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 amphibian photoreceptor rod outer segment contains a guanine nucleotide-binding complex which consists of a 39,000-dalton polypeptide that binds guanine nucleotides (G protein), a 36,000-dalton polypeptide (H protein), and an approximately 6,500-dalton polypeptide. Sensitivity to trypsin proteolysis was utilized as a probe of structure-function relationships for these polypeptides. Digestion of the H protein generated fragments of 26,000 and 15,000 daltons whose proteolytic susceptibility was not altered by guanosine triphosphates, light, or membranes. The approximately 6,500-dalton polypeptide was not trypsin sensitive. When the G protein was eluted from illuminated membranes by GTP, trypsin proteolysis cleaved a terminal 1,000-dalton fragment (G1) to yield a 38,000-dalton fragment (G38). With increased digestion time, a 6,000-dalton fragment (G6) was removed from G38 to yield a 32,000-dalton fragment (G32). G32 was subsequently digested to fragments of 23,000 and 12,000 daltons. However, when the G protein was eluted from illuminated membranes by hydrolysis-resistant analogues of GTP, G32 was protected from further digestion. This is consistent with a GTP-induced conformational change in the G protein which is altered by GTP hydrolysis. Proteolysis of the G protein after covalent labeling with a photoaffinity analogue of GTP demonstrated that the analogue is bound to first G38 and then G32, indicating the GTP-binding site is associated with G32. Fragment G6 was cleaved when the G protein was soluble or bound to unilluminated membranes. However, when bound to illuminated membranes, fragments were generated reflecting the loss of 7,500, 9,000, or 11,000 daltons from the G protein. This light-induced alteration in proteolytic susceptibility indicates there is a light-induced conformational change in the G protein. Fragment G1 was not removed from the G protein when it was membrane bound, suggesting G1 is involved in binding to a membrane structure. These data suggest that the light-induced binding of the G protein to illuminated membranes and the reversal of this binding by GTP are mediated through conformational changes in the G protein and that three conformations exist: 1) a basal, inactive conformation; 2) a primed conformation induced by binding to photolyzed rhodopsin, with a high affinity for GTP; and 3) an active conformation, induced by binding of GTP, which activates the catalytic complex of light-activated phosphodiesterase.
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PMID:Limited trypsin proteolysis of photoreceptor GTP-binding protein. Light- and GTP-induced conformational changes. 632 13

A new, rapid method for purification of calmodulin-stimulated phosphodiesterase from bovine, ovine, and porcine brain using only DEAE-agarose and calmodulin-Sepharose chromatography is described. Purified enzymes from the three species each exhibited a single polypeptide of Mr approximately 59,000 on gel electrophoresis under denaturing conditions. Proteolysis of ovine and bovine enzymes with alpha-chymotrypsin, however, yielded different peptides, indicating that these proteins differ in primary sequence. Homogeneous preparations of bovine and ovine enzymes (purified approximately 5,000- and 2,000-fold, respectively) had different specific activities, although their substrate affinities and activation by calmodulin (8- to 14-fold activation, Kact approximately 1 nM) were very similar. The total amount in ovine was almost twice that in bovine brain. The hydrodynamic properties of bovine and ovine enzymes were indistinguishable with a Stokes radius of 4.35 nm and s20,w of 5.95 S. The calculated frictional ratios of 1.30 to 1.38 suggest a slightly asymmetric molecule. Equilibrium sedimentation data yielded apparent Mr approximately 57,000 in the presence of 6 M guanidine and 124,000 and 112,000 for the native bovine and ovine enzymes, respectively. In addition to the enzyme that was purified to homogeneity (pI approximately 5.6), a major fraction of calmodulin-activated phosphodiesterase with a lower isoelectric point was found in bovine and ovine brain. Whether these represent isozymes, perhaps localized in different types of cells, or whether one is a post-translationally modified form, remains to be determined. The existence of these two otherwise very similar forms of the enzyme has apparently not been previously recognized.
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PMID:Purification and properties of calmodulin-stimulated phosphodiesterase from mammalian brain. 632 22

