guanosine-triphosphate and inositol-1-4-bis(phosphate)

Sugar phosphates are required to maintain active rates of translation in gel-filtered rabbit reticulocyte lysates. They may stimulate polypeptide chain initiation by acting as NADPH generators or by a direct interaction with initiation factor(s). We now provide evidence for the allosteric activation of the purified guanine nucleotide exchange factor (eIF-2B) by sugar phosphates and inositol phosphates. In the presence of microM fructose 1,6-bisphosphate, the rate of eIF-2B-catalyzed GDP/GTP exchange is increased approximately 2-fold. The half-maximal concentration for stimulation of eIF-2B activity (SC50) is 57 microM. The binding of GTP to isolated eIF-2B is stimulated 1.5-fold, whereas GTP-binding to ALP-treated eIF-2B is not affected by sugar phosphates. Inositol 1,4-bisphosphate, like fructose 1,6-bisphosphate, stimulates 2-3-fold the activity of the isolated eIF-2B (SC50, 140 microM).

Topics: Allosteric Regulation; Animals; Fructosediphosphates; Guanine Nucleotide Exchange Factors; Guanosine Diphosphate; Guanosine Triphosphate; Inositol Phosphates; Proteins; Rabbits; Reticulocytes

It is well known that with aging the immune response decreases. Most of the effector functions occur through specific receptors. Thus, we investigated the effects of various stimulants, acting through receptors or directly through the GTP-binding Gi protein, on phosphatidylinositol breakdown in PMNLs of young and elderly subjects and try to modulate it. A marked decrease in inositol phosphate (IP1, IP2, IP3) formation in PMNLs of elderly was found under FMLP stimulation when compared to that of young subjects. Neither GTP gamma S, nor AIF4- could induce an increase of IP3 in PMNLs of elderly comparable to that of young subjects. The results suggest that at least an alteration exists at the GTP-binding Gi protein level, as well as in the mechanism of linkage of the receptor to the G protein.

Topics: Adult; Aged; Aging; Aluminum; Aluminum Compounds; Chloroquine; Fluorides; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; N-Formylmethionine Leucyl-Phenylalanine; Neomycin; Neutrophils; Phosphatidylinositols; Thionucleotides; Virulence Factors, Bordetella

Antigen-mediated exocytosis in intact rat basophilic leukemia (RBL-2H3) cells is associated with substantial hydrolysis of membrane inositol phospholipids and an elevation in concentration of cytosol Ca2+ ([ Ca2+i]). Paradoxically, these two responses are largely dependent on external Ca2+. We report here that cells labeled with myo-[3H]inositol and permeabilized with streptolysin O do release [3H]inositol 1,4,5-trisphosphate upon stimulation with antigen or guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) at low (less than 100 nM) concentrations of free Ca2+. The response, however, is amplified by increasing free Ca2+ to 1 microM. The subsequent conversion of the trisphosphate to inositol 1,3,4,5-tetrakisphosphate is enhanced also by the increase in free Ca2+. Although [3H]inositol 1,4,5-trisphosphate accumulates in greater amounts than is the case in intact cells, [3H]inositol 1,4-bisphosphate is still the major product in permeabilized cells even when the further metabolism of [3H]inositol 1,4,5-trisphosphate is suppressed (by 77%) by the addition of excess (1000 microM) unlabeled inositol 1,4,5-trisphosphate and the phosphatase inhibitor 2,3-bisphosphoglycerate. It would appear that either the activity of the membrane 5-phosphomonoesterase allows virtually instantaneous dephosphorylation of the inositol 1,4,5-trisphosphate under all conditions tested or both phosphatidylinositol 4-monophosphate and the 4,5-bisphosphate are substrates for the activated phospholipase C. The latter alternative is supported by the finding that permeabilized cells, which respond much more vigorously to high (supraoptimal) concentrations of antigen than do intact RBL-2H3 cells, produce substantial amounts of [3H]inositol 1,4-bisphosphate before any detectable increase in levels of [3H]inositol 1,4,5-trisphosphate.

Topics: Animals; Bacterial Proteins; Cell Line; Exocytosis; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Leukemia, Basophilic, Acute; Rats; Streptolysins; Sugar Phosphates; Thionucleotides

fMet-Leu-Phe (fMLP) stimulated the formation of inositol bis- and trisphosphate in the [3H]inositol-labeled plasma membranes from the human leukemic (HL-60) cells differentiated to neutrophil-like cells by dibutyryl cyclic AMP. The stimulatory effect of fMLP was completely dependent on the simultaneous presence of GTP and Ca2+. The fMLP-stimulated formation of the phosphorylated inositols was markedly reduced by the prior ADP-ribosylation of the membranes with pertussis toxin. This toxin ADP-ribosylated a Mr approximately 40,000 protein, presumably the alpha subunit of Gi and/or Go, in the membranes. Reconstitution of the membranes ADP-ribosylated by pertussis toxin with Gi or Go purified from rat brain restored the fMLP-stimulated formation of the phosphorylated inositols. The efficiency of the rat brain Gi and Go in this capacity was roughly equal. The rat brain Gi or Go ADP-ribosylated beforehand by pertussis toxin was inactive in this reconstitution. These results indicate that both rat brain Gi and Go have the potency to couple functionally the fMLP receptor to the phospholipase C-mediated polyphosphoinositide hydrolysis and suggest that Gi or Go may be involved in the mechanism of signal transduction from the fMLP receptor to this reaction in the differentiated HL-60 cells.

Topics: Adenosine Diphosphate Ribose; Calcium; Cell Differentiation; Cell Line; Cell Membrane; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Leukemia, Myeloid, Acute; N-Formylmethionine Leucyl-Phenylalanine; Nucleoside Diphosphate Sugars; Pertussis Toxin; Sugar Phosphates; Virulence Factors, Bordetella