Substrate-induced conformational changes in Plasmodium falciparum guanosine monophosphate synthetase

GMP synthetase is a glutamine amidotransferase that incorporates ammonia derived from glutamine into the nucleotide xanthosine 5'-monophosphate (XMP) to form guanosine 5'-monophosphate (GMP). Functional coordination of domains in glutamine amidotransferases leads to upregulation of glutamine hydrolysis in the presence of acceptor substrates and is a common feature in this class of enzymes. We have shown earlier that binding of substrates to the acceptor domain of Plasmodium falciparum GMP synthetase (PfGMPS) leads to enhancement in both glutaminase activity and rate of glutaminase inactivation, by the irreversible inhibitors acivicin and diazo-oxonorleucine [Bhat JY et al. (2008) Biochem J409, 263-273], a process that must be driven by conformational alterations. In this paper, through the combined use of biochemical assays, optical spectroscopy and mass spectrometry, we demonstrate that PfGMPS undergoes conformational transitions upon binding of substrates to the acceptor domain. Limited proteolysis and hydrogen-deuterium exchange in conjunction with mass spectrometry unveil region-specific conformational changes in the ATP + XMP bound state of PfGMPS. Decreased accessibility of R294 and K428 residues to trypsin in the ATP pyrophosphatase domain and reduced deuterium incorporation in the 143-155 region, pertaining to the glutaminase domain, suggest that in PfGMPS ligand-induced conformational changes are not only local but also transmitted over a long range across the domains. Overall, these results provide a detailed understanding of the substrate-induced changes in PfGMPS that could be essential for the overall catalytic process.     

Competitive peptide antagonists of ANF-induced cyclic guanosine monophosphate production

Atrial natriuretic factor (isoleucine ANF 101-126), cleaved ANF (isoleucine ANF 101-105/106-126) and des (Gln 18, Ser 19, Gly 20, Leu 21, Gly 22) ANF 4-23-NH2 (C-ANF 4-23) stimulated cyclic guanosine monophosphate production (cGMP) by rat aortic vascular smooth muscle cells (VSMC) in culture. Cleaved ANF and ANF C4-23 also antagonised or diminished the response to ANF 101-126. Agonist and antagonist actions of both peptides were dose-related. In contrast, prepro ANF (104-123), an ANF precursor fragment, exhibited no agonist or antagonist effect on cGMP production.     

18O-Labeling of guanosine monophosphate upon hydrolysis of cyclic guanosine 3':5'-monophosphate by phosphodiesterase

The hydrolysis of cGMP by phosphodiesterase was conducted in [18O]water to determine the site of bond cleavage and the stoichiometry of 18O incorporation into 5'-GMP. Three different forms of phosphodiesterase including a calmodulin-calcium-dependent enzyme in its basal and activated states were examined. The hydrolysis of cGMP catalyzed by each of the forms of phosphodiesterase proceeded with incorporation of 1 18O atom recoverable in the phosphate moiety of each molecule of 5'-GMP generated. No molecular species of phosphate deriving from the 5'-GMP generated containing two or three 18O were detectable. These results indicate that the phosphodiesterase-catalyzed hydrolysis of cGMP proceeds by nucleophilic substitution at phosphorus resulting in P-O bond cleavage. The stoichiometry of 18O incorporation indicates that the reaction proceeds without phosphate-water oxygen exchange when the hydrolytic reaction is catalyzed by diverse forms of phosphodiesterase in the basal or activated state. These considerations of the phosphodiesterase reaction help to establish the validity of monitoring the rate of enzyme-catalyzed hydrolysis of cGMP as a function of the rate of 18O-labeling of the phosphate of 5'-GMP when the reaction proceeds in a medium of predetermined 18O enrichment.     

