Purified lipid transfer proteins

Ravnik, S.E., Albers, J.J., and Muller, C.H. (1995). Stimulation of human sperm capacitation by purified lipid transfer protein. Biol. Reprod. J. Exp. Zool. 272 78-83. 

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. 

In the late 1960s, Wirtz and Zilversmit (1968) found a soluble factor in rat liver cytosol that accelerated the exchange of phospholipids between biological membranes. Since this discovery, many lipid transfer proteins have been purified. A list of these and their physical properties is found in Table I. Although there has been considerable speculation concerning the physiological role of lipid transfer proteins, their precise function remains in doubt (Wirtz, 1982). 

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. 

Intracellular transport of phospholipids is thought to require the participation of lipid transfer proteins (LTP) able to facilitate intermembrane lipid movements. These proteins, first detected and partially purified from potato tuber cytosol (Kader, 1985), have been purified to homogeneity from various plant tissues spinach leaf (Kader et al., 1984), maize seedlings (Douady et al., 1982, 1985), and castor bean endosperm (Yamada et al., 1978 Watanabe and Yamada, 1986). These purifications opened perspectives for studying the structure, the mode of action and the molecular biology of this novel category of plant proteins involved in lipid movements. 

Molecular mass - The major lipid-transfer protein purified to homogeneity from plants have a low molecular mass around 9 kDa ( Kader et al., 1984 Douady et al., 1985 Watanabe and Yamada, 1986 Nishida and Yamada, 1986). These values, determined by gel filtration or SDS-electrophoresis, have been confirmed by the determination of the amino-acid sequences (Table 1). In addition to the major protein of castor bean, some isoforms have been recently found with different molecular mass (see Yamada et al., this volume). The molecular mass of... 

A non specific lipid transfer protein (ns-LTP) which enhances the transfer of both phospholipids and galactolipids between biomembranes has been purified from germinated castor bean seeds (Watanabe 1906) and its primary structure has been determined to be 9.3 KD-protein with 92 amino acid residues (Takishima 1986). We now call this nsLTP-9A, because isoforms of nsLTP-9A have been found in the same materials. This paper represents the occurrence of ns-LTP isoforms in germinated castor bean tissues. Materials and Methods... 

Lipid transfer protein (LTP) was purified from extracts of 72-h-old maize seedlings with gel filtration on a Sephadex G 75 column. Activity was assayed by measuring the ability of a protein fraction to stimulate the transfer of H-phosphatidylcholine from liposomes to mitochondria. Purification and assays were done as described by Douady et al. [4]. 

Reconstitution of membranes from a small number of molecular components provides simplified structures to study. Thus, cytochrome oxidase or photosynthetic reaction centers, both electron transfer proteins, may be extracted from their native membranes, purified, and reincorporated at relatively high concentration into a simple well defined lipid bilayer. Diffraction investigation then provides information about the distribution and structure of the protein in the membrane. Understanding the mechanism for electron transport in these proteins will require considerable additional information. One key element of structural informations is the location of the redox centres in the membrane profile. 

In 1993, we reported [19] reversible electron transfer between electrodes and the iron heme protein myoglobin imbedded in cast multi-lameUar liquid crystal films of didodecyldimethylammonium bromide (DDAB). Heretofore, reversible electron transfer from electrodes to myoglobin in solution had been accomplished only for highly purified myoglobin solutions on specially cleaned indium tin oxide electrodes [20,21]. If enhanced electron transfer for proteins in surfactant or lipid films were to prove general, it might help solve longstanding problems in protein electrochemistry. 

Chemical characterization of reticulin is difficult because it usually occurs rather sparsely and mixed with a variety of other tissue components. However, purified reticulin from the cortex of human kidneys has been shown to consist of a combination of collagen, carbohydrate (probably glycoprotein) and possibly lipid. It is thus a complex made up of high molecular weight substances. The lamina densa may influence the transfer of molecules between connective tissue and epithelium and hence would be expected to exert control over the epithelium. It has been suggested that both connective tissue and epithelium collaborate in building up the basement membrane and that the epithelial cells behave atypically by secreting a proline-rich protein into its outer layers. 

Cytochrome C oxidase catalyzes the transfer of electrons from cytochrome C to molecular oxygen and is one of the best investigated intrinsic membrane proteins. The beef-heart enzyme can be purified in an almost lipid-free form and can be functionally reconstituted by incorporation into different lipid systems since the natural lipid composition is usually not required for reconstitution of an active enzyme (see Fig. 9). 

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