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:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An amplifiable eukaryotic expression system, based upon glutamine synthetase, has been applied to the production of a complex integral membrane glycoprotein, the human receptor for the polypeptide hormone thyrotropin (TSH). Production of recombinant protein was achieved in chinese hamster ovary (CHO) cells at levels at least 10-fold higher than has been achieved in any other system. After amplification of the inserted gene, the gene copy number was found to be increased in most (but not all) subclones in the range of 3- to 50-fold; mRNA levels of the individual cell lines broadly followed their gene copy number. The level of protein production (measured both functionally and structurally, by radioligand binding and cytofluorimetry, respectively) also reflected these increases in DNA and RNA, but appeared to be limited to a maximum value which we conclude is the maximum that the cells can tolerate without impairing their viability. The receptor is efficiently coupled to adenylate cyclase (22-45 pM TSH producing a 50% response), although the coupling mechanism appeared to be saturated at higher receptor numbers. The high level of expression has allowed, for the first time, the detection of recombinant TSH receptor by immunochemical means. This expression system should prove very useful, not only in facilitating characterization of the TSH receptor, but also for the production of many other integral membrane proteins in their native form.
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PMID:Characterization of the glutamine synthetase amplifiable eukaryotic expression system applied to an integral membrane protein--the human thyrotropin receptor. 128 93

We report on characterization of a 170,000 Da glycoprotein found exclusively in the PNS. We refer to this protein as the Schwann cell membrane glycoprotein (SAG). SAG contains the HNK-1 carbohydrate, which is considered by some to be a marker of adhesion molecules. Its N-terminal sequence is not similar to previously known polypeptide sequences. SAG is found exclusively in the PNS, is present in rat sciatic nerve prior to myelination, and is in both myelinating and nonmyelinating Schwann cells. Tumors of Schwann cell lineage express SAG where axons are present (neurofibromas) but do not in the absence of axons (schwannomas). Schwannoma cells in culture do not express SAG even when exposed to forskolin, an activator of adenylate cyclase. However, schwannoma cells grown in the presence of a neuronal cell line (PC12) express SAG.
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PMID:SAG: a Schwann cell membrane glycoprotein. 137 75

We have examined the in vitro effects of DN 9693 (piperidinylimidazo-quinazolinone) on various aspects of platelet reactivity. Our results are consistent with its known function as a phosphodiesterase inhibitor in that it increased platelet cyclic AMP, particularly in conjunction with an adenylate cyclase stimulator, and exerted a profound inhibitory effect on platelet aggregation responses to a variety of agonists. DN 9693 also inhibited ristocetin-induced platelet agglutination (RIPA). We therefore examined its effect on ristocetin co-factor assays and on the binding of a monoclonal antibody (McAb) to platelet membrane glycoprotein Ib (GPIb). The drug inhibited the binding of the monoclonal antibody in a dose-dependent manner. This suggests an effect of the drug on the platelet surface membrane with reduced expression of GPIb. Our results indicate that in addition to its anticipated inhibitory effect on platelet aggregation, DN 9693 may also inhibit platelet adhesion.
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PMID:DN 9693: a phosphodiesterase inhibitor with a platelet membrane effect. 254 86

