Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human P2Y1 receptor (P2Y1-R) was purified after high-level expression from a recombinant baculovirus in Sf9 insect cells. Quantification by protein staining and with a radioligand binding assay using the high-affinity P2Y1-R antagonist [3H]MRS2279 ([3H]2-chloro-N6-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bis-phosphate) indicated a nearly homogenous preparation of receptor protein. Ki values determined in [3H]MRS2279 binding assays for antagonists with the purified P2Y1-R were in good agreement with the Ki and KB values determined for these molecules in membrane binding and activity assays, respectively. Availability of P2Y1-R in purified form allowed direct determination of nucleotide agonist affinities under conditions not compromised by nucleotide metabolism/interconversion, and an order of affinities of 2-methylthio-ADP (2MeSADP) > ADP = 2-methylthioATP = adenosine-5'-O-(3-thio)triphosphate = adenosine-5'-O(2-thiodiphosphate) >> ATP was obtained. The signaling activity of the purified P2Y1-R was quantified after reconstitution in proteoliposomes with heterotrimeric G proteins. Steady-state GTP hydrolysis in vesicles reconstituted with P2Y1-R and Galpha(q)beta(1)gamma(2) was stimulated by the addition of either 2MeADP or RGS4 alone and was increased by up to 50-fold in their combined presence. EC50 values of agonists for activation of the purified P2Y1-R were similar to their respective Ki values determined in radioligand binding experiments with the purified receptor. Moreover, ATP exhibited 20-fold higher EC50 and Ki values than did ADP and was a partial agonist relative to ADP and 2MeSADP under conditions in which no metabolism of the nucleotide occurred. Both RGS4 and PLC-beta1 were potent and efficacious GTPase-activating proteins for Galphaq and Galpha11 in P2Y1-R-containing vesicles. These results illustrate that the binding and signaling properties of the human P2Y1-R can be studied with purified proteins under conditions that circumvent the complications that occur in vivo.
Mol Pharmacol 2004 Feb
PMID:Agonist binding and Gq-stimulating activities of the purified human P2Y1 receptor. 1474 85

Pharmacologically important drugs were examined as potential inhibitors or permeants of human concentrative nucleoside transporters 1 (hCNT1)- and 2 (hCNT2)-producing stable transfectants by assessing their abilities to inhibit uridine transport. hCNT1 exhibited high affinities for uridine analogs (5-fluorouridine, 2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, and 5-fluoro-5'-deoxyuridine) with K(i) values of 22 to 33 microM, whereas hCNT2 exhibited moderate affinities for 5-fluoro-2'-deoxyuridine, high affinities for 2'-deoxyuridine and 5-fluorouridine, and low affinity for 5-fluoro-5'-deoxyuridine. The uridine analogs were transported at 2-fold higher rates (at 10 microM) by hCNT1 than by hCNT2. Enantiomeric configuration and the 3'-hydroxyl group of the ribose ring were important determinants for interaction with hCNTs, whereas the 2'-hydroxyl group was less important. Both transporters bound N(6)-(p-aminobenzyl)adenosine with affinities similar to those of adenosine (K(i) = 28-39 microM). Other adenosine receptor ligands, including caffeine, bound better to hCNT1 than to hCNT2 (K(i) = 46 versus 103 microM, respectively), whereas 2-chloroadenosine bound better to hCNT2 than to hCNT1 (K(i) = 37 and 101 microM, respectively). There was a greater than 3-fold difference in binding affinities between hCNT1 and hCNT2 for nicotine (K(i) = 63 versus 227 microM). However, direct measurements of nicotine and caffeine uptake rates (10 microM) failed to demonstrate mediated uptake by either transporter. Although hCNT1 bound several adenosine analogs relatively well, it did not transport 2-chloro-2'-deoxyadenosine (cladribine) or 2-fluoro-9-beta-d-arabinofuranosyladenine (fludarabine), whereas hCNT2 transported both, albeit with low activities. The results indicated that although hCNT1 and hCNT2 possess some overlap in transport of several uridine and adenosine analogs, they also exhibit distinct differences in capacity to interact with some adenosine receptor ligands, adenosine-based drugs, and nicotine.
Mol Pharmacol 2004 Apr
PMID:Interactions of nucleoside analogs, caffeine, and nicotine with human concentrative nucleoside transporters 1 and 2 stably produced in a transport-defective human cell line. 1504 22

