1986;879:345C349. rate. Introduction of the same amount of bad charge into the acceptor vesicle membrane did not impede the transfer rate as efficiently. Also, positive charge in the donor vesicle membrane was not as effective at slowing the transfer rate as was bad charge in the donor vesicle. Increasing the ionic strength of the buffer with NaCl significantly reversed the charge effects. At neutral pH, the transfer protein (p? 9.0) is expected to be positively charged, which may promote association with the negatively charged donor membrane. Based on these and additional experiments, we conclude the transfer process follows first-order kinetics and that the off-rate of the transfer protein from your donor vesicle surface is the rate-limiting step in the transfer process. Glycosphingolipids (GSLs)1 are amphipathic molecules that together with phospholipids and cholesterol constitute the basic lipid core structure of biomembranes. Except for their presence at relatively high amounts in the plasma membranes of neural cells and in the apical membranes of epithelial cells (about 25-30% of total lipids in both membrane types), GSLs are usually minor parts in plasma membranes of eukaryotic cells (about 5%) (1, 2). The prevailing look at has been that newly synthesized GSLs are localized mainly in the outer leaflet of the eukaryotic plasma membrane. This location is consistent with their functions as cell surface markers and as modulators of membrane protein function. Also, particular GSLs function as the surface binding sites for certain bacteria, their toxins, and envelope viruses. For instance, sulfated galactosylceramide (sulfatide), but not galactosylceramide or ganglioside GM1, reportedly functions as the binding site for the envelope glycoprotein gp120 of the human being immunodeficiency computer virus, HIV-1, in cells lacking the CD4 receptor (3). It has also been suggested that the simple monohexosyl sphingolipid glucosylceramide has mitogenic properties Sarcosine that stimulate cell growth, differentiation, and DNA synthesis (4). Moreover, the tendency of GSLs to organize into lateral membrane domains is usually thought to be a key feature, not only in their own intracellular sorting and trafficking but also in the sorting and trafficking of proteins, such as glycosylphosphatidylinositol (GPI)-anchored proteins (5, 6). Given their important roles in various cellular processes, it is clear that this transport and expression of glycolipids within cells must be effectively coordinated and controlled. Glycolipid transfer proteins (GLTPs) have been identified in a wide variety of cell and tissue types, including mammalian brain, liver, kidney, and spleen, as well as in spinach chloroplasts (for review, see refs 7 and 8). These proteins catalyze the in vitro transfer of glycosphingolipids and glycoglycerolipids between donor and acceptor membranes. GLTPs appear to be cytosolic and transfer any glycolipid with a -glucosyl or -galactosyl sugar attached to a hydrophobic ceramide or diglyceride backbone (9). Two other classes of soluble proteins with glycolipid intermembrane transfer activity have been described: (1) glycosidase activator proteins, and (2) nonspecific lipid transfer proteins. Glycosidase activator proteins are lysosomal, and their main function is usually to serve as nonenzymatic cofactors required for the degradation of glycosphingolipids by the acidic glycosidases (10). In the absence of the degrading enzymes, certain activator proteins display in vitro glycolipid transfer activity (11). As a result, secreted forms of certain activator proteins have been proposed to serve as intercellular transporters of glycosphingolipids. A second class of soluble proteins with glycolipid transfer activity is the nonspecific lipid transfer proteins (nsLTPs). Bloj and Zilversmit (12) reported that different neutral glycosphingolipids as well as ganglioside GM1 were transferred by bovine liver nsLTP. Indeed, several nsLTPs identified in both animal and plant sources have been shown to catalyze the in vitro transfer of a wide range of lipids, including glycolipids (13). GLTPs have been purified to apparent homogeneity from porcine and bovine brain, and characterization reveals many shared properties (14, 15). Like porcine brain GLTP, the bovine brain GLTP used in the present study is specific for various glycolipids including neutral glycosphingolipids and gangliosides, but does not stimulate phospholipid or neutral lipid intermembrane transfer (16, 17). Sequencing of the porcine GLTP via Edman degradation revealed 208 amino acids and 1 disulfide bond (18, 19). The bovine GLTP is usually of comparable size with a molecular mass of 23-24 kDa and an isoelectric point near pH 9.0 (15). Several characteristics of bovine and porcine brain GLTPs suggest that these proteins are different from other known lipid transfer proteins. Nearly all of the lipid transfer proteins that show specificity for phosphatidylinositol and/or phosphatidylcholine have molecular masses between 25 and 35 kDa, and the isoelectric points are between 4.6 and 5.9. Most of the nonspecific lipid transfer proteins, on the other hand, have basic isoelectric points, but their molecular masses are in the range of.