Modulation of phosphofructokinase (PFK) from Setaria cervi, a bovine filarial parasite, by different effectors and its interaction with some antifilarials
© Sharma; licensee BioMed Central Ltd. 2011
Received: 8 September 2011
Accepted: 7 December 2011
Published: 7 December 2011
Phosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 126.96.36.199, 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.
The 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.
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.
KeywordsPhosphofructokinase Setaria cervi Nucleotides Specificity Activation Inhibition Antifilarials
Although considerable research has been done in the field of morphology, life cycle and taxonomy of filarial parasites, comparatively little attention has been paid to the physiology and metabolism of the filarial worms and their effects on the host. The basic stumbling block in the design of suitable antifilarial drugs is beset with our poor knowledge about the metabolic activities of adult and various developmental stages of filarial worms as well as the disorders generated in the host harbouring the infection. The non-availability of experimental materials from human filarial parasites and insignificant progress made in culturing them under in vitro condition, have further precluded their study .
Setaria cervi, 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. Furthermore, this worm may be obtained in sufficient quantity from any local abattoir for carrying out enzyme purification and desired experiments towards detailed characterization [2–4].
Phosphofructokinase (ATP: D-fructose-6-phospho-1-phosphotransferase, EC 188.8.131.52, PFK) is a key enzyme which is responsible for catalyzing the transfer of the terminal phosphate of ATP to the C-1 hydroxyl group of Fructose-6-phosphate (F-6-P) to produce
fructose-1,6-diphosphate (FDP). Since, many of the parasites in general and filarial parasites in particular utilize glycolysis as a major source of energy for their survival, the study of this enzyme becomes highly pertinent [2, 4–8]. Filarial worms do not catalyze the complete oxidation of the substrate to CO2 and reduced organic acids as end product of the metabolism [2, 6, 7, 9]. 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].
Earlier reports have indicated comparatively low activity of PFK in S. cervi suggesting thereby that this enzyme may be playing a regulatory role in controlling the operation of the glycolytic pathway . Because of the multiplicity of modifiers, PFK has served as a model in studies of allosteric regulation of enzymes. The enzyme activity appears to be modulated to meet the metabolic needs of the cell, with the metabolites serving as intracellular indicators [12–16]. Although PFK from several parasite and vertebrate sources has been purified and characterized, the information about the regulation of filarial PFK by nucleotides is not well understood. Some of the kinetic characteristics of purified PFK from S. cervi have already been studied and the same have been compared with the analogous enzyme isolated from the mammalian systems [2, 17]. The differences in the kinetic properties of PFK from filarial worms and the mammalian sources indicated that this enzyme could be used as a potential target for design and development of suitable chemotherapeutics against filariasis.
Earlier we reported that this enzyme possesses two different pH optima depending on ATP concentrations, the values being 8.0 at low (0.1 mM) concentration which decreases to pH 7.4 at high ATP (> 0.1 mM) concentration . These results indicated that the activity of filarial PFK was possibly under regulation of ATP levels [14, 16, 17]. The present paper illustrates the influence of different effectors including some nucleotides, thiol group reactants and anthelmintics on the kinetic characteristics of PFK purified from S. cervi. The results indicate that the nucleotides under different assay conditions modulate the enzyme activity differently. Also, the sensitivity of filarial PFK towards antifilarials/anthelmintics radically differs from that of mammalian liver PFK.