Acquired resistance to chemotherapeutic agents is an important clinical problem. One preclinical model, termed multidrug resistance (MDR), is characterized by a complex phenotype of cross-resistance to biochemically unrelated antineoplastic agents, the presence of a high-molecular-weight membrane glycoprotein, and impaired accumulation of drug. To determine whether MDR is mediated in part by altered cyclic 3',5'-adenosine monophosphate (cAMP) levels, the effect of incubation with the adenylate cyclase agonist, forskolin, was investigated in the murine sarcoma S180 cell line and two MDR variants (A5-.8, A5-2.5). Basal cAMP levels in sensitive and MDR lines were not significantly different (range, 0.15 +/- 0.05 to 0.31 +/- 0.09 pmol/mg protein); however, 1-h incubation with forskolin, 10 microM, elevated intracellular cAMP 2-fold in the parent line and 43- and 35-fold in the variants. The adenylate cyclase agonists, prostaglandin E2 and cholera toxin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine had no significant effect on cAMP levels. To determine the effect of forskolin on doxorubicin-induced cell lethality, S180 and MDR lines were incubated with doxorubicin plus forskolin for 1 h and cloned in soft agar. Coincubation with forskolin partially reversed doxorubicin resistance in the MDR lines in a dose-dependent fashion. To determine whether this effect was mediated solely by elevation of intracellular cAMP, the inactive 1,9-dideoxy analogue of forskolin (DF) was used. Incubation with DF resulted in no elevation of cAMP levels in the sensitive or resistant cell lines; however, DF also partially reversed doxorubicin resistance in the MDR variants. Furthermore, coincubation of the A5-2.5 cell line with doxorubicin and 8-bromo cAMP, 1 mM, did not result in reversal of resistance to doxorubicin. To determine whether the reversal of resistance by the diterpenes was associated with alteration of doxorubicin transport, uptake and efflux of [14C]doxorubicin were measured. Coincubation with both forskolin and DF, 10 microM, enhanced [14C]doxorubicin uptake in the resistant cells, while drug efflux was significantly affected only in the cell line exhibiting intermediate resistance. Since both forskolin and its inactive analogue are effective in partially reversing resistance to doxorubicin and augmenting anthracycline uptake, a mechanism other than elevation of cAMP is most likely responsible.
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PMID:Partial reversal of doxorubicin resistance by forskolin and 1,9-dideoxyforskolin in murine sarcoma S180 variants. 282 78

When individually tested, vasoactive intestinal peptide (VIP), cholera toxin, and monoclonal thyroid-stimulating antibodies (TSAbs), but not TSH binding-inhibiting antibodies (TBIAbs), elevate cAMP levels in cultured human thyroid cells. In this study, we tested the effect on cAMP levels in human thyroid cells of the simultaneous presence of VIP and the other ligands noted. Monoclonal TBIAbs (11E8 and 122G3), which interact with a membrane glycoprotein containing the high affinity binding site of the TSH receptor, did not alter VIP-induced cAMP accumulation in the thyroid cells. These data indicate that VIP and TSH bind to distinct sites on the cell membrane. In contrast, monoclonal TSAbs 208F7 and 307H6, which interact with a portion of the TSH receptor other than its high affinity binding site, not only did not have their usual agonist activity, but, instead, caused a marked inhibition of human thyroid cAMP accumulation when VIP was also present. Mutual antagonism by two agonists, i.e. inhibition evident when TSAbs and VIP were mixed, was also found when cholera toxin was coincubated with VIP. The inhibitory effect of mixing cholera toxin and VIP was nearly immediate and was duplicated with mixtures of the beta-subunit of cholera toxin and VIP. The inhibition evident in mixing VIP and the TSAbs or cholera toxin was not duplicated in mixtures of isoproterenol and VIP or in mixtures of forskolin and VIP. These results suggest that the mutual inhibition of VIP and either TSAbs or cholera toxin is at a step that couples the TSH and VIP high affinity receptor-binding sites to the adenylate cyclase complex.
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PMID:Unusual antagonistic actions of mixtures of vasoactive intestinal peptide, thyroid-stimulating antibodies, and cholera toxin on adenosine 3',5'-monophosphate accumulation in normal human thyroid cell cultures. 287 Oct 39

