It has already been shown that influenza virus binds unspecifically to liposomes containing ganglioside GM1 whereas with gangliosides G D l b and G,,, binding occurs in a specific and saturable manner [Slepushkin et al. (1986) Biol. Membr. 3,229 -2351. In the present study the mode of interaction between influenza virus and various gangliosides or phospholipid liposomes containing cholesterol and gangliosides has been investigated.The influence of exogenous gangliosides on the structure of the viral envelope was studied using fluorescent and photoactivatable phospholipids incorporated into the viral membrane. With both types of probes maximal effects of gangliosides were caused by GT1b. Addition of that ganglioside resulted in a marked decrease in the fluorescence polarization (P) of fluorescent labeled virus as well as in substantial changes in the binding of photoactivatable analogues of sphingomyelin and phosphatidylcholine to virus proteins, mainly hemagglutinin.The effects of GTlb and GDlb on P value were comparable, whereas gangliosides with other oligosaccharide chains caused much smaller changes in P. Furthermore GTlb but not Gllll influenced phospholipid-hemagglutinin crosslinking.Interaction of the virus with large unilamellar liposomes was monitored by two fluorescence assays based on resonance-energy transfer from the tryptophans and tyrosines of viral proteins to vesicles labeled with a triacylglycerol (anthrylvinyldioleoyIglycero1) or from these labeled vesicles to virions labeled with a perylenoyl derivative of galactosylcerebroside (PGalSph). A third fluorescence assay was based on relief of self-quenching in PGalSph-labeled virions, upon low-pH-induced virus-liposome fusion. With all three fusion assays the changes of fluorescence caused by GTlb were more pronounced than those induced by GM1. On the other hand, virus-
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...
Affinity resins containing covalently bound phospholipids were used to compare the affinity of the phosphatidylcholine transfer protein from beef liver and a low-specificity lipid transfer protein from rat hepatoma 27 to phosphatidylcholine and sphingomyelin. Binding experiments demonstrated that the beef liver protein associates specifically with immobilized phosphatidylcholine whereas the hepatoma protein showed a preference for sphingomyelin.Purified antiserum raised against the hepatoma sphingomyelin transfer protein was used to determine the presence of that protein in the cytosol of various experimental tumors as well as in those of normal, regenerating and fetal rat liver. The protein was found to be well expressed in all the tumors examined and in fetal liver as determined by immunodiffusion whereas only minute amounts could be detected in normal liver and in 30-h regenerated liver. The presence of the sphingomyelin transfer protein in cytosol was in parallel with the presence of sphingomyelin in the corresponding mitochondria. It is suggested that the occurrence of sphingomyelin in tumor and fetal liver mitochondria may be due to protein-catalyzed sphingomyelin transfer from the endoplasmic reticulum.A number of phospholipid transfer proteins have been purified from a variety of biological tissues (for reviews see [I, 21 and also [3-91). A common feature of all these intracellular transport lipoproteins is that they either do not use sphingomyelin as substrate or catalyze the transfer of sphingomyelin to a much smaller extent than that of phosphatidylcholine. Recently a new lipid transfer protein has been isolated from rat hepatoma 27 which is capable of transferring sphingomyelin at least as efficiently as phosphatidylcholine [lo]. Such a lipid transfer protein could not be detected in rat liver, and the liver postmicrosomal supernatant was not capable of transferring sphingomyelin from rat liver microsomes to mitochondria [lo].Since mitochondria from various tumors frequently show elevated sphingomyelin levels [I 1 -151 it seemed possible that the occurrence of sphingomyelin in tumor mitochondria may be related to the action of sphingomyelin transfer proteins that are absent or less expressed, in normal cells. In this connection it was thought of interest to study further the phospholipid specificity of the rat hepatoma lipid transfer protein and to find out whether the mitochondrial sphingomyelin content in different rat tissues correlates with the presence of sphingomyelin transfer protein in the corresponding cytosol. In this paper we compare the phosphatidylcholine and sphingomyelin binding capacities of the hepatoma lipid transfer protein to those of the specific phosphatidylcholine transfer protein from beef liver using affinity resins containing covalently bound sphingomyelin and phosphatidylcholine. We report further on an immunological examination of the presence of the hepatoma lipid transfer protein in the cytosol of several rat tumors, fetal, regenerating and normal liver. In addition dat...
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