Hostname: page-component-7dd5485656-kp629 Total loading time: 0 Render date: 2025-10-31T10:46:55.320Z Has data issue: false hasContentIssue false
  • English
  • Français

Molecular cloning and characterization of glucose-6-phosphate dehydrogenase from Brugia malayi

Published online by Cambridge University Press:  18 March 2013

ANITA VERMA ,
MANISH K. SUTHAR ,
PAWAN K. DOHAREY ,
SMITA GUPTA ,
SUNITA YADAV ,
PREM M. S. CHAUHAN  and
JITENDRA K. SAXENA
ANITA VERMA
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
MANISH K. SUTHAR
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
PAWAN K. DOHAREY
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
SMITA GUPTA
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
SUNITA YADAV
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
PREM M. S. CHAUHAN
Affiliation:
Division of Medicinal and Process Chemistry, CSIR-Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
JITENDRA K. SAXENA*
Affiliation:
Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India
*
*Corresponding author: Division of Biochemistry, CSIR-Central Drug Research Institute, Lucknow-226001, Uttar Pradesh, India. E-mail: jkscdri@yahoo.com
Get access Rights & Permissions [Opens in a new window]

Summary

Glucose-6-phosphate dehydrogenase (G6PD), a regulatory enzyme of the pentose phosphate pathway from Brugia malayi, was cloned, expressed and biochemically characterized. The Km values for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (NADP) were 0·25 and 0·014 mm respectively. The rBmG6PD exhibited an optimum pH of 8·5 and temperature, 40 °C. Adenosine 5′ [γ-thio] triphosphate (ATP-γ-S), adenosine 5′ [β,γ-imido] triphosphate (ATP-β,γ-NH), adenosine 5′ [β-thio] diphosphate (ADP-β-S), Na+, K+, Li+ and Cu++ ions were found to be strong inhibitors of rBmG6PD. The rBmG6PD, a tetramer with subunit molecular weight of 75 kDa contains 0·02 mol of SH group per mol of monomer. Blocking the SH group with SH-inhibitors, led to activation of rBmG6PD activity by N-ethylmaleimide. CD analysis indicated that rBmG6PD is composed of 37% α-helices and 26% β-sheets. The unfolding equilibrium of rBmG6PD with GdmCl/urea showed the triphasic unfolding pattern along with the highly stable intermediate obtained by GdmCl.

Keywords

Brugia malayiglucose-6-phosphate dehydrogenaseDHEAinhibitorscircular dichroismGdmClureadrug target

Information

Type
Research Article
Information
Parasitology , Volume 140 , Issue 7 , June 2013 , pp. 897 - 906
Copyright
Copyright © Cambridge University Press 2013 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Article purchase