Through the use of specific staining and the analysis of the interaction of pure beta-adrenergic receptor of S49 mouse lymphoma cells with lectins immobilized to insoluble matrices, we establish that this beta-adrenergic receptor is a glycoprotein. The effects of swainsonine (0.2 microgram/ml), an inhibitor of Golgi mannosidase II, as well as those of tunicamycin (0.2 microgram/ml), an inhibitor of N-glycosylation, on the expression and function of this integral membrane glycoprotein were investigated in S49 mouse lymphoma cells grown in culture. Preexisting receptors on the cells were inactivated by alkylation with the beta-adrenergic antagonist ligand N-(2-hydroxy-3-naphthoxylpropyl)-N'-bromoacetyl-ethylenediamine. Swainsonine did not alter the number of beta-receptors measured in intact cells, the Bmax, or Kd of receptors measured in membranes prepared from these cells as assayed by [125I]iodocyanopindolol binding or their functional coupling to adenylate cyclase. Autoradiograms of membranes photoaffinity-labeled with [125I]iodoazidobenzylpindolol and subjected to electrophoresis on polyacrylamide gels reveal a reduction of 6,000 in the Mr of beta-receptors in membranes prepared from swainsonine-treated cells. This form of receptor was sensitive to endoglycosaminidase H, indicating its high mannose hybrid oligosaccharide nature. The number and affinity of beta-receptors in tunicamycin-treated S49 cells were normal. Whereas stimulation of cyclic AMP accumulation in cells or adenylate cyclase in membranes by prostaglandin E1 was essentially abolished by tunicamycin treatment, stimulation by isoproterenol was largely unaffected. The nonglycosylated receptor displays an Mr that is approximately 8,000-11,000 smaller than the native receptor. Thus, N-glycosylation does not affect the expression (steady-state) or function of the beta-adrenergic receptor, whereas prostaglandin E1 receptor function is lost. The role of N-glycosylation in receptor function is not universal among receptors coupled to adenylate cyclase.
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PMID:N-glycosylation in expression and function of beta-adrenergic receptors. 302 55

Some biochemical factors of the iris-ciliary body of the rabbit have been examined for effects induced by water-soluble marihuana-derived material (MDM). Adenylate cyclase activity and sensitivity to beta-adrenergic agonists were unchanged, as measured 4 hours after MDM administration in vivo. Magnesium-dependent and anion-sensitive, but not sodium-potassium, ATPase activities were inhibited 6 hours after MDM administration in vivo, although they were unaffected by in vitro incubation. Topical administration of a potent substance P antagonist had no effect on the time course or magnitude of intravenous MDM-induced ocular effects in rabbit. Intravenously administered sugars antagonized the effects of MDM on intraocular pressure. A variety of drugs which display a range of biochemical effects varying from beta-adrenergic receptor agonism, to alteration of glycoprotein residues were employed. None of the agents employed, ranging from cAMP modifiers to protein synthesis blockers, had any effect on the MDM-induced response. It is apparent that the mechanism underlying the ocular hypotensive effect of MDM does not reside in mediation through adenylate cyclase, ATPase or substance P, but rather through a mechanism mediated by terminal sugar moieties on the molecule. The data suggest that modification of the surface membrane glycoprotein residues on the ciliary epithelium can induce marked alterations in aqueous humor flow rate.
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PMID:Marihuana-derived material: biochemical studies of the ocular responses. 316 May 44

Two groups of mAbs reacting with external domains of a major sea urchin sperm membrane glycoprotein of 210 kD were isolated. Previous studies have shown that group I mAbs inhibit the acrosome reaction induced by egg jelly and also cause large increases in intracellular Ca2+ [( Ca2+]i). Group II mAbs, at comparable levels of cell surface binding, neither inhibit the egg jelly-induced acrosome reaction nor cause increases in [Ca2+]i. In this paper, we investigate the ability of these mAbs to induce the cAMP-dependent phosphorylation of sperm histone H1. Group I mAbs induce H1 phosphorylation to the same level and on the same peptide, as occurs upon treatment of sperm with egg jelly. These mAbs also activate adenylate cyclase to the same extent as egg jelly. Group II mAbs do not induce H1 phosphorylation and are only poor activators of adenylate cyclase. Group I mAbs compete with each other, but not with group II mAbs, for binding to the cell surface. These data indicate that the activation of adenylate cyclase is an initial event in the pathway leading from the binding of mAbs to a specific domain of the 210-kD protein at the cell surface, to the discrete phosphorylation of histone H1 in highly condensed sperm chromatin. The domain on the 210-kD protein recognized by group I mAbs plays a critical role in signal transduction during the early events of fertilization.
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PMID:Monoclonal antibodies to a membrane glycoprotein induce the phosphorylation of histone H1 in sea urchin spermatozoa. 319 82