The control of protein synthesis during maturation in oocytes is mainly exerted through cytoplasmic polyadenylation of stored mRNAs. We first analyzed the polyadenylation status of cyclins A2 and B1 during in vitro maturation (IVM) of bovine oocytes, using Rapid Amplification of cDNA Ends-Polyadenylation Technique (RACE-PAT). An inconstant elongation of the poly(A) tail was observed for cyclin A2 transcripts after maturation, while a constant lengthening was observed for cyclin B1, occurring during the first 12 hr of incubation. We then evaluated the effects of the polyadenylation inhibitor 3'-deoxyadenosine (3'-dA), on polyadenylation and nuclear maturation. The presence of 0.02 mM 3'-dA during the whole incubation period or from 6 hr after its beginning completely prevented meiosis progression in 100% of the oocytes. Polyadenylation of cyclin B1 was also completely prevented when 3'-dA was added at 0 hr, and greatly reduced when added at 6 hr. When 3'-dA was added at 12 hr, around metaphase I (MI), 46.9% of the oocytes have reached metaphase II (MII, vs. 78.8% in the control group) at 24 hr. The use of the same concentration of 3'-deoxyguanosine (3'-dG), that impairs transcription but not polyadenylation, did not affect cyclins polyadenylation, nor nuclear maturation, whatever was the timing of addition. These results suggest that the polyadenylation of cyclin B1 could be related to the first peak of activity of MPF, occurring around MI (10-12 hr after the onset of the maturation period). They also show that, in our culture conditions, inhibition of polyadenylation prevents meiosis progression, especially up to the MI stage, while inhibition of transcription does not.
Mol Reprod Dev 2005 May
PMID:Effects of polyadenylation inhibition on meiosis progression in relation to the polyadenylation status of cyclins A2 and B1 during in vitro maturation of bovine oocytes. 1573 28

2-Chloro-9-(2'-deoxy-2'-fluoro-beta-d-arabinofuranosyl)adenine (Cl-F-ara-A, clofarabine), a purine nucleoside analog with structural similarity to 2-chloro-2'-deoxyadenosine (Cl-dAdo, cladribine) and 9-beta-d-arabinofuranosyl-2-fluoroadenine (F-ara-A, fludarabine), has activity in adult and pediatric leukemias. Mediated transport of the purine nucleoside analogs is believed to occur through the action of two structurally unrelated protein families, the equilibrative nucleoside transporters (ENTs) and the concentrative nucleoside transporters (CNTs). The current work assessed the transportability of Cl-F-ara-A, Cl-dAdo, and F-ara-A in cultured human leukemic CEM cells that were either nucleoside transport-defective or possessed individual human nucleoside transporter types and in Xenopus laevis oocytes and Saccharomyces cerevisiae yeast that produced individual recombinant human nucleoside transporter types. Cells producing hENT1 or hCNT3 exhibited the highest uptake of Cl-F-ara-A, whereas nucleoside transport-deficient cells and cells producing hCNT1 lacked uptake altogether. When Cl-F-ara-A transport rates by hENT1 were compared with those of Cl-dAdo and F-ara-A, Cl-dAdo had the highest efficiency of transport, although Cl-F-ara-A showed the greatest accumulation during 5-min exposures. In cytotoxicity studies with the CEM lines, Cl-F-ara-A was more cytotoxic to cells producing hENT1 than to the nucleoside transport-deficient cells. The efficiency of Cl-F-ara-A transport by oocytes with recombinant transporters was hCNT3 > hENT2 > hENT1 > hCNT2; no transport was observed with hCNT1. Affinity studies with recombinant transporters produced in yeast showed that hENT1, hENT2, and hCNT3 all had higher affinities for Cl-F-ara-A than for either Cl-dAdo or F-ara-A. These results suggest that the nature and activity of the plasma membrane proteins capable of inward transport of nucleosides are important determinants of Cl-F-ara-A activity in human cells.
Mol Pharmacol 2006 Jan
PMID:A comparison of the transportability, and its role in cytotoxicity, of clofarabine, cladribine, and fludarabine by recombinant human nucleoside transporters produced in three model expression systems. 1623 83

