Adenylyl cyclase P-site ligands accelerate differentiation in Ob1771 preadipocytes

Differentiation of Ob1771 preadipocytes to adipocytes wascharacterized by morphological changes and elevated expression of thespecific marker enzyme, glycerol-3-phosphate dehydrogenase. Adifferentiation response substantially more complete and rapid thanthat obtained with insulin and 3,5,3'-triiodothyronine was observed with established inhibitors of adenylyl cyclases:2',5'-dideoxyadenosine (2',5'-dd-Ado),9-(cyclopentyl)adenine (9-CP-Ade), and 9-(arabinofuranosyl)adenine (9-Ara-Ade), coincident with decreased cellular cAMP levels. These ligands inhibit adenylyl cyclases noncompetitively, via a domain referred to as the P-site because of its requirement for an intact purine moiety. Differentiation was not induced by inosine, a nucleoside known not to act at the P-site, or byN⁶-(2-phenylisopropyl)adenosineor 1,3-diethyl-8-phenylxanthine, agonist and antagonist, respectively,for adenosine A₁ receptors. Alsoineffective were IBMX or forskolin, agents that can raise intracellularcAMP levels. Potency of the differentiation response followed the order2',5'-dd-Ado (1-20 µM) > 9-CP-Ade (10-100µM) = 9-Ara-Ade (10-100 µM) >> inosine, consistent withtheir potencies to inhibit adenylyl cyclases. The data suggest thatinhibition of adenylyl cyclase via the P-site and the consequentreduction in cell cAMP levels facilitate the induction ofdifferentiation in Ob1771 cells. The findings raise the questionwhether the known endogenous P-site ligands participate in thedifferentiation response induced by hormones.     

Irreversible inactivation of adenylyl cyclase by the "P"-site agonist 2',5'-dideoxy-,3'-p-fluorosulfonylbenzoyl adenosine

2',5'-Dideoxy,3'-p-fluorosulfonylbenzoyl Adenosine (2',5'-dd3'-FSBA) was synthesized and found to be an agonist and affinity label for the "P"-site of adenylyl cyclase. This compound irreversibly inactivated both a crude detergent-dispersed adenylyl cyclase from rat brain and the partially purified enzyme from bovine brain. The irreversible inactivation by 100 to 200 microM 2',5'-dd3'-FSBA was blocked in a concentration-dependent manner by several established P-site inhibitors of adenylyl cyclase, 2',5'-dideoxyadenosine, 2'-d3'-AMP, adenosine, and 2'-deoxyadenosine, but not by inosine, N6-(phenylisopropyl)adenosine, adenine, 2'-d3':5'-cAMP, or 5'-AMP, agents known not to act at the P-site. Moreover, irreversible inactivation by 2',5'-dd3'-FSBA occurred in the presence of ATP at concentrations up to 3 mM, making it unlikely that inactivation was due to an effect on the enzyme's catalytic site. Adenylyl cyclase was also irreversibly inactivated by 5'-FSBA, although modestly (less than 20%) and apparently nonspecifically. Dithiothreitol protected the enzyme from irreversible inactivation by 2',5'-dd3'-FSBA, but reversible inhibition of the enzyme was still observed, although with reduced potency. When 2 mM dithiothreitol was added after a 30-min preincubation with 2',5'-dd3'-FSBA, the rat brain enzyme was partially (approximately 80%) reactivated. The data suggest that 2',5'-dd3'-FSBA may irreversibly inactivate adenylyl cyclase by reacting with a cysteinyl moiety in proximity to the P-site domain of the enzyme. These data together with results of studies of P-site inhibition kinetics published elsewhere (Johnson, R. A., and Shoshani, I. (1990) J. Biol. Chem. 265, 11595-11600) strongly suggest that the P-site and catalytic site are distinct domains on the enzyme. 2',5'-dd3'-FSBA, and especially its radiolabeled analog, should prove to be a useful probe for structural studies of adenylyl cyclase, particularly with regard to the P-site.     