Sub-cellular localization of activity of S. cervi PFK
Subcellular localization of activity of PFK in adult female S.cervi
% Recovery of protein
Total PFK activity (Units)
% Recovery of PFK activity
Specific Activity (Units/mg protein)
Level of PFK activity in adult female, male and Mf stages of S.cervi
(mg/g wet weight)
PFK activity (Units/g wet weight)
Specific Activity (Units/mg protein)
Distribution of PFK from in intact adult, uteri-free female S.cervi and Mf
PFK activity (Units/g wet weight)
Specific Activity (Units/mg protein)
Nucleotide specificity of S. cervi PFK
Specificity of PFK from S.cervi towards different phosphate group donors
V 2.0 /V 0.2 #
Nucleotides modulate the kinetics of S. cervi PFK
cAMP, AMP and ADP activate the enzyme at the inhibitory concentration of ATP and low concentration of F-6-P (Figures 1A, 2A and 3A). The nucleotide concentrations required for showing half (50%) maximal activation (KA) were found to be 0.1, 0.29 and 2.0 mM for cAMP, AMP and ADP, respectively (Inset of Figures 1B, 2B and 3B).
Effect of cAMP, AMP and ADP on the activity of S.cervi PFK under different conditions
% of control
Effect of thiols and p-chloromercuribenzoate on the activity of S. cervi PFK
Effect of thiols and p-chloromercuribenzoate (p-CMB) on the activity of PFK from S.cervi
Activity remaining (%)
#β-ME + p-CMB
Effect of some antifilarial compounds on the activity of S. cervi PFK
Effect of antifilarial/anthelmintic compounds on the activity of S.cervi PFK
% Residual Activity
S. cervi PFK is cytosolic in nature
Studies on the sub-cellular localization of S. cervi PFK showed that it was mainly present in the soluble fraction of the homogenate of bovine filarial parasite. This is similar to the cytosolic localization of this enzyme observed in the vertebrates  and the parasite, S. mansoni. However, in several Trypanosoma species, PFK has been shown to be present in a new type of sub-cellular membrane bound organelle termed as the glycosome, which contains many of the enzymes of glycolysis [21–23].
Adults of S. cervi contain higher PFK activity than their microfilariae
Comparison of PFK activities in adult (male and female) and microfilarial stages of S. cervi showed highest activity of enzyme in adult (female/male) parasites. The specific activity of S. cervi PFK is close to that of the purified enzyme from human erythrocytes , white adipose tissues of rat  and Onchocerca gutturosa (adults) . However, the specific activity of filarial enzyme was comparatively lower than the values reported for the enzyme purified from rabbit skeletal muscle , and erythrocytes , several other mammalian tissues , yeast  and some parasites such as Echinococcus granulosus and Brugia pahangi adults . The specific activity of S. cervi PFK was higher than that of L. carinii and S. mansoni.
The enzyme from adult female worms has been purified over 100 fold with 30% recovery . The purified PFK from S. cervi showed both similarities and differences when compared with the analogous enzyme from different sources .
S. cervi PFK possess wide specificity towards utilization of nucleotides as phosphate group donors
A study of the nucleotide specificity for S. cervi PFK indicated that UTP, ATP and ATP were the best phosphate donors; ATP showing strongest inhibition at a higher concentration . GTP, GDP and IDP were rather poor phosphate group donors and were also less inhibitory at higher concentrations. These results indicate that S. cervi PFK has a fairly wide specificity for various nucleotides as phosphate group donors. This is similar to the behaviour of the enzyme from vertebrate sources . In the case of pig spleen enzyme, ATP, GTP and ITP are good phosphate group donors, whereas UTP and CTP are less effective . Muscle enzyme can also use several derivatives of purine 5'-triphosphate .
Modulation of S. cervi PFK by nucleotides depends on the concentration of its substrates
cAMP, AMP and ADP activated PFK from S. cervi at inhibitory concentrations of ATP (1.0 mM) and a low concentration of F-6-P (0.5 mM). These nucleotides also activate the enzyme from mammalian tissues [5, 33–35] and a few trypanosomes [23, 36]. In contrast, with the mammalian muscle PFK, it has been shown that the established inhibitors, such as citrate, activate the enzyme activity at low ATP or ITP concentrations while known activators, such as AMP, ADP, and cyclic AMP inhibit at low ATP or ITP concentrations .
