Partial purification and characterization of ornithine decarboxylase from Entamoeba histolytica.
Multiplication of E. histolytica was accompanied by a parallel increase in ornithine decarboxylase (ODC) specific activity up to 72 h of cultivation in TYI-S-33 medium. Thereafter, activity rapidly decayed whereas growth continued for another 24 h before entering into the stationary growth phase. ODC was very unstable. Partial purification (14-fold) of the enzyme was achieved by a three-step procedure involving high-speed centrifugation, gel filtration and adsorption to hydroxylapatite. The partially purified enzyme (Mr 211 kDa) revealed maximum activity at pH 8.5-9.0 and a sigmoidal response to substrate concentration. An S0.5 value of 1.0 mM ornithine was estimated. Although ODC did not exhibit an absolute dependence on pyridoxal phosphate (PLP), addition of PLP increased catalytic activity about 4-fold, with an S0.5 value of 45 microM. Evolution of 14CO2 from ornithine was markedly inhibited by polyamines in the following increasing order of effectiveness: putrescine > spermidine > spermine. The substrate analogs alpha-methylornithine and alpha-difluoromethylornithine had no effect on enzyme activity and cell growth. In contrast, 1,3-diaminopropane and 2,4-diamino-2-butanone, 2 putrescine analogs, severely inhibited both enzyme activity and amoeba multiplication. Results are discussed in terms of the role of ODC in the amoeba proliferation.
Arteaga-Nieto
P
P
Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad Autónoma de Guanajuato, México.
Villagómez-Castro
J C
JC
Calvo-Méndez
C
C
López-Romero
E
E
D000818:Animals; D002455:Cell Division; D004748:Entamoeba histolytica / Q000201:enzymology*; D009955:Ornithine Decarboxylase / Q000302:isolation & purification / Q000378:metabolism*; D065108:Ornithine Decarboxylase Inhibitors; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011700:Putrescine / Q000494:pharmacology; D013095:Spermidine / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology
D065108:Ornithine Decarboxylase Inhibitors; D015800:Protozoan Proteins; D013096:Spermine; D009955:Ornithine Decarboxylase; D013095:Spermidine; D011700:Putrescine