[PubMed] [Google Scholar] 18. pH, the transfer protein (p? 9.0) is expected to be positively charged, which may promote association with the negatively charged donor membrane. Based on these and other experiments, we conclude that this transfer process follows first-order kinetics and that the off-rate of the transfer protein from the donor vesicle surface is the rate-limiting step in the transfer process. Glycosphingolipids (GSLs)1 are amphipathic molecules that together with phospholipids and cholesterol constitute the basic lipid core structure of biomembranes. Except for their presence at relatively high amounts in the plasma membranes of neural tissues and in the apical membranes of epithelial cells (about 25-30% of total lipids in both membrane types), GSLs are usually minor components in plasma membranes of eukaryotic cells (about 5%) (1, 2). The prevailing view has been that newly Sarcosine synthesized GSLs are localized predominantly in the outer leaflet of the eukaryotic plasma membrane. This location is consistent with their roles as cell surface markers and as modulators of membrane protein function. Also, certain GSLs function as the surface binding sites for certain bacteria, their toxins, and envelope viruses. For instance, sulfated galactosylceramide (sulfatide), but not galactosylceramide or ganglioside GM1, reportedly functions as the binding site for the envelope glycoprotein gp120 of the human immunodeficiency virus, HIV-1, in cells lacking the CD4 receptor (3). It has also been suggested that the simple monohexosyl sphingolipid glucosylceramide has mitogenic properties that stimulate cell growth, differentiation, and DNA synthesis (4). Moreover, the tendency of GSLs to organize into lateral membrane domains is usually thought to be a key feature, not only in their own intracellular sorting and trafficking but also in the sorting and trafficking of proteins, such as glycosylphosphatidylinositol (GPI)-anchored proteins (5, 6). Given their important roles in various cellular processes, it is clear that this transport and expression of glycolipids within cells must be effectively coordinated and controlled. Glycolipid transfer proteins (GLTPs) have already been determined in a multitude of cell and cells types, including mammalian mind, liver organ, kidney, and spleen, aswell as with spinach chloroplasts (for review, discover refs 7 and 8). These protein catalyze the in vitro transfer of glycosphingolipids and glycoglycerolipids between donor and acceptor membranes. GLTPs look like cytosolic and transfer any glycolipid having a -glucosyl or -galactosyl sugars mounted on a hydrophobic ceramide or diglyceride backbone (9). Two additional classes of soluble protein with glycolipid intermembrane transfer activity have already been referred to: (1) glycosidase activator protein, and (2) non-specific lipid transfer protein. Glycosidase activator protein are lysosomal, and their primary function can be to serve as non-enzymatic cofactors necessary for the degradation of glycosphingolipids from the acidic glycosidases (10). In the lack of the degrading enzymes, particular activator proteins screen in vitro glycolipid transfer activity (11). Because of this, secreted types of particular activator protein have already been suggested to serve as intercellular transporters of glycosphingolipids. Another course of soluble protein with glycolipid transfer activity may be the non-specific lipid transfer protein (nsLTPs). Bloj and Zilversmit (12) reported that different natural glycosphingolipids aswell as ganglioside GM1 had been moved by bovine liver organ nsLTP. Indeed, many nsLTPs determined in both pet and plant resources have already been proven to catalyze the in vitro transfer of an array of lipids, including glycolipids (13). GLTPs have already been purified.Phys. adverse charge in the donor vesicle. Raising the ionic