Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.5 (5'-nucleotidase)
3,167 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An antiserum was generated against a dodecapeptide whose sequence is found at the C-terminus of a cyclic AMP (cAMP)-specific, type-IVA phosphodiesterase encoded by the rat 'dunc-like' cyclic AMP phosphodiesterase (RD1) cDNA. This antiserum identified a single approximately 73 kDa protein species upon immunoblotting of cerebellum homogenates. This species co-migrated upon SDS/PAGE with a single immunoreactive species observed in COS cells transfected with the cDNA for RD1. Native RD1 in cerebellum was found to be predominantly (approximately 93%) membrane-associated and could be found in isolated synaptosome populations, in particular those enriched in post-synaptic densities. Fractionation of lysed synaptosomes on sucrose density gradients identified RD1 as co-migrating with the plasma membrane marker 5'-nucleotidase. Laser scanning confocal and digital deconvolution immunofluorescence studies done on intact COS cells transfected with RD1 cDNA showed RD1 to be predominantly localized to plasma membranes but also associated with the Golgi apparatus and intracellular vesicles. RD1-specific antisera immunoprecipitated phosphodiesterase activity from solubilized cerebellum membranes. This activity had the characteristics expected of the type-IV cAMP phosphodiesterase RD1 in that it was cAMP specific, exhibited a low Km cAMP of 2.3 microM, high sensitivity to inhibition by 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram) (Ki approximately 0.7 microM) and was unaffected by Ca2+/calmodulin and low concentrations of cyclic GMP. The phosphodiesterase activities of RD1 solubilized from both cerebellum and transfected COS cell membranes showed identical first-order thermal denaturation kinetics at 50 degrees C. Native RD1 from cerebellum was shown to be an integral protein in that it was solubilized using the non-ionic detergent Triton X-100 but not by either re-homogenization or high NaCl concentrations. The observation that hydroxylamine was unable to cause the release of RD1 from either cerebellum or COS membranes and that [3H]palmitate was not incorporated into the RD1 protein immunoprecipitated from COS cells transfected with RD1 cDNA, indicated that RD1 was not anchored by N-terminal acylation. The engineered deletion of the 25 residues forming the unique N-terminal domain of RD1 caused both a profound increase in its activity (approximately 2-fold increase in Vmax) and a profound change in intracellular distribution. Thus, immunofluorescence studies identified the N-terminal truncated species as occurring exclusively ion the cytosol of transfected COS cells. The cDNA for RD1 thus appears to encode a native full-length type-IVA phosphodiesterase that is expressed in cerebellum.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Identification and characterization of the type-IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum. 770 77

A novel plasmid was generated which allowed the expression of the cytosolic bacterial enzyme chloramphenicol acetyl transferase (CAT) in COS-7 cells. Upon transfection, the majority of the novel CAT activity was found in the cytosol fraction of COS cells. Chimeric molecules were made between N-terminal portions of the type IVA cyclic AMP-specific rat 'dunce-like' phosphodiesterase (RD1) (RNPDE4A1A; rPDE-IVA1) fused to CAT at its N-terminus. Expression in COS-7 cells of chimeras formed from 1-100RD1-CAT and 1-25RD1-CAT now showed CAT activity associated with the membrane fraction. In contrast, a chimera formed from 26-100RD1-CAT showed an identical expression pattern to native CAT, with the major fraction of CAT activity occurring in the cytosol fraction. Membrane-bound CAT activity provided by 1-100RD1-CAT and 1-25RD1-CAT was not released by either high-salt or washing treatments but was solubilized in a dose-dependent fashion by the non-ionic detergent Triton X-100. Subcellular fractionation of COS-7 cells showed that, as with RD1, the membrane-bound activity of the RD1-CAT chimera followed that of the plasma membrane marker 5'-nucleotidase. Plasmids containing chimeric cDNAs were exposed to a coupled transcription-translation system that, in addition to the full-length chimeras, was found to generate a range of N-terminal truncated species due to initiation at different methionine residues. Incubation of the mature protein products formed in this system with a COS cell membrane fraction showed that only those chimeric CAT constructs containing the first 25 amino acids of RD1 became membrane-associated. The unique 25 amino acid N-terminal domain of RD1 contains structural information that can confer membrane association upon an essentially soluble protein.
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PMID:Chimeric constructs show that the unique N-terminal domain of the cyclic AMP phosphodiesterase RD1 (RNPDE4A1A; rPDE-IVA1) can confer membrane association upon the normally cytosolic protein chloramphenicol acetyltransferase. 777 57

