Search

L-Amino acid oxidase (LAO) widely exists in snake venoms. Purification of LAO from the Naja naja kaouthia (monocellate cobra) venom has been reported (Tan and Swaminathan, 1992), but its structural characterization and physiological function remained to be determined. The function of snake venom LAOs in hemostasis, especially their effect on platelet aggregation, has been controversial. We determined the N-terminal amino acid sequence of the N. n. kaouthia LAO named K-LAO to be DDRRSPLEECFQQNDYEEFLEIAKNGLKKTxNPKHVXxV (38 residues). The protein data base search revealed that the enzyme had high similarities with other snake venom LAOs. Further, platelet aggregation studies revealed that K-LAO functionally did not induce platelet aggregation in a platelet-rich plasma system, but that it inhibited platelet aggregation induced by agonists such as ADP, collagen and ristocetin in a dose-dependent manner. K-LAO diminished platelet aggregation more intensely under low than high shear stress. This inhibitory activity of K-LAO on either ristocetin-induced or shear-induced platelet aggregation was quenched by addition of catalase. These results indicate that K-LAO functions as an inhibitor to platelet aggregation through the formation of hydrogen peroxide. The enzyme may contribute to the development of a severe hematological disorder due to cobra envenomation.

The aim of this project was to determine the cytotoxic components from the venom of king cobra, Ophiophagus hannah. Venom was purified by a combination of gel-filtration, ion-exchange and reversed-phase chromatographic steps. The biochemical properties of the cytotoxic component were consistent with those of L-amino acid oxidase. The molecular weight of the enzyme was estimated to be 150,000 by gel filtration and 70,000 under the denaturing conditions of SDS-PAGE, indicating a dimer. It has an isoelectric point of 4.5 and is a glycoprotein. The N-terminal sequence of L-amino acid oxidase from the king cobra venom was determined to be SVINLEESFQEPEYE. The cytotoxicity of L-amino acid oxidase was observed in stomach cancer, murine melanoma, fibrosarcoma, colorectal cancer and Chinese hamster ovary cell lines. Cytotoxicity resulted in the loss of ability in attachment and inhibition of cell proliferation. The cytotoxic protein decreased the level of cell proliferation by 74% according to [3H]thymidine uptake assay. The mechanism of enzyme action may be related to the inhibition of thymidine incorporation and an interaction with DNA.