Modulation of phosphofructokinase (PFK) from Setaria cervi, a bovine filarial parasite, by different effectors and its interaction with some antifilarials

<p>Abstract</p> <p>Background</p> <p>Phosphofructokinase (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 <it>Setaria cervi</it>, a bovine filarial parasite having similarity with that of human filarial worms, was isolated, purified and characterized.</p> <p>Results</p> <p>The <it>S. cervi </it>PFK was cytosolic in nature. The adult parasites (both female and male) contained more enzyme activity than the microfilarial (Mf) stage of <it>S. cervi</it>, which exhibited only 20% of total activity. The <it>S. cervi </it>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. <it>S. cervi </it>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 <it>S. cervi </it>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 <it>S. cervi</it>.</p> <p>Conclusions</p> <p>The 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.</p

Cyclodextrin-responsive nanogel as an artificial chaperone for horseradish peroxidase

The thermal stabilization and refolding of horseradish peroxidase (HRP) upon heating were investigated using an artificial molecular chaperone consisting of cholesterol-bearing pullulan (CHP) nanogels. The CHP nanogels inhibited the aggregation of HRP under heating by complexation with the denatured HRP. The enzyme activity of HRP complexed with CHP nanogels was not detected. However, the enzyme activity recovered up to 80% of native HRP after the addition of cyclodextrin (CD) to the complex. The dissociation of CHP nanogels was induced by the formation of an inclusion complex of cholesterol groups of CHP with CD. The enzyme activity of HRP was only significantly recovered by the addition of β-CD or its derivatives. Natural molecular chaperones, such as GroEL/ES, trap, fold, and release the nonnative proteins by changing the hydrophobicity of the specific sites of the molecular chaperone that interact with the nonnative protein. The functional mechanism of the nanogel chaperon system is similar to that of natural molecular chaperones. The nanogel chaperone system is a useful tool to aid the refolding and thermal stabilization of unstable proteins for post-genome research, and in medical and biological applications