To clarify the functional mechanism of the adenosine deaminase-related growth factor (ADGF) particularly in the regulation of insect development, the cDNA encoding a homologue of ADGF proteins was cloned from the cabbage armyworm, Mamestra brassicae, named MbADGF. The purified MbADGF recombinant protein stimulated cell proliferation in a dose-dependent manner of SES-MaBr-4 and NIAS-MaBr-93 cell lines that were derived from fat bodies and haemocytes of M. brassicae. The adenosine deaminase activity of MbADGF was detected using adenosine and 2'-deoxyadenosine as substrates. Northern analysis demonstrated that during the larval development the level of MbADGF in the midgut increased. In situ hybridization showed that MbADGF mRNA was expressed in midgut goblet cells and in the apical cytoplasm of columnar cells, which suggests that MbADGF protein may execute its adenosine deaminase activity at the apical cytoplasm of columnar cells to convert adenosine into inosine.
Insect Mol Biol 2007 Jun
PMID:Molecular characterization of MbADGF, a novel member of the adenosine deaminase-related growth factor in the cabbage armyworm, Mamestra brassicae: the functional roles in the midgut cell proliferation. 1743 45

Adenosine deaminase (ADA; EC 3.5.4.4) activity is responsible for cleaving adenosine to inosine. In this study we described the biochemical properties of adenosine deamination in soluble and membrane fractions of zebrafish (Danio rerio) brain. The optimum pH for ADA activity was in the range of 6.0-7.0 in soluble fraction and reached 5.0 in brain membranes. A decrease of 31.3% on adenosine deamination in membranes was observed in the presence of 5 mM Zn(2+), which was prevented by 5 mM EDTA. The apparent K(m) values for adenosine deamination were 0.22+/-0.03 and 0.19+/-0.04 mM for soluble and membrane fractions, respectively. The apparent V(max) value for soluble ADA activity was 12.3+/-0.73 nmol NH(3) min(-1) mg(-1) of protein whereas V(max) value in brain membranes was 17.5+/-0.51 nmol NH(3) min(-1) mg(-1) of protein. Adenosine and 2'-deoxyadenosine were deaminated in higher rates when compared to guanine nucleosides in both fractions. Furthermore, a significant inhibition on adenosine deamination in both soluble and membrane fractions was observed in the presence of 0.1 mM of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). The presence of ADA activity in zebrafish brain may be important to regulate the adenosine/inosine levels in the CNS of this species.
Comp Biochem Physiol B Biochem Mol Biol 2008 Sep
PMID:Kinetic characterization of adenosine deaminase activity in zebrafish (Danio rerio) brain. 1858 89

Immunogenicity caused by the use of nonhuman enzymes in antibody-directed enzyme prodrug therapy has limited its clinical application. To overcome this problem, we have developed a mutant human purine nucleoside phosphorylase, which, unlike the wild-type enzyme, accepts (deoxy)adenosine-based prodrugs as substrates. Among the different mutants of human purine nucleoside phosphorylase tested, a double mutant with amino acid substitutions E201Q:N243D (hDM) is the most efficient in cleaving (deoxy)adenosine-based prodrugs. Although hDM is capable of using multiple prodrugs as substrates, it is most effective at cleaving 2-fluoro-2'-deoxyadenosine to a cytotoxic drug. To target hDM to the tumor site, the enzyme was fused to an anti-HER-2/neu peptide mimetic (AHNP). Treatment of HER-2/neu-expressing tumor cells with hDM-AHNP results in cellular localization of enzyme activity. As a consequence, harmless prodrug is converted to a cytotoxic drug in the vicinity of the tumor cells, resulting in tumor cell apoptosis. Unlike the nonhuman enzymes, the hDM should have minimal immunogenicity when used in antibody-directed enzyme prodrug therapy, thus providing a novel promising therapeutic agent for the treatment of tumors.
Mol Cancer Ther 2009 Jan
PMID:Humanized ADEPT comprised of an engineered human purine nucleoside phosphorylase and a tumor targeting peptide for treatment of cancer. 1913 28

