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Use of Digitonin. Print E-mail

Use of Digitonin in mitochondria isolation.

     In earlier studies, digitonin treatment had frequently been used to isolate mitochondria from cells and tissue. One of the most efficient protocols for mitochondria isolation from small amounts of brain tissue (~50 mg of adult mouse brain) combines Percoll gradient and digitonin treatment of tissue homogenates [1]. The rationale for using digitonin to improve mitochondria recovery is that it selectively disrupts the lipid membranes enriched in sterols. Digitonin complexes with most unesterified 3-b-hydroxysterols, including cholesterol, the principal free sterol present in vertebrate tissues. As the molecular weight of digitonin is about three times that of cholesterol, the intercalation of digitonin into cell membranes significantly disrupts the packing of lipids in the bilayer and facilitates its rupture.
    Therefore, digitonin greatly facilitates the disruption of cholesterol-rich cellular membranes and synaptosomal membranes and dissolves fragments of endoplasmic reticulum and cellular plasmalemma. This improves purity of the mitochondrial preparations and increases their yield. However, the outer mitochondrial membrane also contains susbtantial amount of cholesterol and can easily be disrupted by digitonin (e.g., see [2, 3]). Moreover, inner mitochondrial membrane, although devoid of cholesterol, can incorporate digitonin [4] and eventully get lysed by it. This makes digitonin-based methods cumbersome and prone to artifacts.
    It had been reported that digitonin-isolated guinea pig brain mitochondria exhibited non-standard patterns of response to the respiratory chain inhibitors [5] and that rat brain mitochondria isolated with digitonin exhibited altered cytochrome c release and Ca2+-handling properties [6]. Digitonin was also shown to alter the mobility of the mitochondrial respiratory complexes and the rates of electron traansfer [4]. A partial rupture of the outer mitochondrial membrane induced by digitonin during the isolation of mitochondria is difficult to detect. However, it likely would result in serious non-physiological alterations of the mitochondrial functions. Many critically important proteins such as cytochrome c, creatine kinase, sulfite oxidase, proteases and several pro-apoptotic proteins are located in the intermembrane space and could become depleted during the isolation of mitochondria if their outer membrane is ruptured. When a study requires functional structurally intact mitochondria, digitonin–based mitochondria isolation protocols such as described in [1, 7, 8] should better be avoided.

[1]. Anderson MF, Sims NR. Improved recovery of highly enriched mitochondrial fractions from small brain tissue samples. Brain Res Brain Res Protoc. 2000 5(1):95-101. PMID: 10719270
[2]. Crémel G, Filliol D, Jancsik V, Rendon A. Cholesterol distribution in rat liver and brain mitochondria as determined by stopped-flow kinetics with filipin. Arch Biochem Biophys. 1990 278(1):142-7. PMID: 2321954
[3]. Dorbani L, Jancsik V, Linden M, Leterrier JF, Nelson BD, Rendon A. Subfractionation of the outer membrane of rat brain mitochondria: evidence for the existence of a domain containing the porin-hexokinase complex. Arch Biochem Biophys. 1987 252(1):188-96. PMID: 3028256
[4]. Hochman J, Ferguson-Miller S, Schindler M. Mobility in the mitochondrial electron transport chain. Biochemistry. 1985 24(10):2509-16. PMID: 2990530
[5]. Tretter L, Mayer-Takacs D, Adam-Vizi V. The effect of bovine serum albumin on the membrane potential and reactive oxygen species generation in succinate-supported isolated brain mitochondria. Neurochem Int. 2007 50(1):139-47.
[6]. Brustovetsky N, Jemmerson R, Dubinsky JM. Calcium-induced Cytochrome c release from rat brain mitochondria is altered by digitonin. Neurosci Lett. 2002 332(2):91-4.
[7]. Moreadith RW, Fiskum G. Isolation of mitochondria from ascites tumor cells permeabilized with digitonin. Anal Biochem. 1984 137(2):360-7. PMID: 6731817
[8]. Rosenthal RE, Hamud F, Fiskum G, Varghese PJ, Sharpe S. Cerebral ischemia and reperfusion: prevention of brain mitochondrial injury by lidoflazine. J Cereb Blood Flow Metab. 1987 7(6):752-8. PMID: 3693430

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