Lipid transfer activity

Nakanishi T, Tahara D, Akazawa S, Miyake S, Nagataki S (1990) Plasma lipid transfer activities in hyper-high-density lipoprotein cholesterolemic and healthy control subjects. Metabolism 39 225-230... 

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). 

Several functions of MTP have been identified all have been implicated in coordinating successful lipoprotein assembly (Fig. 27-1). MTP transfers lipids between vesicles in vitro, and this activity is likely to be its major function. MTP can pick up lipids from membrane (step A) or vesicles and droplets (step B) and transfer them to the nascent apoB. In addition, the lipid transfer activity of MTP has been implicated in the accretion of neutral lipids from the cytosol into the ER lumen (step C). Compounds that inhibit in vitro transfer activity of MTP decrease apoB secretion by cells, indicating that this activity is essential for apoB lipoprotein secretion. Apart from transferring lipids, MTP has been shown to interact physically with apoB (step D). This activity... 

Why has the use of MTP lipid transfer activity inhibitors been unsuccessful as... 

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). 

In addition to the assays described in this section, other assays involving the separation of substrates have been used to quantitate lipid transfer activity. These assays differ primarily in their choice of substrates and the method of separation. Additional biological membranes, which have been used to quantitate transfer activity, include chloroplasts (Tanaka et al., 1980), retinol rod disc membranes (Dudley and Anderson, 1978), intra-cytoplasmic membrane derived from Rhodopseudomonas sphaeroides (Cohen et al., 1979), myelin (Ruenwongsa et al., 1979), and lamellar bodies (Tsao, 1979). Biological membranes have been isolated by sucrose... 

Lipid transfer activities are generally determined by assays involving separation of donor and acceptor particles. These techniques have some distinct disadvantages. The time-consuming process of separating donors and acceptors is especially troublesome in a kinetic analysis of a transfer reaction when it is necessary to measure the time dependence of lipid transfer reactions. In addition, the separation of donor and acceptor membranes requires that the two particles differ in some respect. 

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... 

Many different assays are used to quantitate lipid transfer activity. Some are more suited for routine assays because they are easy to perform and versatile. Other assays more effectively answer questions about the mech-... 

Lipid Transfer Activity, Quantitation of (Wetterau and Zilversmit). 30 199... 

Quantitation of Lipid Transfer Activity. By John R. Wetterau... 

Specific inhibitors of the lipid transfer activity of MTP have been developed as a potential treatment for atherosclerosis based on the premise that inhibition of MTP reduces VLDL secretion. Indeed, MTP inhibitors effectively reduce plasma cholesterol levels by up to 80% in rats, hamsters, and rabbits [13]. However, heterozygosity for MTP deficiency in humans does not diminish plasma lipids or lipoproteins, indicating that a modest reduction in MTP activity does not limit VLDL production. On the other hand, adenovirus-mediated over-expression of MTP in mouse liver increased the secretion, and plasma levels, of TG, apo B100, and apo B48 (D.J. Rader, 1999). MTP inhibitors are not currently used therapeutically, probably because they induce some storage of TG in the liver (steatosis) as a result of the blockage in VLDL secretion. 

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. 

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