A Toxoplasma gondii phosphoinositide phospholipase C (TgPI-PLC) with high affinity for phosphatidylinositol.
The Toxoplasma gondii phosphoinositide-specific phospholipase C gene (TgPI-PLC) was cloned, sequenced and expressed in Escherichia coli and its enzymatic characteristics were investigated. TgPI-PLC is present in the genome as a single-copy gene consisting of 22 exons interrupted by 21 introns, and encodes a polypeptide of 1097 amino acids with a predicted molecular mass of 121 kDa. In addition to the conserved catalytic X and Y domains, TgPI-PLC contains an apparent N-terminal PH domain, an EF hand motif and a C-terminal C2 domain. When compared with mammalian delta-type PI-PLC, TgPI-PLC has an additional extended N-terminus and two insertions in the region between the X and Y domains, with a 31-35% identity over the whole sequence. Recombinant TgPI-PLC, as well as the native enzyme obtained from crude membrane extracts of the parasite, was more active with phosphatidylinositol than with phosphatidylinositol 4,5-bisphosphate as substrate. Indirect immunofluorescence analysis using an affinity-purified antibody against TgPI-PLC revealed that this enzyme localizes in the plasma membrane of the parasites.
Fang
Jianmin
J
Department of Cellular Biology, and Center for Tropical and Emerging Global Diseases, The University of Georgia, Athens, GA 30602, USA.
Marchesini
Norma
N
Moreno
Silvia N J
SN
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium; D004795:Enzyme Stability; D005786:Gene Expression Regulation; D006863:Hydrogen-Ion Concentration; D008274:Magnesium; D008969:Molecular Sequence Data; D043265:Phosphatidylinositol Diacylglycerol-Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D054801:Phosphoinositide Phospholipase C; D021381:Protein Transport; D013379:Substrate Specificity; D013696:Temperature; D014122:Toxoplasma / Q000166:cytology / Q000201:enzymology*
D010716:Phosphatidylinositols; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D008274:Magnesium; D002118:Calcium