Hormone-sensitive adenylyl cyclase in preadipocytes cultured from adipose tissue: comparison with 3T3-L1 cells and adipocytes

The adenylyl cyclase system of preadipocytes derived from the stromal vascular fraction of perirenal rat fat pads was characterized. Unlike mature adipocytes, preadipocyte adenylyl cyclase was only weakly stimulated by catecholamines and adrenocorticotrophic hormone, but was stimulated by guanine nucleotides. Parathyroid hormone and 2-chloroadenosine also stimulated preadipocyte adenylyl cyclase. The adenylyl cyclase system of preadipocytes resembled that of undifferentiated 3T3-L1 cells. However, agents which induced the differentiation of the 3T3-L1 cell adenylyl cyclase system did not have a similar effect on preadipocytes. A medium (CDM6) which induced some differentiation of preadipocyte adenylyl cyclase was developed. The observations that the adenylyl cyclase system of preadipocytes and undifferentiated 3T3-L1 cells are similar, that preadipocyte adenylyl cyclase can be induced to develop along lines similar to early differentiation of 3T3-L1 cells, and that the adenylyl cyclase system of fully-differentiated 3T3-L1 cells has characteristics intermediate between preadipocytes and adipocytes, suggest that the differentiation of preadipocyte and 3T3-L1 adenyly cyclase in vitro mimics adipose adenylyl cyclase development in vivo. The increased catecholamine and ACTH stimulation, and reduced GTP and adenosine sensitivities of adipocytes compared to preadipocytes suggest that a number of genes affecting adenylyl cyclase-associated regulatory and receptor proteins are coordinately repressed and derepressed during development.     

Phenyl phosphotriester derivatives of AZT: variations upon the SATE moiety

Synthesis, in vitro anti-HIV activity, stability studies as well as potential for oral absorption of some novel phenyl S-acyl-2-thioethyl (SATE) phosphotriester derivatives of AZT (zidovudine; 3'-azido-2',3'-dideoxythymidine) are described herein. These pronucleotides are characterized by the presence of polar functions on the SATE biolabile phosphate protections. Whereas derivatives incorporating an amino residue in the vicinity of the thioester functionality display low chemical stability, the introduction of one or two hydroxyl groups on the SATE moieties confers high resistance of the resulting prodrugs towards esterase hydrolysis. Thus, one of these pronucleotides, the monohydroxylated SATE derivative of AZT 2, is able to cross a Caco-2 cell monolayer mainly in intact form, probing that further development is warranted as a possible HIV-pronucleotide candidate.     

Synthesis and use of 3'-(azidoiodosalicyl) derivatives of 2', 5'-dideoxyadenosine as photoaffinity ligands for adenylyl cyclase

3'-[(4-Azidosalicyl)glycyl]-2',5'-dideoxyadenosine (1), 3'- [(4-azidosalicyl)-gamma-aminobutyryl]-2',5'-dideoxyadenosine (2), and the (125)I-labeled mono- and diiodinated analogs of 1 were synthesized and tested as photoaffinity probes for adenylyl cyclases. Kinetics for inhibition of purified type I enzyme by 1 was noncompetitive with respect to Mn(*)5'-ATP in the absence of light, implying a P-site mechanism of inhibition. In a UV-dependent manner both 1 and 2 and the iodinated derivative of 1 irreversibly inactivated membrane-bound and purified forms of recombinant type I bovine adenylyl cyclase expressed in ovarian cells of either the fall armyworm (Sf9) or Trichoplasia ni (High Five). Irreversible inactivation was independent of 5'-ATP and was prevented by 2', 5'-dideoxyadenosine. Adenylyl cyclase, whether purified from bovine brain or in membranes from High Five cells expressing type I enzyme, when subjected to UV irradiation in the presence of (125)I-labeled 1 resulted in radioactive incorporation into protein migrating at approximately 116 kDa. The cross-linking of 1 and its iodinated derivative with adenylyl cyclase suggests potential for such compounds to be useful in structural studies of adenylyl cyclases or of other proteins for which adenine nucleosides are substrates or allosteric regulators.     