Hepatic 5'-nucleotidases of vertebrates were investigated for localization in the lysosomes and the plasma membrane, microheterogeneity of the glycosylphosphatidylinositol (GPI)-anchor moiety and minimal requirement of the C-terminal signal peptide for GPI attachment. Using PIPLC of Bacillus thuringiensis and subcellular fractionation by Percoll gradient centrifugation, we found that chicken liver 5'-nucleotidase can be transferred from plasma membrane to lysosomes in the GPI-anchored or soluble form. Bovine liver ecto 5'-nucleotidase was solubilized by PIPLC, purified to a homogeneous state, and analyzed for the structures of GPI-anchor isoforms by HPLC and ESI-MS in combination with glycosidase treatments, after peptide-bond cleavage by CNBr or trypsin. Several isomers of the GPI anchor were thus characterized; major components contained two phosphorylethanolamine residues, whereas the component containing three phosphorylethanolamine residues was present only as a small percentage of the total. The cleavage/attachment site of the GPI anchor in the C-terminal of 5'-nucleotidase was shown to be Ser523. The peptide region cleaved off at the posttranslational processing has a length of 25 amino acid residues which contains a hydrophobic stretch of 17 amino acids. By site-directed mutagenesis, we determined the minimal length of the hydrophobic peptide to be 13 amino acids for expression of 5'-nucleotidase as a GPI-anchored form on the COS cell surface. When peptide length was shortened to less than 13 amino acids, the expressed enzyme was not sorted to the cell surface but present within, or secreted out of the cells.
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PMID:Studies on the GPI-anchored enzyme, hepatic 5'-nucleotidase. Microheterogeneity of the anchor, processing and localization. 808 Dec 55

In order to address the minimum domain of the COOH-terminal hydrophobic region responsible for GPI modification of bovine liver 5'-nucleotidase, we constructed a series of the deletion mutants of the COOH-terminus and expressed them in COS cells. Cells transfected by the deletion mutant of 6 amino acids (-IIILYQ) from the hydrophobic domain (-FSLIFLSVLAVIII-LYQ) did not show any elevation of cell surface-associated 5'-nucleotidase activity, whereas the 2 (-YQ) or 4 (-ILYQ) amino acid deletion mutant retained the bovine liver-derived activity on the cell surface as a GPI-anchored protein. Loss of half the hydrophobic domain (6 or 8 amino acids) resulted in accumulation of the activity in the cell. On the other hand, deletion of the whole hydrophobic domain (17 amino acids) or the entire cleaved-off domain (25 amino acids) made the product secreted into the medium. In conclusion, the hydrophobicity of 13 amino acids in length was enough for the GPI modification of the bovine liver 5'-nucleotidase.
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PMID:Mutational analysis of the COOH-terminal hydrophobic domain of bovine liver 5'-nucleotidase as a signal for glycosylphosphatidylinositol (GPI) anchor attachment. 814 26

