Purification and Properties of the L-Amino Acid Oxidase from Malayan Pit Viper (Calloselasma rhodostoma) Venom

Ponnudurai, Gnanajothy; Chung, Maxey C.M.; Tan, Nget-Hong

doi:10.1006/abbi.1994.1401

Cited by 100 publications

(88 citation statements)

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“…5). According to a protein sequence data base search, it was revealed that the sequence has a close similarity (75% identity) to LAO from Malayan pit viper (Calloselasma rhodostoma) venom and a moderate similarity (45% identity) to LAO from king cobra (Ophiophagus hannah) venom (19) (Fig. 5).…”

Section: Resultsmentioning

confidence: 99%

“…5). The LAO from the Malayan pit viper is a homodimeric glycoprotein composed of a 66-kDa polypeptide as determined by SDS-polyacrylamide gel electrophoresis (19), resembling apoxin I. Therefore, we measured the LAO activity of the purified apoxin I. Apoxin I oxidized L-leucine in a dose-and time-dependent manner, whereas it did not oxidize D-leucine (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Apoxin I, a Novel Apoptosis-inducing Factor with L-Amino Acid Oxidase Activity Purified from Western Diamondback Rattlesnake Venom

Torii

Naito

Tsuruo

1997

Journal of Biological Chemistry

172

108

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Venom of the western diamondback rattlesnake (Crotalus atrox) induces apoptosis in human umbilical vein endothelial cells, which could result in hemorrhage in tissues bitten by the snake. To identify the hemorrhagic factor, we purified a novel protein, apoxin I, from rattlesnake venom. Apoxin I induced apoptosis in human umbilical vein endothelial, human promyelocytic leukemia HL-60, human ovarian carcinoma A2780, and mouse endothelial KN-3 cells. Amino acid sequence analysis of the apoxin I showed close similarity to L-amino acid oxidase from the Malayan pit viper (Calloselasma rhodostoma). The purified apoxin I oxidized L-leucine but not D-leucine to produce H 2 O 2 . The apoxin I-induced apoptosis was inhibited by catalase, a H 2 O 2 scavenger. These results indicate that the H 2 O 2 produced by L-amino acid oxidation by apoxin I is involved in the apoxin I-induced apoptosis and in hemorrhage caused by rattlesnake venom.Apoptosis is a physiological process by which cells undergo controlled cell death, accompanied by nuclear condensation and fragmentation prior to loss of membrane integrity (1-3). Many kinds of stimuli have been reported to induce apoptosis in a variety of cell systems. These include ligation of Fas and tumor necrosis factor receptors by their ligands (4 -8), deprivation of growth factors (9), and cytotoxic stimuli such as anticancer drugs (10 -12) and ionized radiation (13). In addition to these well characterized stimuli, hemorrhagic snake venoms from the western diamondback rattlesnake (Crotalus atrox), mamushi (Agkistrodon halys blomhottii), puff adder (Bitis arietans), and habu (Trimeresurus flavoviridis) induce apoptosis in vascular endothelial cells (14), although the active component in the venom is not identified. We report here the identification of the apoptosis-inducing factor apoxin I from venom of the western diamondback rattlesnake that has Lamino acid oxidase (LAO, 1 EC 1.4.3.2) activity. EXPERIMENTAL PROCEDURESMaterials-Lyophilized crude venom from the western diamondback rattlesnake was purchased from Sigma. Benzyloxycarbonyl-Val-AlaAsp-CH 2 OC(O)-2,6-dichlorobenzene (Z-VAD) and benzyloxycarbonylAsp-CH 2 OC(O)-2,6-dichlorobenzene (Z-Asp) were synthesized at Kirin Brewery Co. Ltd (Gunma, Japan) as described (15) and dissolved in Me 2 SO at 10 mg/ml. Trolox (Aldrich) was dissolved in 1 M NaHCO 3 at a concentration of 300 mM, and the pH was adjusted to 7.0. For the experiments, the solution was diluted to the desired concentration with medium. Catalase and all other chemicals were purchased from Wako Pure Chemicals (Tokyo, Japan) and Sigma.Purification of Apoxin I-Lyophilized crude venom (100 mg) from the western diamondback rattlesnake (Sigma) was dissolved in 5 ml of distilled water, and the insoluble material was removed by centrifugation (1,000 ϫ g) for 10 min at 4°C. The supernatant was applied to Sephadex G-100 column (Pharmacia Biotech Inc.; 3 ϫ 60 cm) equilibrated with 50 mM sodium phosphate buffer containing 0.15 M sodium chloride (pH 7.0), and the proteins were eluted ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Apoxin I, a Novel Apoptosis-inducing Factor with L-Amino Acid Oxidase Activity Purified from Western Diamondback Rattlesnake Venom

Torii

Naito

Tsuruo

1997

Journal of Biological Chemistry

172

108

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“…The existence of specific paths for H 2 O 2 has previously been shown in catalases, where peroxide acts as a substrate. 20 Comparison of the residues that line the catalase peroxide channel and the observed channel in LAAO reveal predominantly hydrophobic residues or the aliphatic portions of polar residues, as expected to suit the less polar H 2 O 2 molecule (dipole moment = 1.57 D). Interestingly, the path suggested for peroxide release in LAAO is positioned near to the glycosylation site at Asn172.…”

