Purification and characterization of phenoloxidase from crab Charybdis japonica

Liu, Guangxing; Yang, Lingling; Fan, Ting-Jun; Cong, Rongjuan; Tang, Zhe; Sun, Wenjie; Meng, Xia; Zhu, Lingyu

doi:10.1016/j.fsi.2005.03.012

Cited by 68 publications

(56 citation statements)

References 32 publications

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“…The R. prolixus trypanolytic factor's biological activity remained stable at room temperature during parasite counting (10 h) or at 7 to 10.5 pH, possibly due to the protein's chemical or physical stability not being affected, thereby conserving its native state. Lytic activity against T. rangeli KP1(-) continued in EGTA and EDTA chelate agent-treated hemolymph; on the other hand, we deduced that the trypanolytic factor did not belong to the metalloprotein family or any lectin requiring divalent cations for carrying out its normal biological activity as described by Ashida et al (1990, Yang et al (2000), Konozy et al (2003), Liu et al (2006).…”

Section: Discussionmentioning

confidence: 82%

Preliminary characterization of a Rhodnius prolixus hemolymph trypanolytic protein, this being a determinant of Trypanosoma rangeli KP1(+) and KP1(-) subpopulations' vectorial ability

Pulido

Pérez

Vallejo

2008

Mem. Inst. Oswaldo Cruz

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Rhodnius prolixus is the main Trypanosoma rangeli vector in several Latin-American countries and is susceptible to infection with KP1(+) strains; however, it presents an invasion-resistant response to KP1(-) strains. The present work has identified a trypanolytic protein against T. rangeli KP1(-) in the R. prolixus hemolymph which was fractioned with ammonium sulfate (following dialysis). The results revealed a protein component which did not depend on divalent cations for its biological function whilst keeping its trypanolytic activity at temperatures ranging from -20ºC to 37ºC, at 7.0 to 10.5 pH. The protein was partially purified by gel filtration chromatography and ionic exchange chromatography. The major component presented a molecular weight of around 79 kDa and an isoelectric point between 4.9 and 6.3 and may be directly related to hemolymph trypanolytic activity against T. rangeli KP1(-) populations.Key words: Rhodnius prolixus -Trypanosoma rangeli KP1(+) -Trypanosoma rangeli KP1(-) -trypanolytic factorvectorial ability -insect immunity Trypanosoma rangeli is a hemoflagellate which is apparently apathogenous for humans, presenting pathogenicity for the invertebrate host. T. rangeli is of great interest due to its presence in mixed infection with Trypanosoma cruzi in vertebrates and invertebrate hosts, thereby interfering with a clear diagnosis of Chagas' disease and the epidemiology of these parasites in different Latin-American countries .T. rangeli vectors become infected by ingesting blood from wild or domestic reservoirs in natural conditions. The parasite develops in the digestive tract and then invades the hemolymph where it is recognized by the vector's immune system; however, some strains may escape host defense mechanisms, reaching the salivary glands where they become infective forms or metacyclic trypomastigotes (D'Alessandro-Bacigalupo & Gore-Saravia 1992). Salivary gland invasion is a necessary step for transmission, being mediated by one or several ligandreceptor interactions (Basseri et al. 2002).Previous work has revealed two phylogenetically different groups in T. rangeli called T. rangeli KP1(-) and T. rangeli KP1(+), these being mainly transmitted by the 16 Rhodnius species described so far (Vallejo et al. 2002(Vallejo et al. , 2007. Specificity between the vector and the parasite strain which it transmits has been pointed out; each Rhodnius species seems to select the T. rangeli subpopulation to which it is susceptible in nature so that it can then be transmitted to the vertebrate host via insect bite . Nevertheless, a close association has been observed between T. rangeli KP1(+) and vectors from the prolixus group (which includes Rhodnius prolixus and Rhodnius neglectus) and between T. rangeli KP1(-) and vectors from the pallescens group (including Rhodnius pallescens, Rhodnius colombiensis and Rhodnius ecuadoriensis) , Urrea et al. 2005. Recent parasite-vector interaction studies have shown a trypanolytic factor in R. prolixus hemolymph impeding the development of T. rangeli KP1(...

