Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.6.1.1 (adenylate cyclase)
 19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The initial step in TSH action reflects binding of the hormone to specific receptor sites on the plasma membrane. Such binding has been studied using plasma membranes, homogenates, isolated thyroid cells grown in culture, and thyroid slices. 3-H- and iodinated TSH preparations have been used; the latter have been prepared using both chloramine-T and lactoperoxidase. Some of the discrepancies reported in the literature might reflect the different thyroid and hormone preparations and the variable incubation conditions which have been used. In general, good correlation exists between binding of TSH and activation of adenylate cyclase in thyroid plasma membranes. Data is reviewed related to activation of protein kinase in intact thyroid cells by TSH. Although there is impressive evidence for cyclic AMP mediation of effects of TSH on the thyroid, some data that are inconsistent with this concept are considered, especially in relationship to 32-P incorporation into phospholipid. The role of cyclic GMP in thyroid function is discussed.
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PMID:Thyroid-stimulating hormone and cyclic adenosine 3',5'-monophosphate in the regulation of thyroid gland function. 16 59

The radioreceptor assay system for TSH is considered to be useful in quantitating the hormone and analyzing the mechanism of its action. The assay was established, and the interaction of abnormal thyroid stimulators in Graves' patients was evaluated in this assay system. A 10,000xg fraction of human thyroid homogenates was used as the receptor. Human TSH supplied from NIH was iodinated by using lactoperoxidase. The binding of 125I-TSH to the receptor was small, and 125I-TSH was further purified by the receptor binding. The receptor (25mg equivalent), purified 125I-TSH, and standard TSH or a sample were incubated at 37 degrees C for 60 min in a final volume of 300 microliters. The binding of 125I-TSH to the receptor was time- and temperature-dependent with optimal binding under the conditions described above. The binding was completely inhibited by the addition of human, bovine and ovine TSH and partially inhibited by high concentrations of HCG, FSH-LH. However, there was no cross reactivity with insulin, prostaglandin E1, E2, T3, T4 and Nal. The assay was sensitive enough to detect 5 to 50 microU of TSH. The amount of TSH bound to the receptor was almost parallel to the TSH concentration which is necessary to stimulate human thyroid adenylate cyclase activity. Studies of dissociation kinetics and Scatchard plot indicated that there were two classes of TSH receptors in the human thyroid. A higher association constant was calculated as 1.5 x 10(8)M-1. LATS-IgG from a patient with Graves' disease completely inhibited the binding of 125I-TSH to the receptor, and studies of Lineweaver-Burk, plot suggested that TSH and LATS-IgG shared common binding sites. The radioreceptor assay of TSH appears to be useful in evaluating the abnormal thyroid stimulators present in Graves' disease.
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PMID:[Studies on the radioreceptor assay of TSH: the binding of 125I-TSH to the human thyroid receptor and the interaction of LATS-IgG (author's transl)]. 22 97

