Purification and properties of mitochondrial adenosine triphosphatase of hamster brown adipose tissue.
1. Oligomycin-insensitive ATPase (ATP phosphohydrolase, EC 3.6.1.3) was purified from brown adipose tissue mitochondria. It had a specific activity of 50 units/mg which could be increased up to 85 units/mg by KHCO3. The isolated enzyme represented less than 0.5% of the initial membrane proteins.2. The enzyme had a molecular weight equal to beef heart ATPase and was composed of five subunits with molecular weights of 56 200, 54 300, 33 500, 13 400 and 9500 respectively. 3. Isolated ATPase was labile while cold and was activated by the divalent cations Mn2+, Mg2+, Co2+ and Cd2+. The optimum ATP/Mg2+ ratio found was 1.58 and the enzyme had a maximum activity at pH 8.5; the Km was 220 micrometer. 4. The ATPase activity was 55% inhibited by aurovertin. The isolated enzyme enhanced the fluorescence of aurovertin, quenched by ATP and Mg2+ and enhanced by ADP. 5. Oligomycin sensitivity and cold stability of isolated ATPase was restored by its reconstitution with both brown adipose tissue and beef heart particles depleted of ATPase. 6. The results presented demonstrate that the low ATPase activity of brown adipose tissue mitochondria is due to a reduced content of ATPase.
D000064:Acclimatization; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002001:Adipose Tissue, Brown / Q000201:enzymology*; D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D003080:Cold Temperature; D006224:Cricetinae; D004355:Drug Stability; D004789:Enzyme Activation; D007700:Kinetics; D046911:Macromolecular Substances; D008647:Mesocricetus; D008928:Mitochondria / Q000201:enzymology; D008931:Mitochondria, Muscle / Q000201:enzymology; D008970:Molecular Weight; D009840:Oligomycins / Q000494:pharmacology; D012995:Solubility
D002413:Cations, Divalent; D046911:Macromolecular Substances; D009840:Oligomycins; D000251:Adenosine Triphosphatases