Bacterially synthesized c-Ha-ras protein (Ras) was incubated with guanosine triphosphatase (GTPase) activating (GA) protein in the presence of various phospholipids. The stimulation of Ras GTPase activity by GA protein was inhibited in some cases. Among the lipids most active in blocking GA protein activity were lipids that show altered metabolism during mitogenic stimulation. These included phosphatidic acid (containing arachidonic acid), phosphatidylinositol phosphates, and arachidonic acid. Other lipids, including phosphatidic acid with long, saturated side chains, diacylglycerols, and many other common phospholipids, were unable to alter GA protein activity. The interaction of lipids with GA protein might be important in the regulation of Ras activity during mitogenic stimulation.
We analyzed carbohydrate chains of human, bovine, sheep, and rat alpha1-acid glycoprotein (AGP) and found that carbohydrate chains of AGP of different animals showed quite distinct variations. Human AGP is a highly negatively charged acidic glycoprotein (pKa = 2.6; isoelectic point = 2.7) with a molecular weight of approximately 37,000 when examined by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and contains di-, tri-, and tetraantennary carbohydrate chains. Some of the tri- and tetraantennary carbohydrate chains are substituted with a fucose residue (sialyl Lewis x type structure). In sheep AGP, mono- and disialo-diantennary carbohydrate chains were abundant. Tri- and tetrasialo-triantennary carbohydrate chains were also present as minor oligosaccharides, and some of the sialic acid residues were substituted with N-glycolylneuraminic acid. In rat AGP, very complex mixtures of disialo-carbohydrate chains were observed. Complexity of the disialo-oligosaccharides was due to the presence of N, O-acetylneuraminic acids. Triantennary carbohydrate chains carrying N,O-acetylneuraminic acid were also observed as minor component oligosaccharides. We found some novel carbohydrate chains containing both N-acetylneuraminic acid and N-glycolylneuraminic acid in bovine AGP. Interestingly, triantennary carbohydrate chains were hardly detected in bovine AGP, but diantennary carbohydrate chains with tri- or tetrasialyl residues were abundant. Furthermore the major sialic acid in these carbohydrate chains was N-glycolylneuraminic acid. It should be noted that these sialic acids are attached to multiple sites of the core oligosaccharide and are not present as disialyl groups.
A cytoplasmic protein has been identified that inhibits the guanosine triphosphatase (GTPase) activity of bacterially synthesized, cellular H-Ras protein. This GTPase inhibiting protein is able to counteract the activity of GTPase activating protein (GAP), which has been postulated to function as a negative regulator of Ras activity. The potential biological importance of the GTPase inhibiting protein is further supported by its interaction with lipids. Phospholipids produced in cells as a consequence of mitogenic stimulation increase the activity of the GTPase inhibiting protein, as well as inhibit the activity of GAP. The interaction of such lipids with each of these two regulatory proteins would, therefore, tend to increase the biological activity of Ras and stimulate cell proliferation.
Certain lipids were found to inhibit the interaction between rho and R-ras proteins and their respective GTPase-activating proteins (GAP). Inhibitory lipids were similar for each protein but differed significantly from those previously found to inhibit the interaction between ras protein and GAP activity. These data raise the possibility that ras and related proteins are controlled biologically by interactions between lipids and GAP molecules.R-ras and rho are related to the proto-oncogene ras by sequence homology as well as by their ability to bind and hydrolyze GTP (11,12). Ras is part of a signal transduction mechanism leading to cellular proliferation (14), but no function is known for either R-ras or rho. In contrast to control of classical G proteins involved in signal transduction (8), the biological and biochemical control of ras and ras-related proteins is poorly understood. A GTPase-activating protein (GAP) which is a potential negative regulator of ras activity has been characterized (16), but because ras-GAP remains active in proliferating cells its potential role in the control of ras (and therefore cellular proliferation) is unclear (10, 16). Recently, however, certain lipids whose metabolism is known to be altered in mitogenically stimulated cells were shown to disrupt the effect of ras-GAP upon purified ras protein (17). It is now clear that ras-GAP interacts with R-ras, whereas the rho protein interacts with a distinct smaller molecule, rho-GAP (6). In this study, a similar but nonidentical group of lipids is shown to disrupt the interaction between R-ras and GAP and between rho and rho-GAP. The biological significance of these observations is not yet clear, but the data raise the possibility that lipids might normally function to control the activity of ras-related proteins through interactions with GAP molecules.The ras-related proteins R-ras and rho were purified from a bacterial expression system (6), and their GTPase activities were assayed by measuring the loss of radiolabeled _y32p from protein-bound GTP. The GTPase activities were relatively slow for rho and R-ras proteins in the absence of added GAP activity as previously observed for ras protein.Addition of GAP activity as a crude cytoplasmic extract increased the GTPase rate severalfold (Fig. 1). The presence of a 29-kilodalton protein with GAP activity specific for rho has been demonstrated in such extracts, along with the 125-kilodalton ras-GAP activity (6, 7). Previous studies indicate that the ability of ras-GAP to stimulate GTPase activity of ras protein was inhibited by certain lipids (17). We were anxious to test the possibility that lipids might also inhibit the GAP activity associated with other ras-related proteins. For this analysis, a variety of lipids were coincubated with rho protein and GAP-containing cell lysates. Certain of the lipids tested were able to inhibit the ability of rho-GAP to stimulate GTPase activity of rho protein (Table 1). In particular, 60 ,ug of added phosphatidic acid per ml * Corresponding author....
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