Mitochondrial adenosine triphosphatase from human placenta--purification and catalytic properties.
1. The purification of ATPase (EC 3.6.1.3) from human placental mitochondria is described. The yield based on mitochondrial enzyme activity was about 70% and the purification was 380-fold. 2. The rate of Mg-ATP hydrolysis was 85 mumole per min per mg of protein under optimum conditions. 3. Nucleoside triphosphates were hydrolyzed by the purified enzyme at decreasing rates in the following order: GTP greater than ITP greater than ATP greater than epsilon-ATP greater than UTP greater than CTP in Tris-HCl buffer (pH 8.0), and in the order: ATP greater than GTP greater than or equal to ITP greater than epsilon-ATP greater than UTP greater than CTP in Tris-bicarbonate buffer at pH 8.0. 4. The values of kinetic parameters are reported. The ATPase reaction deviated from typical Michaelis-Menten kinetics in Tris-HCl buffer but not in Tris-bicarbonate. Eadie-Hofstee plots for Mg-ATP hydrolysis were biphasic in Tris-HCl (Km = 0.2 mM, 0.09 mM) and monophastic in Tris-becarbonate medium (Km = 0.16 mM). 5. In the presence of Mg-ITP or Mg-GTP as substrates no curvature of the reciprocal plots was observed. 6. The results presented reflect the fact that multiple conformations of the enzyme molecule do exist and are probably involved in its regulatory functions. 7. The existence of two kinetically distinct classes of catalytic sites and of an anion-binding site on the placental ATPase is proposed.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002458:Cell Fractionation; D005260:Female; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011487:Protein Conformation; D012265:Ribonucleotides / Q000378:metabolism; D013942:Thymine Nucleotides / Q000378:metabolism
D012265:Ribonucleotides; D013942:Thymine Nucleotides; D000251:Adenosine Triphosphatases; D008274:Magnesium