Temporarily unavailable

References

REFERENCES

Bautista, J. M., Manson, J. M. and Luzzatto, L. (1992). Purification and properties of human glucose-6-phosphate dehydrogenase made in E. coli. Biochemica et Biophysica Acta 1119, 74–80.CrossRefGoogle ScholarPubMed
Betke, K., Beutler, E., Brewer, G. J., Kirkman, H. N., Luzzatto, L., Motulsky, A. G., Ramot, B. and Siniscalco, M. (1967). Standardization of Procedures for the Study of Glucose-6-Phosphate Dehydrogenase: Report of a World Health Organization Scientific Group. WHO Technical Report Serial No. 366. World Health Organization, Geneva, Switzerland.Google Scholar
Bockarie, M. J. and Deb, R. M. (2010). Elimination of lymphatic filariasis: do we have the drugs to complete the job. Current Opinion in Infectious Diseases 23, 617–620.CrossRefGoogle ScholarPubMed
Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.CrossRefGoogle Scholar
Ciftci, M., Beydemir, S., Yilmaz, H. and Altikat, S. (2003). Purification of glucose-6-phosphate dehydrogenase from Buffalo (Bubalus bubalis) erythrocytes and investigation of some kinetic properties. Protein Expression and Purification 29, 304–310.CrossRefGoogle ScholarPubMed
Cordeiro, A. T., Thiemann, O. H. and Michels, P. A. M. (2009). Inhibition of Trypanosoma brucei glucose-6-phosphate dehydrogenase by human steroids and their effects on the viability of cultured parasites. Bioorganic and Medicinal Chemistry 17, 2483–2489.CrossRefGoogle ScholarPubMed
Critchley, J., Addiss, D., Ejere, H., Gamble, C., Garner, P. and Gelband, H. (2005). Albendazole for the control and elimination of lymphatic filariasis: systematic review. Tropical Medicine and International Health 10, 818–825.CrossRefGoogle ScholarPubMed
Fico, A., Paglialunga, F., Cigliano, L., Abrescia, P., Verde, P., Martini, G., Iaccarino, I. and Filosa, S. (2004). Glucose-6-phosphate dehydrogenase plays a crucial role in protection from redox-stress-induced apoptosis. Cell Death and Differentiation 11, 823–831.CrossRefGoogle Scholar
Filosa, S., Fico, A., Paglialunga, F., Balestrieri, M., Crooke, A., Verde, P., Abrescia, P., Bautista, J. M. and Martini, G. (2003). Failure to increase glucose consumption through the pentose phosphate pathway results in the death of glucose-6-phosphate dehydrogenase gene deleted mouse embryonic stem cells subjected to oxidative stress. Biochemical Journal 370, 935–943.CrossRefGoogle ScholarPubMed
Gupta, S., Igoillo-Esteve, M., Michels, P. A. M. and Cordeiro, A. T. (2011). Glucose-6-phosphate dehydrogenase of trypanosomatids: characterization, target validation and drug discovery. Molecular Biology International 2011, 135701.CrossRefGoogle ScholarPubMed
Gordon, G., Mackow, M. C. and Levy, H. R. (1995). On the mechanism of interaction of steroids with human glucose-6-phosphate dehydrogenase. Archives of Biochemistry and Biophysics 318, 25–29.CrossRefGoogle ScholarPubMed
Ibraheem, O., Adewale, I. O. and Afolayan, A. (2005). Purification and properties of glucose 6-phosphate dehydrogenase from Aspergillus aculeatus. Journal of Biochemistry and Molecular Biology 5, 584–590.Google Scholar
Igoillo-Esteve, M. and Cazzulo, J. J. (2006). The glucose-6-phosphate dehydrogenase from Trypanosoma cruzi: its role in the defense of the parasite against oxidative stress. Molecular and Biochemical Parasitology 149, 170–181.CrossRefGoogle ScholarPubMed
Klenova, E. M., Nicolas, R. H., Sally, U., Carne, A. F., Lee, R. E., Lobanenkov, V. V. and Goodwin, G. H. (1997). Molecular weight abnormalities of the CTCF transcription factor: CTCF migrates aberrantly in SDS–PAGE and the size of the expressed protein is affected by the UTRs and sequences within the coding region of the CTCF gene. Nucleic Acids Research 25, 466–473.CrossRefGoogle ScholarPubMed
Kohn, W. D., Kay, C. M. and Hodges, R. S. (1995). Protein destabilization by electrostatic repulsions in the two stranded alpha helical coiled/coil leucine zipper. Protein Science 4, 237–250.CrossRefGoogle ScholarPubMed
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.CrossRefGoogle ScholarPubMed
Little, C., Sannera, T. and Pihl, A. (1969). Stimulation of fructose 1,6-diphosphatase by sulfhydryl reagents and interaction between the different enzymic sites in the molecule. European Journal of Biochemistry 8, 229–236.CrossRefGoogle ScholarPubMed
