Adenyl Imidodiphosphate Research Articles

Crystal Structures of the Lyn Protein Tyrosine Kinase Domain in Its Apo- and Inhibitor-bound State

The Src-family protein-tyrosine kinase (PTK) Lyn is the most important Src-family kinase in B cells, having both inhibitory and stimulatory activity that is dependent on the receptor, ligand, and developmental context of the B cell. An important role for Lyn has been reported in acute myeloid leukemia and chronic myeloid leukemia, as well as certain solid tumors. Although several Src-family inhibitors are available, the development of Lyn-specific inhibitors, or inhibitors with reduced off-target activity to Lyn, has been hampered by the lack of structural data on the Lyn kinase. Here we report the crystal structure of the non-liganded form of Lyn kinase domain, as well as in complex with three different inhibitors: the ATP analogue AMP-PNP; the pan Src kinase inhibitor PP2; and the BCR-Abl/Src-family inhibitor Dasatinib. The Lyn kinase domain was determined in its "active" conformation, but in the unphosphorylated state. All three inhibitors are bound at the ATP-binding site, with PP2 and Dasatinib extending into a hydrophobic pocket deep in the substrate cleft, thereby providing a basis for the Src-specific inhibition. Analysis of sequence and structural differences around the active site region of the Src-family PTKs were evident. Accordingly, our data provide valuable information for the further development of therapeutics targeting Lyn and the important Src-family of kinases.

The interaction of fluorescein isothiocyanate with the ryanodine receptor/Ca2+ release channel of sarcoplasmic reticulum.

Fluorescein 5'-isothiocyanate (FITC) markedly inhibited ryanodine binding to rabbit skeletal muscle junctional sarcoplasmic reticulum. Half-maximal inhibition was obtained with about 20 microM and complete inhibition by 80 microM FITC. Inhibition was enhanced in the presence of high salt and at alkaline pH. The pH dependence of the inactivation of ryanodine binding suggested that FITC bound to a very reactive lysine epsilon-amino group with a pK alpha of about 7.5 or above. Kinetic analysis of the time course of inactivation of ryanodine binding by various concentrations of FITC suggested that the inactivation resulted from the modification of 2 or more amino acid residues. The inhibition of ryanodine binding by FITC was partially prevented by ATP, ADP, adenyl imidodiphosphate, and 3-O-(benzoyl)-benzoyl-ATP (Bz-ATP) but not by AMP. FITC modification of sarcoplasmic reticulum membranes inhibited the photoaffinity labeling by [alpha-32P]Bz-ATP of the 450-kDa protein and the ryanodine receptor with half-maximal inhibition at about 100 microM. The results suggest that the inhibition of ryanodine binding is mainly due to FITC modification of sites which are not involved in ATP binding. The FITC moiety is bound to the 160-, 96-, 76-, and 60-kDa ryanodine receptor tryptic fragments, and the FITC site is apparently on the 21.5-, 18-, and 17-kDa fragments which are formed by the V8 protease. Covalent modification by FITC dramatically affected the activity of single Ca2+ release channels incorporated into planar lipid bilayers. FITC caused a marked increased in channel open probability mainly to a noisy approximately 60% subconductance state. FITC-modified channels were no longer affected by ryanodine but were still abolished by Mg2+ and ruthenium red. We suggest that FITC modifies reactive lysine residues involved in channel activation by transmembrane charge movement in the t-tubular system.

Subclasses of purinoceptors in feline bladder

Purines have been shown to inhibit and excite feline detrusor smooth muscle through P 1 and P 2 receptor activation. Several recent studies have demonstrated differences in agonist potency orders for subclasses of purinoceptors, including P 2Y and nucleotide, or P 2U receptors. The current studies were performed to determine the presence of such receptor subtypes in feline detrusor smooth muscle. Cats were surgically prepared for monitoring detrusor smooth muscle contractions as increases in intravesical pressure. Contractions were induced by pelvic nerves stimulation (PNS), ATP, and ATP analogs, such as β, γ-methylene ATP (APPCP), 5′ adenylimido diphosphate (AMP-PNP) and 2-metthylthio (2-MeSATP), ATPγS, UTP, CTP and GTP. These agents all produced contractions and had an agonist potency order of AMP-PNP = APPCP > ATP γS = 2-MeSATP ⪢ ATP > UTP = CTP = GTP. The agonist potency order for inhibition of PNS nerve-evoked bladder contractions was APPCP = AMP-PNP = ATP γS > 2-MeSATP = ATP > UTP = CTP = GTP. Reactive Blue 2 and Coomassie's Brilliant Blue G , two putative P 2Y receptor antagonists, antagonized purine-induced actions. This antagonism and the agonist potency orders suggest the possible presence of novel receptors in detrusor smooth muscle and/or the presence of multiple receptors in detrusor smooth muscle.

P2-purinoceptor induced prostaglandin synthesis in primary rat astrocyte cultures.

