Abstract
Plasmodium spp. solely rely on glycolysis for their energy needs during asexual multiplication in human RBCs, making the enzymes of this pathway potential drug targets. We have cloned, over-expressed and purified Plasmodium falciparum glyceraldehyde-3-phosphate dehydrogenase (PfGapdh) for its kinetic and structural characterization. ∼30-40mg pure recombinant enzyme with a specific activity of 12.6units/mg could be obtained from a liter of Escherichia coli culture. This enzyme is a homotetramer with an optimal pH∼9. Kinetic measurements gave KmNAD=0.28±0.3mM and KmG3P=0.25±0.03mM. Polyclonal antibodies raised in mice showed high specificity as was evident from their non-reactivity to rabbit muscle Gapdh. Western blot of Plasmodium yoelii cell extract showed three bands at MW ∼27, ∼37 and ∼51kDa. Presence of PyGapdh in all the three bands was confirmed by LC-ESI-MS. Interestingly, the ∼51kDa form was present only in the soluble fraction of the extract. Subcellular distribution of Gapdh in P. yoelii was examined using differential detergent fractionation method. Each fraction was analyzed on a two-dimensional gel and visualized by Western blotting. All four subcellular fractions (i.e., cytosol, nucleus, cytoskeleton and cell membr...Continue Reading
References
May 7, 1976·Analytical Biochemistry·M M Bradford
Aug 1, 1992·The Journal of Experimental Medicine·V Pancholi, V A Fischetti
Sep 1, 1964·The Biochemical Journal·W Ferdinand
Aug 15, 1970·Nature·U K Laemmli
Nov 30, 1982·Biochemical and Biophysical Research Communications·E F RothR L Nagel
Jul 17, 1999·Biochimica Et Biophysica Acta·M A Sirover
Apr 18, 2000·Gene·C A DaubenbergerG Pluschke
May 16, 2003·The Journal of Biological Chemistry·Naomi CampanaleLeann Tilley
Dec 4, 2003·Acta Crystallographica. Section D, Biological Crystallography·Sandra W Cowan-JacobMarkus G Grütter
Mar 25, 2004·Infection and Immunity·Simone BergmannSven Hammerschmidt
Sep 21, 2004·Proteomics·Limei Meng OkkelsPeter R Jungblut
Dec 21, 2004·European Journal of Biochemistry·Ipsita Pal-BhowmickGotam K Jarori
Jun 14, 2005·Nature Cell Biology·Makoto R HaraAkira Sawa
Nov 4, 2005·Nature·Douglas J LaCountRobert E Hughes
Apr 18, 2009·Microbiology·Guang-Tao LuJi-Liang Tang
May 12, 2011·Molecular & Cellular Proteomics : MCP·Bernardo Javier FothZbynek Bozdech
Sep 9, 2011·Memórias do Instituto Oswaldo Cruz·Anil Kumar Ghosh, Marcelo Jacobs-Lorena
Sep 7, 2013·PloS One·Saudamini ShevadeGotam K Jarori
Citations
Aug 24, 2016·The Journal of Experimental Medicine·Sung-Jae ChaMarcelo Jacobs-Lorena
Feb 27, 2020·Biology Open·David B PirovichPatrick J Skelly
Jun 30, 2021·MBio·Emily S MathewsAudrey R Odom John