The plasmalemma and hyaline ectoplasm together constitute the sensory and motor organ of macrophages. The purpose of this study was to isolate this cell fraction in order to analyze it biochemically and functionally. Brief sonification of warmed rabbit lung macrophages caused release of heterodisperse hyaline blebs and filopodia, which were easily collected by differential centrifugation. Viewed in the electron microscope, these structures consisted of membrane-bounded sacs principally containing actin filaments. Some contained secondary lysosomes. They were enriched threefold over whole cell homogenates in specific adenylate cyclase activity and in trichloroacetic-acid-precipitable (125)I when derived from cells labeled with 125(I) by means of a lactoperoxidase-catalyzed reaction. These markers were found to have identical isopycnic densitites when macrophage homogenates were subjected to sedimentation in a focusing sucrose density gradient system, and these markers had densities distinct from those of other cytoplasmic organelles. These markers were therefore assumed to be associated with macrophage plasma membranes. The specific beta- glucuronidase activity of the bleb fraction was similar to that of homogenates, but the blebs had considerably lower specific succinic dehydrogenase activity and RNA content, and DNA was undetectable. Electrophoresis of blebs solubilized in sodium dodecyl sulfate on polyacrylamide gels revealed polypeptides co-migrating with macrophage actin-binding protein, myosin, and actin; blebs also had EDTA-activated adenosine triphosphatase activity characteristic of myosin. The concentrations of actin-binding protein and myosin were higher in blebs than in cells or cytoplasmic extracts, whereas actin concentrations were similar (relative to extracts) or only slightly greater (than in cells). Blebs and intact cells had high lactate dehydrogenase activities in the presence but not the absence of Triton X-100. Blebs and cells oxidased 1-[(14)C]glucose, and the rate of glucose oxidation was increased substantially in the presence of latex beads. We conclude that intact sacs of plasmalemma encasing contractile proteins and cytoplasmic enzymes can be isolated from macrophages. They are enriched in myosin and actin-binding protein, indicating that the contractile apparatus is regulated in the cell periphery. These structures have the capacity to respond to environmental signals. We suggest the name "podosomes" for them because of their resemblance to macrophage pseudopodia. We propose that podosome formation results from rapid dissolution of the cortical gel when the membrane is in an actively extended configuration.
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PMID:Peripheral hyaline blebs (podosomes) of macrophages. 92 88

A new and simplified method is described for preparation of turkey erythrocyte membranes which are essentially devoid of supernatant or nuclear contamination, but retain catecholamine-sensitive adenylate cyclase activity. These membranes have been solubilized in sodium dodecyl sulfate and analyzed by polyacrylamide gel electrophoresis and the major protein components identified. The turkey erythrocyte membranes exhibit a protein profile very similar to that of the human erythrocyte membrane, but contain a protein component of apparent molecular weight of 50000 which is not present in the human membranes. Three surface glycoprotein components of the turkey erythrocyte membranes (apparent molecular weights of 90000, 41000, and 26000) have been identified by periodic acid-Schiff staining of polyacrylamide gels and by cell surface 125I labeling using lactoperoxidase followed by polyacrylamide gel electrophoresis. After deoxycholate solubilization of membranes prepared from iodinated cells, glycoprotein with molecular weights of 90000 and 41000 bind to an infinity column of concanavalin A-Sepharose 4B and elute upon application of methyl alpha-Dmannopyrannoside. The lowest molecular weight glycoprotein component, however does not bind to the insolubilized concanavalin A.
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PMID:Proteins of the turkey erythrocyte membrane. 93 39

131I-TSH prepared by the lactoperoxidase method was used to study the binding of hormone to bovine thyroid plasma membrane. Specific binding was obtained using as little as 0.12 mU/ml 131I-TSH. Half-maximal binding occurred with 17.1 plus or minus 3.5 mU/ml and saturation at approximately 40 mU/ml. Scatchard plot analysis revealed two classes of binding sites, with association constants of 1.1 plus or minus 0.06 x 10(8) M(-1) and 1.4 x 10(7) M(-1) for the high- and low-affinity sites, respectively. Binding of 131I-TSH was linearly related to the amount of thyroid plasma membrane protein. Other polypeptide hormones and prostaglandin E1 did not inhibit specific TSH binding. Identical results were obtained using two TSH preparations of different biologic specific activity. 12.5 mU/ml unlabeled TSH decreased 131I-TSH binding 50%, and 156 mU/ml caused complete inhibition. After equilibrium of 131I-TSH binding was established, maximal displacement was achieved by 120 min using about 300 mU/ml TSH. However, only about one-half of the 131I-TSH was displaced. Although GTP potentiated the stimulation of adenylate cyclase by TSH, it inhibited binding of 131I-TSH. Binding of TSH correlated very well with activation of adenylate cyclase.
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PMID:Studies of thyroid-stimulating hormone binding to bovine thyroid plasma membranes. 114 91