strength from the buffer with NaCl reversed the charge results significantly. At natural pH, the transfer proteins (p? 9.0) is expected to end up being charged positively, which might promote association using the negatively charged donor membrane. Predicated on these and additional tests, we conclude how the transfer process comes after first-order kinetics which the off-rate from the transfer proteins through the donor vesicle surface area may be the rate-limiting part of the transfer procedure. Glycosphingolipids (GSLs)1 Sarcosine are amphipathic substances that as well as phospholipids and cholesterol constitute the essential lipid core framework of biomembranes. Aside from their existence at fairly high quantities in the plasma membranes of neural cells and in the apical membranes of epithelial cells (about 25-30% of total lipids in both membrane types), GSLs are often minor parts in plasma membranes of eukaryotic cells (about 5%) (1, 2). The prevailing look at continues to be that recently synthesized GSLs are localized mainly in the external leaflet from the eukaryotic plasma membrane. This area is in keeping with their tasks as cell surface area markers so that as modulators of membrane proteins function. Also, particular GSLs function as surface area binding sites for several bacteria, their poisons, and envelope infections. For example, sulfated galactosylceramide (sulfatide), however, not galactosylceramide or ganglioside GM1, apparently features as the binding site for the envelope glycoprotein gp120 from the human being immunodeficiency disease, HIV-1, in cells missing the Compact disc4 receptor (3). It has additionally been recommended that the easy monohexosyl sphingolipid glucosylceramide offers mitogenic properties that promote cell development, differentiation, and DNA synthesis (4). Furthermore, the inclination of GSLs to arrange into lateral membrane domains can be regarded as an integral feature, not merely within their personal intracellular sorting and trafficking however in the sorting and trafficking of protein also, such as for example glycosylphosphatidylinositol (GPI)-anchored protein (5, 6). Provided their important tasks in various mobile processes, it really is clear how the transport and manifestation of glycolipids within cells should be efficiently coordinated and managed. Glycolipid transfer protein (GLTPs) have already been determined in a multitude of cell and cells types, including mammalian mind, liver organ, kidney, and spleen, aswell as with spinach chloroplasts (for review, discover refs 7 and 8). These protein catalyze the in vitro transfer of glycosphingolipids and glycoglycerolipids between donor and acceptor membranes. GLTPs look like cytosolic and transfer any glycolipid having a -glucosyl or -galactosyl sugars mounted on a hydrophobic ceramide or diglyceride backbone (9). Two additional classes of soluble protein with glycolipid intermembrane transfer activity have already been referred to: (1) glycosidase activator protein, and (2) non-specific lipid transfer protein. Glycosidase activator protein are lysosomal, and their primary function can be to serve as non-enzymatic cofactors necessary for the degradation of glycosphingolipids from the acidic glycosidases (10). In the lack of the degrading enzymes, particular activator proteins screen in vitro glycolipid transfer activity (11). Because of this, secreted types of particular activator protein have already been suggested to serve as intercellular transporters of glycosphingolipids. Another course of soluble protein with glycolipid transfer activity may be the non-specific lipid transfer protein (nsLTPs). Bloj and Zilversmit (12) reported that different natural glycosphingolipids aswell as ganglioside GM1 had been moved by bovine liver organ nsLTP. Indeed, several nsLTPs recognized in both animal HEY1 and plant sources have been shown to catalyze the in vitro transfer of a wide range of lipids, including glycolipids (13). GLTPs have been purified to apparent homogeneity from porcine and bovine mind, and characterization reveals many shared properties (14, 15). Like porcine mind GLTP, the bovine mind GLTP used in the present study is specific for numerous glycolipids including neutral glycosphingolipids and gangliosides, but does not stimulate phospholipid or neutral lipid intermembrane transfer (16, 17). Sequencing of the porcine GLTP via Edman degradation exposed 208 amino acids and 1 disulfide relationship (18, 19). The bovine GLTP is definitely of related size Sarcosine having a molecular mass