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A novel ATP-diphosphohydrolase from human term placental mitochondria
O Flores-Herrera1, A Uribe, J P Pardo
1Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, Coyoacán, México, D. F., 04510, México.
Abstract:
This report describes an ATP-diphosphohydrolase activity associated with the inner membrane of human term placental mitochondria. An enriched fraction containing 30 per cent of the total protein and 80 per cent of the total ATP-diphosphohydrolase activity was obtained from submitochondrial particles. ATP-diphosphohydrolase activity was characterized in this fraction. The enzyme had a pH optimum of 8 and catalysed the hydrolysis of triphospho- and diphosphonucleosides other than ATP or ADP. Pyrophosphate was also hydrolysed, but AMP or other monoester phosphates were not. The activity of ATP-diphosphohydrolase was dependent on Mg(2 + ), Ca(2 + )or Mn(2 + )and the enzyme substrate was the cation-nucleotide complex. An excess of free cation produced inhibition.ATP-diphosphohydrolase activity was stimulated at micromolar concentrations of calcium or magnesium in the presence of La-PPi. Negative cooperativity kinetics was observed with all substrates tested. The V(max)ranged from 150 to 300nmol of Pi released/mg/min. The [S](0.5)for nucleotides was 1-10m m and 182m m for PPi. The enzyme was inhibited by orthovanadate, but not by l -phenylalanine, oligomycin, sodium azide, P(1),P(5)-di(adenosine-5')pentaphosphate or sodium fluoride.The experimental evidence showing absence of inhibition by sodium azide and sodium fluoride, hydrolysis of pyrophosphate but not of monoester phosphates, and negative cooperativity suggested that this enzyme was a novel ATP-diphosphohydrolase.
Insights
Researchers identified a novel ATP-diphosphohydrolase enzyme in human placental mitochondria. This enzyme hydrolyzes nucleoside triphosphates and diphosphates, playing a key role in mitochondrial energy metabolism.
Area of Science:
- Biochemistry
- Mitochondrial Biology
- Enzymology
Background:
- Human term placental mitochondria possess ATP-diphosphohydrolase activity.
- Submitochondrial particles were used to isolate and characterize this enzymatic activity.
Purpose of the Study:
- To characterize the biochemical properties of the ATP-diphosphohydrolase activity in human placental mitochondria.
- To determine if this activity represents a novel enzyme.
Main Methods:
- Enrichment of ATP-diphosphohydrolase activity from submitochondrial particles.
- Enzyme kinetics studies including pH optimum, substrate specificity, and metal ion dependence.
- Inhibition studies using various chemical agents.
Main Results:
- The enzyme exhibited optimal activity at pH 8 and hydrolyzed nucleoside triphosphates and diphosphates, but not AMP.
- Activity was dependent on divalent cations (Mg2+, Ca2+, Mn2+) with inhibition by excess cations.
- Negative cooperativity kinetics were observed, and the enzyme was inhibited by orthovanadate but not by sodium azide or fluoride.
Conclusions:
- The characterized ATP-diphosphohydrolase activity demonstrates unique properties, including pyrophosphate hydrolysis and distinct inhibition patterns.
- These findings suggest the presence of a novel ATP-diphosphohydrolase in human placental mitochondria.