Parathyroid hormone-like proteins (PTHLP) display actions in the kidney which are similar to those of parathyroid hormone (PTH). We compared the binding properties of PTHLP and PTH in canine renal cortical membranes to determine if they interacted with the same or different receptors. Radioiodination to high specific activity (greater than 400 microCi/micrograms) of [Nle8,18,Tyr34]human PTH-(1-34)amide and [Tyr36]PTHLP-(1-36)amide was performed using the lactoperoxidase method. Complete enzymatic digestion of both radioligands demonstrated that the peptides were monoiodinated. Both radioligands retained full biological activity in the renal adenylate cyclase assay, and neither was significantly degraded during incubation with highly purified canine renal membranes under binding assays conditions. Specific binding reached equilibrium by 20 min at 20 degrees C. Competition binding studies using unlabeled [Nle8,18,Tyr34]human PTH-(1-34)amide, [Tyr36] PTHLP-(1-36)amide, and bovine PTH-(1-34) with either radioligand revealed similar binding affinities for all three peptides. Biologically inactive PTHLP fragments did not show significant displacement. In contrast to its similar binding affinity, [Tyr36]PTHLP-(1-36)amide was 6-15-fold less potent than bovine PTH-(1-34) in the renal adenylate cyclase assay, suggesting less efficient receptor-effector coupling. Photoaffinity cross-linking using either radioligand in canine renal membrane labeled indistinguishable 70,000-dalton proteins. In the presence of multiple protease inhibitors, binding to an 85-kDa component was observed. Labeling of both receptor forms was specifically abolished by an excess of either cold peptide and dose-response curves using affinity cross-linked membranes corroborated the apparent binding affinities determined by conventional radioligand binding assays. We conclude that PTHLP-(1-36) and amino-terminal PTH analogues bind to indistinguishable receptors in canine renal cortical membranes, but display differential coupling to post-receptor events.
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PMID:Characterization of canine renal receptors for the parathyroid hormone-like protein associated with humoral hypercalcemia of malignancy. 253 69

Parathyroid hormone (PTH) receptors have been described in renal tissue from several species, but not in the rat. In this study, radioligand binding techniques were used to identify and characterize PTH receptors in rat kidney cortical membranes. The sulfur-free PTH analog [Nle8,18Tyr34]bovine PTH-(1-34)amide was iodinated using the iodogen method. This ligand was suitable for use in identifying PTH receptors in canine renal membranes, but not rat renal membranes. Synthetic, unsubstituted rat PTH-(1-34) was iodinated using the milder, lactoperoxidase technique and was purified by HPLC on a C8 column. [125I]rat PTH-(1-34) bound rapidly to both rat and dog renal membranes. At 22 degrees C reaction reached steady state within 20 minutes, and this level was maintained for at least 3 h. Specific binding was routinely greater than 90% for rat kidney and greater than 95% for dog kidney. Similar results were obtained at 4 degrees C with a longer time required to attain steady state (approximately 45 minutes). Binding was reversible as demonstrated by dissociation of bound ligand after either infinite dilution or displacement with excess nonradioactive PTH. Binding was saturable and of high affinity (rat kidney: Bmax = 2.3 pmol/mg protein, Kd = 3.1 nM, dog kidney: Bmax = 2.1 pmol/mg protein, Kd = 3.7 nM). Rat renal cortical adenylate cyclase activity was stimulated by rat PTH in a dose-dependent manner with an EC50 of 4 nM, a value in good agreement with the binding data. This study demonstrates the feasibility of identifying and characterizing parathyroid hormone receptors in rat renal cortical plasma membranes using radioligand binding techniques.
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PMID:Identification and characterization of parathyroid hormone receptors in rat renal cortical plasma membranes using radioligand binding. 255 87