S. cervi PFK exhibits presence of a thiol group at its active site
S. cervi PFK was activated by some thiol compounds such as cysteine and β-ME, and inhibited by p-CMB. The partial reversal of p-CMB inhibition by addition of cysteine or β-ME suggests the functioning of -SH group at the active site of the enzyme molecule. The -SH groups have also been implicated in the catalytic activity of PFKs from some mammalian systems [5, 38].
S. cervi PFK is more sensitive towards antifilarials than the mammalian PFK
Among the different antifilarials tested, suramin was most effective in inhibiting PFK activity. Centperazine, DEC, levamisole and the compound 72/70 (synthesized at CDRI-Lucknow) inhibited this enzyme at higher (mM) concentrations. The inhibition of S. cervi PFK by suramin was non-competitive with respect to F-6-P. Suramin was also found to inhibit PFK of rat liver but at 40 times higher concentration (than that required for S. cervi enzyme), showing that the drug is comparatively more toxic to the parasite than the host. Suramin also has a strong inhibitory effect on lactic and malic dehydrogenases of T. immitis[39, 40]; Onchocerca volvulus and S. cervi[41–44], protein kinase of O. volvulus and S. cervi, β-D-glucosaminidase of S. cervi and phosphatidylglycero-phosphate synthetase of O. volvulus and rat liver . Furthermore, the results presented by Bronsvoort et al  indicated the potential of β-tubulin, the binding site of benzimidazoles, as a key molecular target for rational drug design of macrofilaricides. Very recently, Johnston et al  have reported that globomycin, a signal peptidase II (LspA) inhibitor in Gram-negative bacteria, is effective in reducing the motility and viability of adult B. malayi in vitro.
Unlike other parasites, S. cervi PFK was present in its cytosolic fraction. The adult female S. cervi showed more enzyme activity than the microfilarial stage (Mf) of the parasite, suggesting presence of PFK in the musculature of the worm. The enzyme displayed a wide range of specificity towards utilization of nucleotides as phosphate group donors. However, the response of the enzyme to different nucleotides was dependent on the concentrations of F-6-P and ATP. The enzyme contains a thiol group at its active site and the inhibition of PFK by p-CMB could be protected to a significant extent by pretreatment with cysteine or β-ME. S. cervi PFK exhibited 40 times higher sensitivity towards suramin than that of mammalian PFK, thereby suggesting that this enzyme could be used as a potential chemotherapeutic target against filariasis.
Materials and methods
Motile adult female worms (average length 6.0 ± 1.0 cm, average weight 35 ± 6.0 mg) and males (average length 4.0 ± 0.8 cm, average weight 6.0 ± 1.5 mg) of S. cervi were collected from the peritoneal folds of freshly slaughtered naturally infected Indian water buffaloes (Bubalus bubalis Linn.) at a local abattoir during early morning hours. The worms were brought to the laboratory in the Ringer's solution  within 2 h of slaughtering. The worms were thoroughly washed three-four times with lukewarm isotonic saline to remove the adhering contaminants. The worms were either frozen at -20°C until a week or used a fresh for this study.
Isolation of microfilariae (Mf)
The microfilariae (Mf) of S. cervi were collected by dissection of gravid females and by incubating the distal portion of the uteri (1 cm) for 3-4 h at 37 ± 1°C in Ringer's solution containing penicilline-G (1000 U/ml) and streptomycin sulfate (1000 U/ml). The Mf released into the medium were removed by low speed centrifugation and separated from the embryos and other tissues and washed twice with isotonic saline. The intact Mf could remain alive and active for 2 days at 4°C. The wet weight of one million Mf was about 36 mg.
D-fructose-6-phosphate (F-6-P), adenosine-3', 5'-triphosphate (ATP), α-glycerophosphate dehydrogenase (GDH), D-fructose-1,6-diphosphate (FDP), triosephosphate isomerase (TPI), aldolase and phosphoenolpyruvate (PEP) were purchased from Sigma Chemical Co.-USA. Nicotinamide adenine dinucleotide reduced (NADH) was obtained from CSIR Centre of Biochemical Technology, New Delhi. Other reagents used were analytical grade.