Rhodopsin-detergent micelles aggregate upon activation of cyclic guanosine monophosphate phosphodiesterase

In the presence of G protein and phosphodiesterase, GTP induces aggregation of phospholipid-free rhodopsin-detergent micelles or rhodopsin reconstituted in phospholipid vesicles. The net electrical charge of the vesicle is not critical to the aggregation process since this phenomenon is not altered by reconstitution with phospholipids with different charge. The aggregation process is observed by monitoring changes in the light-scattering properties of the detergent micelles or vesicle suspension and by phase-contrast microscopy. The lowest light intensity which triggers the aggregation process and concomitant light-scattering changes in a rhodopsin-detergent micellar suspension bleaches 6% rhodopsin. Under these conditions, the signal saturates at 30% rhodopsin bleaching. The aggregation process appears likely to depend on the protein-protein interaction, and the presence of a disk membrane is not necessary for this process.     

Estrogen synthetase in choriocarcinoma cell culture. stimulation by dibutyryl cyclic adenosine monophosphate and theophylline

The stimulation of estrogen biosynthesis by N⁶, O² -dibutyryl adenosine 3':5'-cyclic monophosphate and theophylline (dbT) in cultures of the JAr line of choriocarcinoma cells was investigated by measuring the specific activity and kinetic constants of estrogen synthetase (aromatase) in the various subcellular fractions after differential centrifugation of homogenized cells in isotonic sucrose. The low speed (900x$\text{g}$) pellet,from cells grown with or without dbT and homogenized in isotonic sucrose,contains the majority of the aromatase activity and the highest aromatase specific activity. The aromatase specific activity in the homogenate of cells grown with dbT and in the various subcellular fractions is 4- to 10-fold higher than in cells grown without dbT. The Vmax of androstenedione (4-androstene-3,17-dione) aromatization in homogenates from dbT-stimulated cells (6.9 pmol estrogen/min per mg protein) is significantly increased over that measured in the absence of dbT (1.5 pmol estrogen/min per mg protein); the Km values, however, are not significantly different (average of 43.8nM in dbT-stimulated fractions; 53.2nM in control fractions). These results suggest that the increased aromatase specific activity in dbT-stimulated cells results from an increase in amount of active enzyme, rather than from an increase in affinity of the enzyme for its substrate.     

Optogenetic manipulation of cyclic guanosine monophosphate to probe phosphodiesterase activities in megakaryocytes

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.     

Induction of lysozyme activity by adenosine 3':5' cyclic monophosphate in cultured mouse myeloid leukemic cells.

Mouse myeloid leukemic cells(Ml) could be induced by glucocorticoids to form Fc receptors, phagocytize, migrate in agar, induce lysosomal enzyme activities, and change into forms that were morphologically similar to macrophages and granulocytes. Adenosine 3′:5′ cyclic monophosphate also induced lysosomal enzyme activities, but not the other differentiation-associated properties. The induction of lysozyme activity was marked, the activity reaching about 400 times the initial activity at 5 days after treatment. This suggests that adenosine 3′:5′ cyclic monophosphate may be important in induction of lysozyme activity during differentiation of the cells.     

Improved measurement of thymidylate synthetase activity by a modified tritium-release assay

Accurate quantitation of thymidylate synthetase activity using a tritium-release assay is dependent upon measurement of only that tritium released from deoxy[5-³H]uridine monophosphate ([³H]dUMP) during the biosynthesis of thymidylate. Removal of remaining [³H]dUMP on completion of the assay by charcoal adsorption and correction for the nonenzymatic release of tritium are necessary. Although over 99% of [³H]dUMP is removed immediately following addition of charcoal, these studies demonstrate that sufficient [³H]dUMP can remain to prevent accurate measurement of low levels of thymidylate synthetase activity. By delaying measurement of radioactivity for at least 24 h following addition of charcoal, this problem is minimized. To account for nonenzymatic release of tritium, a blank containing enzyme extract with omission of $\text{±,}\text{l}\text{-5,10-}\text{methylenetetrahydrofolate}$ is demonstrated to be more effective than the commonly used blank in which water is substituted for enzyme extract. In samples containing 5-fluoro-2′-deoxyuridine monophosphate (FdUMP), a potent inhibitor of thymidylate synthetase activity, an alternative blank containing a high concentration of FdUMP (approximately 1mM) is useful in demonstrating a theoretical maximal or complete inhibition of thymidylate synthetase activity.     

