Proteins, lipid transfer activity

ApoD is found in association with LCAT and with apoA-I in the HDL fraction. Albers et al. used a specific antibody to apoD to remove all apoD by immunoadsorption chromatography from plasma about 64% of LCAT activity and 11% of apoA-I were also removed from plasma (A14). Purified apoD has an apparent Mr of 32,500, and appears as three isoforms on isoelectric focusing (pi 5.20, 5.08, and 5.00) (A14). An HDL apolipoprotein, Mr 35,000, has been thought to be apoD, and to be a cholesteryl ester transfer protein (i.e., to transfer newly synthesized esterified cholesterol from HDL to LDL) (C8). Cholesteryl ester transfer activity in plasma was removed by polyclonal immunoglobulin to apoD (C8, F10). However, Morton and Zilversmit (M41) were able to separate apoD and lipid transfer protein (i.e., the cholesteryl ester transfer protein, or lipid transfer protein I) by chromatography, and they showed that the removal of apoD from plasma by precipitation with specific antisera did not remove any lipid transfer activity. Albers et al. (A14) also showed that immunoadsorption with antibody specific for apoD removed all the apoD from plasma without removing any cholesteryl ester transfer activity. 

LTP-I may be part of a 150,000-Da molecular complex that includes LCAT (12). The presence of such a complex in plasma might account for the observation that in some cases lipid transfer activity on gel permeation chromatography elutes in a fraction characteristic of large-molecular-weight proteins (Mr > 100,000) (B47, R3, Z8). 

To quantitate the lipid transfer activity of a protein, one measures the movement of labeled lipids from one membrane, the donor, to a second membrane, the acceptor. Typically, the donor and acceptor membranes are incubated in the presence and absence of transfer protein. After the incubation, the particles are separated and either the loss of radiolabeled lipids from the donor particles or the appearance of radiolabeled lipids in the acceptor particles is quantitated. The rate of lipid transfer in the presence of protein minus the transfer that occurs in the absence of protein is a measure of the lipid transfer activity of the protein. The transfer activity is expressed as a percent of the donor lipid transferred or the number of nmols lipid transferred per unit of time. To determine if the rate of lipid transfer also represents the rate of exchange, it must first be established that lipid exchange occurs between donors and acceptors. Exchange occurs when the rate of lipid transfer from donor to acceptor equals the rate of transfer from acceptor to donor or when the chemical composition of the donor and acceptor membranes does not change during the transfer reaction. 

The transfer of phospholipids between mitochondria and microsomes in vitro was first used to measure the activity of lipid transfer proteins (Wirtz and Zilversmit, 1968). In this assay, isolated mitochondria and microsomes are incubated with an appropriate amount of transfer protein. Either particle may be radiolabeled and serve as the donor particle. The exchange reaction is terminated by sedimenting the mitochondria by centrifugation. The change in the radioactivity of either the donor or acceptor particles can be used to calculate the lipid transfer activity. 

The transfer of radiolabeled phospholipids between vesicles and erythrocyte membranes could be used to assay lipid transfer activity. Intact erythrocytes are not an ideal substrate for routine measurements of transfer activity because some transfer proteins do not readily accelerate the transfer of phospholipids from these membranes. Van Meer et al. (1980) found that a very high concentration of the phosphatidylcholine-specific transfer protein was necessary to exchange the phosphatidylcholine of intact red blood cells. Erythrocyte ghosts are a more active substrate for this protein (Bloj and Zilversmit, 1976). However, the nonspecific transfer protein from bovine liver accelerates the exchange of phospholipid between intact erythrocytes and phosphatidylcholine vesicles (Crain and Zilversmit, 1980c). 

Similar approaches may be used to determine the contribution of the phosphatidylinositol- and phosphatidylcholine-specific transfer proteins to the total phosphatidylinositol and phosphatidylcholine transfer activity. The lipid transfer activity of specific transfer proteins has been shown... 

Seedorf, U., Brysch, P., Engel, T., Schrage, K. Assmann, G. (1994) J. Biol Chem. 269, 21277-21283. Sterol carrier protein X is peroxisomal 3-oxoacyl coenzyme A thiolase with intrinsic sterol carrier and lipid transfer activity. 

Lipid transfer peptides and proteins occur in eukaryotic and prokaryotic cells. In vitro they possess the ability to transfer phospholipids between lipid membranes. Plant lipid transfer peptides are unspecific in their substrate selectivity. They bind phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and glycolipids. Some of these peptides have shown antifungal activity in vitro The sequences of lipid transfer proteins and peptides contain 91-95 amino acids, are basic, and have eight cysteine residues forming four disulfide bonds. They do not contain tryptophan residues. About 40% of the sequence adopts a helical structure with helices linked via disulfide bonds. The tertiary structure comprises four a-helices. The three-dimensional structure of a lipid transfer peptide from H. vulgare in complex with palmitate has been solved by NMR. In this structure the fatty acid is caged in a hydrophobic cavity formed by the helices. 

The subcellular distribution of lipid-dependent, glycosylation reactions has also been investigated in a number of plant systems. In plant cells, the situation is, however, more complicated, as their membranes often have the capability to transfer activated sugars, not only to lipid-bound saccharides201-203 and to proteins,4B-204-2,)li but also to cell-wall... 

Studies on the physiological effect of LTP-I have been assisted by the recognition that there are marked species differences in activity. Rabbit plasma contains between two and three times the activity in human plasma, and plasma from rats, sheep, and pigs, for instance, contains less than 20% of the activity of human plasma (HI). Whereas rat plasma is deficient in cholesteryl ester and triglyceride transfer activity, facilitated phospholipid transfer activity is not impaired (II, 12, T4). The reason is unknown it is possible that rat plasma contains a different lipid transfer protein (T2), perhaps homologous to the LTP-2 reported in human plasma (A17). 

The species differences observed in lipid transfer protein activity may, in part, be due to the presence of an inhibitor which markedly reduces CE and TG transfer and can be separated from LTP-I in human plasma (M42). Inhibitory activity has also been demonstrated in lipoprotein-free plasma from rat, pig, goat, chicken, and cow, but not in rabbit lipoprotein-free plasma. The levels of inhibitor in the species studied were not quantitated, but it seems possible that the level of inhibitor in the plasma of different species may be an important factor in determining LTP-I activity (M42). 

The rate of cholesterol esterification in plasma is not correlated with HDL concentration (A12, R17, S45, S58, Wl, W2) but is correlated with the concentration of VLDL or triglyceride (A12, P8, R17, S58, T7, Wl, W2). Although HDL is the major substrate for LCAT, VLDL and indirectly LDL are the major recipients of the esterfied cholesterol, transferred (it is thought) by lipid transfer protein. Accumulation of esterified cholesterol in the recipient lipoproteins is associated with a decrease in LCAT activity (C7, Fll, F13) that can be relieved by the addition of recipient lipoproteins but not by addition of LCAT substrate (Fll). Hopkins and Barter (H32, H33) have explained these observations by showing that the depletion of HDL esterified cholesterol by transfer to VLDL enhances the capacity of HDL to act as a substrate for LCAT. 

A2. Abbey, M., Savage, J. K., Macldnnon, A. M., Barter, P. J., and Calvert, G. D., Detection of lipid transfer protein activity in rabbit liver perfusate. Biochim. Biophys. Acta 793, 481-484 (1984). 

Jung, H.W., Kim, W. and Hwang, B.K., 2003, Three pathogen-inducible genes encoding lipid transfer protein from pepper are differentially activated by pathogens, abiotic and environmental stresses. Plant Cell Environ. 26 915-928. 

---