A glycosylphosphatidylinositol (GPI)-anchored protein, 5'-nucleotidase [EC 3.1.3.5], was released from the membrane of bovine liver by use of phosphatidylinositol-specific phospholipase C (PI-PLC) of Bacillus thuringiensis and purified by several column chromatographies to a homogeneous state. The purified protein has an apparent molecular mass of 61 kDa, as estimated by SDS-polyacrylamide gel electrophoresis. From the partial amino acid sequence of a tryptic peptide, mixed oligonucleotides were synthesized and used to screen a lambda gt11 liver cDNA library, and one positive clone, pE1, was isolated. Since the insert of the clone lacked the NH2-terminal coding region, another lambda gt11 liver cDNA library was screened by using a synthetic probe corresponding to the 5' region of the insert of pE1. Three additional cDNA clones were obtained. Sequencing of these cDNAs revealed an open reading frame that encodes a 574-residue polypeptide with a calculated mass of 63,084 Da. The predicted structure showed two highly hydrophobic stretches at both ends of the protein, like those of rat and human 5'-nucleotidases. The NH2-terminal 26 residues comprise a signal peptide and the COOH-terminal hydrophobic stretch may serve as a signal for the posttranslational GPI modification. An expression vector of the cDNA, pSVNT, was constructed in a mammalian expression vector pSVL and the 5'-nucleotidase activity was transiently expressed in COS-1 cells. The expressed activity was about 8 times higher than the pSVL-transfected control activity. PI-PLC released 45% of the transiently expressed 5'-nucleotidase activity, indicating that the cDNA isolated here encodes this enzyme expressed as a GPI-anchored protein.
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PMID:Purification and cDNA cloning of bovine liver 5'-nucleotidase, a GPI-anchored protein, and its expression in COS cells. 834 Mar 54

Pneumocystis carinii pneumonia is a hallmark disease associated with AIDS. An abundant glycoprotein, termed gpA, on the surface of P. carinii is considered an important factor in host-parasite interactions. The primary structure of ferret P. carinii gpA contains a carboxyl-terminal sequence characteristic of a signal for glycosylphosphatidylinositol (GPI) anchors. Here we report the capacity for this gpA carboxyl sequence to direct attachment of a secreted protein, human growth hormone (hGH), to the membranes of COS cells. A control fusion protein (hGHDAF37) was obtained which, under the direction of the GPI signal from decay accelerating factor, directs hGH cell surface expression. A construct (phGH2-1A30) was created similar to hGHDAF37 by fusing hGH to the putative GPI signal sequence encoded in the terminal 30 residues from a ferret P. carinii gpA cDNA clone. By indirect immunofluorescent staining, hGH was detected on the surface of COS cells transfected with phGH2-1A30; this surface location was confirmed by confocal laser cytometry. Metabolic labeling with [3H]ethanolamine and subsequent immunopurification of hGH from cells transfected with phGH2-1A30 confirmed that a lipid moiety characteristic of a conventional GPI anchor was linked covalently to hGH, and cell surface hGH2-1A30 fusion protein was sensitive to enzymatic cleavage by phosphatidylinositol-phospholipase C. Furthermore, hGH2-1A30 recombinant protein cofractionated with 5'-nucleotidase, a classical GPI-anchored membrane marker. Together, these results indicate that the carboxyl-terminal residues of ferret P. carinii gpA constitute a biologically functional GPI consensus domain, thus providing a potential mechanism for antigenic variation of P. carinii gpA during P. carinii pneumonia.
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PMID:The carboxyl terminus of Pneumocystis carinii glycoprotein A encodes a functional glycosylphosphatidylinositol signal sequence. 974 3

Adenosine increases blood flow and decreases excitatory nerve firing. In the heart, it reduces rate and force of contraction and preconditions the heart against injury by prolonged ischemia. Based on indirect kinetic arguments, an AMP-selective cytosolic 5'-nucleotidase designated cN-I has been implicated in adenosine formation during ATP breakdown. The molecular identity of cN-I is unknown, although an IMP/GMP-selective cytosolic 5'-nucleotidase (cN-II) and an ecto-5'-nucleotidase (e-N) have been cloned. We utilized the high abundance of cN-I in pigeon heart to purify a 40-kDa subunit for partial protein sequencing and subsequent cDNA cloning. We obtained a full-length clone encoding a novel 40-kDa peptide, unrelated to cN-II or e-N, that was most abundant in heart, brain, and breast muscle. Immunolocalization in heart showed a striated cytoplasmic location, suggesting association with contractile elements. Transient expression in COS-7 cells, generated a 5'-nucleotidase that catalyzed adenosine formation from AMP, which was increased during ATP catabolism. In conclusion, the cloning and expression of cN-I provides definitive evidence of its ability to produce adenosine during ATP breakdown.
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PMID:The mechanism of adenosine formation in cells. Cloning of cytosolic 5'-nucleotidase-I. 1036 22

