An extracellular monoADP-ribosyl transferase activity in Entamoeba histolytica trophozoites |
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Authors: | Delgado-Corona Patricia Martínez-Cadena Guadalupe Alvarez Angel H Torres-Calzada Horacio E Avila Eva E |
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Institution: | Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, P O Box 187, Guanajuato, Gto., México CP36000. |
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Abstract: | Due to the important role of monoADP-ribosyl transferases in physiological and pathological events, we investigated whether the protozoan parasite Entamoeba histolytica had monoADP-ribosyl transferase activity. Reactions were initiated using ameba-free medium as the source of both enzyme and ADP-ribosylation substrate(s) and 32P]NAD+ as source of ADP-ribose. Proteins were analyzed by electrophoresis, and 32P]-labeled proteins were detected by autoradiography. Using the crude extracellular medium, a major labeled product of Mr 37.000 was observed. The yield of this product was reduced markedly using medium from Brefeldin A-treated trophozoites, indicating that the extracellular monoADP-ribosyl transferase and/or its substrate depended on vesicular transport. The labeling of the 37-kDa substrate was dependent on reaction time, temperature, pH, and the ratio of unlabeled NAD+ to 32P]NAD+. After two purification steps, several new substrates were observed, perhaps due to their enrichment. The reaction measured ADP-ribosylation since 14C-carbonyl]NAD+ was not incorporated into ameba substrates and a 75-fold molar excess of ADP-ribose caused no detectable inhibition of the monoADP-ribosyl transferase reaction. On the basis of sensitivity to NH2OH, the extracellular monoADP-ribosyl transferase of E. histolytica may be an arginine-specific enzyme. These results demonstrate the existence in E. histolytica of at least one extracellular monoADP-ribosyl transferase, whose localization depends upon a secretion process. |
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Keywords: | ADP-ribosylation ameba Brefeldin A C3 exoenzyme protozoa Rho protein |
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