We compared receptor binding and adenylate cyclase stimulation of intact bovine parathyroid hormone (bPTH)-(1-84) and the synthetic amino-terminal fragments, bPTH-(1-34) and rat PTH (rPTH)-(1-34). Radioligands for binding studies were prepared by the lactoperoxidase technique and purified by high-pressure liquid chromatography. In both canine renal membranes and cloned rat osteosarcoma cells the amino-terminal fragments bound to a single order of sites; the affinity of rPTH-(1-34) exceeded that of bPTH-(1-34), correlating with its higher potency in stimulating adenylate cyclase. In studies with oxidized bPTH-(1--84), the middle and carboxyl regions of intact PTH were found to bind to both tissues but with higher affinity to osteosarcoma cells than to renal membranes. Our results demonstrate that rPTH-(1--34) is the most favorable probe of amino-terminal PTH binding and the most potent of the PTH peptides in stimulating renal and osseous adenylate cyclase. The results also show that midregion and carboxyl determinants within intact PTH contribute to hormone binding, which does not correlate with adenylate cyclase activation and appears more significant for skeletal than for renal binding.
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PMID:Comparison of renal and osseous binding of parathyroid hormone and hormonal fragments. 386 82

A plasma membrane-enriched vesicle fraction has been prepared from Trypanosoma brucei by sonication and differential centrifugation on sucrose gradients. This fraction is enriched 5-fold in the plasma membrane marker enzymes adenyl cyclase (EC 4.6.1.1) and ouabain-inhibitable, (Na+ +K+)-dependent adenosine triphosphatase (EC 3.6.1.3). It is also enriched up to 14-fold in iodinated surface proteins, and up to 4-fold in (3H-mannose-labeled glycoproteins, of which the major variable surface coat glycoprotein is the main constituent. Proteins of the plasma membrane fraction and other subcellular fractions have been identified by electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gradient slab gels. Several high molecular weight surface glycopeptides have been selectively investigated and partially characterized by a combination of metabolic labeling with [3H]mannose, lactoperoxidase-catalyzed surface iodination, and affinity chromatography on Con A-Sepharose. In addition to the major variable surface coat glycoprotein (estimated Mr = 58000), there are several minor surface glycopeptides (Mr = 76000, 86000 and 92000-100000) which are apparent extrinsic membrane components, and two surface glycopeptides (Mr = 42000 and 130000) which are intrinsic membrane components.
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PMID:Identification and partial characterization of plasma membrane polypeptides of Trypanosoma brucei. 628 66

Iodinated native bovine parathyroid hormone (bPTH(1-84)) was separated from uniodinated hormone by reversed-phase liquid chromatography techniques after lactoperoxidase labeling. Analysis of iodinated residues after enzymatic digestion indicated that the major labeled product was largely monoiodinated on the sole tyrosine residue. This material retained full bioactivity in an in vitro renal adenylate cyclase assay. Binding of 125I-bPTH(1-84) to rabbit renal membranes at 4 degrees C was proportional to membrane protein concentration and was saturable and dissociable. Radioligand binding was inhibited by concentrations of unlabeled bPTH(1-84) required to stimulate adenylate cyclase in the same membrane preparation but was not inhibited by non-PTH peptides other than adrenocorticotropin at high concentrations (greater than 10 microM). Synthetic NH2-terminal analogues of bPTH(1-84) all elicited approximately equivalent inhibition of radioligand binding which was, however, less potent than unlabeled bPTH(1-84), suggesting a role for the carboxyl region of the molecule in the interaction of bPTH(1-84) with its receptor. Activity of the NH2-terminal agonists was similar to bPTH(1-84) in stimulating adenylate cyclase. Although substitution in sequence position one, of serine in human PTH(1-34) for alanine in bPTH(1-34), reduced activity in the adenylate cyclase assay, inhibition of 125I-bPTH(1-84) binding by both peptides and by an analogue of bPTH(3-34) was equivalent, consistent with a minimal contribution of the first 2 residues for receptor binding of the NH2-terminal region of PTH. The results illustrate the utility of the radiolabeled preparation of native bPTH we have developed and emphasize the importance of probing the PTH receptor with an intact hormone to maximize information concerning the mechanism of PTH action.
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PMID:Characterization of the rabbit renal receptor for native parathyroid hormone employing a radioligand purified by reversed-phase liquid chromatography. 629 18


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