Adenosine triphosphatase of rat liver mitochondria: Detergent solubilization of an oligomycin- and dicyclohexylcarbodiimide-sensitive form of the enzyme

John W. Soper, Peter L Pedersen

Research output: Contribution to journalArticle

Abstract

The hydrolytic activity of the ATPase bound to purified inner membrane vesicles of rat liver mitochondria can be increased threefold by washing extensively with a high ionic strength phosphate buffer. The specific ATPase activities of such phosphate-washed membranes are the highest reported to date for a mitochondrial membrane preparation (21-24 μmol of ATP hydrolyzed min-1 mg-1 in bicarbonate buffer at 37°C). Deoxycholate (0.1 mg/mg of protein) extracts from these membranes a soluble, cold-stable ATPase complex which exhibits a specific activity under optimal assay conditions of 12 μmol of ATP hydrolyzed min-1 mg-1. This complex is not sedimented by centrifugation at 201 000g for 90 min, and readily passes through a 250-Å Millipore filter. The ATPase activity of the soluble complex is inhibited 95% by 2.4 μM oligomycin. In addition, inhibitions of 60% or better are obtained in the presence of 1-8 μM dicyclohexylcarbodiimide, p-chloromercuribenzoate, venturicidin, and aurovertin. While a similar complex may be extracted with Triton X-100, this preparation is always lower in both specific activity and in inhibitor sensitivities than the complex extracted with deoxycholate. Detergents of the Tween and Brij series and other detergents of the Triton series are also much less effective than deoxycholate in solubilizing the oligomycin-sensitive ATPase complex of rat liver. It is concluded that deoxycholate is superior to other detergents as an extractant of the oligomycinsensitive ATPase complex of rat liver mitochondria, and that the complex extracted with deoxycholate possesses a closer similarity to the membrane-associated ATPase than does the complex extracted with Triton X-100. These studies document the first report of a detergent-solubilized, oligomycin-sensitive ATPase preparation from rat liver mitochondria.

Original languageEnglish (US)
Pages (from-to)2682-2690
Number of pages9
JournalBiochemistry®
Volume15
Issue number12
StatePublished - 1976

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Dicyclohexylcarbodiimide
Oligomycins
Mitochondria
Liver Mitochondrion
Deoxycholic Acid
Detergents
Liver
Adenosine Triphosphatases
Rats
Membranes
Enzymes
Octoxynol
Aurovertins
Buffers
Adenosine Triphosphate
Phosphates
Micropore Filters
Chloromercuribenzoates
Centrifugation
Polysorbates

ASJC Scopus subject areas

  • Biochemistry

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Adenosine triphosphatase of rat liver mitochondria : Detergent solubilization of an oligomycin- and dicyclohexylcarbodiimide-sensitive form of the enzyme. / Soper, John W.; Pedersen, Peter L.

In: Biochemistry®, Vol. 15, No. 12, 1976, p. 2682-2690.

Research output: Contribution to journalArticle

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abstract = "The hydrolytic activity of the ATPase bound to purified inner membrane vesicles of rat liver mitochondria can be increased threefold by washing extensively with a high ionic strength phosphate buffer. The specific ATPase activities of such phosphate-washed membranes are the highest reported to date for a mitochondrial membrane preparation (21-24 μmol of ATP hydrolyzed min-1 mg-1 in bicarbonate buffer at 37°C). Deoxycholate (0.1 mg/mg of protein) extracts from these membranes a soluble, cold-stable ATPase complex which exhibits a specific activity under optimal assay conditions of 12 μmol of ATP hydrolyzed min-1 mg-1. This complex is not sedimented by centrifugation at 201 000g for 90 min, and readily passes through a 250-{\AA} Millipore filter. The ATPase activity of the soluble complex is inhibited 95{\%} by 2.4 μM oligomycin. In addition, inhibitions of 60{\%} or better are obtained in the presence of 1-8 μM dicyclohexylcarbodiimide, p-chloromercuribenzoate, venturicidin, and aurovertin. While a similar complex may be extracted with Triton X-100, this preparation is always lower in both specific activity and in inhibitor sensitivities than the complex extracted with deoxycholate. Detergents of the Tween and Brij series and other detergents of the Triton series are also much less effective than deoxycholate in solubilizing the oligomycin-sensitive ATPase complex of rat liver. It is concluded that deoxycholate is superior to other detergents as an extractant of the oligomycinsensitive ATPase complex of rat liver mitochondria, and that the complex extracted with deoxycholate possesses a closer similarity to the membrane-associated ATPase than does the complex extracted with Triton X-100. These studies document the first report of a detergent-solubilized, oligomycin-sensitive ATPase preparation from rat liver mitochondria.",
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