Whether or not the large molecular size of native Asp-NAT was an artifact of the enzyme preparation methods was tested by using different homogenization and solubilization conditions. A buffer that is commonly used in mitochondrial protein purification (Tris-sucrose medium: Tris-HCl, 50 mM; 1 mM EDTA; 0.32 M sucrose; 1 mM DTT; protease inhibitor cock-tail added according to the quantity of processed tissue; pH adjusted to 7.4) was employed to obtain a crude mitochondrial pellet from rat brains. Various detergents were tested to solubilize the pellet: deoxycholate (DC) (negatively charged and one of the most commonly used detergents in mitochondrial studies), hexadecyl trimethyl ammonium bromide (positively charged, commonly called CTAB), Triton X-100 (non-ionic), laurylmaltoside (LM) (non-ionic, commonly used in mitochondrial protein reconstitution experiments) and CHAPS.
Crude mitochondrial pellets were obtained by homogenizing frozen rat brain tissue in the Tris-sucrose medium using a glass-teflon Potter-Elvehjem homogenizer, followed by two centrifugation steps: the first at low speed (1350g- for 5 min) to separate the debris and the second at high speed (18,000g for 15 min) to obtain a crude mitochondrial pellet. The pellet was incubated in Tris-sucrose medium with various concentrations of detergents for 1h on ice with periodic agitation, followed by separation of the solubilized
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