Myogenic effects of cyclic guanosine monophosphate in smooth muscle cells. Role of protein kinase C

Heterogeneity of the cGMP-dependent contractility effects of the smooth cells (SMC) was shown in guinea pig ureter by the methods of the double sucrose gap junction. Sodium nitroprusside (SN, 0.1-100 microM) relaxed the high-K+ depolarisationprecontracted SMC but strengthened the SMC constriction triggered by electrical stimulation. In taenia coli, SN or nitroglycerin in the same concentration ranges depressed the electrical or mechanical activity of the SMC and relaxed the SMC, precontracted by depolarization in high-K+ medium. The inhibitor of the phosphodiesterases vinpocetine (1 microM) contributed to the activating effect of SN; the inhibitor of the soluble guanilatcyclase Methilen Blue (10 microM) depressed it. Histamine and mesotone (1-10 microM) increased the action potential and constriction of the SMC triggered by electrical stimulation but decreased the effect of SN. The activator of the protein kinase C (PK-C) phorbol miristoyl-13-acetyl (0.5 microM) changed direction of the SN effects inhibiting both the parameters of an action potential and of the SMC constriction. The pre-treatment with the inhibitor of PK-C calphostin C (0.1 microM) additionally depressed the effects of SN, increasing SMC constriction, especially in the presence of histamine and mesatone. We suggest that c-GMP depressed activity of the PK-C by independent mechanisms operating in SMC.     

Neuropeptide-stimulated cyclic guanosine monophosphate immunoreactivity in the neurosecretory terminals of a neurohemal organ

The neuropeptide eclosion hormone acts on the nervous system of the tobacco hornworm, Manduca sexta, to increase cyclic guanosine monophosphate (cGMP) levels. In this study I describe the localization of some of the sites where these increases occur. Prior to pupal ecdysis, eclosion hormone stimulates an increase in cGMP in a network of fibers in the transverse nerve of each abdominal ganglion. Double-label experiments with propidium iodide suggest that the cGMP immunoreactivity is primarily localized in neurosecretory nerve endings. The time course of the increase in cGMP immunoreactivity and its requirement for lipid metabolism is similar to that of the cGMP increase measured by radioimmunoassay. The cGMP response in the transverse nerve is stage-specific, occurring prior to pupal ecdysis and not prior to larval or adult ecdysis. © 1996 John Wiley & Sons, Inc.     

Quenching of thymidylate synthetase fluorescence by substrate analogs.

Quenching of fluorescence occurs when $\text{Lactobacillus casei}$ thymidylate synthetase is titrated with fluorodeoxyuridylate in the presence of 1-L-methylenetetrahydrofolate to form a ternary complex. Neither fluorodeoxyuridylate nor 1-L-methylenetetrahydrofolate added singly has any effect on enzyme fluorescence but d-L-methylenetetrahydrofolate alone causes quenching. Thus ternary complex formation and interaction with d-L-methylenetetrahydrofolate alter the environment of tryptophan residues in thymidylate synthetase in a similar manner.     

Assay of intracellular thymidylate synthetase activity and inhibition by 5-fluoro-2'-deoxyuridine in lymphocytes.