Enzymatic synthesis of unlabeled and beta-(32)P-labeled beta-L-2', 3'-dideoxyadenosine-5'-triphosphate as a potent inhibitor of adenylyl cyclases and its use as reversible binding ligand

beta-L-2',3'-Dideoxyadenosine-5'-triphosphate (beta-L-2', 3'-dd-5'-ATP) was prepared enzymatically from the corresponding monophosphate by the use of adenylate kinase, creatine phosphate, and creatine kinase in a single step. The beta-(32)P-labeled analog was prepared similarly, but in a two step reaction. beta-L-2', 3'-dd-5'-ATP inhibited adenylyl cyclase from rat brain competitively with respect to substrate (5'-ATP.Mn(2+)) and exhibited an IC(50) approximately 24 nM. The labeled ligand was used in the development of a reversible binding assay for adenylyl cyclases. Binding of beta-L-2',3'-dd-[beta-(32)P]5'-ATP was saturable with increasing concentrations of ligand and increased in proportion to membrane protein, and was enhanced by Mn(2+) to a greater extent than by Mg(2+). Binding was displaced with adenine nucleotides known to be either competitive or noncompetitive inhibitors but not by agents known not to act on the cyclase, or by 3-isobutyl-1-methylxanthine, creatine phosphate, or creatine kinase. Binding was rapid, with a half-time for the on-rate <1.8 min and for the off-rate <0.8 min. The potency and mechanism of the inhibition of this ligand and the pattern of agents that displace binding suggest an interaction with adenylyl cyclase per se and to a configuration of the enzyme consistent with an interaction at the catalytic active site. The data suggest that this is a pretransition state inhibitor and contrasts with the equipotent 2',5'-dd-3'ATP, a post-transition state noncompetitive inhibitor.     

Prostaglandin-sensitive adenylyl cyclase of cultured preadipocytes and mature adipocytes of the rat: probable role of Gi in determination of stimulatory or inhibitory action

Preadipocytes of rats were obtained from the stromal-vascular fraction of collagenase-digested perirenal fat pads and grown in serum-containing medium. By day 8 of culture the cells reached confluence and by 12 days were lipid-laden. The adenylyl cyclase of the plasma membranes was compared to that of mature fat cells. Unlike the membranes from adipocytes, the preadipocytes showed adenylyl cyclase activity that was stimulated by GTP. Stimulation of preadipocyte membranes by Gpp(NH)p, NaF, and forskolin was comparable to that of membranes from adipocytes, but the response to epinephrine and isoproterenol was minimal (approximately 1.5-fold for preadipocytes vs. 4-5-fold for adipocytes). In contrast, GTP-dependent stimulation of adenylyl cyclase of preadipocytes by PGE1 was nearly 8-fold. Stimulation occurred even in the presence of both GTP and 140 mM NaCl, a condition that leads to inhibition by PGE1 of adenylyl cyclase in membranes of adipocytes. Other characteristics of the adenylyl cyclase of preadipocyte membranes that differ from those of adipocytes include lack of inhibition by GTP of forskolin-activated activity, and, following treatment with pertussis toxin, enhanced stimulation by PGE1. ADP-ribosylation of Gi and Gs with pertussis and cholera toxins, respectively, indicated that the membranes of preadipocytes contained only 5-11% of the Gi of adipocytes and a much lower ratio of Gi:Gs. These findings suggest that cultured preadipocytes have an incompletely developed Gi pathway that may account for the stimulatory effect of prostaglandins on the adenylyl cyclase of these cells as opposed to the inhibitory action of PG in mature fat cells.     

Inhibition of the ras-dependent mitogenic pathway by phosphopeptide prodrugs with antiproliferative properties

Phosphopeptide prodrugs bearing two S-acyl-2-thioethyl (SATE) biolabile phosphate protections were developed. They are capable to inhibit the Shc/Grb2 interaction and MAP kinases (ERK1 and ERK2) phosphorylation in cellular assay. The S-acetyl-2-thioethyl (MeSATE) analogue showed an IC50 of 1 microM in the inhibition of the colony formation of tumor cell line NIH3T3/HER2.     