Section: Possible Inlet and Outlet For Oxygen And Hydrogen Peroxidementioning

confidence: 94%

“…LAAO from C.rhodostama was purified from isolated snake venom as outlined by Ponnudari et al 20 Prior to crystallization, the enzyme, stored at −80 o C, was thawed and subjected to a reactivation process by incubating at 30 o C after dialysis against 200 mM acetate buffer (pH 4.6). The enzyme activity was confirmed using a horseradish peroxidase coupled assay as described in the purification paper.…”

Section: Protein Purification and Crystallizationmentioning

confidence: 99%

Crystal Structure of LAAO from Calloselasma rhodostoma with an l-Phenylalanine Substrate: Insights into Structure and Mechanism

Moustafa

Foster

Lyubimov

et al. 2006

Journal of Molecular Biology

139

169

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L-amino acid oxidase (LAAO) is a dimeric glycosylated flavoenzyme, a major constituent of the snake-venom from Calloselasma rhodostoma. The enzyme exhibits apoptosis-inducing effects as well as antibacterial and anti-HIV activities. The structure of LAAO with its substrate (Lphenylalanine) has been refined to a resolution of 1.8 Å. The complex structure reveals the substrate bound to the reduced flavin (FAD red ). Alternate conformations for the key residues: His223 and Arg322 are evident, suggesting a dynamic active site. Furthermore conformational changes are also apparent for the isoalloxazine ring; the three ring system exhibits more bending around the N5-N10 axis compared to the oxidized flavin. The implications of the observed dynamics on the mechanism of catalysis are discussed. Inspection of buried surfaces in the enzyme reveals a Y shaped channel system extending from the external surface of the protein to the active site. One portion of this channel may serve as the entry path for O 2 during the oxidative half reaction. The second region, separated from the proposed O 2 channel by the N-terminus (residues 8 -16) of the protein, may play a role in H 2 O 2 release. Interestingly, the later portion of the channel would direct the H 2 O 2 product to the exterior surface of the protein, near to the glycan moiety, thought to anchor the enzyme to the host cell. This channel location may explain the ability of the enzyme to localize H 2 O 2 to the targeted cell inducing the apoptotic effect.

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“…The process of necrosis could be related to the direct action of hydrogen peroxide on the cell plasma membrane, since the mechanism of apoptosis in the development of morphological and biochemical changes leads to cell death (Ande et al, 2006). Therefore, most of the biological effects of LAAO may be due to the secondary effect of hydrogen peroxide generated during the specific catalytic activity (Ponnudurai et al, 1994). This hypothesis can be supported when catalase is added to the medium in experimental conditions, when the toxic activity is totally abolished (Toyama et al, 2006;Izidoro et al, 2006;Rodrigues et al, 2009;Ciscotto et al, 2009).…”

Section: Discussionmentioning

confidence: 99%

Bothrops pirajai snake venom L-amino acid oxidase: in vitro effects on infection of Toxoplasma gondii in human foreskin fibroblasts

Izidoro¹,

Alves²,

Rodrigues³

et al. 2011

Rev. bras. farmacogn.

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Abstract:The effect of an L-amino acid oxidase isolated from Bothrops pirajai snake venom (BpirLAAO-I) was investigated on infection of Toxoplasma gondii in human foreskin fibroblasts (HFF). The cytotoxic activity of BpirLAAO-I on HFF cells showed a dose-dependent toxicity with median cytotoxic dose (TD50) of 11.8 µg/mL. BpirLAAO-I induced considerable dose-dependent decrease in the T. gondii infection index under two different conditions, treatment of tachyzoites before infection or treatment of HFF cells after infection. A maximal inhibition of infection (56%) was found for treatment before infection, with a median inhibitory dose (ID50) at 1.83 µg/mL and selectivity index (SI) at 6.45. For treatment after infection, it was observed a maximal inhibition of infection at 65%, ID50 of 1.20 µg/mL and SI of 9.83. The treatment before infection was also effective to reduce intracellular parasitism up to 62%, although presenting higher values of ID50 (3.14 µg/mL) and lower values of SI (3.76). However, treatment after infection was not effective, suggesting that the enzyme seems to have no effect on the parasite intracellular replication for this condition. In conclusion, BpirLAAO-I was more effective to inhibit the infection of neighboring cells and consequently parasite dissemination than primary infection and parasite replication. Thus, the effect of BpirLAAO-I described herein could be taken into account for the development of new synthetic anti-parasite therapeutic agents.

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Purification and Properties of the L-Amino Acid Oxidase from Malayan Pit Viper (Calloselasma rhodostoma) Venom

Cited by 100 publications

References 0 publications

Apoxin I, a Novel Apoptosis-inducing Factor with L-Amino Acid Oxidase Activity Purified from Western Diamondback Rattlesnake Venom

Apoxin I, a Novel Apoptosis-inducing Factor with L-Amino Acid Oxidase Activity Purified from Western Diamondback Rattlesnake Venom

Crystal Structure of LAAO from Calloselasma rhodostoma with an l-Phenylalanine Substrate: Insights into Structure and Mechanism

Bothrops pirajai snake venom L-amino acid oxidase: in vitro effects on infection of Toxoplasma gondii in human foreskin fibroblasts

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