Biochemical and molecular characterization of nucleoside triphosphate hydrolase isozymes from the parasitic protozoan Toxoplasma gondii.
We have previously reported the presence of a novel nucleoside triphosphate hydrolase (NTPase) in the rapidly multiplying tachyzoite form of a virulent strain (RH) of Toxoplasma gondii. On further examination, it was found that the purified enzyme was not a single enzyme but was a mixture of two isozymes termed NTPase-I and NTPase-II. The two isozymes had similar molecular masses of approximately 240-270 kDa by gel filtration and contained four identical subunits of molecular mass 66-67 kDa by SDS-polyacrylamide gel electrophoresis. Both forms of the NTPase were activated by dithiothreitol, and NTPase-I had a specific activity 4.5-fold higher than NTPase-II in hydrolysis of ATP. The primary difference between these isozymes lies in their ability to hydrolyze nucleoside triphosphate versus diphosphate substrates. While NTPase-II hydrolyzed ATP to ADP and ADP to AMP at almost the same rate, NTPase-I hydrolyzed ADP to AMP at a much slower rate (0.7% of the rate for ATP). The complete cDNAs for NTPase-I and NTPase-II were sequenced and found to encode the same size predicted open reading frame of which only 16 of 628 amino acids differed between the two isozymes. Both forms of the NTPase contained an NH2-terminal hydrophobic signal peptide, consistent with our previous findings that these enzymes are secreted into the host cell vacuole occupied by the parasite. The gene encoding NTPase-II was found in all strains of Toxoplasma, while the NTPase-I was confined only to virulent strains. Expression of this highly active ATPase (NTPase-I) may contribute to intracellular survival and virulence of T. gondii.
Asai
T
T
Department of Tropical Medicine and Parasitology, Keio University School of Medicine, Tokyo, Japan.
Miura
S
S
Sibley
L D
LD
Okabayashi
H
H
Takeuchi
T
T
D017766:Acid Anhydride Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002850:Chromatography, Gel; D018076:DNA, Complementary; D004229:Dithiothreitol / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D043583:Nucleoside-Triphosphatase; D016366:Open Reading Frames; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D014122:Toxoplasma / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D007527:Isoenzymes; D046911:Macromolecular Substances; D017766:Acid Anhydride Hydrolases; D043583:Nucleoside-Triphosphatase; D004229:Dithiothreitol