209:etiology; D005342:Fibrinolysis / Q000187:drug effects; D007610:Kallikreins / Q000037:antagonists & inhibitors; D008297:Male; D008969:Molecular Sequence Data; D010314:Partial Thromboplastin Time; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D012639:Seeds; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology
D000894:Anti-Inflammatory Agents, Non-Steroidal; C408183:L1TI protein, Leucaena leucocephala; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; D007610:Kallikreins; D001920:Bradykinin