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Section: Discussionmentioning

confidence: 82%

Preliminary characterization of a Rhodnius prolixus hemolymph trypanolytic protein, this being a determinant of Trypanosoma rangeli KP1(+) and KP1(-) subpopulations' vectorial ability

Pulido

Pérez

Vallejo

2008

Mem. Inst. Oswaldo Cruz

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“…Several earlier reports suggest a wide range of molecular mass for proPO in arthropods. It exists as a monomeric protein weighing 113.9 kDa in P. californiensis [7], a tetramer having 300 kDa in crayfish [20], 37 kDa in Procambarus clarkii [21] and 69.5 kDa in Charybdis japonica [22].…”

Section: Discussionmentioning

confidence: 99%

Properties of phenoloxidase from haemocytes of Scylla tranquebarica

2016

J App Biol Biotech

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The presence of phenoloxidase (PO) activity in the haemocytes of Scylla tranquebarica was electrophoretically and spectrophotometrically studied. Majority of the enzyme was located as proenzyme, prophenoloxidase (proPO) in the haemocytes. The enzyme prefers L-dihydroxyphenylalanine (L-dopa) as its substrate than phenol and is optimally active at pH 8.0. Besides trypsin, the proPO was also activated by both Gram positive and Gram negative microbes in vitro while, chemicals such as sodium azide, thiourea and EDTA significantly inhibited the enzyme expression. The protein needs considerable levels of divalent cations like calcium (20 mM as CaCl2) or magnesium (20-50 mM as MgCl2) for its activity. The gel filtration chromatography of haemocyte lysate supernatant showed a single major peak of protein having PO activity. Electrophoresis of purified PO by native PAGE revealed a single prominent band of approximately 167.2 kDa which was further resolved to three bands having molecular mass of approximately 77.1, 56.9 and 30.2 kDa respectively, on SDS-PAGE.

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“…Meanwhile, PO was measured to be 78.5 kDa of molecular mass that corresponds with reports on POs of other insects 2 . Liu et al 16 believed that different biochemical properties of POs like pH, response to xenobiotic and isoforms could be attributed to species and its survival conditions that appeared in in vitro studies. PO is one of the sensitive enzymes in insects.…”

Section: Discussionmentioning

confidence: 99%

“…Although purified PO from hemocytes of 17,18 . These differences could be correlated with pH of hemolymph that indicate type of species and survival conditions 16,19 . Enzymes have temperature-dependent activities that are correlated with enzymatic stability and temperature of biochemical media.…”

Section: Discussionmentioning

confidence: 99%

“…Increased activity of the PO from hemocytes of E. kuehniella demonstrate that the enzyme is a metalloprotein that and small changes at the metal center can amplify its biological role. Moreover, metal ions (Mainly divalent cations) increase PO activity by changing its protein folding 16,20 . In fact, binding of metal ions significantly affect secondary structure of some peptides depending on their nature 20 .…”

Section: Discussionmentioning

confidence: 99%

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Purification and characterization of a phenoloxidase in the hemocytes ofEphestia kuehniellaZeller (Lepidoptera: Pyralidae): effects of insect growth regulators and endogenous inhibitors

Delkash-Roudsari

Zibaee

Bigham

2014

Journal of Enzyme Inhibition and Medicinal Chemistry

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A phenoloxidase was extracted and purified from hemocytes of Ephestia kuehniella by using ammonium sulfate, Sepharyl G-100 and DEAE-Cellulose fast flow chromatographies. At the final stage of purification, a protein was purified by molecular mass of 78.5 kDa, specific activity of decreased activity of the purified PO but Ca 2+ and Cu 2+ increased the enzymatic activity. EDTA (General chelating agent), DTC (Copper chelating agent) and EGTA (Calcium chelating agent) significantly decreased PO activity but TTHA (Magnesium chelating agent) showed no statistically significant effects. Kinetic parameters of the purified enzyme showed the highest V max when L-DOPA was used as substrate but no significant differences were observed in case of K m for used L-DOPA, pyrocatechol and hydroquinone. In vitro inhibition of the purified PO by using two insect growth regulators, Hexaflumuron and Pyriproxyfen, revealed IC 50 of 96.41 and 38.59 mg/ml for these compounds, respectively. Kinetic studies using different concentrations of L-DOPA and IC 50 concentrations of the two IGRs revealed the increase of K m value versus control and competitive inhibition. Finally, column chromatography of hemolymph revealed peak III showing endogenous inhibitors of phenoloxidase by molecular weight of 27.3 that showed competitive inhibition on the PO.

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Purification and characterization of phenoloxidase from crab Charybdis japonica

Cited by 68 publications

References 32 publications

Preliminary characterization of a Rhodnius prolixus hemolymph trypanolytic protein, this being a determinant of Trypanosoma rangeli KP1(+) and KP1(-) subpopulations' vectorial ability

Preliminary characterization of a Rhodnius prolixus hemolymph trypanolytic protein, this being a determinant of Trypanosoma rangeli KP1(+) and KP1(-) subpopulations' vectorial ability

Properties of phenoloxidase from haemocytes of Scylla tranquebarica

Purification and characterization of a phenoloxidase in the hemocytes ofEphestia kuehniellaZeller (Lepidoptera: Pyralidae): effects of insect growth regulators and endogenous inhibitors

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