THE ADENOSINE-TRIPHOSPHATASE ACTIVITY OF ADRENAL CHROMAFFIN GRANULES.
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ABSTRACT:
SUBMITTER: BANKS P
PROVIDER: S-EPMC1214348 | biostudies-other | 1965 May
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Glycoprotein II (GP II) is a protein found in the membranes of chromaffin granules from adrenal medulla. Immunoblotting (one- and two-dimensional) revealed that this antigen is also present in liver and in kidney. Subcellular fractionation of the latter organ indicated that GP II was present in lysosomes. This was confirmed by immunoelectron microscopy. The antiserum against GP II immunolabelled the membranes of organelles which could be identified as lysosomes by the labelling of their contents with an antiserum against cathepsin D. Thus GP II is an antigen common to secretory vesicles and lysosomes.
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Project description:1. The lipid composition of microsomes, mitochondria and chromaffin granules, obtained from homogenates of bovine adrenal medulla, has been investigated. 2. The three types of particle showed characteristic differences of phospholipid and cholesterol content. The lipid composition of microsomes and mitochondria resembled that of corresponding particles from other tissues. The chromaffin granules contained 19% of the cholesterol and 14% of the phospholipids of the low-speed supernatant. 3. Thin-layer chromatography indicated the presence of these phospholipids in extracts from each particle: lecithin, lysolecithin, phosphatidylethanolamine (partly plasmalogen), phosphatidylserine, phosphatidylinositol and sphingomyelin. 4. On quantitative analysis of the phospholipids, chromaffin granules were found to contain a high concentration of lysolecithin (17% of the lipid phosphorus). Mitochondria and microsomes, on the other hand, contained very little lysolecithin (less than 2% of the lipid phosphorus).
| S-EPMC1270618 | biostudies-other
Project description:The association of adenosine triphosphatase and ADP/ATP isotope-exchange activities with chromaffin-granule membranes was shown by sucrose-density-gradient centrifugation. The two activities were solubilized, and separated by differential sedimentation.
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Project description:Chromaffin granules isolated from bovine adrenal gland were incubated with (3)H-labelled nucleotides and [(14)C]noradrenaline to study the uptake of these substances. [(3)H]ATP, [(3)H]ADP and [(3)H]AMP are taken up by these organelles by the same temperature-dependent mechanism. The apparent K(m) for ATP and ADP is 1.4mm, and for AMP it is 2.9mm. The uptake of ATP has a flat pH optimum, whereas the catecholamine uptake increases with more alkaline pH. Atractyloside and carboxyatractyloside are competitive and specific inhibitors of nucleotide uptake, whereas reserpine inhibits only that for catecholamines. Mg(2+) ions activate uptake of both catecholamine and nucleotides, whereas EDTA and N-ethylmaleimide inhibit these processes. Nucleotide and catecholamine uptakes are inhibited by uncouplers of oxidative phosphorylation and by two ATP analogues. NH(4) (+) ions and nigericin in the presence of KCl inhibit only catecholamine uptake. It is concluded that nucleotide uptake, as proposed previously for catecholamine uptake, depends on an electrochemical proton gradient produced by a proton-translocating adenosine triphosphatase localized in the membrane of chromaffin granules. Furthermore, as suggested by the effect of NH(4) (+) and nigericin, catecholamine uptake apparently depends on the chemical part of this gradient, whereas the results for nucleotide uptake are consistent with its dependence on the electrical component.
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Project description:We studied the participation of adrenal medulla (AM) chromaffin cells in hypercapnic chemotransduction. Using amperometric recordings, we measured catecholamine (CAT) secretion from cells in AM slices of neonatal and adult rats perfused with solutions bubbled with different concentrations of CO2. The secretory activity augmented from 1.74 +/- 0.19 pC/min at 5% CO2 to 6.36 +/- 0.77 pC/min at 10% CO2. This response to CO2 was dose dependent and appeared without changes in extracellular pH, although it was paralleled by a drop in intracellular pH. Responsiveness to hypercapnia was higher in neonatal than in adult slices. The secretory response to hypercapnia required extracellular Ca2+ influx. Both the CO2-induced internal pH drop and increase in CAT secretion were markedly diminished by methazolamide (2 microm), a membrane-permeant carbonic anhydrase (CA) inhibitor. We detected the presence of two CA isoforms (CAI and CAII) in neonatal AM slices by in situ hybridization and real-time PCR. The expression of these enzymes decreased in adult AM together with the disappearance of responsiveness to CO2. In patch-clamped chromaffin cells, hypercapnia elicited a depolarizing receptor potential, which led to action potential firing, extracellular Ca2+ influx, and CAT secretion. This receptor potential (inhibited by methazolamide) was primarily attributable to activation of a resting cationic conductance. In addition, voltage-gated K+ current amplitude was also decreased by high CO2. The CO2-sensing properties of chromaffin cells may be of physiologic relevance, particularly for the adaptation of neonates to extrauterine life, before complete maturation of peripheral and central chemoreceptors.
| S-EPMC6725439 | biostudies-literature
Project description:1. The influence of various substances on the uptake of [3H]ATP and [14C]-noradrenaline into isolated bovine chromaffin granules was investigated. The carrier-mediated [3H]ATP uptake is specifically inhibited by SO42-, PO43- and phosphoenolpyruvate. Compounds with carboxylic acid or sulphonic acid groups had no significant inhibitory effects on either uptake. 2. 35SO42-, 32PO43- and phosphoenol[14C]pyruvate are taken up into chromaffin granules by a temperature-dependent process that is inhibited by atractyloside, uncouplers of oxidative phosphorylation and lipid-permeant anions. The apparent Km of 35SO42- uptake is 0.4 mM. 3. These results indicate that the nucleotide carrier in chromaffin granules has a broad specificity, transporting compounds with two strong negative charges. 4. Amino acid probes influence the uptake of ATP and catecholamines differently. Pyridoxal phosphate inhibits both uptake processes, 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid preferentially blocks ATP uptake, whereas phenylglyoxal blocks only ATP transport. It is suggested that the nucleotide carrier possesses arginine residues in a functionally important position. 5. The significance of these results obtained on isolated granules for the function of chromaffin granules within the cell is discussed.
