Purification and characterization of phosphoenolpyruvate carboxykinase from Raillietina echinobothrida, a cestode parasite of the domestic fowl.
Phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) is an essential regulatory enzyme of glycolysis in helminths in contrast to its role in gluconeogenesis in their host. Previously we have reported that phytochemicals from Flemingia vestita (Family: Fabaceae), genistein in particular, have vermifugal action and are known to affect carbohydrate metabolism in the cestode, Raillietina echinobothrida. In order to determine the functional differences of PEPCK from the parasite and its avian host (Gallus domesticus), we purified the parasite enzyme apparently to homogeneity, and characterized it. The native PEPCK is a monomer with a subunit molecular weight of 65 kDa. The purified enzyme displayed standard Michaelis-Menten kinetics with Km value of 42·52 μM for its substrate PEP. The Ki for the competitive inhibitors GTP, GMP, ITP and IMP for the carboxylation reaction were determined and discussed. In order to identify putative modulators from plant sources, phytochemicals from F. vestita and Stephania glabra were tested on the purified PEPCK, which resulted in alteration of its activity. From our results, we hypothesize that PEPCK may be a potential target site for anthelmintic action.
Department of Zoology, North Eastern Hill University, Shillong-793022, India.
B
B
Das
V
V
Tandon
J K
JK
Saxena
S
S
Joshi
A R
AR
Singh
D000818:Animals; D002589:Cestoda / Q000201:enzymology*; D002645:Chickens / Q000469:parasitology; D004789:Enzyme Activation / Q000187:drug effects; D007887:Fabaceae / Q000737:chemistry; D006019:Glycolysis; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000494:pharmacology; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010936:Plant Extracts / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D031607:Stephania / Q000737:chemistry
D007477:Ions; D008670:Metals; D010936:Plant Extracts; C069767:phosphoenolpyruvate carboxylase kinase; D017346:Protein Serine-Threonine Kinases