Phosphohydrolytic activity in Paramecium caudatum at neutral pH.
Phosphohydrolytic activity was cytochemically characterized in Paramecium caudatum, a ciliated protozoa, at neutral pH. We stained cells in the presence of several mononucleotides as substrates, namely adenosine 5'-monophosphate (5'-AMP), adenosine 2'-monophosphate, guanosine 5'-monophosphate (5'-GMP) and beta-glycerophosphate (beta-GLP) using a lead capture method at 37 degrees C. Cells were also incubated in the presence of 5'-AMP with the inhibitor for alkaline phosphatase, tetramisole. In all cases, varying amounts of final reaction product, lead sulfide, was observed in Paramecium cytoplasm. Tetramisole did not have any effect on Paramecium 5'-AMP hydrolytic activity. The phosphohydrolytic activity was measured as the increase in total absorbance of "test minus control" reactions at 440 nm per unit time after 20 min of incubation using a microphotometric system for image analysis that has been developed by us. From the relationship between the concentrations of 5'-AMP and activity, an apparent K(m) value was estimated to be 0.20 mM. These results suggest that mononucleotides and phosphate monoesters are hydrolyzed by one or more enzymes with wide substrate specificity in P. caudatum. All the activity distribution patterns in Paramecium cultures, that were tested, were monomodal. The mean activity for 5'-AMP hydrolysis widely varied in these cultures. To investigate substrate affinity, distribution patterns and mean activity with 5'-AMP as substrate were compared with those in the presence of 2 other substrates, 5'-GMP and beta-GLP. Affinity of the enzyme(s) was similar for 5'-AMP and 5'-GMP and lower for beta-GLP.
Chikamori
K
K
Department of Chemistry, Naruto University of Education, Takashima, Tokushima, Japan.
Araki
T
T
Sato
K
K
D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D005994:Glycerophosphates / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007091:Image Processing, Computer-Assisted; D010247:Paramecium / Q000201:enzymology*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D013379:Substrate Specificity; D013773:Tetramisole / Q000494:pharmacology
D005994:Glycerophosphates; D000249:Adenosine Monophosphate; D006157:Guanosine Monophosphate; D013773:Tetramisole; D010744:Phosphoric Monoester Hydrolases; C031463:beta-glycerophosphoric acid