Catabolism of AMP during ATP breakdown produces adenosine, which restores energy balance. Catabolism of IMP may be a key step regulating purine nucleotide pools. Two, cloned cytosolic 5'-nucleotidases (cN-I and cN-II) have been implicated in AMP and IMP breakdown. To evaluate their roles directly, we expressed recombinant pigeon cN-I or human cN-II at similar activities in COS-7 or H9c2 cells. During rapid (more than 90% in 10 min) or slower (30-40% in 10 min) ATP catabolism, cN-I-transfected COS-7 and H9c2 cells produced significantly more adenosine than cN-II-transfected cells, which were similar to control-transfected cells. Inosine and hypoxanthine concentrations increased only during slower ATP catabolism. In COS-7 cells, 5'-nucleotidase activity was not rate-limiting for inosine and hypoxanthine production, which was therefore unaffected by cN-II- and actually reduced by cN-I- overexpression. In H9c2 cells, in which 5'-nucleotidase activity was rate-limiting, only cN-II overexpression accelerated inosine and hypoxanthine formation. Guanosine formation from GMP was also increased by cN-II. Our results imply distinct roles for cN-I and cN-II. Under the conditions tested in these cells, only cN-I plays a significant role in AMP breakdown to adenosine, whereas only cN-II breaks down IMP to inosine and GMP to guanosine.
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PMID:Distinct roles for recombinant cytosolic 5'-nucleotidase-I and -II in AMP and IMP catabolism in COS-7 and H9c2 rat myoblast cell lines. 1076 85

Adenosine production catalysed by cytosolic 5'-nucleotidase (cN-I) regulates diverse physiological processes. We report here a mouse cN-I (mcN-I) cloned from heart and testis. The open reading frame contains several potential translation initiation sites, which yield similarly active 5'-nucleotidases. Using overexpression in COS-7 cells we showed that mcN-I, like the previously cloned pigeon cN-I, is activated by ADP and catalyses adenosine formation during ATP breakdown. The N- and C-termini of mcN-I and pcN-I are divergent. Deletion of the 12 C-terminal amino acids or the first 19 N-terminal amino acids of pcN-I does not diminish activity, although deletion of the first 31 N-terminal amino acids reduces activity by 70%. Overall mcN-I is only 66% identical to pcN-I or the recently cloned human cN-I (hcN-I), while hcN-I and pcN-I are 85% identical. We report here a partial hcN-I sequence that is only 70% identical with the published hcN-I amino acid sequence but is 87% identical with mcN-I. Both hcN-I sequences have perfect matches to distinct human genome sequences. Our data imply the existence of at least two genes for cN-I, cN-I(A), previously cloned from pigeon and human, and cN-I(B) that we report here from mouse and partially from human.
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PMID:Cloning of a mouse cytosolic 5'-nucleotidase-I identifies a new gene related to human autoimmune infertility-related protein. 1169 Jun 31

5'-Nucleotidase specific towards dCMP and AMP was isolated from avian breast muscle and characterized. It was found to be similar to a type-I form (cN-I) identified earlier as the AMP-selective 5'-nucleotidase responsible for adenosine formation during ATP breakdown in transfected COS-7 cells. Expression pattern of the cN-I gene in pigeon tissues indicated breast muscle as a rich source of the transcript. We purified the enzyme from this source using two-step chromatography and obtained an active homogenous preparation, free of ecto-5'-nucleotidase activity. The tissue content of the activity was calculated at 0.09 U/g wet weight. The specific activity of the enzyme preparation was 4.33 U/mg protein and it preferred dCMP and AMP to dAMP and IMP as a substrate. Its kinetic properties were very similar to those of the enzyme purified earlier from heart tissue. It was strongly activated by ADP. Inhibition by inorganic phosphate was more pronounced than in heart-isolated cN-I. Despite this difference, a similar physiological function is suggested for cN-I in both types of muscle.
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PMID:Isolation and characterization of pigeon breast muscle cytosolic 5'-nucleotidase-I (cN-I). 1594 Mar 49


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