Inhibition of Bordetella pertussis and Bacillus anthracis adenylyl cyclases by polyadenylate and "P"-site agonists

Inhibition of adenylyl cyclases from Bacillus anthrasis and Bordetella pertussis by polyadenylate and by the most potent "P"-site agonists was investigated. These bacterial adenylyl cyclases differed in their sensitivity to inhibition by nominal "P"-site agents and in the effect of divalent cations on this inhibition. The enzyme from Bordetella pertussis was relatively insensitive to inhibition by "P"-site agonists, exhibiting a rank order of potency of 2'd3'AMP greater than 3'-AMP greater than 2',5'-ddAdo approximately Ado approximately 2'-dAdo, with IC50 values for 2'd3'AMP and 3'-AMP of 1-3 mM. Inhibition by 2'd3'AMP, however, was not affected by divalent cation, making it distinct from "P"-site-mediated inhibition of most mammalian adenylyl cyclases. The sensitivity to these nucleosides was comparable with potency for inhibition of bovine sperm adenylyl cyclase but was 3 orders of magnitude less potent than for activated enzyme from bovine or rat brain. The Bordetella pertussis enzyme was similarly insensitive to inhibition by polyadenylate, with 16 microM inhibiting less than 20%. By comparison, Bacillus anthrasis adenylyl cyclase was more potently inhibited by 2'd3'AMP (IC50 approximately 85 microM) but not by the other nucleosides (less than 15% inhibition at 1 mM), and inhibition by 2'd3'AMP was optimally enhanced by 5-10 mM Mg2+ or Mn2+, as is typical for inhibition by "P"-site agonists. The Bacillus anthrasis enzyme was potently inhibited by polyadenylate (IC50 approximately 0.3 microM), comparable to inhibition of brain adenylyl cyclases. Sensitivity of Bacillus anthrasis adenylyl cyclase to poly(A) was diminished somewhat by Ca2+/calmodulin (to IC50 approximately 1 microM) although Ca2+/calmodulin was without effect on inhibition by 2'd3'AMP. In contrast to inhibition of mammalian adenylyl cyclases via the "P"-site, inhibition of both bacterial adenylyl cyclases by 2'd3'AMP was competitive with respect to substrate MgATP. The data indicate basic differences in susceptibilities of these bacterial adenylyl cyclases to inhibition by poly(A), by adenosine analogs, and the effects of divalent cations. Although the potency of 2'd3'AMP and the metal-dependent nature of inhibition of Bacillus anthrasis adenylyl cyclase shared characteristics of "P"-site-mediated inhibition, the fact that inhibition of both bacterial adenylyl cyclases was competitive with respect to substrate strongly suggests that this inhibition was at the catalytic site and that these bacterial enzymes do not contain a distinct "P"-site.     

An adenylyl cyclase pseudogene in Mycobacterium tuberculosis has a functional ortholog in Mycobacterium avium

A number of genes similar to mammalian Class III nucleotide cyclases are found in mycobacteria, and biochemical characterization of some of these proteins has indicated that they code for adenylyl cyclases, with properties similar to the mammalian enzymes. Our earlier bioinformatic analysis had predicted that the Rv1120c gene in Mycobacterium tuberculosis is a pseudogene, while analysis of the genome of Mycobacterium avium indicated the presence of a functional ortholog. We therefore cloned and expressed Rv1120c and its ortholog from M. avium, Ma1120, in Escherichia coli, and find that while the protein from M. tuberculosis is misfolded and found in inclusion bodies, Ma1120 is expressed to high levels as a functional adenylyl cyclase. Sequence analysis of Ma1120 indicates interesting variations in critical amino acids that are known to be important for catalytic activity. Ma1120 is maximally active in the presence of MnATP as substrate ((app)Km approximately 400 microM), and is inhibited by P-site inhibitors (IC50 of 2',5'-dideoxy-3'-adenosine triphosphate approximately 730 nM) and tyrphostins (IC50 approximately 36 microM) in a manner similar to the mammalian enzymes. This therefore represents the first Class III cyclase biochemically characterized from M. avium, and the absence of a functional ortholog in M. tuberculosis suggests a unique role for this enzyme in M. avium.     

