Abstract
BackgroundPhosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11, PFK) is of primary importance in the regulation of glycolytic flux. This enzyme has been extensively studied from mammalian sources but relatively less attention has been paid towards its characterization from filarial parasites. Furthermore, the information about the response of filarial PFK towards the anthelmintics/antifilarial compounds is lacking. In view of these facts, PFK from Setaria cervi, a bovine filarial parasite having similarity with that of human filarial worms, was isolated, purified and characterized.ResultsThe S. cervi PFK was cytosolic in nature. The adult parasites (both female and male) contained more enzyme activity than the microfilarial (Mf) stage of S. cervi, which exhibited only 20% of total activity. The S. cervi PFK could be modulated by different nucleotides and the response of enzyme to these nucleotides was dependent on the concentrations of substrates (F-6-P and ATP). The enzyme possessed wide specificity towards utilization of the nucleotides as phosphate group donors. S. cervi PFK showed the presence of thiol group(s) at the active site of the enzyme, which could be protected from inhibitory action of para-chloromercuribenzoate (p-CMB) up to about 76% by pretreatment with cysteine or β-ME. The sensitivity of PFK from S. cervi towards antifilarials/anthelmintics was comparatively higher than that of mammalian PFK. With suramin, the Ki value for rat liver PFK was 40 times higher than PFK from S. cervi.ConclusionsThe results indicate that the activity of filarial PFK may be modified by different effectors (such as nucleotides, thiol group reactants and anthelmintics) in filarial worms depending on the presence of varying concentrations of substrates (F-6-P and ATP) in the cellular milieu. It may possess thiol group at its active site responsible for catalysis. Relatively, 40 times higher sensitivity of filarial PFK towards suramin as compared to the analogous enzyme from the mammalian system indicates that this enzyme could be exploited as a potential chemotherapeutic target against filariasis.
Highlights
Phosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11, PFK) is of primary importance in the regulation of glycolytic flux
Sub-cellular localization of activity of S. cervi PFK In order to ascertain the sub-cellular localization of the activity of PFK from S. cervi, the enzyme was assayed in different sub-cellular fractions of the homogenate of the parasite
Nucleotide specificity of S. cervi PFK Several nucleotides di- and tri-phosphates have been studied as phosphate group donors in the phosphorylation of F-6-P catalyzed by PFK of S. cervi
Summary
Phosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11, PFK) is of primary importance in the regulation of glycolytic flux. A bovine filarial parasite, dwelling in the lymphatics and intraperitoneal folds of naturally infected Indian water buffaloes (Bubalus bubalis Linn.), serves as a unique experimental model for such studies as it resembles human filarial worms in nocturnal periodicity, metabolic pathways, antigenic make up and sensitivity towards antifilarials, and anthelmintic compounds. This worm may be obtained in sufficient quantity from any local abattoir for carrying out enzyme purification and desired experiments towards detailed characterization [2,3,4]. The filarial nematodes are known to utilize a limited quantity of oxygen, when available and possess rudimentary and unusual electron transport chains that catalyze limited terminal oxidation with generation of little energy [2,6,10,11]
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