In Escherichia coli, expression of the glyoxylate bypass operon appears to be controlled, in part, by the product of icIR+. Mutations (17,19). This bypass is essential for growth on acetate, since it yields C4 acids while avoiding the net loss of the acetate carbons as carbon dioxide in the Krebs cycle (Fig. 1). After induction, the flow of isocitrate through the glyoxylate bypass is regulated, in part, by the phosphorylation of isocitrate dehydrogenase (IDH), the Krebs cycle enzyme that competes with isocitrate lyase (8,12,24). During growth on acetate, ca. 70% of the IDH is maintained in the inactive phosphorylated form (22,23,32), reducing the activity of this enzyme and so forcing isocitrate through the bypass (24, 32). The phosphorylation and dephosphorylation of IDH are catalyzed by a single bifunctional enzyme, IDH kinase/phosphatase (20, 21).The metabolic and regulatory proteins of the glyoxylate bypass reside in the same operon, which maps at 91 min on the E. coli chromosome (4,5,23,26 When required, ampicillin (200 ,ug/ml) tetracycline (12.5 ,ug/ml), or kanamycin (50 ,ug/ml) was included in the growth media.Measurement of enzymatic activities. Cultures were grown at 37°C in a gyratory incubator to mid-log phase and were then harvested by centrifugation at 4,000 x g for 10 min. The cells were suspended in 10 ml of extraction buffer (25 mM N-morpholinepropanesulfonate [pH 7.5], 2 mM ,-mercaptoethanol, 1 mM EDTA) and then pelleted again by centrifugation. Cell pellets were stored at -80°C. For assay, the samples were thawed, suspended in 5 ml of extraction buffer, and disrupted by sonication. Cellular debris was removed by centrifuged at 22,000 x g for 20 min, and the samples were assayed for IDH phosphatase activity. Samples derived from cultures harboring plasmid were also assayed for ,-lactamase activity to ensure that the plasmid had not been lost during growth.The activity of IDH phosphatase was measured by monitoring the release of [32P]phosphate from [32P]phospho-IDH,

In serum-starved NIH 3T3 clone 7 fibroblasts, choline phosphate (ChoP) (0.5-1 mM) and insulin synergistically stimulate DNA synthesis. Here we report that ATP also greatly enhanced the mitogenic effects of ChoP (0.1-1 mM) both in the absence and presence of insulin; maximal potentiating effects required 50 -100 M ATP. The co-mitogenic effects of ATP were mimicked by adenosine 5-O-(3-thiotriphosphate), adenosine 5-O-(2-thiodiphosphate), ADP, and UTP, but not by AMP or adenosine, indicating the mediatory role of a purinergic P 2 receptor. Externally added ChoP acted on DNA synthesis without its detectable uptake into fibroblasts, indicating that ChoP can be a mitogen only if it is released from cells. Extracellular ATP (10 -100 M) induced extensive release of ChoP from fibroblasts. ChoP had negligible effects, even in the presence of ATP or insulin, on the activity state of p42/p44 mitogen-activated protein kinases, while in combination these agents stimulated the activity of phosphatidylinositol 3-kinase (PI 3-kinase). Expression of a dominant negative mutant of the p85 subunit of PI 3-kinase or treatments with the PI 3-kinase inhibitor wortmannin only partially (ϳ40 -50%) reduced the combined effects of ChoP, ATP, and insulin on DNA synthesis; in contrast, the pp70 S6 kinase inhibitor rapamycin almost completely inhibited these effects. ATP and insulin also potentiated, while rapamycin strongly inhibited, the mitogenic effects of sphingosine 1-phosphate (S1P). Furthermore, even maximally effective concentrations of ChoP and S1P synergistically stimulated DNA synthesis. The results indicate that in the presence of extracellular ATP and/or S1P, ChoP induces mitogenesis through an extracellular site by mechanisms involving the activation of pp70 S6 kinase and, to a lesser extent, PI 3-kinase.

Glycosphingolipids (GSLs) are important constituents of lipid rafts and caveolae, are essential for the normal development of cells, and are adhesion sites for various infectious agents. One strategy for modulating GSL composition in lipid rafts is to selectively transfer GSL to or from these putative membrane microdomains. Glycolipid transfer protein (GLTP) catalyzes selective intermembrane transfer of GSLs. To enable effective use of GLTP as a tool to modify the glycolipid content of membranes, it is imperative to understand how the membrane regulates GLTP action. In this study, GLTP partitioning to membranes was analyzed by monitoring the fluorescence resonance energy transfer from tryptophans and tyrosines of GLTP to N-(5-dimethyl-aminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phospho-ethanolamine present in bilayer vesicles. GLTP partitioned to POPC vesicles even when no GSL was present. GLTP interaction with model membranes was nonpenetrating, as assessed by protein-induced changes in lipid monolayer surface pressure, and nonperturbing in that neither membrane fluidity nor order were affected, as monitored by anisotropy of 1,6-diphenyl-1,3,5-hexatriene and 6-dodecanoyl-N,N-dimethyl-2-naphthylamine, even though the tryptophan anisotropy of GLTP increased in the presence of vesicles. Ionic strength, vesicle packing, and vesicle lipid composition affected GLTP partitioning to the membrane and led to the following conclusion: Conditions that increase the ratio of bound/unbound GLTP do not guarantee increased transfer activity, but conditions that decrease the ratio of bound/unbound GLTP always diminish transfer. A model of GLTP interaction with the membrane, based on the partitioning equilibrium data and consistent with the kinetics of GSL transfer, is presented and solved mathematically.

Glycolipid transfer protein (GLTP) is a soluble 24 kDa protein that selectively accelerates the intermembrane transfer of glycolipids in vitro. Little is known about the GLTP structure and dynamics. Here, we report the cloning of human GLTP and characterize the environment of the three tryptophans (Trps) of the protein using fluorescence spectroscopy. Excitation at 295 nm yielded an emission maximum (lambda(max)) near 347 nm, indicating a relatively polar average environment for emitting Trps. Quenching with acrylamide at physiological ionic strength or with potassium iodide resulted in linear Stern-Volmer plots, suggesting accessibility of emitting Trps to soluble quenchers. Insights into reversible conformational changes accompanying changes in GLTP activity were provided by addition and rapid dilution of urea while monitoring changes in Trp or 1-anilinonaphthalene-8-sulfonic acid fluorescence. Incubation of GLTP with glycolipid liposomes caused a blue shift in the Trp emission maximum but diminished the fluorescence intensity. The blue-shifted emission maximum, centered near 335 nm, persisted after separation of glycolipid liposomes from GLTP, consistent with formation of a GLTP-glycolipid complex at a glycolipid-liganding site containing Trp. The results provide the first insights into human GLTP structural dynamics by fluorescence spectroscopy, including global conformational changes that accompany GLTP folding into an active conformational state as well as more subtle conformational changes that play a role in GLTP-mediated transfer of glycolipids between membranes, and establish a foundation for future studies of membrane rafts using GLTP.