Kreb's Ringer Bicarbonate (KRB) solution
This solution was prepared essentially according to the DeLuca and Cohen . NaCl (9 g), KCl (0.42 g), glucose (0.50 g), NaCO3 (0.25 g) and CaCl2 (0.42 g) were added to the distilled water, made up to 1 L and the solution was sterilized by filtering through Millipore membrane filters (0.22 μm pore size).
Preparation of tissue extract and purification of S. cervi PFK
PFK from adult female, male or Mf of S. cervi was isolated in the Tris-HCl buffer (50 mM, pH 8.0) containing ammonium sulfate (300 mM), β-mercaptoethanol (β-ME, 100 μM) and ATP (100 μM). A 10% (w/v) tissue homogenate of adult female parasite was prepared. The Mfs were treated with ultrasonic cell disrupter (Heat system, Ultrasonics Inc.Ltd., N.Y. W-220-F) on ice. The tissue extracts were centrifuged at 105,000 g for 60 min at 4°C and the cytosolic fractions were collected. The enzyme from adult female S. cervi was purified to electrophoretic homogeneity using very simple procedures and the activity was stabilized using suitable reagents .
S. cervi PFK was assayed using an enzyme coupled reaction method described by Racker  with slight modification as described by Sharma et al; . In this method, we measured the formation of the D-fructose-1,6-diphosphate (FDP) using aldolase, TPI, GDH and NADH. The reaction mixture (3 ml) contained Tris HCl buffer (50 mM, pH 8.0), F-6-P (3.3 mM), ATP (0.1 mM), MgCl2 (3.3 mM), NADH (0.04 mM), GDH (0.66 Units/ml), TPI (5.6 Units/ml), aldolase (0.21 Units/ml) and suitable amount of enzyme protein (10-20 μg). The reaction was always started by adding substrate to the reaction mixture and the change in absorbance (oxidation of NADH to NAD+) after every 30 sec interval was measured spectrophotometrically at 340 nm. The extinction coefficient of NADH 6.22 × 103M-1cm-1 was used to calculate the amount of oxidized pyridine nucleotide (NAD+). The three auxiliary enzymes such as aldolase, GDH and TPI were added in excess so that the overall reaction was governed by PFK activity present in the assay mixture. The concentration of Mg2+ was kept higher than that of ATP (unless stated otherwise) for generating Mg-ATP complex (the substrate for the enzyme) and avoiding presence of free ATP molecules, which are known to be inhibitory in nature to PFK from other sources .
The reaction for PFK assay
Determination of activation constant (Ka)
The Ka value for different activators were calculated from their corresponding double reciprocal plots using 1/V-V0 and 1/ [Nucleotide] on Y-and X-axes, respectively, where V and V0 represent the rates of PFK catalyzed reaction in the presence and absence of the effectors. The intersection point of the straight line at the negative abscissa of the X-axis was observed as -1/ Ka.
Determination of the inhibition constant (Ki)
The Ki value for a non-competitive inhibitor was determined from the formula: Slope of inhibited reaction = Km / Vmax.(1+ [Inhibitor]/Ki). The Hill coefficient (n) value for the inhibitor has been determined from the slope of the Hill plot having Log V0-Vi / Vi and Log [Inhibitor] values on Y-and X-axes. V0 and Vi represent the rate of PFK catalyzed reaction in the absence and presence of the suramin.
The author is grateful to Prof. O.P. Malhotra, BHU-Varanasi and late Dr. S.N. Ghatak, CDRI, Lucknow for their valuable inputs. The financial assistance to BS in the form of a fellowship from CSIR-New Delhi is acknowledged.
