Identification of an essential lysine residue in the phosphatidylcholine-transfer protein from bovine liver by modification with phenylisothiocyanate.

Modification by phenylisothiocyanate inhibits the phosphatidylcholine-transfer protein from bovine liver. Inhibition by this apolar reagent was greatly enhanced in the presence of vesicles, indicating that an effective modification of an essential lysine residue(s) from the interface may occur. Labeling with [14C]phenylisothiocyanate demonstrated that Lys55 was the major site of modification. We propose that Lys55 is part of the peptide segment that interacts with the membrane.

Prediction of secondary structural elements in the phosphatidylcholine-transfer protein from bovine liver.

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 alpha-helix and beta-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.

Modification of the phosphatidylcholine-transfer protein from bovine liver with butanedione and phenylglyoxal. Evidence for one essential arginine residue.

1. Modification of arginine residues with 2,3-butanedione and phenylglyoxal completely inhibits the transfer activity of the phosphatidylcholine transfer protein from bovine liver. Removal of borate and butanedione leads to a slow reactivation of the protein. 2. Both alpha-dicarbonyl reagents modify three of the ten arginine residues present per protein molecule. The extent of modification is linearly related to the loss of activity. 3. Inactivation with butanedione is greatly diminished when the protein is bound to strongly negatively charged vesicles. Under these conditions a rapid modification of two arginine residues is observed. This suggests that the transfer protein contains one arginine residue essential for activity, probably as a binding site for the negatively charged phosphate group of the phosphatidylcholine molecule. 4. This study provides convincing evidence that arginine residues may play an essential role in phospholipidprotein interactions.

A new high-yield procedure for the purification of the non-specific phospholipid transfer protein from rat liver.

Rat liver contains a non-specific phospholipid transfer protein that transfers phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol and sphingomyelin as well as cholesterol between membranes (Bloj, B. and Zilversmit, D.B. (1977) J. Biol. Chem. 252, 1613-1619). The present paper describes a new high-yield procedure for the purification of this protein which includes fractionation on DEAE-cellulose, Sephadex G-50 and hydroxyapatite. Starting from a pH 5.1 supernatant, a homogeneous protein was obtained after a 1 540-fold purification at a yield of 50%. The protein has a molecular weight of 14 800 as estimated by electrophoresis on polyacrylamide gels in the presence of SDS. It has a blocked N-terminal amino acid and a tryptophanyl fluorescence emission maximum at 335 nm. Its amino acid composition has been determined and compared to data published by others on similar proteins.

Phospholipid transfer activities in Morris hepatomas and the specific contribution of the phosphatidylcholine exchange protein.

Phospholipid transfer activities for phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were measured in three hepatomas of increasing growth rate and degree of dedifferentiation, the hepatomas of 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 phosphatidylinositol 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 o f a phosphatidylcholine exchange protein specific antiserum. To this end a new procedure for the purification of the phosphatidylcholine exchange protein from rat 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.

The primary structure of the phosphatidylcholine-exchange protein from bovine liver. Isolation and characterization of the staphylococcal protease peptides and the amino-acid sequence of the N-terminal half (residues 1--122).

The phosphatidylcholine exchange protein from bovine liver consists of a single polypeptide chain and has a blocked N terminus. The protein contains an estimated 244 amino acid residues in accordance with a determined molecular weight of 28000. The protease from mouse submaxillaris gland cleaved the citraconylated and S-carboxymethylated derivative of the exchange protein at one specific site (Arg14-Glu15) close to the N terminus. Analysis of the two resulting peptides showed that N-acetyl-methionine was the N-terminal residue and gave the sequence of the first 41 residues. The modified protein was also fragmented with the protease from Staphylococcus aureus. The peptides isolated represented 88% of the protein; their sequences were determined by manual and automated Edman degradation. Alignment of a number of these peptides gave the complex sequence of the N-terminal half up to position 122.