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:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The switching on of the cGMP phosphodiesterase (PDE) in retinal rod outer segments by activated transducin (T alpha-GTP) is a key step in visual excitation. The finding that trypsin activates PDE (alpha beta gamma) by degrading its gamma subunit and the reversal of this activation by gamma led to the proposal that T alpha-GTP activates PDE by relieving an inhibitory constraint imposed by gamma (Hurley and Stryer: J. Biol. Chem. 257:11094-11099, 1982). We report here studies showing that the addition of gamma subunit also reverses the activation of PDE by T alpha-GTP-gamma S. A procedure for preparing gamma in high yield (50-80%) is presented. Analyses of SDS polyacrylamide gel slices confirmed that inhibitory activity resides in the gamma subunit. Nanomolar gamma blocks the activation of PDE by micromolar T alpha-GTP gamma S. The degree of activation of PDE depends reciprocally on the concentrations of gamma and T alpha-GTP gamma S. gamma remains bound to the disk membrane during the activation of PDE by transducin. The binding of gamma to the alpha beta subunits of native PDE is very tight; the dissociation constant is less than 10 pM, indicating that fewer than 1 in 1,700 PDE molecules in rod outer segments are activated in the absence of T alpha-GTP.
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PMID:Reciprocal control of retinal rod cyclic GMP phosphodiesterase by its gamma subunit and transducin. 283 61

A low-Km cyclic nucleotide phosphodiesterase solubilised from rat liver membranes by mild proteolysis with chymotrypsin has been purified to apparent homogeneity. The purification included chromatography on cellulose phosphate, Ecteola-cellulose, hydroxyapatite, a theophylline affinity matrix and HPLC on a DEAE-substituted column. The purified enzyme has linear kinetic plots with a Km of 0.24 microM and a Vmax of 6.2 mumol mg-1 min-1 with cyclic AMP as a substrate. It also hydrolyses cyclic GMP with a Km of 0.17 microM and a Vmax which is about a third of that with cyclic AMP. Cyclic GMP is also a competitive inhibitor of cyclic AMP hydrolysis with a Ki of 0.18 microM. The proteolytically solubilised enzyme has a subunit molecular mass of 73 kDa by SDS gel electrophoresis and of 130 kDa by HPLC size-exclusion chromatography, suggesting that it exists as a dimer. A partially purified preparation of this enzyme was used to raise antiserum in a sheep. The antiserum immunoprecipitated activity from liver and adipose tissue of rat and mouse. It had little activity against phosphodiesterase from other rat tissues or other species. Insulin-activated phosphodiesterase from both adipocytes and hepatocytes was immunoprecipitated by the antiserum suggesting that the purified enzyme was an insulin-sensitive phosphodiesterase.
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PMID:Purification of an insulin-sensitive cyclic AMP phosphodiesterase from rat liver. 283 72

ARPB is an age-related protein in the human brain which is present in individuals younger than approximately 40 years of age, but disappears or becomes remarkably decreased in individuals above this age (Yasuda, T. and Kishi, K. (1985) Proc. Japan Acad. 61B, 273-276). ARPB was isolated from four different cerebra obtained from subjects 0, 15, 35 and 37 years old, and purified to an electrophoretically homogeneous state. Its molecular weight was estimated to be 17,000 and 36,000-38,000 by SDS-polyacrylamide gel electrophoresis and gel filtration, respectively. The amino acid composition of the purified ARPB, containing an excess of acidic amino acid residues (about 33%), proved to be very similar to that of calmodulin. ARPB exhibited some of the properties characteristic of calmodulin, such as a Ca2+-dependent mobility change upon electrophoresis, and activation of calmodulin-deficient phosphodiesterase activity in a Ca2+-dependent manner. However, it was possible to distinguish ARPB and human calmodulin with regard to their kinetic parameters for the activation reaction of phosphodiesterase activity, despite a close similarity in their reaction mode. It can be concluded that ARPB belongs to one of the calmodulin group proteins, although it remains unknown whether ARPB is identical to calmodulin.
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PMID:Purification and properties of the age-related protein (ARPB) detected in human brain: comparison with human brain calmodulin. 283 38

In the inherited retinal degeneration of rd mice, cyclic GMP accumulates in affected rod photoreceptors prior to their degeneration. A deficiency in the activity of the visual cell phosphodiesterase apparently results in the accumulation of cyclic GMP. The cyclic GMP phosphodiesterase (PDE) of normal mouse photoreceptors is a heteromeric protein complex of about 170 kDa, consisting of the alpha beta catalytic unit and the gamma inhibitory unit. The isolated complex has low enzyme activity but it can be activated by incubation with histone. Affinity-purified polyclonal antibodies against the PDE complex of bovine rod outer segments were prepared and used to identify in retinas of both normal and rd mice PDE-immunoreactive polypeptides which comigrated on SDS-polyacrylamide gels with the large subunits (88 kDa) of the normal PDE complex. During development of normal retinas, the 88 kDa immunoreactive component of the PDE complex were detected by day 7, with immunoreactivity increasing throughout the second postnatal week. In rd retinas, the 88 kDa immunoreactivity increased after 9 postnatal days, decreased during rod photoreceptor degeneration, and was undetectable in mature rd retinas. Under nondenaturing conditions, the PDE-immunoreactive polypeptide of rd retinas sedimented on sucrose gradients with a sedimentation coefficient of 5.6S and an apparent molecular mass of about 105 kDa; no associated histone-activated PDE activity was detected. These findings show that PDE-immunoreactive polypeptides are synthesized in immature rd photoreceptors and that the PDE-immunoreactive polypeptides fail to form a PDE complex which is comparable to that of normal photoreceptors.
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PMID:Characterization of a phosphodiesterase-immunoreactive polypeptide from rod photoreceptors of developing rd mouse retinas. 284 77

