Preparation of rat enterocyte mitochondria

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Abstract
Rat enterocyte mitochondria were prepared with respiratory control ratios of 4 or 5 and occasionally 6. When EGTA [ethylene glycol-bis(.beta.-aminoethyl ether)N,N,N'',N''-tetraacetic acid] was excluded from the mitochondrial incubation medium the calculated P/O ratios were high, especially those based on the 1st addition of ADP. These ratios were lowered by increasing the EGTA concentration from 1 mM to 2 mM in the mitochondrial preparation medium and including 1 mM EGTA in the incubation medium. The use of EDTA in the enterocyte isolation medium led to the mitochondria requiring added cytochrome c. Substituting EGTA for EDTA abolished this requirement. The mitochondrial fraction consisted of 2 components, an upper cream-colored layer rich in DNA and a lower brown-colored layer poor in DNA. Both components were capable of oxidative phosphorylation with succinate or the glutamate/malate couple as substrates. The mitochondrial yield was assessed by assaying succinate dehydrogenase activity, and the contamination of the mitochondrial fraction by other cell organelles was assessed by assays for appropriate marker enzymes.