- Ramachandran CP, Sivanandan S: Inoculation of infective larvae of sub-periodic Brugia malayi into domestic cats by various routes. SE Asian J Trop Med Pub Hlth. 1970, 1: 150-Google Scholar
- Sharma B, Ghatak S, Malhotra OP, Kaushal NA: Stabilization and characterization of phosphofructokinase purified from Setaria cervi, a filarial bovine filarial parasite. Helminthologia. 1995, 32: 15-23.Google Scholar
- Sharma B: Phosphofructokinase from Setaria cervi: mode of action of certain anthelmintics /chemotherapeutics. Helminthologia. 1998, 35: 12-14.Google Scholar
- Sharma B: Kinetic characterisation of phosphofructokinase purified from Setaria cervi, a bovine filarial parasite. Enzyme Research. 2011,Google Scholar
- Bloxham DP, Lardy HA: Phosphofructokinase. The enzymes. Edited by: Boyer PD. 1973, Academic Press, San Diego, 8: 339-Google Scholar
- Ahmad R, Srivastava AK: Biochemical composition and metabolic pathways of filarial worms Setaria cervi: search for new antifilarial agents. J Helminthol. 2007, 81: 261-280.View ArticlePubMedGoogle Scholar
- Barrett J: Forty years of helminth biochemistry. Parasitology. 2009, 136: 1633-1642. 10.1017/S003118200900568X.View ArticlePubMedGoogle Scholar
- Gupta S, Srivastava AK: Biochemical targets in filarial worms for selective antifilarial drug design. Acta Parasitologica. 2005, 50: 1-18.Google Scholar
- Barrett J: Biochemistry of filarial worms. Helminthol Abstr. 1983, Ser. A 52: 1-18.Google Scholar
- Bryant C, Behme CA: Biochemical adaptations in Parasites. 1989, London: Chapman and HallGoogle Scholar
- Saz HJ: Biochemical aspects of filarial parasites. TIBS. 1981, 6: 117-119.Google Scholar
- Cleland WW: The kinetics of enzyme catalyses reactions with two or more substrates and products, nomenclature and rate equations. Biochim Biophys Acta. 1963, 67: 173-187.View ArticlePubMedGoogle Scholar
- Cleland WW: The kinetics of enzyme catalyzed reactions with two or more substrates and products II, Inhibition, nomenclature and theory. Biochim Biophys Acta. 1963, 67: 187-203.Google Scholar
- Ogush S, Lawson JW, Dobson GP, Weech RL, Uyeda K: A new transient activator of phosphofructokinase during initiation of rapid glycolysis in brain. J Biol Chem. 265: 10943-10949.Google Scholar
- Storey KB: Phosphofructokinase. Methods in Enzymol. 1982, 90: 39-44.View ArticleGoogle Scholar
- Uchida Y, Koyama T, Hachimori A: Stabilization and conformation of porcine phosphofructokinase M and L. Comp Biochem Physiol. 1990, 96: 399-404.View ArticleGoogle Scholar
- Sharma B, Kaushal NA, Ghatak S: Phosphofructokinase from bovine filarial parasite, Setaria cervi. Indian J Parasitol. 1987, 11: 5-8.Google Scholar
- Reinhart DG, Lardy HA: Rat liver phosphofructokinase: kinetic activity under near-physiological conditions. Biochem. 1980, 19: 417-1484.Google Scholar
- Daunway GA, Weber G: Rat liver phospho- fructokinase isozymes. Archs Biochem. Biophys. 1974, 620-628. 162Google Scholar
- Shapiro TA, Talalay P: Schistosoma mansoni: Mechanisms in regulation of glycolysis. Exp Parasitol. 1982, 54: 379-390. 10.1016/0014-4894(82)90047-9.View ArticlePubMedGoogle Scholar