The thyrotropin (TSH) receptor has been proposed to be composed of a membrane glycoprotein and a membrane ganglioside, the former important in high affinity recognition, the latter vital for message coupling to the adenylate cyclase system. The present study used two approaches, formation of antireceptor monoclonal antibodies and reconstitution, to validate the model and further examine the role of the ganglioside. Three kinds of monoclonal antireceptor antibodies are defined. One group which inhibits TSH binding and TSH functions, i.e., TSH-stimulated adenylate cyclase activity, iodide uptake, and thyroid hormone release, is shown to be directed against the glycoprotein component of the receptor. The second group includes antibodies which mimic TSH in all stimulatory actions, are competitive agonists of TSH, are equivalent to thyroid stimulating antibodies in the sera of patients with Graves' disease, and are directed against the ganglioside component of the receptor. These stimulating monoclonal antibodies are directed against a minor ganglioside membrane component which fractionates as a disialoganglioside. When this ganglioside is incorporated into 1-8 thyroid cells which have a correlated ganglioside deficiency and TSH receptor defect, reconstitution of TSH stimulated adenylate cyclase activity occurs. Whereas the first group of antibodies inhibits TSH-stimulated function, they do not inhibit the stimulatory antibodies which mimic TSH, an observation consistent with the 2 component hypothesis of the receptor model. The third group of antibodies have a mix of properties from the first two groups and suggests that the TSH receptor in situ is an actual complex of the two components or that there are common carbohydrate determinants in the functional sites of each receptor component. Implications of a TSH receptor structure in which its ganglioside and glycoprotein components are in equilibrium with pools of free components and, in turn, components important for cholera toxin, tetanus toxin and interferon receptors are discussed. In regard to the pathogenesis of Graves' disease, the data indicate that thyroid stimulating autoantibodies are autoimmune equivalents of cholera toxin with respect to the importance of ganglioside function. Since antiidiotype studies of antibodies against TSH confirm a structural relationship between receptors for thyrotropin, cholera toxin, and thyroid stimulating autoantibodies, the data establish an unequivocal role for the ganglioside in TSH receptor structure which facilitates interpretation of in vitro experiments aimed at understanding the mechanism of ganglioside-ligand interactions.
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PMID:Gangliosides, the thyrotropin receptor, and autoimmune thyroid disease. 633 Nov 33

In aggregometry studies employing platelet-rich plasma, monoclonal antibody 6D1 (6D1) binds to platelet membrane glycoprotein 1b (GP1b) and has been shown to abolish agglutination with ristocetin (1.5 mg/ml), with little effect on first-phase and slight diminution of second-phase aggregation with adenosine diphosphate (20 mumol/L). In perfusion studies with polyethylene microconduits (PE-100; interior diameter, 0.86 mm; length, 5 cm), platelet deposition (platelets/cm2) is restricted to a patchy monolayer when platelets are treated with 6D1 (10 micrograms/ml), providing a new model to study surface platelet adhesion isolated from platelet aggregation. The power of low-molecular-weigh dextran (DEX; mild inhibitor of fibrin formation and polymerization and von Willebrand factor-platelet interaction), aspirin (ASA; cyclooxygenase inhibitor), and prostaglandin E1 (PGE1; adenylate cyclase stimulator) to inhibit platelet adhesion on PE-100 was tested with this model. PE-100 segments were perfused with citrated human blood (5 ml, hematocrit, 35% +/- 5%) containing 6D1-treated, 111indium (111In-)labeled platelets (2.0 +/- 0.5 x 10(5) platelets/microliters) in a customized perfusion chamber (37 degrees C; flow, 1.2 ml/min; shear, 312 s-1). After a buffer flush under the same conditions, platelet deposition was measured by 111In-scintigraphy of the perfused conduits.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The inability of low-molecular-weight dextran, aspirin, and prostaglandin E1 to inhibit monolayer platelet adhesion to polyethylene. 768 20


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