Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Intact secretory granules isolated from bovine adrenal medulla express tyrosine hydroxylase (TH) activity. Granule-associated TH sediments on continuous sucrose gradients with dopamine beta-hydroxylase, a marker for granule membranes, indicating that TH is associated with chromaffin granules. Membranes prepared from lysed granules retain TH, whereas granule contents are free of the enzyme. TH immunoreactivity was detected in granule membranes by immunoblot analysis using a polyclonal antiserum against TH. TH immunoreactivity cannot be removed from membranes by washes in high ionic strength buffers and is only partially removed from membranes by treatment with either urea or Na2CO3. TH can be removed from granule membranes by the detergents Nonidet P-40, Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Treatment of membranes with a phosphatidylinositol-specific phospholipase C did not remove TH, ruling out the possibility of a glycosyl phosphatidyl anchor. Fractionation of granule membranes by temperature-induced phase separation in Triton X-114 revealed that TH is recovered in phases in which integral (detergent phase) and hydrophobic (phospholipid phase) membrane proteins are typically found. By contrast, TH from adrenal cytosol fractionated exclusively into the aqueous phase along with other soluble proteins. Digestion of granules with various protease enzymes revealed that TH is resistant to degradation, suggesting that the enzyme is embedded within membranes. TH becomes phosphorylated when intact granules are exposed to the catalytic subunit of the cAMP-dependent protein kinase, indicating that at least the N-terminal region of TH is exposed on the cytoplasmic surface of granules. These results establish that a fraction of TH is an integral component of bovine granule membranes. The association of TH with granule membranes may play a role in coordinating TH activity and catecholamine release.
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PMID:Tyrosine hydroxylase in secretory granules from bovine adrenal medulla. Evidence for an integral membrane form. 196 7

The mode of attachment of the membrane-bound form of the chromaffin granule glycoprotein dopamine beta-hydroxylase has been investigated; specifically, the possibility of a covalently attached glycosyl phosphatidylinositol anchor has been examined in detail. Incubation of fragmented chromaffin granule membranes with phosphatidylinositol-specific phospholipase C led to no solubilization of dopamine beta-hydroxylase activity. Further, chemical analyses of the inositol and phosphate content of the soluble form of dopamine beta-hydroxylase indicate that the enzyme contains no covalently attached phosphate or inositol. The results of these studies allow elimination of phosphatidylinositol as a source of membrane anchoring for dopamine beta-hydroxylase and, coupled with previously available data, argue against a post-translational covalent modification of the enzyme as the mechanism of membrane attachment. Possible alternative models for the anchoring of membranous dopamine beta-hydroxylase are discussed.
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PMID:Bovine membranous dopamine beta-hydroxylase is not anchored via covalently attached phosphatidylinositol. 341 Aug 39

Metal selectivity of exocytosis was analyzed by comparing the effects of polyvalent metal cations Ca2+, Ba2+, Sr2+, Pb2+, La3+, Cd2+, Co2+, Tb3+, Mn2+, and Zn2+ on the release of norepinephrine (NE) from staphylococcal alpha-toxin-permeabilized bovine chromaffin cells. Pb2+, La3+, Cd2+, Sr2+, and Ba2+ activated NE secretion accompanied by the release of intragranular dopamine beta-hydroxylase but not cytosolic lactate dehydrogenase, indicating the activation of the mechanism of exocytosis. The release triggered by saturating concentrations of Pb2+, La3+, Cd2+, and Sr2+ was nonadditive with Ca2+, indicating a common site of action. In contrast, the Ba2(+)-evoked NE release was additive with Ca2+ and the Ca2+ agonists Pb2+, La3+, Cd2+, and Sr2+, suggesting that Ba2+ activates secretion at a site distinct from the Ca2+ receptor. In distinction to the NE release evoked by Pb2+, La3+, Cd2+, and Ba2+, the Sr(2+)-evoked NE release was associated with a significant elevation of Ca2+ concentration in the medium and abolished by Ca2+ chelation. This indicates that the secretagogue effect of Sr2+ was indirect and secondary to the displacement of bound Ca2+, Co2+ and Mn2+ inhibited the NE release evoked by Ca2+, Sr2+, Pb2+, La3+, and Cd2+ but had no effect on the Ba(2+)-dependent secretion. Tb3+ and Zn2+ were without effect on exocytosis.
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PMID:Metal selectivity of exocytosis in alpha-toxin-permeabilized bovine chromaffin cells. 859 35

Pituitary adenylate cyclase-activating polypeptide (PACAP)i a potent stimulant of catecholamine secretion, increased catecholamine production in cultured porcine adrenal medullary chromaffin cells. PACAP induced dose-and time-dependent increases in mRNAs for the catecholamine synthesizing enzymes, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH), with maximal 6- and 4-fold increases occurring at 8-16 h, respectively. The half-maximally and maximally effective PACAP concentrations for stimulation of TH and DBH gene expression were 0.5 and 3 nM, respectively. The TH protein level also showed an increase over the unstimulated basal level at 16-24 h in PACAP-stimulate cells. We previously demonstrated that PACAP activates both phospholipase C and adenylate cyclase in adrenal medullary cells. Addition of forskolin alone induced increases in mRNA expression of both TH and DBH. The phosphodiesterase inhibitor 3- isobutyl-1-methylxanthine potentiated the induction of TH and DBH mRNAs by PACAP. Addition of the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also caused increases in TH and DBH mRNA levels. In protein kinase C-downregulated cells pretreated with PMA for 24 h, the stimulatory effect of PACAP on TH and DBH gene expression was diminished. These results suggest that cAMP and protein kinase C mediate the PACAP-induced TH and DBH gene expression. Removal of extracellular Ca2+ with EGTA enhanced the PACAP-induced increases in both cellular cAMP and mRNA levels of TH and DBH, suggesting that Ca2+ has an inhibitory effect on the induction of TH and DBH mRNAs. In conclusion, the present study indicates that PACAP coordinately upregulates the gene expression of both TH and DBH by activating the cAMP and protein kinase C signaling pathways, leading to simulation of cate-cholamine synthesis, while Ca2+ negatively regulates TH and DBH gene expression in porcine adrenal medullary cells.
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PMID:Pituitary adenylate cyclase-activating polypeptide induces gene expression of the catecholamine synthesizing enzymes, tyrosine hydroxylase and dopamine beta hydroxylase, through 3',5'-cyclic adenosine monophosphate- and protein kinase C-dependent mechanisms in cultured porcine adrenal medullary chromaffin cells. 877 59