HT 29, a cell line derived from a human colonic adenocarcinoma, is highly responsive to the vasoactive intestinal peptide (VIP) as shown by a more than 100-fold intracellular cAMP increase (Ka = 0.3 nM), the stimulations of protein kinase A (Ka = 0.1 nM) and the low-Km cAMP phosphodiesterase (Ka = 40 nM). Remarkably, adenylate cyclase, cAMP-dependent kinase and cAMP-specific phosphodiesterase are activated in a sequential manner. Binding studies with [125I]-labeled VIP indicate a high affinity site with a Kd value (0.5 nM) close to the activation constant value (Ka) of the three enzymes. The molecular structure of the VIP receptor was studied by immunological and chemical approaches. A monoclonal antibody (mAb 109-10-16) which partially decreased the binding of VIP to its receptor allowed the characterization of Mr = 53,000 and Mr = 48-49,000 polypeptides. More precise identification of protein components of the VIP receptor resulted from covalent cross-linking on intact HT 29 cells by four bifunctional reagents: dithiobis-(succinimidyl propionate) and its non-cleavable analog disuccinimidyl suberate, the photoactivable azido phenyl glyoxal and dimethylpimelimidate. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated a major band of Mr = 67,000 regardless of which cross-linker was used. The same band and an Mr = 49,000 species were found in experiments using a crude membrane fraction of HT 29 cells. Assuming one molecule of VIP (Mr = 3326) linked per polypeptide, these observations suggest that an Mr = 64,000 species belongs to the VIP specific plasma membrane receptor. This protein contains an Mr = 20,000 N-linked sialic acid rich oligosaccharidic moiety.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:HT 29, a model cell line: stimulation by the vasoactive intestinal peptide (VIP); VIP receptor structure and metabolism. 284 4

Membrane-associated, Type II (cGMP-activatable) cyclic nucleotide phosphodiesterase (PDE) from rabbit brain, representing 75% of the total homogenate Type II PDE activity, was purified to apparent homogeneity. The enzyme was released from 13,000 x g particulate fractions by limited proteolysis with trypsin and fractionated using DE-52 anion-exchange, cGMP-Sepharose affinity and hydroxylapatite chromatographies. The enzyme showed 105 kDa subunits by SDS-PAGE and had a Stokes radius of 62.70 A as determined by gel filtration chromatography. Hydrolysis of cAMP or cGMP showed positive cooperativity, with cAMP kinetic behavior linearized in the presence of 2 microM cGMP. Substrate concentrations required for half maximum velocity were 28 microM for cAMP and 16 microM for cGMP. Maximum velocities were approx. 160 mumol/min per mg for both nucleotides. The apparent Kact for cGMP stimulation of cAMP hydrolysis at 5 microM substrate was 0.35 microM and maximal stimulation (3-5-fold) was achieved with 2 microM cGMP. Cyclic nucleotide hydrolysis was not enhanced by calcium/calmodulin. The purified enzyme can be labeled by cAMP-dependent protein kinase as demonstrated by the incorporation of 32P from [gamma-32P]ATP into the 105 kDa enzyme subunit. Initial experiments showed that phosphorylation of the enzyme did not significantly alter enzyme activity measured at 5 microM [3H]cAMP in the absence or presence of 2 microM cGMP or at 40 microM [3H]cGMP. Monoclonal antibodies produced against Type II PDE immunoprecipitate enzyme activity, 105 kDa protein and 32P-labeled enzyme. The 105 kDa protein was also photoaffinity labeled with [32P]cGMP. The purified Type II PDE described here is physicochemically very similar to the isozyme purified from the cytosolic fraction of several bovine tissues with the exception that it is predominantly a particulate enzyme. This difference may reflect an important regulatory mechanism governing the metabolism of cyclic nucleotides in the central nervous system.
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PMID:Purification and partial characterization of membrane-associated type II (cGMP-activatable) cyclic nucleotide phosphodiesterase from rabbit brain. 284 74

