Stabilization of rat liver mitochondrial F1-adenosine triphosphatase during chloroform-induced solubilization.
1. Isolation of ATPase from rat liver submitochondrial particles by chloroform treatment requires the presence of ATP or ADP during enzyme solubilization. In the absence of adenine nucleotides the enzyme activity is very low although all protein components of F1-ATPase are released. The low concentrations of ATP or ADP required (5 microM) indicate that the high affinity nucleotide-binding sites are involved in enzyme stabilization. Other nucleotides tested (ITP, GTP, UTP, CTP) were found to be less effective. 2. Polyacrylamide gel electrophoresis and immunodiffusion in agar plates revealed that in the absence of adenine nucleotides a fraction of F1-ATPase released by chloroform treatment is split into fragments. The part of the dissociated enzyme molecule has a molecular weight identical with that of a beta-subunit of F1-ATPase. 3. Dissociation of the F1-ATPase molecule could also be prevented by aurovertin. 4. Crude F1-ATPase solubilized by chloroform treatment can be further purified by Sepharose 6B gel filtration. Specific ATPase activity of the purified enzyme was 90 mumol Pi/min per mg protein and the enzyme was composed of five protein subunits (alpha, beta, gamma, delta, epsilon) with molecular weights 58 000, 55 000, 28 000, 13 000 and 8000, respectively. 5. Chloroform-released F1-ATPase from rat liver mitochondria displayed immunochemical cross-reactivity with that isolated from beef heart mitochondria.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002725:Chloroform; D004355:Drug Stability; D005779:Immunodiffusion; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010086:Oxidative Phosphorylation Coupling Factors / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D012995:Solubility; D013367:Submitochondrial Particles / Q000201:enzymology
D010086:Oxidative Phosphorylation Coupling Factors; D002725:Chloroform; D000251:Adenosine Triphosphatases