Opioid-Inhibited Adenylyl Cyclase in Rat Brain Membranes: Lack of Correlation with High-Affinity Opioid Receptor Binding Sites

Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone), irreversible antagonists [naloxonazine, beta-funaltrexamine, and beta-chlornaltrexamine (beta-CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled D-alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (mu), 3H-labeled 2-D-serine-5-L-leucine-6-L-threonine enkephalin (delta), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity mu, delta, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only beta-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of beta-CNA was not due to nonspecific interactions with G(i), Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by beta-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity mu, delta, or EKC sites identified in brain by traditional binding studies.     

Multiple cyclic AMP receptors are linked to adenylyl cyclase in Dictyostelium.

cAMP receptor 1 and G-protein alpha-subunit 2 null cell lines (car1- and g alpha 2-) were examined to assess the roles that these two proteins play in cAMP stimulated adenylyl cyclase activation in Dictyostelium. In intact wild-type cells, cAMP stimulation elicited a rapid activation of adenylyl cyclase that peaked in 1-2 min and subsided within 5 min; in g alpha 2- cells, this activation did not occur; in car1- cells an activation occurred but it rose and subsided more slowly. cAMP also induced a persistent activation of adenylyl cyclase in growth stage cells that contain only low levels of cAMP receptor 1 (cAR1). In lysates of untreated wild-type, car1-, or g alpha 2- cells, guanosine 5'-O-'(3-thiotriphosphate) (GTP gamma S) produced a similar 20-fold increase in adenylyl cyclase activity. Brief treatment of intact cells with cAMP reduced this activity by 75% in control and g alpha 2- cells but by only 8% in the car1- cells. These observations suggest several conclusions regarding the cAMP signal transduction system. 1) cAR1 and another cAMP receptor are linked to activation of adenylyl cyclase in intact cells. Both excitation signals require G alpha 2. 2) cAR1 is required for normal adaptation of adenylyl cyclase. The adaptation reaction caused by cAR1 is not mediated via G alpha 2. 3) Neither cAR1 nor G alpha 2 is required for GTP gamma S-stimulation of adenylyl cyclase in cell lysates. The adenylyl cyclase is directly coupled to an as yet unidentified G-protein.     

Molecular basis for P-site inhibition of adenylyl cyclase

P-site inhibitors are adenosine and adenine nucleotide analogues that inhibit adenylyl cyclase, the effector enzyme that catalyzes the synthesis of cyclic AMP from ATP. Some of these inhibitors may represent physiological regulators of adenylyl cyclase, and the most potent may ultimately serve as useful therapeutic agents. Described here are crystal structures of the catalytic core of adenylyl cyclase complexed with two such P-site inhibitors, 2'-deoxyadenosine 3'-monophosphate (2'-d-3'-AMP) and 2',5'-dideoxyadenosine 3'-triphosphate (2',5'-dd-3'-ATP). Both inhibitors bind in the active site yet exhibit non- or uncompetitive patterns of inhibition. While most P-site inhibitors require pyrophosphate (PP(i)) as a coinhibitor, 2',5'-dd-3'-ATP is a potent inhibitor by itself. The crystal structure reveals that this inhibitor exhibits two binding modes: one with the nucleoside moiety bound to the nucleoside binding pocket of the enzyme and the other with the beta and gamma phosphates bound to the pyrophosphate site of the 2'-d-3'-AMP.PP(i) complex. A single metal binding site is observed in the complex with 2'-d-3'-AMP, whereas two are observed in the complex with 2', 5'-dd-3'-ATP. Even though P-site inhibitors are typically 10 times more potent in the presence of Mn(2+), the electron density maps reveal no inherent preference of either metal site for Mn(2+) over Mg(2+). 2',5'-dd-3'-ATP binds to the catalytic core of adenylyl cyclase with a K(d) of 2.4 microM in the presence of Mg(2+) and 0.2 microM in the presence of Mn(2+). Pyrophosphate does not compete with 2',5'-dd-3'-ATP and enhances inhibition.     