Adenosine deaminase (ADA; EC 3.5.4.4) is a purine catabolic enzyme causing hydrolytic deamination of adenosine and 2'-deoxyadenosine to inosine and 2'-deoxyinosine. In the present study, the normal endogenous activity level of ADA was investigated in different regions of the gastrointestinal tract (GIT) during postnatal development of chicken. The effects of corticosterone and dibutyryl cAMP (Bt(2)-cAMP) were studied at two selected postnatal ages. The results indicated a significantly high level of ADA at day 1 in all the regions of GIT, which then declined (-34% in esophagus, -35% in crop, and -48% in small intestine) at day 10 and remained fairly constant till day 90. While in the proventiculus, the activity of ADA decreased (-30%) at day 30 and showed further decline (-52%) at day 90 as compared to day 1. Corticosterone was seen to significantly decrease (-23 to 79%) the activity level, depending on the regions of GIT studied except proventriculus. The magnitude of decline was more pronounced at day 60 compared to day 10. Bt(2)-cAMP, on the other hand, caused a significant increase (+21 to 67%) in the activity level of ADA again depending on the regions of GIT studied except crop. Western blot analyses also depicted that the decrease and/or increase, respectively, of ADA activity by corticosterone and Bt(2)-cAMP was at the ADA protein level. In conclusion, the study suggests that the ADA activity level is highest at day 1 in all the regions of chicken GIT and could be reduced or enhanced by corticosterone and dibutyryl cAMP, respectively, in an age-specific manner.
Mol Cell Biochem 2009 Jul
PMID:Antithetical effects of corticosterone and dibutyryl cAMP on adenosine deaminase in the gastrointestinal tract of chicken during postnatal development. 1922 93

The synthesis of 8-azido-2'-deoxyadenosine-5'-triphosphate is described. The photoreactive dATP analog was characterized by thin layer chromatography and UV spectroscopy. Its photoreactivity upon UV irradiation was studied. After incorporation of this dATP analog by nick translation into DNA containing the tet operator sequence the investigation of the interactions between tet operator DNA and Tet repressor becomes possible. Photocrosslinking of protein to DNA was demonstrated by the reduced migration of the DNA protein crosslinks in SDS polyacrylamide gel electrophoresis.
Methods Mol Biol 2009
PMID:Ultraviolet crosslinking of DNA protein complexes via 8-azidoadenine. 1937 78

GS-9148 ([5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl]-phosphonic acid) is a dAMP (2'-deoxyadenosine monophosphate) analog that maintains its antiviral activity against drug-resistant HIV. Crystal structures for HIV-1 reverse transcriptase (RT) bound to double-stranded DNA, ternary complexes with either GS-9148-diphosphate or 2'-deoxyadenosine triphosphate (dATP), and a post-incorporation structure with GS-9148 translocated to the priming site were obtained to gain insight into the mechanism of RT inhibition. The binding of either GS-9148-diphosphate or dATP to the binary RT-DNA complex resulted in the fingers subdomain closing around the incoming substrate. This produced up to a 9 A shift in the tips of the fingers subdomain as it closed toward the palm and thumb subdomains. GS-9148-diphosphate shows a similar binding mode as dATP in the nucleotide-binding site. Residues whose mutations confer resistance to nucleotide/nucleoside RT inhibitors, such as M184, Y115, L74, and K65, show little to no shift in orientation whether GS-9148-diphosphate or dATP is bound. One difference observed in binding is the position of the central ring. The dihydrofuran ring of GS-9148-diphosphate interacts with the aromatic side chain of Y115 more than does the ribose ring of dATP, possibly picking up a favorable pi-pi interaction. The ability of GS-9148-diphosphate to mimic the active-site contacts of dATP may explain its effective inhibition of RT and maintained activity against resistance mutations. Interestingly, the 2'-fluoro moiety of GS-9148-diphosphate was found in close proximity to the Q151 side chain, potentially explaining the observed moderately reduced susceptibly to GS-9148 conferred by Q151M mutation.
J Mol Biol 2010 Apr 09
PMID:Visualizing the molecular interactions of a nucleotide analog, GS-9148, with HIV-1 reverse transcriptase-DNA complex. 2015 54


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