Adenosine triphosphate (ATP) is one of the cotransmitters that are commonly released at catecholaminergic and cholinergic nerve terminals. The glial cell type most closely associated with the synapse is the astrocyte and, thus, is the next cellular element beside the postsynaptic neuron to face the transmitters released. This report gives evidence of P2-purinoceptors on cultured astroglial cells. Upon stimulation with nucleoside triphosphates and nucleoside diphosphates, the cells respond with synthesis of prostaglandins of the D2 type, which is the predominant prostaglandin made in rat brain. Nucleoside triphosphate analogues, such as 5'-adenyl-imido diphosphate, beta,gamma-methylene, or alpha,beta-methylene ATP were less effective than ATP or its non-hydrolysable analogue ATP [gamma S]. The receptor was desensitized by ATP [gamma S] within 15 min, whereas desensitization by alpha,beta-methylene ATP was significantly delayed. 8-phenyl-theophylline (10(-4) M) had no influence on ATP-stimulated prostaglandin synthesis. Adenosine 5'-monophosphate (AMP) and adenosine were unable to stimulate prostaglandin D2 formation. According to the common nomenclature for purinoceptors, the described astroglial receptor would fulfill the characteristics of a P2-purinoceptor. Furthermore, it is shown that pertussis toxin sensitive G-proteins influence some early step in prostaglandin synthesis. The inactivation of these proteins results in reduced prostaglandin formation. It is assumed that ATP serves as an important mediator in the cross-talk between neurons and astroglial cells at the synaptic cleft.

Localization of adenylate cyclase activity in Populus: its relation to pollen-pistil recognition and incompatibility

Adenylate cyclase has been localized cytochemically in female and male parents as well as during the pollen-stigma interaction with an original technique employing strontium as the capture ion and adenyl imidodiphosphate as the specific substrate. The specificity of the reaction was checked by using several controls. No final specific reaction product was detected in unpollinated P. deltoides stigmas or in the P. deltoides or P. alba pollen grains used for compatible and incompatible pollinations. In the compatible cross between P. deltoides × P. deltoides, fine dense precipitates were observed in the dictyosomes and the plasma membrane and exterior to the exine of hydrated pollen grains adhering to the stigma surface. Labeling of the stigmatic pellicle was also observed after pollen adhesion and hydration. This was accompanied by a strong reactivity of the cell wall and plasmalemma of the stigma papillae at the sites of pollen tube germination on the stigma surface and at the sites of penetration of pollen tubes between adjacent papillae. In the incompatible cross between P. deltoides x P. alba, adenylate cyclase activity was still present but reduced at the stigma surface following adhesion, hydration, and germination of P. alba pollen. This activity was completely abolished after the penetration of pollen tubes between stigma papillae. These findings suggest that in Populus, adenylate cyclase activity is correlated to pollen adhesion, hydration, and germination at the stigma surface, and that the abolition of this enzyme activity could be one of the cellular events governing the gametophytic phenotype of incompatibility in the cross between P. deltoides and P. alba.

Stable complexes of axoplasmic vesicles and microtubules: protein composition and ATPase activity.

Fast transport of axonal vesicles and organelles is a microtubule-associated movement (Griffin, J. W., K. E. Fahnestock, L. Price, and P. N. Hoffman, 1983, J. Neuroscience, 3:557-566; Schnapp, B. J., R. D. Vale, M. P. Sheetz, and T. S. Reese, 1984, Cell, 40:455-462; Allen, R. D., D. G. Weiss, J. H. Hayden, D. T. Brown, H. Fujiwake, and M. Simpson, 1985, J. Cell Biol., 100:1736-1752). Proteins that mediate the interactions of axoplasmic vesicles and microtubules were studied using stable complexes of microtubules and vesicles (MtVC). These complexes formed spontaneously in vitro when taxol-stabilized microtubules were mixed with sonically disrupted axoplasm from the giant axon of the squid Loligo pealei. The isolated MtVCs contain a distinct subset of axoplasmic proteins, and are composed primarily of microtubules and attached membranous vesicles. The MtVC also contains nonmitochondrial ATPase activity. The binding of one high molecular mass polypeptide to the complex is significantly enhanced by ATP or adenyl imidodiphosphate. All of the axoplasmic proteins and ATPase activity that bind to microtubules are found in macromolecular complexes and appear to be vesicle-associated. These data allow the identification of several vesicle-associated proteins of the squid giant axon and suggest that one or more of these polypeptides mediates vesicle binding to microtubules.

Kinetics of ATP Synthesis in Pea Cotyledon Submitochondrial Particles

A kinetic study of oxidative phosphorylation by pea submitochondrial particles gave two K(m) values for ADP, one low, the other high. The high value probably reflected a damaged site or a population of leaky mitochondria. Only the high affinity site with a low K(m) for ADP was involved in ATP synthesis. alpha,beta-Methylene ADP was found to be a competitive inhibitor of ATP synthesis. The inorganic phosphate analog, thiophosphate, decreased the apparent K(m) of ADP while the rate of the reaction remained approximately the same. Adenyl imidodiphosphate, a specific inhibitor of ATP hydrolysis activity, had little effect on oxidative phosphorylation. A slight decrease in the K(m) of the high affinity binding site for ADP was noted. Aurovertin was found to be a potent inhibitor of oxidative phosphorylation in pea submitochondrial particles. The K(m) of the high affinity site was increased 10-fold. Also, the inhibition normally exerted by ADP on ATPase activity was severely reduced by aurovertin. In contrast, increasing the concentration of aurovertin only slightly affected the level of inhibition caused by adenyl imidodiphosphate on ATP hydrolysis.