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Project description:Highly purified chromaffin granules can be obtained from homogenates of either ox, pig, horse or rat adrenal medullae by ultracentrifugation of the large-granule fraction layered on 1.6m-sucrose solution, by using angle-head rotors. The chromaffin granules are obtained as a pink sediment that is only slightly contaminated by mitochondria and lysosomes.
| S-EPMC1270431 | biostudies-other
Project description:1. A soluble protein has been purified from an aqueous extract of bovine adrenal chromaffin granules by chromatography on Sephadex G-200. This protein comprises 25% of the total protein of the granules and gave a single band on gel electrophoresis. 2. The protein is unusually rich in acidic amino acids, notably glutamic acid (26.0%, w/w); it is also relatively rich in proline (8.6%, w/w) but poor in cystine (0.35%, w/w). 3. A molecular weight of 77000 was obtained from sedimentation and diffusion measurements on the protein, and approach-to-equilibrium measurements gave apparent molecular weights of the same order. 4. A molecular weight 7 times that given above was estimated from the results of chromatography on a column of Sephadex G-200 that had been calibrated with globular proteins. However, good agreement between the ultracentrifuge and Sephadex experiments was obtained on the assumption that Sephadex chromatography depends on the effective hydrodynamic radii of proteins and not on their molecular weights. 5. The hydrodynamic properties of the protein differed from those of a typical globular protein. Thus the protein had a high intrinsic viscosity, a high frictional ratio and a large effective hydrodynamic volume. 6. The hydrodynamic properties of the protein, but not its molecular weight, were dependent on the ionic strength of the solvent. Increasing the ionic strength caused an increase in the sedimentation and diffusion coefficients, but a decrease in the intrinsic viscosity and in the frictional ratio of the protein. 7. Optical-rotatory-dispersion measurements indicated that only a small part of the polypeptide chain was in an alpha-helical conformation. 8. These results are compatible with the protein's having a conformation approaching that of a random-coil polypeptide, the volume occupied by the molecule being determined by electrostatic repulsion between the excess of negative charges.
| S-EPMC1270432 | biostudies-other
Project description:The pro-inflammatory cytokines, tumor necrosis factor-?, and interleukin-1?/? modulate catecholamine secretion, and long-term gene regulation, in chromaffin cells of the adrenal medulla. Since interleukin-6 (IL6) also plays a key integrative role during inflammation, we have examined its ability to affect both tyrosine hydroxylase activity and adrenomedullary gene transcription in cultured bovine chromaffin cells. IL6 caused acute tyrosine/threonine phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), and serine/tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3). Consistent with ERK1/2 activation, IL6 rapidly increased tyrosine hydroxylase phosphorylation (serine-31) and activity, as well as up-regulated genes, encoding secreted proteins including galanin, vasoactive intestinal peptide, gastrin-releasing peptide, and parathyroid hormone-like hormone. The effects of IL6 on the entire bovine chromaffin cell transcriptome were compared to those generated by G-protein-coupled receptor (GPCR) agonists (histamine and pituitary adenylate cyclase-activating polypeptide) and the cytokine receptor agonists (interferon-? and tumor necrosis factor-?). Of 90 genes up-regulated by IL6, only 16 are known targets of IL6 in the immune system. Those remaining likely represent a combination of novel IL6/STAT3 targets, ERK1/2 targets and, potentially, IL6-dependent genes activated by IL6-induced transcription factors, such as hypoxia-inducible factor 1?. Notably, genes induced by IL6 include both neuroendocrine-specific genes activated by GPCR agonists, and transcripts also activated by the cytokines. These results suggest an integrative role for IL6 in the fine-tuning of the chromaffin cell response to a wide range of physiological and paraphysiological stressors, particularly when immune and endocrine stimuli converge.
| S-EPMC5177545 | biostudies-literature
Project description:Washed membranes of bovine adrenal chromaffin granules contained most of the cholesterol and phospholipids of the particle and 22% of the total protein. The protein/lipid ratio was about 0.45 (w/w). Dopamine(3,4-dihydroxyphenethylamine)beta-hydroxylase, Mg(2+)-activated nucleoside triphosphatase and cytochrome b-559 activities were present in the membrane. ATP was the best substrate for the nucleoside triphosphatase, whose pH optimum was 6.4, K(m) 7x10(-4)m and V(max.) 1.8mumol/h per mg of protein. Treatment of the membranes with various detergents caused a preferential solubilization of protein compared with lipids. Membranes dissolved in sodium dodecyl sulphate or phenol-acetic acid-urea were subjected to polyacrylamide-gel electrophoresis at alkaline and acid pH respectively. The electrophoretic patterns given by the proteins of the chromaffin granule membrane were distinct from those given by the chromogranins, and from those given by mitochondrial and microsomal membrane proteins.
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