Employing the recombinant runaway replication plasmid pDPK13 [sbcB+], an exonuclease I-overproducing derivative of Escherichia coli K12 has been constructed. The strain SK4258 has exonuclease I activity 140-400-fold higher than wild type control levels. A new purification procedure has been developed such that the protein can be purified to near homogeneity and is free of endonuclease and RNase activities. The specific activity of the purified enzyme is 10-fold higher than reported previously (Ray, R.K., Reuben, R., Molineux, I., and Gefter, M. (1974) J. Biol. Chem. 249, 5379-5381). Native exonuclease I is a single polypeptide having Mr = 55,000 with a Stokes radius of 3.12 nm.
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PMID:Amplification and purification of exonuclease I from Escherichia coli K12. 634 75

During growth and maturation of the tapeworm, Hymenolepis diminuta, significant decreases occur in the brush border membrane-bound alkaline phosphatase, phosphodiesterase, 5'-nucleotidase, adenosine triphosphatase and ribonuclease activities. These decreases are accompanied by qualitative and quantitative changes in the polypeptide profiles of the brush border membrane fraction. Gradients of enzymatic activities and polypeptide profiles are also demonstrable when mature tapeworms are cut into pieces and the brush border membrane of each piece analyzed individually. In fully developed tapeworms the enzymatic activities and polypeptide profiles of membrane preparations reflect mainly the contributions of the more mature proglottids; these proglottids constitute most of the tapeworm biomass. The most anterior sections of these fully developed worms are biochemically similar to young, developing worms.
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PMID:Alterations in brush border membrane proteins and membrane-bound enzymes of the tapeworm, Hymenolepis diminuta, during development in the definitive host. 663 65

TRH was found to rapidly influence 32PO4 incorporation into phospholipids of PRL-secreting GH pituitary cells. Analogs of TRH were found to exert similar effects, with potencies related to receptor-binding affinity. Additional PRL-releasing agents were also tested. Bombesin exerted a similar effect, whereas vasoactive intestinal polypeptide, 8-bromo cAMP, phosphodiesterase inhibitors, 50 mM K+, and scorpion venom toxin had no influence. Cationophore A23187 stimulated phospholipid labeling in a manner distinguishable from that of TRH. Chromatographic analysis showed the action of TRH to be restricted to the labeling of phosphatidylinositol and phosphatidic acid. Kinetic studies indicated a rapid influence of TRH on phosphatidylinositol breakdown, with subsequent accelerated 32PO4 incorporation into phosphatidylinositol and phosphatidic acid. These studied identified a rapid, receptor-mediated, cAMP-independent action of TRH on phospholipid metabolism. Similar effects of other hormones are believed to be involved in promoting cellular Ca2+ translocation. The rapid onset of the response reported here suggests that this event may play a role in mediating the PRL-releasing effects of TRH and bombesin in GH cells.
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PMID:Thyrotropin-releasing hormone (TRH) selectively and rapidly stimulates phosphatidylinositol turnover in GH pituitary cells: a possible second step of TRH action. 680 Jul 71

The amphiphatic polypeptide melittin migrates as an equimolar complex with bovine brain calmodulin when monitored by gel disc electrophoresis or gel filtration in the presence of Ca2+, even in 4M-urea. The complex disassociates in the presence of EDTA and urea. The affinity is of the same order as that of calmodulin for its target enzymes, and more than 1000-fold higher than that of calmodulin for basic peptide hormones or hydrophobic drugs. The activation of brain phosphodiesterase by calmodulin is inhibited by melittin. The kinetics of inhibition suggest competition between the enzyme and melittin for calmodulin. The calmodulin-melittin interaction may constitute a model for that existing between calmodulin and its target enzymes.
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PMID:Ca2+-dependent high-affinity complex formation between calmodulin and melittin. 684 15

The calcium-dependent regulator protein, calmodulin, is a 17,000 molecular weight polypeptide which binds calcium and has been shown to confer calcium sensitivity on contractile and other proteins. In the present study, we have examined the presence and subcellular distribution of this protein in preparations of human platelets. Calmodulin was quantified using a two-stage phosphodiesterase assay. Whole platelets contained 1.33 +/- 0.06 units calmodulin per 10(6) platelets or 26.5 +/- 3.4 fg calmodulin per platelet. The distribution of calmodulin in the platelet was predominantly soluble with over 80 percent of calmodulin activity in the soluble fraction of the cell. There was no apparent difference in the distribution of calmodulin between soluble and particulate compartments in recalcified platelet homogenates compared to homogenates in EDTA. Indirect immunofluorescent studies with monospecific antisera to dinitrophenylated calmodulin showed intense staining of platelets in a diffuse pattern. The identification of calmodulin in platelets raises the possibility that this protein may participate in calcium-dependent reactions important in platelet aggregation and release.
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PMID:Platelet calcium-dependent proteins: identification and localization of the calcium-dependent regulator, calmodulin, in platelets. 701 28

In this paper we demonstrate the presence and localization of calmodulin, a calcium-dependent regulatory protein, in the ciliated protozoan Paramecium tetraurelia. Calmodulin is demonstrated by several criteria: (a) the ability of whole cell Paramecium extracts to stimulate mammalian phosphodiesterase activity, (b) the presence of an acidic, thermostable, 17,000-dalton polypeptide whose mobility shifts in SDS polyacrylamide gel electrophoresis in the presence of Ca2+, and (c) the affinity of antibodies against mammalian calmodulin for a Paramecium component as demonstrated by both indirect immunofluorescent localization and radioimmunoassay. Indirect immunofluorescence studies reveal that Paramecium calmodulin is distributed in three distinct regions of the cell, i.e., (a) large, spherical cytoplasmic organelles representing perhaps the food vacuoles or other vacuolar inclusions of the cell, (b) along the entire length of oral and somatic cilia, and (c) along a linear punctate pattern corresponding to the kinetics (basal bodies) of the cell.
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PMID:Presence and indirect immunofluorescent localization of calmodulin in Paramecium tetraurelia. 701 19

The 10 S DNA polymerase alpha from calf thymus (Masaki, S., and Yoshida, S. (1978) Biochim. Biophys. Acta 521, 74-88) has been purified to near homogeneity. The most purified fraction obtained by repeated sucrose rate-zonal centrifugation contained three large polypeptides of 150,000, 145,000, and 140,000 daltons and three to four smaller polypeptides ranging from 43,000 to 50,000 daltons. A good resolution of these polypeptides was achieved on a sodium dodecyl sulfate-polyacrylamide linear gradient gel (5-20%) which was stained by the silver stain method. The three large polypeptides were also observed in the more crude fractions prepared in the presence of three kinds of protease inhibitors. By a peptide mapping analysis, it was revealed that these three polypeptides have a similar primary structure. Treatments of the enzyme with alkaline phosphatase, phosphodiesterase, and neuraminidase did not affect the gel pattern. These results indicate that the 10 S DNA polymerase alpha of calf thymus has a microheterogeneity in terms of the large polypeptide component. Among these three large polypeptides, the two polypeptides of 150,000 and 145,000 daltons disappeared by keeping the sucrose gradient fraction at 4 degrees C in the absence of glycerol, while the 140,000-dalton polypeptide was well preserved. The poly(rA)oligo(dT)-dependent activity of 10 S DNA polymerase alpha was selectively lost under this condition.
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PMID:10 S DNA polymerase alpha of calf thymus shows a microheterogeneity in its large polypeptide component. 708 21

Plasma membranes were isolated from lactating bovine mammary gland. Two crude membrane fractions; medium/d 1.033 (light membrane) and 1.033/1.053 interfaces (heavy membrane), were obtained by Ficoll density gradient centrifugation of osmotically washed microsomal fraction. Two crude membranes were further purified separately by sucrose density gradient centrifugation. Both light and heavy membranes banded at a sucrose density of 1.14. The purified membranes appeared as heterogeneous smooth membrane vesicles on electron microscopy. The contaminating suborganelles were not detected. The yield of the purified membranes relative to the homogenate was 1.2%. The degree of purity of the membranes was shown by a great increase in the specific activity of 5'-nucleotidase over the homogenate of 20-fold for light membrane and of 16-fold for heavy membrane. The relative activities of Mg2+-ATPase, (Na+ + K+)-ATPase, gamma-glutamyl transpeptidase, phosphodiesterase I, alkaline phosphatase and xanthine oxidase were also high (12-18-times) and nearly 20% of these enzymes was recovered. The activity of marker enzyme for mitochondria, endoplasmic reticulum and Golgi apparatus was very low, while that of acid phosphatase for lysosome was relatively high (5-times). DNA and RNA contents were very low. The major polypeptides rich in other suborganelles were not detected profoundly in the membrane fraction and the polypeptide composition in both light and heavy membranes were similar upon SDS-polyacrylamide gel electorphoresis.
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PMID:Isolation and characterization of plasma membrane from lactating bovine mammary gland. 720 55


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