Effect of adenosine versus adenine nucleotides on evoked potentials in a rat hippocampal slice preparation

A marked depression of evoked CA1 potentials was observed with the nucleotide analogues a,b-methylene ADP (AOPCP) and adenylimido-diphosphate (AIP) and with 2′ -adenosine monophosphate (2′ -AMP). While the depression elicited by 5′ -nucleotides was completely antagonized by the action of adenosine deaminase, AOPCP and2 ′ -AMP were only partially antagonized. The findings indicate that nucleotides on their own are capable of modulating synaptic transmission but that the physiologically more prevalent 5′ -AMP is mediating its effect via adenosine. By producing this membrane permeable compound and allowing its re-uptake, the 5′ -nucleotidase may determine the time course of purinergic action.

Extracellular ATP, ecto-ATPase and calcium influx in Dictyostelium discoideum cells

There is evidence that a protein kinase is present on the surface of Dictyostelium discoideum cells [l-3] and is capable of phosphorylating certain plasma membrane proteins [3]. Such phosphorylation may be involved in membrane permeability and transport, cell movement, and the activation of cyclic AMP receptors which occurs during early differentiation [4]. External ATP induces cell aggregation, decreases the chemotactic sensitivity of cells to a cyclic AMP gradient and inhibits intercellular contact sites outside the aggregation center [ 1,5]. We found that ATP (0.1-0.8 PM produced at 1.5 X lo7 cells/ml) is present extracellularly in cell suspensions. This reflected a steady state concentration since a Mg”-dependent ecto-ATPase was continually hydrolyzing extracellular ATP. The ectoATPase was inhibited by suramin and I-ethyl-3(3-dimethyl-aminopropyl)carbodiimide (EDAC), but not by oligomycin or ouabain. Ecto-ADPase, -AMPase and nucleotide diphosphokinase activities were also detected. Calcium influx was inhibited by suramin, l-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDAC), KCN, dinitrophenol, carbonyl-mchlorophenylhydrazone (CCCP), adenyl imidodiphosphate, adenyl methylene diphosphate, and by adding hexokinase or apyrase to split extracellular ATP. Addition of ATP (optimum level 10e6 M) to cell suspensions stimulated calcium influx and partially overcame the KCN-induced inhibition of this influx. Glucose uptake was not affected by suramin or apyrase.

Regulation of the shape of unsealed erythrocyte membranes By Mg-ATP and Ca 2+

In the presence of MgCl 2 and ATP, the specific viscosity of suspensions of unsealed freezethawed erythrocyte membranes decreased slowly with time at 37 °C. The decrease in viscosity was found to be an index of Mg-ATP-specific induced folding of these membranes. Mg-ATP-dependent shape or viscosity changes were found to be highly temperature dependent and the viscosity of these membranes did not decrease in the presence of 2 m m 5′-adenyl imidodiphosphate and MgCl 2. Cyclic AMP, NaCl, or KCl did not have any effect on the rate of Mg-ATP-induced viscosity decreases. The Mg-ATP-dependent viscosity decreases were inhibited 100% by 1 m m chlorpromazine or 1 m m N-ethylmaleimide. Mg-ATP-dependent viscosity decreases were half-maximally inhibited by 1 μ m Ca 2+ and completely inhibited by 3–5 μ m Ca 2+. Ca 2+ (5 μ m) also inhibited Mg 2+-dependent phosphorylation 25 to 30% in these membranes. However, if these membranes were preincubated in the absence of Ca 2+ for greater than 10 min at 37 °C, 5 μ m Ca 2+ no longer inhibited Mg-ATP-dependent viscosity decreases and only inhibited Mg 2+-dependent phosphorylation 5% in these preincubated membranes. Preincubation of these membranes at 37 °C for 10 min in the absence of Ca 2+ also resulted in the loss of approximately 40 to 50% of the high-Ca 2+ affinity Ca + Mg-ATPase activity. The presence of 5 μ m Ca 2+ in the preincubation medium protected against the loss of the inhibitory effect of Ca 2+ on Mg 2+-dependent phosphorylation and Mg-ATP-dependent viscosity decreases. The presence of Ca 2+ in the preincubation medium also protected against the loss of Ca + Mg-ATPase activity in these membranes. It is hypothesized that freeze-thawed erythrocyte membranes contain a Ca 2+ phosphatase activity which is temperature labile in the absence of Ca 2+ and that this Ca 2+ phosphatase activity may be involved in the regulation of shape of these membranes. Also discussed is the possible relationship of this Ca 2+ phosphatase with Ca + Mg-ATPase activity and the problems inherent in studying Ca 2+-regulated functions in freeze-thawed erythrocyte membranes.