Gamma-glutamylcysteine synthetase from Plasmodium berghei.
Gamma-glutamylcysteine synthetase (L-glutamate-L-cysteine ligase, gamma-GCS, EC 6.3.2.2.), the rate limiting enzyme in glutathione biosynthetic pathway has been analysed in the asexual erythrocytic stages of rodent malaria parasite, Plasmodium berghei and its host erythrocytes. Cell-free parasite isolated by saponin lysis contained about 2 and 8 times higher activity of gamma-GCS compared to P. berghei-infected and normal mice erythrocytes respectively. Subcellular fractionation revealed that the enzyme was mainly confined to the cytosolic part of the parasite. gamma-GCS from P. berghei was purified employing ammonium sulphate precipitation, Sephadex G-200 gel filtration and anionic exchange chromatography on DEAE-cellulose. There was 51.6 fold purification of enzyme and its specific activity was 39.5 U/mg. SDS-PAGE showed P. berghei gamma-GCS as a heterodimer dissociating into two non-identical sub-units of 66 kDa and 57 kDa. The enzyme was observed as white band of activity on native polyacrylamide gel stained for specific gamma-GCS activity. Km values for L-Cys, ATP and L-Glu were 0.53 mM, 0.92 mM and 0.75 mM, respectively. The inhibition of gamma-GCS activity by glutathione was found to be competitive with respect to glutamate (Ki=1.53 mM) and non competitive to ATP and cysteine. Antimalarial drugs did not show any significant effect on parasite gamma-GCS. Parasite enzyme induced humoral response in mice demonstrated by ELISA, IFA and immunoblotting and exhibited partial protection against P. berghei infection suggesting a significant role of P. berghei gamma-GCS in malaria control.
Sharma
S K
SK
Laboratory of Parasitology and Immunology, Department of Biosciences, Himachal Pradesh University, Shimla-171005, India. sanjaysharma_bio@yahoo.com
Banyal
H S
HS
D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay; D004912:Erythrocytes / Q000469:parasitology*; D005455:Fluorescent Antibody Technique; D005721:Glutamate-Cysteine Ligase / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D007114:Immunization; D015151:Immunoblotting; D007700:Kinetics; D008288:Malaria / Q000276:immunology / Q000517:prevention & control; D051379:Mice; D010962:Plasmodium berghei / Q000201:enzymology* / Q000276:immunology / Q000472:pathogenicity; D013347:Subcellular Fractions
D000913:Antibodies, Protozoan; D005721:Glutamate-Cysteine Ligase