Thymidylate synthetase catalyses the formation of thymidine monophosphate from deoxyuridine monophosphate. Purified thymidylate synthetase can be assayed radiochemically using labelled deoxyuridine monophosphate as substrate, but cells are impervious to deoxyuridine monophosphate and so intracellular thymidylate synthetase activity cannot be assayed in this way. In this paper we describe the assay of intracellular thymidylate synthetase activity in intact cells using labelled 2'-deoxyuridine. The assay showed linear kinetics with respect to time, concentration of 2'-deoxyuridine, and cell concentration. 5-fluoro-2'-deoxyuridine inhibited intracellular thymidylate synthetase activity measured with this assay by 50% at 5 nM. Cell growth was inhibited by 50% at 6 nM 5-fluoro-2'-deoxyuridine. The assay was specific for thymidylate synthetase and enabled measurement of thymidylate synthetase activity in situ in intact cells.     

Essential arginyl residues in thymidylate synthetase.

Thymidylate synthetase from amethopterin-resistant $\text{Lactobacillus}$$\text{casei}$ is rapidly and completely inactivated by 2,3-butanedione in borate buffer, a reagent that is highly selective for the modification of arginyl residues. The reversible inactivation follows pseudo-first order kinetics and is enhanced by borate buffer. dUMP and dTMP afford significant protection against inactivation while (±)-5,10-methylenetetrahydrofolate and 7,8-dihydrofolate provide little protection. Unlike native enzyme, butanedione-modified thymidylate synthetase is incapable of interacting with 5-fluoro-2′-deoxyuridylate and 5,10-(+)-methylenetetrahydrofolate to form stable ternary complex. The results suggest that arginyl residues participate in the functional binding of dUMP.     

Correlated effects of acetylcholine and cyclic guanosine monophosphate on membrane properties of mammalian neocortical neurons.

The effects of successive extracellular iontophoresis of acetylcholine (ACh) and atropine, and intracellular hyperpolarizing iontophoresis of cyclic GMP (cGMP) were studied in single neurons of the coronal-pericruciate cortex of awake cats. (a) Fifty-seven percent of the neurons that were tested responded to ACh with an increase in neuronal input resistance (Rm) and 50% responded to ACh with an increase in firing rate; 65% responded to cGMP with an increase in Rm and 60% responded to cGMP with an increase in firing rate. (b) After application of atropine, increases in Rm and firing rate associated with iontophoresis of ACh failed to recur. (c) Persistent increases in Rm following application of ACh accompanied by current-induced neuronal discharge were not diminished by subsequent application of atropine. (d) Atropine did not prevent increases in Rm and firing rate associated with intracellular iontophoresis of cGMP. (e) All cells tested with both ACh and cGMP that were shown initially to respond to extracellular ACh with increases in Rm were later shown to have comparable responses to cGMP.     

Immunohistochemical localization of cyclic guanosine monophosphate (cGMP) on mouse fetal chromosomes

In previous immunohistochemistry studies, cyclic guanosine monophosphate (cGMP) has been found in polytene chromosomes of D. melanogaster, cGMP has not been found in mammalian metaphase chromosomes, but this could be due to loss of cGMP during staining. Thus the effect of different fixation techniques on the immunohistochemically detectable cGMP associated with metaphase chromosomes from mouse fetal tissue was examined. In chromosomes from cells fixed in 2% formalin, or unfixed cells dropped on slides preheated to 60 degrees C, there was diffuse cGMP staining. When cells were fixed in methanol:glacial acetic acid, 3:1, no chromosomal cGMP immunofluorescence was observed, whereas chromosomes from cells fixed in methanol:glacial acetic acid, 6:1, had different patterns of cGMP immunofluorescence depending on the temperature of the slides onto which the fixed cells were dropped. On slides prechilled to 4 degrees C, cGMP immunofluorescence outlined the chromosomes; on room temperature slides, faint chromosomal cGMP staining was observed, and on slides preheated to 68 degrees C or room temperature slides blown dry with hot air, the chromosomes had more intense diffuse cGMP immunofluorescence or distinct symmetrical bands of cGMP immunofluorescence. We have demonstrated the presence of cGMP in mammalian metaphase chromosomes. The different patterns of cGMP immunofluorescence observed may reflect variable preservation of chromosomal proteins that have binding sites for cGMP.