Synthesis of branched ribonucleotides

A synthetic strategy for branched ribonucleotides, which have recently been discovered, was described. A fully protected adenosine unit (5) having the tris (4,5-dichlorophthalimido)trityl (CPTr), bis(anilino)phosphoryl (BAP), and bis(phenylthio)phosphoryl (BPTP) groups as the 5'-, 2'-, and 3'-hydroxyl protecting groups, respectively, was synthesized from adenosine by a five-step reaction involving a new method for the 2'-O-phosphorylation by the use of hexaethylphosphorous triamide. The selective deprotection of appropriate protecting groups from 5 followed by stepwise condensation with two different ribonucleoside derivatives (7 and 10) gave a protected branched ribonucleotide (11) via a 3'-phosphorylated 2'-5' dinucleotide (8). Deprotection of 11 and 8 gave a branched trinucleotide (12) and 3'-phosphorylated dinucleotide (13).     

Structure-based redesign of an edema toxin inhibitor

Edema factor (EF) toxin of Bacillus anthracis (NIAID category A), and several other toxins from NIAID category B Biodefense target bacteria are adenylyl cyclases or adenylyl cyclase agonists that catalyze the conversion of ATP to 3',5'-cyclic adenosine monophosphate (cAMP). We previously identified compound 1 (3-[(9-oxo-9H-fluorene-1-carbonyl)-amino]-benzoic acid), that inhibits EF activity in cultured mammalian cells, and reduces diarrhea caused by enterotoxigenic Escherichia coli (ETEC) at an oral dosage of 15μg/mouse. Here, molecular docking was used to predict improvements in potency and solubility of new derivatives of compound 1 in inhibiting edema toxin (ET)-catalyzed stimulation of cyclic AMP production in murine monocyte-macrophage cells (RAW 264.7). Structure-activity relationship (SAR) analysis of the bioassay results for 22 compounds indicated positions important for activity. Several derivatives demonstrated superior pharmacological properties compared to our initial lead compound, and are promising candidates to treat anthrax infections and diarrheal diseases induced by toxin-producing bacteria.     

Structural requirements for the binding of AMP to the allosteric site of NAD-specific isocitrate dehydrogenase from bakers' yeast

The specificity of yeast NAD-specific isocitrate dehydrogenase for the structures of the allosteric effector 5'-AMP was examined with analogues modified in the purine ring, pentosyl group, and 5'-phosphate group. An unsubstituted 6-amino group was essential for activation as was the phosphoryl group at the 5'-position. Activity was retained when an oxygen function of the 5'-phosphoryl was replaced by sulfur (Murry & Atkinson, 1968) or by nitrogen (phosphoramidates). 2-NH2-AMP, 2-azido-AMP, and 8-NH2-AMP were active; 8-azido-AMP and 8-Br-AMP were inactive. The configuration or nature of substituents about carbons 2' and 3' of the pentosyl portion of AMP was not critical for allosteric activation since AMP analogues containing, e.g., 2',3'-dideoxyribose or the bulky 2',3'-O-(2,4,6-trinitrocyclo-hexadienylidene) substituent (TNP-AMP) were active. TNP-AMP was bound to the enzyme with fluorescence enhancement and had an S0.5 for activation similar to the S0.5 for AMP. Positive effector activity was decreased when the pentosyl moiety of 5'-AMP was replaced by the six-membered nitrogen-containing morpholine group, indicating that the pentosyl group may be critical as a spacer for the proper geometry of binding to enzyme at the 6-amino and 5'-phosphoryl groups of 5'-AMP. A comparison of molecular models of 5'-AMP with 8,5'-cycloAMP suggests that the species of 5'-AMP required for binding to the enzyme contains the purine and ribose moieties in an anti conformation and positioning of the 5'-phosphate trans with respect to carbon 4'.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzymatic preparation of 32P-labeled beta-L-2',3',-dd-5'ATP and its use as a high-affinity, conformation-specific ligand for labeling adenylyl cyclases.