- Aguilar Z, Urbina JA: The phosphofructokinase of Trypanosoma cruzi: purification and kinetic mechanisms. Mol Biochem Parasitol. 1986, 21: 103-111. 10.1016/0166-6851(86)90013-7.View ArticlePubMedGoogle Scholar
- Cronin CN, Tipton KF: The role of Mg2+ ions in the reaction catalyzed by PFK from T. brucei. Biochem J. 1987, 247: 41-46.PubMed CentralView ArticlePubMedGoogle Scholar
- Opperdoes FR, Borst P: Localisation of nine glycolytic enzymes in a microbody-like organelle in Trypanosoma brucei: the glycosome. FEBS Lett. 1977, 80: 360-364. 10.1016/0014-5793(77)80476-6.View ArticlePubMedGoogle Scholar
- Lee DL: The fine structure of the excretory system in adult Nippostrongylus brasiliensis. Tissue and Cell. 1970, 2: 225-231. 10.1016/S0040-8166(70)80017-9.View ArticlePubMedGoogle Scholar
- Sale EM, Denton RM: Adipose-tissue phosphofructokinase. Rapid purification and regulation by phosphorylation in vitro. Biochem J. 1985, 232: 897-904.PubMed CentralView ArticlePubMedGoogle Scholar
- McManus DP: Developmental aspects of metabolism in parasites. Int J Parasitol. 1986, 17: 79-95.View ArticleGoogle Scholar
- Paetkau V, Lardy HA: Phosphofructokinase. Correlation of physical anti enzymatic properties. J Biol Chem. 1967, 242: 2035-2042.PubMedGoogle Scholar
- Kono N, Uyeda K: Chicken liver phosphofructokinase. Biochem Biophys Res Commun. 1972, 42: 1095-1100.View ArticleGoogle Scholar
- Wilgus H, Pringle JR, Stellwagen E: The molecular weight of polypeptide chains of yeast phosphofructokinase. Biochem Biophys Res Commun. 1971, 44: 89-93. 10.1016/S0006-291X(71)80162-6.View ArticlePubMedGoogle Scholar
- McManus DP, Smyth JD: Intermediary carbohydrate metabolism in protoscoleces of Echinococcus granulosus (horse and sheep strains) and E.multilocularis. Parasitol. 1982, 84: 351-366. 10.1017/S0031182000044899.View ArticleGoogle Scholar
- Ramp T, Kohler P: Glucose and puruvate catabolism in Litomosoides carinii. Parasitol. 1984, 89: 229-224. 10.1017/S0031182000001268.View ArticleGoogle Scholar
- Muntz JH: Partial purification and some properties of brain phosphofructokinase. Arch Biochim Biophys. 1953, 42: 435-445. 10.1016/0003-9861(53)90371-3.View ArticleGoogle Scholar
- Hiclman PE, Freidemann MJ: The purification and properties of Trypanosoma cruzi phosphofructokinase: purification and kinetic mechanisms. Biochem J. 1975, 151: 327-336.View ArticleGoogle Scholar
- Secrist JA, Barrio JR, Leonard NJ: A fluorescent modification of adenosine triphosphate with activity in enzyme systems: 1,N 6-ethenoadenosine triphosphate. Science. 1972, 175: 646-647. 10.1126/science.175.4022.646.View ArticlePubMedGoogle Scholar
- Mansour TE: Studies on heart phosphofructokinase: purification, inhibition and activation. J Biol Chem. 1963, 238: 2285-2292.Google Scholar
- Cronin CN, Tipton KF: Purification and regulatory properties of phosphofructokinase from Trypanosoma (Trypanozoon) brucei brucei. Biochem J. 1985, 227: 113-124.PubMed CentralView ArticlePubMedGoogle Scholar
- Kemp RG, Tsai MY, Colombo G: A kinetic model for phosphofructokinase based on the paradoxical action of effectors. Biochem Biophys Res Commun. 1976, 68: 942-948. 10.1016/0006-291X(76)91236-5.View ArticlePubMedGoogle Scholar
