Influence of mitochondria on phospholipid synthesis in preparations from rat liver

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Abstract
1. The addition of mitochondria to an incubation system containing the soluble and microsomal fractions of rat liver enhances severalfold the incorporation of each of ethanolamine, phosphorylethanolamine and CDP-ethanolamine into phosphatidylethanolamine. 2. In the presence of microsomal, mitochondrial and soluble fractions, CDP-ethanolamine exhibits the greatest initial rate of incorporation (approx. 6nmol/h per mg of protein), being slightly faster than that of phosphorylethanolamine (approx. 5nmol/h per mg of protein). Incorporation of ethanolamine proceeds very slowly for the first 20min and only after 30min gives rates approaching those of the other two precursors. 3. By using a substrate ‘dilution’ technique it was shown that in the reconstituted system the affinity of each of the enzymes for their respective substrates is very high: 10μm for ethanolamine, 25μm for phosphorylethanolamine and 5μm for CDP-ethanolamine. 4. Isolation of the mitochondrial and microsomal fractions from the medium after incubation together with phosphorylethanolamine showed that about 70% of the total radioactivity was present in the microsomal fraction and about 30% in the mitochondria after only 20min. Similar experiments with ethanolamine as precursor revealed that after 20min only about 15% of the total radioactivity was present in the mitochondria but that after 40min about 30% was present in this fraction. 5. Heating and phospholipase treatment of mitochondria, but not freeze-thawing, eliminated the stimulatory effect of mitochondria on phospholipid synthesis. 6. The reconstituted system exhibits an absolute requirement for Mg2+(2mm gave maximal rates) and is inhibited by very low concentrations of Ca2+(100μm-Ca2+produced half-maximal inhibition with 3mm-Mg2+). Further addition of Mg2+overcame the Ca2+inhibition, suggesting that the inhibitory effect is readily reversible. 7. The concept that modification of the Mg2+/Ca2+ratio is a means of controlling the rate of cellular phospholipid synthesis is introduced.