Lipid transfer activity particles

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. 

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. 

PLTP is responsible for the majority of phospholipid transfer activity in human plasma. Specifically, it transfers surface phospholipids from VLDL to HDL upon lipolysis of triglycerides present in VLDL. The exact mechanism by which PLTP exerts its activity is yet unknown. The best indications for an important role in lipid metabolism have been gained from knockout experiments in mice, which show severe reduction of plasma levels of HDL-C and apoA-I. This is most likely the result of increased catabolism of HDL particles that are small in size as a result of phospholipid depletion. In addition to the maintenance of normal plasma HDL-C and apoA-I concentration, PLTP is also involved in a process called HDL conversion. Shortly summarized, this cascade of processes leads to fusion of HDL... 

Fig. 8. Role of lipophorin in DG delivery to flight muscle. Adipokinetic hormone (AKH) is released from the corpus cardiacum and binds to the fat body, where it cause production of cAMP and entry of Ca . These second messengers activate lipolysis of triacylglycerol (TG) and production of diacylglycerol (DG). The DG leaves the fat body with the assistance of a lipid transfer particle (LTP) and is taken up by HDLp. The capacity of HDLp to carry DG is increased by binding of apoLp-HI to the surface. Ultimately, LDLp is formed and moves to the flight muscle, where a lipoprotein lipase hydrolyzes the DG to produce fatty acid (FA) and regenerate HDLp and apoLp-IIl. The FA enters the flight muscle, where it is oxidized to produce the ATP required to power flight. HDLp and apoLp-HI circulate back to the fat body to complete the cycle.

Several mechanisms of lipid metabolism have now been linked to reverse cholesterol transport, including the lipid transfer protein (LTP) reaction, the activity of leci-thinxholesterol acyltransferase (LCAT), and the removal of cholesterol from cells (cholesterol efflux). Reports in the literature link the function of Apo A-IV to these three processes. Weinberg and Spector [42] concluded from their experiments that Apo A-IV could function as, or in concert with, a Upid transfer factor. Lagrost et al. [22] showed that a small HDL-like particle was formed in human serum in vitro upon incubation with partially purified Upid transfer protein and Apo A-IV. Furthermore, Apo A-IV was shown to activate LCAT in vitro [5, 33, 34], and the plasma distribution of Apo A-IV appeared to be dependent on the LCAT reaction in rats [6] and humans [3]. Evidence also exists to link Apo A-IV to the LCAT reaction in vivo. This is supported by the observation that plasma of Apo A-I-deficient patients [13] contains normal amounts of cholesteryl ester. 

Fig. 5.2.1 The major metabolic pathways of the lipoprotein metabolism are shown. Chylomicrons (Chylo) are secreted from the intestine and are metabolized by lipoprotein lipase (LPL) before the remnants are taken up by the liver. The liver secretes very-low-density lipoproteins (VLDL) to distribute lipids to the periphery. These VLDL are hydrolyzed by LPL and hepatic lipase (HL) to result in intermediate-density lipoproteins (IDL) and low-density lipoproteins (LDL), respectively, which then is cleared from the blood by the LDL receptor (LDLR). The liver and the intestine secrete apolipoprotein AI, which forms pre-jS-high-density lipoproteins (pre-jl-HDL) in blood. These pre-/ -HDL accept phospholipids and cholesterol from hepatic and peripheral cells through the activity of the ATP binding cassette transporter Al. Subsequent cholesterol esterification by lecithinxholesterol acyltransferase (LCAT) and transfer of phospholipids by phospholipid transfer protein (PLTP) transform the nascent discoidal high-density lipoproteins (HDL disc) into a spherical particle and increase the size to HDL2. For the elimination of cholesterol from HDL, two possible pathways exist (1) direct hepatic uptake of lipids through scavenger receptor B1 (SR-BI) and HL, and (2) cholesteryl ester transfer protein (CfiTP)-mediated transfer of cholesterol-esters from HDL2 to chylomicrons, and VLDL and hepatic uptake of the lipids via the LDLR pathway...

Release of VLDLs VLDLs are secreted directly into the blood by the liver as nascent VLDL particles containing apolipoprotein B-100. They must obtain apo C-ll and apo E from circulating HDL (see Figure 18.17). As with chylomicrons, apo C-ll is required for activation of lipoprotein lipase. [Note Abetalipoproteinemia is a rare hypolipoproteinemia caused by a defect in triacylglycerol transfer protein, leading to an inability to load apo B with lipid. As a consequence, no chylomicrons or VLDLs are formed, and tria-cylglycerols accumulate in the liver and intestine.]... 

Lipid/DNA particles represent a nonliposomal but lipid-based delivery system for gene transfer. Monomeric or micellar lipids are allowed to interact with DNA in the presence of detergent or some other surface-active agent that is then removed by dialysis. As the surface-active agent diffuses out, solid, condensed particles of lipid and DNA form (17). These can be prepared such that they are smaller and more homogeneous than liposome/DNA complexes yet transfect cells equally well (F. Wong, unpublished observations). 

The advantage of reconstituted lipoproteins over the native lipoproteins is that the model particles can be made with a single apolipoprotein and one or a few defined lipid components to study each component individually. In addition, particles of uniform size can be isolated where high-resolution structural analysis is potentially possible. Also the reconstituted lipoproteins lend themselves for the study of structure-function relationships. In fact, reconstituted lipoproteins display all the known functions ascribed to native lipoproteins, including enzyme activation, receptor binding, and uptake and transfer of lipids. 

HDL particle. Apo A1 is a cofactor for LCAT (A. Jonas, 2000) and the conversion of cholesterol to CE (Section 2.2) induces the formation of a neutral lipid core and the concomitant change in particle shape. Triacylglycerol molecules are introduced into the core of spherical HDL as a consequence of CE transfer protein activity. The apo A1 amphipathic a-helices are embedded among the PL molecules on the surface of spherical HDL (Fig. 7) but the detailed conformations of the apo A1 molecules are not known. Spherical HDL in plasma can be remodeled via a fusion event into large and small particles by PL transfer protein (K.A. Rye, 2001). Some HDL particles contain both apo A1 and apo A2 molecules and the interactions between these two proteins need to be understood. The presence of apo A2 inhibits HDL remodeling by CE transfer protein and the dissociation of apo A1 molecules to create pre-p-HDL. It seems that apo A2 interacts with apo A1 and reduces the ability of the latter to desorb from the HDL particle surface (K.A. Rye, 2003). 

The adverse effects of trans fatty acids on serum lipids and lipoproteins are thought to be mediated by alterations in lipid catabolism and metabolism. Trans fatty acids increase the catabolism rates of apolipoprotein A-I and decrease apolipoprotein B catabolism rates (Matthan et al., 2004), reduce LDL-C particle size (Mauger et al., 2003), and can increase cholesteryl ester transfer protein (CETP) activity (van Tol et al., 1995). CETP mediates the transfer of cholesterol esters from HDL- to LDL- and very-low-density lipoprotein (VLDL)-C, thereby offering a potential explanation for the LDL-C-raising and HDL-C-lowering effect of trans fatty acids. 

In the process of maturation, the nascent HDL particles accumulate phospholipids and cholesterol from cells lining the blood vessels. As the central hollow core of nascent HDL progressively fills with cholesterol esters, HDL takes on a more globular shape to eventually form the mature HDL particle. The transfer of lipids to nascent HDL does not require enzymatic activity. 

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