- Gilbert HF: Biological disulfides: the third messenger? Modulation of phosphofructokinase activity by thiol/disulfide exchange. J Biol Chem. 1982, 257: 12086-12091.PubMedGoogle Scholar
- Walter RD, Mühlpfordt H, Königk E: Comparative studies of the desoxythymidylate synthesis in Plasmodium chabaudi, Trypanosoma gambiense and Trypanosoma lewisi. Z Tropenmed Parasitol. 1970, 21 (4): 347-57.PubMedGoogle Scholar
- Walter RD: Inhibition of lactate dehydrogenase activity from Dirofilaria immitis by suramin. Tropenmed Parasitol. 1979, 30: 463-465.PubMedGoogle Scholar
- Walter RD, Schulzkey H: Onchocerca volvulus: Effect of suramin on lactate dehydrogenase and malate dehydrogenase. Tropenmed Parasitol. 1980, 31: 55-58.PubMedGoogle Scholar
- Saxena JK, Bharadwaj N, Kaushal NA, Ghatak S: Purification and characterization of malate dehydrogenase from Setaria cervi. Indian J Parasitol. 1986, 10: 93-100.Google Scholar
- Saxena JK, Bharadwaj N, Rathaur S, Ghatak S: Purification and characterization of lactate dehydrogenase from Setaria cervi. Indian J Med Res. 1986, 84: 264-269.PubMedGoogle Scholar
- Bharadwaj N, Saxena JK, Ghatak S, Singh C: Effect of suramin on the activities of lactic and malic dehydrogenase of S. cervi. Biol Mem. 1987, 13: 29-36.Google Scholar
- Walter RD, Schulzkey H: Interaction of suramin with protein kinase I from Onchocerca volvulus. The Host-Invader Interplay. Edited by: Van den Bossche H. 1980, Elsevier/North-Holland, Amsterdam, 709-712.Google Scholar
- Saxena JK, Srivastava AK, Murti PK, Chatterjee RK, Ghatak S, Walter RD: Protein kinases in different life stages of Brugia malayi and other filarial worms. TropenMed Parasitol. 1984, 35: 174-176.PubMedGoogle Scholar
- Singh RP, Saxena JK, Ghatak S: Effect of suramin on the activity of β-D-glucosaminidase of S.cervi . Proc Asian Congress of Parasitol. 1986, 65-Google Scholar
- Srivastava AK, Walter RD: Effect of suramin on the activity of phosphatidylglycerophosphate synthetase of O.volvulus and rat liver. Med Sci Res. 1987, 15: 435-436.Google Scholar
- deC Bronsvoort, Makepeace BL, Renz A, Tanya VN, Fleckenstein L, Ekale D, Trees AJ: UMF-078: A modified flubendazole with potent macrofilaricidal activity against Onchocerca ochengi in African cattle. P&V. 2008, 1: 18-Google Scholar
- Johnston KL, Wu B, Guimarães A, Ford L, Slatko BE, Taylor MJ: Lipoprotein biosynthesis as a target for anti-Wolbachia treatment of filarial nematodes. P&V. 2010, 3: 99-Google Scholar
- Singhal KC, Madan BR, Saxena PN: Studies on the use of Setaria cervi for in vitro antifilarial screenings. Japan J Pharmacol. 1973, 23: 793-797. 10.1254/jjp.23.793.View ArticleGoogle Scholar
- DeLuca HF, Cohen PP: Suspending media for animal tissues. Manometric Techniques. Edited by: Umbreit WW, Burris RH, Staufer JF. 1964, Burgess Publishing Co., Minneapolis, 133-Google Scholar
- Racker E: Spectrophotometric measurement of hexokinase and phosphofructokinase activity. J Biol Chem. 1947, 167: 843-858.PubMedGoogle Scholar
- Horecker BL, Kornberg A: The extinction coefficients of the reduced band of pyridine nucleotides. J Biol Chem. 1948, 175: 385-390.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.