An enzymatic method was developed for the preparation of unlabeled and [beta-32P]-labeled beta-L-2',3'-dd-5'ATP from the monophosphate with near quantitative yields. beta-L-2',3'-dd-5'ATP was a competitive and potent inhibitor of adenylyl cyclases (IC5 approximately 30 nM). Upon uv-irradiation beta-L-2',3'-dd-[beta-32P]-5'ATP directly crosslinked to a chimeric construct of this enzyme. Data suggest that this is a pre-transition state inhibitor and contrasts with the equipotent 2',5'-dd-3'ATP, a post-transition state, noncompetitive inhibitor.     

Divergent effects of cations on follicle-stimulating hormone- and forskolin-activated adenylyl cyclase in granulosa cells

The divalent cations magnesium, calcium and manganese, and the monovalent cation, potassium, but not sodium, enhance binding of [125I]iodo-porcine follicle-stimulating hormone to follicle-stimulating hormone (FSH) receptors in membranes of porcine granulosa cells via an increase in the apparent number of binding sites. The objective of the present studies was to determine if increased binding of FSH to its receptor causes increased adenylyl cyclase activity in response to FSH, or conversely, if enhancement of the cyclase or one of its components causes increased binding, or if the two processes are modulated independently. MgCl2 and CaCl2, which both enhance binding in intact cells and in cell-free membrane preparations, had opposite effects on cyclase-MgCl2 stimulatory, CaCl2 inhibitory. In isotonic NaCl, MgCl2 did not enhance binding, but it did increase FSH-stimulated production of cyclic adenosine 3',5'-monophosphate (cAMP). NaCl did not enhance FSH binding and it did not enhance cyclase in cell-free membranes, but it did increase FSH- and forskolin-stimulated cAMP production in intact cells. In intact cells, maximally effective concentrations of MgCl2 and KCl were additive in enhancing cAMP production whereas the effects of NaCl and KCl together were synergistic. The results indicate that although cationic effects on FSH binding are not causally related to effects on cyclase, the cationic microenvironment of the granulosa cell membrane is critical to both FSH receptor and adenylyl cyclase functions.     

A novel action of human apurinic/apyrimidinic endonuclease: excision of L-configuration deoxyribonucleoside analogs from the 3' termini of DNA

beta-l-Dioxolane-cytidine (l-OddC, BCH-4556, Troxacitabine) is a novel unnatural stereochemical nucleoside analog that is under phase II clinical study for cancer treatment. This nucleoside analog could be phosphorylated and subsequently incorporated into the 3' terminus of DNA. The cytotoxicity of l-OddC was correlated with the amount of l-OddCMP in DNA, which depends on the incorporation by DNA polymerases and the removal by exonucleases. Here we reported the purification and identification of the major enzyme that could preferentially remove l-OddCMP compared with dCMP from the 3' termini of DNA in human cells. Surprisingly, this enzyme was found to be apurinic/apyrimidinic endonuclease (APE1) (), a well characterized DNA base excision repair protein. APE1 preferred to remove l- over d-configuration nucleosides from 3' termini of DNA. The efficiency of removal of these deoxycytidine analogs were as follows: l-OddC > beta-l-2',3'-dideoxy-2', 3'-didehydro-5-fluorocytidine > beta-l-2',3'-dideoxycytidine > beta-l-2',3'-dideoxy-3'-thiocytidine > beta-d-2',3'-dideoxycytidine > beta-d-2',2'-difluorodeoxycytidine > beta-d-2'-deoxycytidine >/= beta-d-arabinofuranosylcytosine. This report is the first demonstration that an exonuclease can preferentially excise l-configuration nucleoside analogs. This discovery suggests that APE1 could be critical for the activity of l-OddC or other l-nucleoside analogs and may play additional important roles in cells that were not previously known.