of 23-24 kDa and an isoelectric point near pH 9.0 (15). Several characteristics of bovine and porcine mind GLTPs suggest that these proteins are different from additional known lipid transfer proteins. Nearly all of the lipid transfer proteins that display specificity for phosphatidylinositol and/or phosphatidylcholine have molecular people between 25 and 35.Phys. NaCl significantly reversed the charge effects. At neutral pH, the transfer protein (p? 9.0) is expected to be positively charged, which may promote association with the negatively charged donor membrane. Based on these and additional experiments, we conclude the transfer process follows first-order kinetics and that the off-rate of the transfer protein from your donor vesicle surface is the rate-limiting step in the transfer process. Glycosphingolipids (GSLs)1 are amphipathic molecules that together with phospholipids and cholesterol constitute the basic lipid core structure of biomembranes. Except for their presence at relatively high amounts in the plasma membranes of neural cells and in the apical membranes of epithelial cells (about 25-30% of total lipids in both membrane types), GSLs are usually minor parts in plasma membranes of eukaryotic cells (about 5%) (1, 2). The prevailing look at has been that newly synthesized GSLs are localized mainly in the outer leaflet of the eukaryotic plasma membrane. This location Sarcosine is consistent with their functions as cell surface markers and as modulators of membrane protein function. Also, particular GSLs function as the surface binding sites for certain bacteria, their toxins, and envelope viruses. For instance, sulfated galactosylceramide (sulfatide), but not galactosylceramide or ganglioside GM1, reportedly functions as the binding site for the envelope glycoprotein gp120 of the human being immunodeficiency computer virus, HIV-1, in cells lacking the CD4 receptor (3). It has also been suggested that the simple monohexosyl sphingolipid glucosylceramide offers mitogenic properties that activate cell growth, differentiation, and DNA synthesis (4). Moreover, the inclination of GSLs to organize into lateral membrane domains is definitely thought to be a key feature, not only in their personal intracellular sorting and trafficking but also in the sorting and trafficking of proteins, such as glycosylphosphatidylinositol (GPI)-anchored proteins (5, 6). Given their important functions in various cellular processes, it is clear the transport and manifestation of glycolipids within cells must be efficiently coordinated and controlled. Glycolipid transfer proteins (GLTPs) have been recognized in a wide variety of cell and cells types, including mammalian mind, liver, kidney, and spleen, as well as with spinach chloroplasts (for review, observe refs 7 and 8). These proteins catalyze the in vitro transfer of glycosphingolipids and glycoglycerolipids between donor and acceptor membranes. GLTPs look like cytosolic and transfer any glycolipid having a -glucosyl or -galactosyl sugars attached to a hydrophobic ceramide or diglyceride backbone (9). Two additional classes of soluble proteins with glycolipid intermembrane transfer activity have been explained: (1) glycosidase activator proteins, and (2) nonspecific lipid transfer proteins. Glycosidase activator proteins are lysosomal, and their main function is definitely to serve as nonenzymatic cofactors required for the degradation of glycosphingolipids from the acidic glycosidases (10). In the absence of the degrading enzymes, particular activator proteins display in vitro glycolipid transfer activity (11). As a result, secreted forms of particular activator proteins have been proposed to serve as intercellular transporters of glycosphingolipids. A second class of soluble proteins with glycolipid transfer activity is the nonspecific lipid transfer proteins (nsLTPs). Bloj and Zilversmit (12) reported that different neutral glycosphingolipids as well as ganglioside GM1 were transferred by bovine liver nsLTP. Indeed, several nsLTPs recognized in both animal and plant resources have already been proven to catalyze the in vitro transfer of an array of lipids, including glycolipids (13). GLTPs have already been purified to obvious homogeneity from porcine and bovine human brain, and characterization reveals many distributed properties (14, 15). Like porcine human brain GLTP, the bovine human brain GLTP found in the present research is particular for different glycolipids including natural glycosphingolipids and gangliosides, but will not stimulate phospholipid.