Marks, P. A. and Banks, J. (1960). Inhibition of mammalian glucose-6-phosphate dehydrogenase by steroids. Proceedings of the National Academy of Sciences, USA 46, 447–452.CrossRefGoogle ScholarPubMed
Monera, O. D., Kay, C. M. and Hodges, R. S. (1994). Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions. Protein Science 3, 1984–1991.CrossRefGoogle ScholarPubMed
Osei-Atweneboana, M. Y., Eng, J. K., Boakye, D. A., Gyapong, J. O. and Prichard, P. K. (2007). Prevalence and intensity of Onchocerca volvulus infection and efficacy of ivermectin in endemic communities in Ghana: a two-phase epidemiological study. Lancet 369, 2021–2029.CrossRefGoogle Scholar
Ramzy, R. M., El Setouhy, M., Helmy, H., Ahmed, E. S., Abd Elaziz, K. M., Farid, H. A., Shannon, W. D. and Weil, G. J. (2006). Effect of yearly mass drug administration with diethylcarbamazine and albendazole on bancroftian filariasis in Egypt: a comprehensive assessment. Lancet 367, 992–999.CrossRefGoogle ScholarPubMed
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA.Google Scholar
Saxena, J. K., Pandya, U., Gupta, D. and Shukla, O. P. (1996). Antioxidant system in adults and microfilariae of the bovine filarial parasite Setaria cervi. Journal of Parasitic Diseases 20, 159–162.Google Scholar
Schulz, T. J., Zarse, K., Voigt, A., Urban, N., Birringer, M. and Ristow, M. (2007). Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress. Cell Metabolism 6, 280–293.CrossRefGoogle ScholarPubMed
Schwab, A. E., Churcher, T. S., Schwab, A. J., Basanez, M. G. and Prichard, R. K. (2007). An analysis of the population genetics of potential multi-drug resistance in Wuchereria bancrofti due to combination chemotherapy. Parasitology 134, 1025–1040.CrossRefGoogle ScholarPubMed
Singh, A. R., Joshi, S., Arya, R., Kayastha, A. M., Srivastava, K. K., Tripathi, L. M. and Saxena, J. K. (2008). Molecular cloning and characterization of Brugia malayi hexokinase. Parasitology International 57, 354–361.CrossRefGoogle ScholarPubMed
Singh, S., Anand, A. and Srivastava, P. K. (2012). Regulation and properties of glucose-6-phosphate dehydrogenase: a review. International Journal of Plant Physiology and Biochemistry 4, 1–19.Google Scholar
Smith, J. E. and Anwer, M. S. (1970). Studies on glucose-6-phosphate dehydrogenase. Experientia 27, 835–836.CrossRefGoogle Scholar
Tian, W. N., Braunstein, L. D., Apse, K., Pang, J., Rose, M., Tian, X. and Stanton, R. C. (1999). Importance of glucose-6-phosphate dehydrogenase activity in cell death. American Journal of Physiology – Cell Physiology 276, C1121–C1131.CrossRefGoogle ScholarPubMed
Van Raamsdonk, J. M. and Hekimi, S. (2010). Reactive oxygen species and aging in Caenorhabditis elegans: causal or casual relationship. Antioxidants and Redox Signaling 13, 1911–1953.CrossRefGoogle ScholarPubMed
Wang, X. T. and Engel, P. C. (2009). An optimised system for refolding of human glucose-6-phosphate dehydrogenase. BMC Biotechnology 9, 19–29.CrossRefGoogle ScholarPubMed
Winiarska, K., Drozak, J., Wegrzynowicz, M., Jagielski, A. K. and Bryla, J. (2003). Relationship between gluconeogenesis and glutathione redox state in rabbit kidney-cortex tubules. Metabolism 52, 739–746.CrossRefGoogle ScholarPubMed
World Health Organization (2009). Global programme to eliminate lymphatic filariasis. Weekly Epidemiological Record 84, 437–444.Google Scholar
Wrigley, N. G., Heather, I. V., Bonsignore, A. and De Flore, A. (1972). Human erythrocyte glucose-6-phosphate dehydrogenase: electron microscope studies on structure and interconversation of tetramers, dimers and monomers. Journal of Molecular Biology 68, 483–499.CrossRefGoogle Scholar
Zhang, Z., Liew, C. W., Handy, D. E., Zhang, Y., Leopold, J. A., Hu, J., Lili Guo, L., Kulkarni, R. N., Loscalzo, J. and Stanton, R. C. (2010). High glucose inhibits glucose-6-phosphate dehydrogenase, leading to increased oxidative stress and β-cell apoptosis. FASEB Journal 24, 1497–1505.CrossRefGoogle ScholarPubMed
Supplementary material: Image

Verma supplementary figures

Verma supplementary figures

Download Verma supplementary figures(Image)
Image 2.2 MB