Monoclonal antibodies to Neurospora crassa cyclic nucleotide phosphodiesterase (PDE I) were selected by their capacity to inhibit the enzyme activity. The monoclonal immunoglobulin, coupled to Sepharose 4B, was used for the affinity purification of PDE I activity. After SDS-polyacrylamide gel electrophoresis the affinity purified PDE I fractions showed a single polypeptide band of about 41 kDa. This band reacted in Western blots with the above mentioned monoclonal immunoglobulin.
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PMID:Cyclic nucleotide phosphodiesterase activity in Neurospora crassa. Purification by immunoaffinity chromatography and characterization. 284 23

The particulate, cGMP- and cilostamide-inhibited "low Km" cAMP phosphodiesterase was purified greater than 100,000-fold in good yield (approximately 20%) from bovine omental fat by a procedure similar to that utilized for purification of an analogous enzyme from rat epididymal adipose tissue; ten-fold more enzyme protein (20-30 micrograms) could be prepared from bovine omentum (25 kg) than from rats (approximately 900 fat pads). Kinetic parameters (all similar to those for the rat enzyme) were: for cAMP, Km and Vmax = 0.3 microM and 2.5 mumol/min/mg, respectively; for cGMP, 0.8 microM and 1.6 mumol/min/mg. For inhibition of cAMP hydrolysis, IC50 values were: for cGMP = 0.6 microM and for OPC 3911, milrinone, CI 930, 0.1-2.0 microM. The purified enzyme, the activity of which eluted from Sephadex G-200 with Mr(app) = 110,000 and was associated with a single protein band during non-denaturing electrophoresis, exhibited on SDS-PAGE silverstained bands of 62 (probably a 61-63 doublet) and 77 kDa, perhaps due to proteolytic nicking. On Western blots, each of the polypeptides cross-reacted with a monoclonal antibody toward the bovine cardiac low Km cAMP and polyclonal rabbit antibody generated toward the purified bovine omental phosphodiesterase. Rabbit anti-phosphodiesterase IgG, which inhibited bovine and rat phosphodiesterase enzymatic activity, did not cross-react with purified bovine retina cGMP-binding or bovine cGMP-stimulated phosphodiesterase.
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PMID:Purification, properties and polyclonal antibodies for the particulate, low Km cAMP phosphodiesterase from bovine adipose tissue. 285 60

8-Azido cAMP photoaffinity labeling of cAMP-dependent protein kinase regulatory subunits (R1 = 49 K;R2 = 55K) was done on spermatozoa from species lacking, and species containing an epididymis. Spermatozoa from sea urchin and trout contained only R1, while rat caudaepididymal spermatozoa contained both R1 and R2 subunits. This was established by the Mr value of the 8-azido cAMP photolabeled moieties, and a biochemical analysis based on the known differences of protein-nucleotide interactions of Type I and II cAMP-dependent protein kinases. Sea urchin and trout sperm R1 subunits were similar to mammalian sperm R1 subunits in co-migration on SDS-polyacrylamide gels and in both saturation and specificity of nucleotide binding. Calcium enhanced photoprobe binding to rat R1 and R2 subunits and to sea urchin R1 subunit without revealing a sea urchin R2 subunit. Likewise, phosphodiesterase incubation of sea urchin and trout spermatozoa prior to photolabeling did not reveal R2 subunits. These data suggest that the cAMP regulation of sperm physiology may require R1 subunit in species both with and without an epididymis. Further taxonomic study is necessary to determine whether evolutionary acquisition of the epididymis and internal fertilization may have created unique environments favoring the addition of sperm R2 regulatory subunits of cAMP-dependent protein kinase.
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PMID:A comparative analysis of cAMP-dependent protein kinase regulatory subunits in sea urchin and rat spermatozoa. 299 17

Nucleotide pyrophosphatase was purified from human placenta to near homogeneity with a specific activity of about 500-fold over the Triton extract of the homogenate. Purification was achieved most effectively by successive chromatographic steps with AMP-agarose and ADP-agarose columns, based on the affinity of the enzyme towards 5'-adenylate and adenosine 3',5'-diphosphate, and a lectin-Sepharose column, based on the glycoprotein nature of the enzyme. The purified enzyme was found to be essentially homogeneous on SDS-polyacrylamide gel electrophoresis with a mobility corresponding to 130K. The purified enzyme was found to hydrolyze a wide variety of nucleotides, i.e. 3'-phosphoadenosine 5'-phosphosulfate (PAPS), adenosine 5'-phosphosulfate (APS), NADH, ATP, nucleotide sugars, oligonucleotides, and p-nitrophenyl-thymidine 5'-phosphate (PNTP). From the oligonucleotides, the enzyme produced 5'-phosphates. Mg2+ was required for full activity. Glycine and sulfhydryl compounds such as 2-mercaptoethanol and 2,3-dimercapto-1-propanol were inhibitory. Most of these properties are common to nucleotide pyrophosphatases [EC 3.6.1.9] and type I (5'-phosphate forming) phosphodiesterases [EC 3.1.4.1] from various sources. The relevance of this enzyme to a unique genetic disease, Lowe's syndrome, is discussed.
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PMID:Purification and properties of nucleotide pyrophosphatase from human placenta. 300 Oct 38


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