The complete primary structure of the phosphatidylcholine transfer protein from bovine liver consists of: Ac-Met

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SummaryPhospholipid transfer activities for phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were measured in three hepatomas of increasing growth rate and degree of dedifferentiation, the hepatomas 7787, 9633 and 7777, and compared to the activities found in normal and host liver. A 2--3-fold increase was found in the phosphatidylcholine and phosphatidyinositol transfer activities in the fast-growing 7777 hepatoma, while these activities were moderately or not increased in the 7787 and 9633 hepatomas. Phosphatidylethanolamine transfer was found to be extremely low in all three hepatomas. The possible significance of these findings with respect to the altered phospholipid content and composition of the hepatoma membranes is discussed.The contribution of the phosphatidylcholine specific exchange protein to the total phosphatidylcholine transfer activity was determined in normal and host liver and in the hepatomas 7777 and 9633 with the aid of a phosphatidylcholine exchange protein specific antiserum. To this end a new procedure for the purification of the phosphatidylcholine exchange protein from mt liver was developed which leads to a final purification factor of 5300 and a high overall yield of 17%. In addition, this protein was chemically and immunologically characterized and its properties were compared to those of the bovine phosphatidylcholine exchange protein purified in our laboratory previously.

Secondary structural elements of the phosphatidylcholine-transfer protein from bovine liver have been predicted from its primary structure with the aid of two computerized methods. The predicted a-helix and /]-strand content have been compared with the values derived from circular dichroism spectra. The hydrophobicity profile (Rose plot) of the protein indicated that the supposed lipid-binding site occurs in the most hydrophobic region. The predicted secondary structural elements have been folded in a tentative model of the protein molecule according to its hydrophobicity profile.Eukaryotic cells contain proteins which mediate the transfer of phospholipid between membranes [l -71. The protein, the most extensively investigated to date, is the phosphatidylcholine-transfer protein from bovine liver, which acts as a specific carrier of phosphatidylcholine [S -141. Upon interaction with the membrane the protein can both insert and bind a phosphatidylcholine molecule. Binding implies that a transient protein-membrane complex is formed in which it is energetically favourable for phosphatidylcholine to leave the bilayer. This is very interesting and suggests that the tertiary structure of this transfer protein has some unique features.In order to understand the mode of action of the phosphatidylcholine-transfer protein the primary structure has been elucidated [IS]. Here we present a tentative model of the protein molecule based on the secondary structure as predicted by the computerized methods of Chou and Fasman [16, 171 and Lim [18,19], and the hydrophobicity profile derived from the primary structure by the methods of Rose [20,21]. It will be shown that this protein has some extremely hydrophobic peptide segments, which may be involved in the interaction of the protein with the interface and its binding of a phosphalidylcholine molecule. MATERIALS AND METHODSThe phosphatidylcholine-transfer protein was purified from bovine liver as described previously [1,22]. The amino acid sequence was elucidated by manual and automated Edman degradation (for details see [15,23]). Circular dichroism spectra were recorded on a Dichrograph I1 (Jouan) with automatic slit control at room temperature under a constant nitrogen flush. Protein concentrations were determined by measuring the ultraviolet absorbance = 2.42). Estimation of the secondary structure was performed by conformation analysis according to Chen et al. [24]. RESULTS A N D DISCUSSION Primary StructureThe amino acid sequence of the phosphatidylcholinetransfer protein is presented in Fig. 1. The protein consists of a single peptide chain of 213 amino acids of which N-acetylmethionine forms the blocked N terminus. It contains two disulfide bonds at C y~" -C y s~~ and CysY3-Cyszo7. Recent studies with photolabeled phosphatidylcholine have indicated that the hydrophobic peptides Val'71-Phe-Met-Tyr-Tyr-Phe'76 and Trp186-Val-Ile-Asn-Trp1yo form the lipid-binding site ([26,27], unpublished work). It follows from the sequence that there is one other extremely hydrophobic pept...