Purification and properties of the adenosine triphosphatase released from the liver mitochondrial membrane by chloroform.
1. Soluble ATPase (adenosine triphosphatase) activity is released when rat liver submitochondrial particles are shaken with chloroform, provided that ATP or glycerol is present in the suspending medium. The extraction is very rapid and appears to be complete. 2. The ATPase of the chloroform extract is about 50% pure and can be readily purified to a specific activity of 60-70mumol/min per mg of protein by (NH(4))(2)SO(4) fractionation and column chromatography on Sephadex G-200. 3. The particulate and soluble ATPases have many similar properties, including their K(m) values for ATP, activation by various metal ions, hydrolytic activity with other nucleotides and stimulation by bicarbonate ions. 4. Unlike the particulate enzyme, the soluble enzyme is cold-labile and insensitive to oligomycin. 5. The molecular weight indicated by the mobility of the soluble ATPase on Sepharose 6B is 360000. 6. The soluble ATPase combines very readily with liver submitochondrial particles depleted of ATPase by salt extraction, and oligomycin-sensitivity is restored. Very little recombination of the enzyme occurs with chloroform-extracted particles. 7. The soluble enzyme contains orcinol-reactive material, suggesting that it may be a glycoprotein. The carbohydrate content was estimated to be 1-2% by weight. 8. It is concluded that the liver ATPase obtained by the chloroform extraction method of Beechey, Hubbard, Linnett, Mitchell & Munn [(1975) Biochem. J.148, 533-537] is similar to other preparations described previously and that this method is superior in simplicity and speed.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002725:Chloroform; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008722:Methods; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D051381:Rats; D013367:Submitochondrial Particles / Q000201:enzymology; D013696:Temperature
D002241:Carbohydrates; D002725:Chloroform; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases