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 uveitogenicity of several protein fractions of the bovine retinal pigment epithelium (RPE) was studied in Lewis rats, and a major pathogenic fraction was selected. Fresh RPE cells were carefully isolated and purified in order to minimize the presence of rod outer segments (ROS). The buffer-insoluble part of the cells was extracted by Triton X-100. Most uveitogenicity was found in the Triton-insoluble pigment and cytoskeleton-containing fraction of RPE (RPE-TI). The S-antigen and opsin contents of RPE-TI were too low to induce an inflammatory response, while transducin, IRBP and cGMP-phosphodiesterase were absent. Hence, a hitherto unknown uveitogenic RPE protein, called PEP-X, evoked the pathogenic response. A typical dose-dependent experimental autoimmune anterior uveitis (EAAU) developed when the rats were immunized with RPE-TI. Initially, mononuclear cells infiltrated the anterior segment. In subsequent severe stages polymorphonuclear cells predominated in the anterior chamber. EAAU differed in particular from the known forms of EAU induced by photoreceptor proteins in that the inflammation remained exclusively anterior and the photoreceptor cells and the pineal gland were not affected. In immunized rats the immune responses to ROS proteins were very low. In contrast, there were consistently high cellular and humoral immune responses to RPE-TI. As in experimental autoimmune (uveo)retinitis (EAU), the development of EAAU could be inhibited by cyclosporin treatment indicating T-cell-dependency. A combination of histopathological, immunological and biochemical results indicates that PEP-X is an intrinsic RPE protein that is highly pathogenic. In view of its characteristics, EAAU may be a valuable model for human acute anterior uveitis, the most prevalent form of uveitis.
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PMID:Experimental autoimmune anterior uveitis (EAAU), a new form of experimental uveitis. I. Induction by a detergent-insoluble, intrinsic protein fraction of the retinal pigment epithelium. 203 26

Isolated CNS myelin membranes were extracted with Triton X-100 under conditions previously established for the isolation of cytoskeletal proteins. Treated myelin retained much of its characteristic lamellar structure despite the removal of most of the major myelin basic protein (18.5 kDa) and the proteolipid protein, which together normally constitute 60% of the total myelin protein. The SDS-PAGE profile of this extract residue demonstrated an enrichment in proteins of Mr 30 to 60 kilodaltons (the Wolfgram group). The major myelin proteins were identified by antibodies on Western immunoblots, as were the 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNP), actin, tubulin, myelin-associated glycoprotein (MGP) and the 21.5 kDA MBP. The overall behavior of CNP, the 21.5 kDa MBP, MGP and tubulin towards Triton extraction is reminiscent of the behavior of other membrane-skeletal complexes, supporting the idea that these and other minor myelin proteins might be part of heteromolecular complexes with interactions spanning several lamellae of the myelin sheath.
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PMID:Triton X-100 extractions of central nervous system myelin indicate a possible role for the minor myelin proteins in the stability in lamellae. 245 78

A method for the isolation of plasma membrane fractions from Xenopus oocytes has been developed, and the membranes have been characterized biochemically and morphologically. Plasma membrane complexes prepared by this procedure consisted of large sheets of the membrane, with associated vitelline envelope (a nonmembranous meshwork of fibers) and cortical (secretory) granules still attached. The morphology of cell surface microvilli and coated pits was well preserved. Cortical granules were removed by gentle homogenization in a low ionic strength medium, and integral and peripheral membrane proteins were then separated from vitelline envelopes by detergent extraction and phase separation in Triton-X-114. Biochemical characterization of the plasma membrane fractions indicated substantial levels of 5'-nucleotidase and alkaline phosphodiesterase activity associated with the oocyte cell surface, with 44-66% recovery of these markers in the final membrane preparations. Lectin blotting and lectin affinity chromatography with Concanavalin A and wheat germ agglutinin were used to characterize the major glycoprotein species associated with the plasma membrane complexes. Plasma membrane fractions prepared by this procedure should be very useful in both biochemical and morphological studies of membrane protein sorting in the Xenopus oocyte system.
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PMID:Isolation of plasma membrane complexes from Xenopus oocytes. 271 44

Phosphatases and phosphodiesterases that hydrolyse polyphosphoinositides are described in both membrane and cytosol fractions of human, pig, rat, rabbit, and sheep erythrocytes using exogenous substrates. With suitably optimized assay conditions, Ca2+-dependent phosphatidylinositol bisphosphate (PIP2) phosphodiesterase activity was found in the hemoglobin-free cytosol fraction, as well as the membrane. Membrane activity is completely dependent upon Triton X-100 and salt and inhibited by cetyltrimethylammonium bromide (CTAB), while the soluble activity requires CTAB and is inhibited by Triton. A low Ca2+-dependent PIP2 phosphatase activity, not present in other tissues, was also detected. The cation-independent phosphatidylinositol phosphate (PIP) phosphatase is localized in the membrane in most species, while the diesterase and the PIP2 phosphatases (both Mg2+ and Ca2+ dependent) are localized in the cytosol. Rat and rabbit erythrocytes are atypical in having a substantial proportion of their Mg2+-dependent PIP2 phosphatase activities in the membrane. All activities are lowest in sheep erythrocytes, except the PIP phosphatase, most of which is soluble in this species. Ca2+-dependent PIP2 phosphatase activity is not correlated with the activity or subcellular distribution of any of the other hydrolases and seems to be a separate enzyme. All the phosphoinositide hydrolase activities, particularly the diesterase, are orders of magnitude lower in erythrocytes than in other tissues. Both soluble and membrane diesterase activities are lost as erythrocytes age. Soluble polyphosphoinositide diesterase does not seem to be active with membrane-bound substrate, since pig and sheep erythrocytes that have negligible membrane activity do not respond to Ca2+ loading, yet have substantial diesterase activity in the cytosol. This supports the view that the diesterase is not physiologically functional in normal erythrocytes.
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PMID:Soluble and membrane-bound polyphosphoinositide phosphohydrolases in mammalian erythrocytes. 283 56

This study shows that the membrane-permeable stereospecific 1-oleoyl-2-acetyl-sn-glycerol (OAG), which is the analog of the natural 1,2-diacylglycerol (DAG), can stimulate the growth of ascites tumor cells. OAG can fully replace high serum concentrations in the culture medium and stimulates DNA synthesis in a dose-dependent manner. Investigation of the protein kinase C (PKC) isolated from a Triton extract of a 100,000g membrane pellet revealed that OAG can directly activate this enzyme. Concomitantly the phosphorylation of several cytosolic proteins with the molecular weights of 26, 33, 49, 55, 64, and 90 kDa is observed which is also found in serum-stimulated cells. Since DAG as a second messenger molecule originates from the hydrolysis of phosphoinositides we have investigated the metabolism of these lipids after labeling the cells with [3H]inositol. In detail, we have measured the amount of radioactive inositol trisphosphate (IP3) and the phosphodiesterase hydrolyzing phosphatidylinositol-4,5-bisphosphate (PIP2). The decreased radioactivity level of IP3 in OAG-stimulated cells as compared to non-growing cells (1-2% serum) indicates a feedback regulation of PIP2 hydrolysis which is substantiated by a profound reduction of PIP2-specific phospholipase C activity. The reduced IP3 formation has apparently no inhibitory effect on the cytoplasmic free Ca2+ concentration of OAG-stimulated cells, suggesting that the Ca2+ release is not directly correlated to the amount of IP3, which is also demonstrated for the non-growing cells. These data indicate that OAG apparently has a duel effect on the inositol phospholipid-mediated signal transfer system.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Differential effect on inositol-phospholipid hydrolysis, cytosolic-free Ca2+ concentration, protein kinase C activity and protein phosphorylation of 1-oleoyl-2-acetyl-sn-glycerol growth-stimulated ascites tumor cells. 284 1

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

1. Endocytosis of formaldehyde-treated bovine serum albumin by rat liver sinusoidal cells has been followed by injecting rats with the protein labelled with 125I-tyramine cellobiose (125I-TCfBSA). 125I-TCfBSA is quickly taken up by the liver; the radioactivity present in the organ reaches a plateau 5-10 min after injection and is maintained for up to at least 180 min. During the first 5 min most of radioactivity remains acid-precipitable. After which, labelled acid-soluble components are produced at a constant rate for up to 30-40 min. 2. Differential centrifugation shows that radioactivity is first recovered mainly in the microsomal fraction. Within a few minutes it exhibits a distribution pattern similar to that of lysosomal enzymes, being chiefly located in the mitochondrial fractions. 3. Isopycnic centrifugation in a sucrose gradient of the microsomal fraction isolated 1 min after injection indicates a similar distribution for radioactivity and alkaline phosphodiesterase. Later, the microsomal radioactivity distribution curve is shifted towards higher densities and becomes distinct from that of the plasma-membrane enzyme. After isopycnic centrifugation in a sucrose gradient of the total mitochondrial fraction a considerable overlapping of acid-precipitable and acid-soluble radioactivity distributions is observed without significant changes with time. The same is observed in a Percoll gradient except that after a relatively long time (greater than 30 min) of injection a marked shift of radioactivity distribution towards higher densities occurs. 4. A pretreatment of rats with Triton WR 1339, a density perturbant of liver lysosomes, causes a striking shift of acid-soluble radioactivity distribution in a sucrose gradient towards lower densities while having markedly less influence on the acid-precipitable distribution. As a result, a distinction between the distribution of both kinds of radioactivity becomes clearly apparent. A preinjection of yeast invertase, modifies the acid-soluble distribution without having a significant effect on the acid-precipitable distribution up to 30 min after 125I-TCfBSA injection. 5. Glycyl-1-phenylalanine-2-naphthylamide largely releases acid-soluble radioactivity associated with the mitochondrial fraction, whatever the time after 125I-TCfBSA injection. On the other hand the proportion of acid-precipitable radioactivity present in the fraction that can be released is almost zero at 10 min after injection, and it later increases. 6. The results presented here are best explained by supposing that, after being trapped in small pinocytic vesicles, 125I-TCfBSA is quickly delivered to the endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Uptake and intracellular transport in rat liver of formaldehyde-treated bovine serum albumin labelled with 125I-tyramine-cellobiose. 339 Nov 77

Sea urchin spermatozoa demembranated with Triton X-100 in the presence of EGTA, termed potentially asymmetric, generate asymmetric bending waves in reactivation solutions containing EGTA. After they are converted to the potentially symmetric condition by extraction with Triton and millimolar Ca++, they generate symmetric bending waves in reactivation solutions containing EGTA. In the presence of EGTA, their asymmetry can be restored by addition of brain calmodulin or the concentrated supernatant obtained from extraction with Triton and millimolar Ca++. These extracts contain calmodulin, as assayed by gel electrophoresis, radioimmunoassay, activation of brain phosphodiesterase, and Ca++-dependent binding of asymmetry-restoring activity to a trifluorophenothiazine-affinity resin. Conversion to the potentially symmetric condition can also be achieved with trifluoperazine substituted for Triton during the exposure to millimolar Ca++, which suggests that the calmodulin-binding activity of Triton is important for this conversion. These observations suggest that the conversion to the potentially symmetric condition is the result of removal of some of the axonemal calmodulin and provide additional evidence for axonemal calmodulin as a mediator of the effect of Ca++ on the asymmetry of flagellar bending.
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PMID:Modulation of the asymmetry of sea urchin sperm flagellar bending by calmodulin. 392 94

A phosphodiesterase activity is shown to copurify with the plasma membrane fraction prepared by the two-phase partition method. The enrichment in phosphodiesterase parallels that of alkaline phosphatase, which is thought to be a typical membranous enzyme. Up to 66% of the phosphodiesterase activity can be solubilized by a treatment with 0.2% Triton X-100. Higher doses were ineffective in solubilizing more activity. Analysis by native gel electrophoresis showed that an activity extracted by 2 M NaCl migrated at the same position as 'soluble' phosphodiesterase of cytosolic or extracellular origin. In contrast, the Triton-solubilized enzyme had an apparently higher molecular weight. When subjected to charge shift electrophoresis on agarose gels in the presence of an ionic detergent, the Triton-solubilized phosphodiesterase displayed a hydrophobic character. This behaviour contrasts with that of 'soluble' phosphodiesterases, the electrophoretic mobility of which is unaffected by the presence of an anionic detergent. The hydrophobic character of the membranous enzyme was lost after gentle hydrolysis by papain.
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PMID:The hydrophobic character of the membrane-bound phosphodiesterase from Dictyostelium discoideum. 626 Feb 58

The ability of nonionic detergents to solubilize the membrane-bound enzymes of the brush-border plasma membrane of Hymenolepis diminuta was investigated. Of the detergents tested (Triton X-100, Tween 80, Brij 35, Lubrol PX and WX, W-1, and beta-octyl-D-glucoside), only Triton was an effective solubilizing agent. Optimal solubilization was achieved by incubating an isolated fraction of the brush-border membrane in the presence of 1% Triton X-100 for 60 min at 37 C, followed by centrifugation at 100,000 g for 60 min at 25 C. This treatment resulted in solubilization of 94% of the alkaline phosphohydrolase, 91% of the phosphodiesterase and ribonuclease, and 88% of the 5'-nucleotidase activities. The pH optima for enzymes solubilized in nonionic and ionic detergents (Triton and sodium dodecyl sulfate, respectively) did not differ. Isoelectric focusing of the Triton-solubilized material demonstrated the presence of at least 14 polypeptides, a majority of which had isoelectric points below pH 7.
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PMID:Solubilization of the membrane-bound enzymes of the brush-border plasma membrane of Hymenolepis diminuta (Cestoda) using nonionic detergents. 628 6


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