An extracellular monoADP-ribosyl transferase activity in Entamoeba histolytica trophozoites.
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.
Delgado-Corona
Patricia
P
Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, P O Box 187, Guanajuato, Gto., México CP36000.
Martínez-Cadena
Guadalupe
G
Alvarez
Angel H
AH
Torres-Calzada
Horacio E
HE
Avila
Eva E
EE
D036002:ADP Ribose Transferases / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001905:Botulinum Toxins / Q000378:metabolism; D020126:Brefeldin A / Q000494:pharmacology; D003470:Culture Media; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity
D003470:Culture Media; D020126:Brefeldin A; D000246:Adenosine Diphosphate Ribose; D001120:Arginine; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins