guanosine-diphosphate and Leukemia--Myeloid

Membranes of myeloid differentiated human leukemia (HL 60) cells contain receptors for the chemotactic peptide, fMet-Leu-Phe (fMet, N-formylmethionine), interacting with pertussis-toxin-sensitive guanine-nucleotide-binding proteins (G proteins). Agonist activation of the receptors increases binding of the GTP analog, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), to membrane G proteins, at 30 degrees C only in the presence of exogenous GDP. In contrast, at 0 degrees C fMet-Leu-Phe stimulated binding of GTP[S] to G proteins maximally without addition of GDP. Under conditions resulting in marked degradation of membrane-bound GDP, control binding of GTP[S] measured at 0 degrees C was significantly increased, whereas the extent of agonist-stimulated binding was reduced. Furthermore, there was a rapid spontaneous release of membrane-bound GDP at 30 degrees C, but not at 0 degrees C. The data suggest that in intact membranes of HL 60 cells G proteins are initially in a GDP-liganded form, which state allows the receptor-induced exchange of bound GDP for GTP[S] at low temperature. In contrast, at or near physiological temperature, bound GDP is rapidly released (and degraded), resulting in unligated G proteins to which GTP[S] will bind independently of agonist-activated receptors.

Topics: Alkaline Phosphatase; Cell Membrane; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Leukemia, Myeloid; N-Formylmethionine Leucyl-Phenylalanine; Receptors, Formyl Peptide; Receptors, Immunologic; Temperature; Tumor Cells, Cultured

High-affinity agonist binding to formyl peptide receptors in membranes of myeloid differentiated human leukemia (HL 60) cells is known to be regulated by guanine nucleotides, most potently by the GTP analog, guanosine-5'-O-(3-thiotriphosphate) (GTP[S]). Here we analyzed whether nucleoside diphosphokinase present in these membranes and capable of forming GTP[S] from GDP and adenosine-5'-O-(3-thiotriphosphate) (ATP[S]) can contribute to nucleotide regulation of agonist receptor binding. Using GDP and ATP[S] at concentrations causing by themselves only small reductions in receptor binding of the labelled formyl peptide, N-formyl-methionyl-leucyl-phenylalanine ([3H]FMLP), a marked potentiation (up to 30-fold) was observed when both nucleotides were combined. Under conditions in which the combination of GDP and ATP[S] induced 70-90% of maximal inhibition of [3H]FMLP binding, a total concentration of about 7 nM GTP[S] formed was measured. The synergistic effect of GDP and ATP[S] on [3H]FMLP binding was not seen in the presence of UDP (1 mM), which blocked formation of GTP[S] from GDP and ATP[S]. Furthermore, no potentiation was observed when instead of GDP and ATP[S], guanosine-5'-O-(2-thiodiphosphate) and adenylyl-5'-imidodiphosphate, respectively, were used. Finally, regulation of [3H]FMLP binding by ATP[S] plus GDP (or GTP) was a time-dependent process, reaching maximal inhibition after 20-30 min of incubation at 25 degrees C. The data indicate that nucleoside diphosphokinase present in membranes of HL 60 cells can transfer the thiophosphate group of ATP[S] to GDP leading to formation of GTP[S] and that the GTP[S] thus formed efficiently binds to G proteins interacting with formyl peptide receptors and thereby regulates their agonist binding affinity.

Topics: Adenosine Triphosphate; Binding Sites; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Leukemia, Myeloid; N-Formylmethionine Leucyl-Phenylalanine; Nucleoside-Diphosphate Kinase; Receptors, Formyl Peptide; Receptors, Immunologic; Tumor Cells, Cultured; Uridine Diphosphate

Myeloid-differentiated HL-60 cells were used to study the activation of G-proteins by receptor agonists. Following incubation of membranes with the photoreactive GTP analog. [alpha-32P]GTP azidoanilide, and subsequent exposure to ultraviolet light (254 nm), photolabeling of 40 kDa proteins comigrating with the Gi2 alpha-subunit was observed. Photolabeling in the absence or presence of the chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (FMLP), absolutely required Mg2+; FMLP stimulated photolabeling at all Mg2+ concentrations employed (up to 30 mM). Addition of GDP (3-50 microM) reduced basal photolabeling to a greater extent than photolabeling stimulated by FMLP. FMLP did not stimulate photolabeling of proteins modified by pertussis toxin. Leukotriene B4 and C5a also stimulated photolabeling of 40 kDa proteins. The results indicate that (i) the major G-protein in HL-60 cells, Gi2, requires Mg2+ for basal and receptor-stimulated activity, (ii) effective receptor-mediated activation of G-proteins is observed at mM concentrations of Mg2+, and (iii) receptor agonists apparently reduce the affinity of G-proteins for GDP.

Topics: Affinity Labels; Binding Sites; Complement C5a; Drug Synergism; GTP-Binding Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Leukemia, Myeloid; Leukotriene B4; Magnesium; Membrane Proteins; N-Formylmethionine Leucyl-Phenylalanine; Photic Stimulation; Receptors, Cell Surface; Ultraviolet Rays

Myeloid differentiated human leukaemia (HL-60) cells contain a soluble phospholipase C that hydrolysed phosphatidylinositol 4.5-bisphosphate and was markedly stimulated by the metabolically stable GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]). Half-maximal and maximal (up to 5-fold) stimulation of inositol phosphate formation by GTP[S] occurred at 1.5 microM and 30 microM respectively. Other nucleotides (GTP, GDP, GMP, guanosine 5'-[beta-thio]diphosphate. ATP, adenosine 5'-[gamma-thio]triphosphate, UTP) did not affect phospholipase C activity, GTP[S] stimulation of inositol phosphate accumulation was inhibited by excess GDP, but not by ADP. The effect of GTP[S] on inositol phosphate formation was absolutely dependent on and markedly stimulated by free Ca2+ (median effective concn. approximately 100 nM). Analysis of inositol phosphates by anion-exchange chromatography revealed InsP3 as the major product of GTP[S]-stimulated phospholipase C activity. In the absence of GTP[S], specific phospholipase C activity was markedly decreased when tested at high protein concentrations, whereas GTP[S] stimulation of the enzyme was markedly enhanced under these conditions. As both basal and GTP[S]-stimulated inositol phosphate formation were linear with time whether studied at low or high protein concentration, these results suggest that (a) phospholipase C is under an inhibitory constraint and (b) GTP[S] relieves this inhibition, most likely by activating a soluble GTP-binding protein.

Topics: Cytosol; Deoxycholic Acid; Enzyme Activation; Granulocytes; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Humans; Hydrogen-Ion Concentration; Hydrolysis; Inositol Phosphates; Kinetics; Leukemia, Experimental; Leukemia, Myeloid; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phospholipids; Stimulation, Chemical; Tumor Cells, Cultured; Type C Phospholipases

Fucosyltransferase activity was measured in plasma samples obtained from patients with chronic granulocytic leukemia in different disease stages. Total enzyme activity was generally elevated in untreated and in stable disease; a marked further elevation was noted 30 to 60 days before development of blast crisis. Electrofocusing studies indicate the presence of three major enzymes. The level of enzyme which focused at pH 5.6 was elevated in untreated disease and rose markedly during blastic transformation. A second enzyme focused at pH 5.1 and appeared to be related to marrow erythroid turnover. A third enzyme with pI = 4.7 was elevated only during marrow regeneration which followed myelosuppressive therapy. These data suggest the enzyme with pI = 5.6 to be a specific marker in chronic granulocytic leukemia.

Topics: Fucosyltransferases; Guanosine Diphosphate; Hexosyltransferases; Humans; Hydrogen-Ion Concentration; Isoelectric Focusing; Kinetics; Leukemia, Myeloid; Time Factors