Evidence for the presence and role of tightly bound adenine nucleotides in phospholipid-free purified Micrococcus lysodeikticus adenosine triphosphatase.
[32P]-labeled ATPase was isolated in a highly purified state from Micrococcus lysodeikticus strain PNB grown in medium supplemented with [32P]orthophosphate. Selective extraction procedures allowed us to determine that at least 25% of the firmly bound label belonged to adenine nucleotides, ATP and ADP being present in equimolar amounts. However, no 32P label was found to be part of phospholipids. This was confirmed by purification of the ATPase from cells fed with [2-3H]glycerol. Using the luciferin-luciferase assay we estimated that ATPase freshly isolated by Sephadex chromatography (specific activity 10-14 micromole substrate transformed x min(-1) x mg protein(-1)) contained 2 moles ATP/mole of enzyme. The ratio fell with the age of enzyme and its purification by gel electrophoresis and this was paralleled by a loss of ATPase activity. The endogenous nucleotides were readily exchanged by added ADP or ATP. This result suggests that the sites for tight binding of adenine nucleotides are equivalent, although ADP seems to have a higher affinity for them. The last properties represent a peculiar characteristic of this bacterial ATPase as compared with other bacterial and organelle energy-transducing proteins.
Muñoz
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Instituto de Inmunología y Biología Microbiana, Membrane Biochemistry Section, Velázquez, 144, Madrid-6, Spain.
Palacios
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Muñoz
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D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002845:Chromatography; D003911:Dextrans; D008156:Luciferases; D008837:Micrococcus / Q000201:enzymology*; D010743:Phospholipids / Q000378:metabolism; D010761:Phosphorus Radioisotopes
D000227:Adenine Nucleotides; D003911:Dextrans; D010743:Phospholipids; D010761:Phosphorus Radioisotopes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; C025614:sephadex; D008156:Luciferases; D000251:Adenosine Triphosphatases