Free fatty acids in carrot-tissue preparations and their effect on isolated carrot mitochondria

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Abstract
The concentrations of free fatty acids in carrot homogenates, prepared with or without added albumin and kept at 4[degree] for 25 minutes were 0.013 and 0.015 mM, respectively, rising after 60 minutes to 0.041 and 0.11 mM, respectively; the concentrations of free fatty acids in the mito-chondrial suspensions prepared from these homogenates were 1.1 and 0.56 mM, respectively. The main components of these fractions were oleic acid, stearic acid, palmitic acid, and an unidentified short-chain compound whose concentration rose considerably on standing; the mitochondrial fraction (prepared with added albumin) also contained relatively high concentrations of polyunsaturated C18 fatty acids. The total fatty acids of tissue lipid and mitochondrial lipid consisted primarily of linoleic acid and palmitic acid; large porportions of octodeca-6,9-dienoic acid and an unidentified fatty acid (RT 2.81) were also present in mitochondrial lipid. Carrot mitochondria, prepared with added albumin and incubated with succinate, malate, citrate, a-oxogluta-rate, and NADH2, gave P:O ratios of 1.6, 1.9, 2.0, 2.0 and 1.3 respectively; comparable preparations isolated without albumin give P:O values of less than 0.1, and, with the same substrates, qO2 values 15, 64, 43, 5, 9, and 47% respectively of the corresponding values for mitochondria prepared with added albumin. Oleic acid (0.1, 1, and 10 mM) inhibited oxygen uptake with succinate to a greater extent than with malate. Laurie acid, oleic acid, linolenic acid, myristic acid, and linoleic acid inhibited oxygen uptake of carrot mitochondria, prepared with added albumin and incubated with succinate, by more than 70%; smaller inhibitions were observed with palmitic acid (57%), stearic acid (32%), arachldic acid (17%), and butyric acid (nil). The fatty acids most effective in inhibiting oxygen uptake were also most effective in lowering the P:O ratios; the inhibition of phosphorylation and oxygen uptake increased markedly with an increase in chain length from C8 to C12. Malate-dehydrogenase activity was not inhibited by 1 mM-stearate or 1 mM-oleate; succinate-dehydrogenase activity was inhibited 39% by both fatty acids; dihydronicotinamide-adenine dinucleotide dehydrogenase was inhibited by 85% and 92% by 1 mM-stearate and 1 mM-oleate, respectively. The results are discussed in relation to the general problems of isolating "intact" plant mitochondria and the action of fatty acids in innibiting mitochondrial functions.