A Universal Lipid Exchange Protein from Rat Hepatoma

Abstract

The postmicrosomal protein fraction from rat hepatoma 27 adjusted to pH 5.1 stimulates phospholipid exchange between rat liver microsomes and mitochondria with higher rates and in a less specific way than the corresponding fraction from rat liver. A phospholipid exchange protein has been purified to homogeneity from the hepatoma pH-5.1 supernatant by gel filtration on Sephadex (3-75 and ion-exchange chromatography on carboxymethylcellulose. The isolated protein had a molecular weight of 1 1 200 as determined by electrophoresis on polyacrylamide in the presence of dodecyl sulfate a i d of 1 1 168 as calculated from the amino acid composition. Isoelectric focusing showed a single band at pH 5.2.In the assay system rat liver microsomes + mitochondria the protein exhibits a complete lack of substrate specificity transferring all the major microsomal phospholipids to about the same extent. The possible role of the isolated phospholipid exchange protein in the chemical dedifferentiation of hepatoma cell membranes is discussed.Previous work has shown that mitochondria, microsomes, plasma membranes and nuclei from fastgrowing rat hepatomas have similar phospholipid patterns [ 11. whereas the corresponding subcellular fractions from liver and other mammalian tissues differ markedly in their phospholipid composition [1,2]. A particularly important feature of the mitochondria and microsomes of rapidly growing hepatomas was that. unlike those from normal liver, they always contained significant amounts of sphingomyelin and cardiolipin, respectively. It was demonstrated that the 'lipid dedifferentiation' of hepatoma cell membrane fractions was not caused by cross contamination [ l ] or by phospholipid redistribution during isolation of the subcellular fractions [ 3 ] and that it depended on the malignancy of the tumor [4,5]. The fact that hepatoma mitochondria, in contrast to those from the liver, contain appreciable amounts of sphingomyelin has been confirmed independently by a number of authors [5-71.Since mitochondria usually do not synthesize their own cholinephospholipids [8] it seemed probable that the sphingomyelin present in the hepatoma mitochondria is synthesized in the endoplasmic reticulum and then transferred to the mitochondria by some phospholipid exchange protein which is absent or inhibited in the normal liver.In the present paper we describe the lipid exchange properties of the pH-5.1 fractions of the postmicrosoma1 supernatants from rat liver and hepatoma 27 and report the isolation from the hepatoma of an individual protein transferring sphingomyelin from microsomes to mitochondria. The protein appeared to differ from the known phospholipid exchange proteins by its ability to transfer the same relative amount of any microsomal phospholipid in similar time from rat liver microsomes to mitochondria. I t thus appears that the activity of this universal phospholipid exchange protein in hepatoma cells is responsible for the lipid dedifferentiation of the hepatoma cell membranes. MATERIALS AND METHODS Isolatio...

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