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1.
Characterization of hormone and protein release from alpha-toxin-permeabilized chromaffin cells in primary culture 总被引:14,自引:0,他引:14
M F Bader D Thiersé D Aunis G Ahnert-Hilger M Gratzl 《The Journal of biological chemistry》1986,261(13):5777-5783
Addition of Staphylococcus aureus alpha-toxin to adult bovine chromaffin cells maintained in primary culture causes permeabilization of cell membrane as shown by the release of intracellular 86Rb+. The alpha-toxin does not provoke a spontaneous release of either catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However the addition of micromolar free Ca2+ concentration induced the co-release of noradrenaline and chromogranin A. In alpha-toxin-treated cells, the released chromogranin A could not be sedimented and lactate dehydrogenase was still associated within cells, which provides direct evidence that secretory product is liberated by exocytosis. By contrast, permeabilization of cells with digitonin caused a Ca2+-dependent but also a Ca2+-independent release of secretory product, a dramatic loss of lactate dehydrogenase, as well as release of secretory product in a sedimentable form. Ca2+-dependent exocytosis from alpha-toxin-permeabilized cells required Mg2+-ATP and did not occur in the presence of other nucleotides. Thus alpha-toxin is a convenient tool to permeabilize chromaffin cells, and has the advantage of keeping intracellular structures, specifically the exocytotic machinery, intact. 相似文献
2.
High capacity, low affinity Ca2+ binding of chromogranin A. Relationship between the pH-induced conformational change and Ca2+ binding property 总被引:1,自引:0,他引:1
Chromogranin A, the major intravesicular protein of adrenal chromaffin granules, bound Ca2+ in a pH-dependent manner. Both the maximal binding and affinity of chromogranin A for Ca2+ were dependent on pH. Chromogranin A bound 670 nmol of Ca2+/mg (32 mol/mol) and 1150 nmol of Ca2+/mg (55 mol/mol) at pH 7.5 and 5.5, respectively, with dissociation constants (Kd) of 2.7 and 4 mM. This pH dependence probably reflects different conformations of the protein at the two pH values. Conformational differences of chromogranin A at two different pH values were demonstrated by limited tryptic digestion patterns confirming previous results obtained by circular dichroism spectroscopy (Yoo, S. H., and Albanesi, J. P. (1990) J. Biol. Chem. 265, 14414-14421). Sedimentation equilibrium studies revealed the native molecular mass of chromogranin A to be 100 kDa at pH 7.5 and 192 kDa at pH 5.5, indicating dimeric and tetrameric states of the protein at the two pH levels. We postulate that the pH- and Ca2(+)-induced conformational changes of chromogranin A may have a role both in the regulation of Ca2+ release of chromaffin granules and in the early stages of secretory vesicle biogenesis. 相似文献
3.
Chromogranin A is a high capacity, low affinity Ca2+ binding protein which undergoes Ca2+- and pH-dependent conformational changes, and has recently been suggested to play a Ca2+-buffering role in the secretory vesicle of adrenal medullary chromaffin cell, the major inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store of chromaffin cell (Yoo, S.H., and Albanesi, J.P. (1990) J. Biol. Chem. 265, 13446-13448). In the present study, it is shown that chromogranin A exists in a monomer-dimer equilibrium at pH 7.5 and in a monomer-tetramer equilibrium at pH 5.5. The pH appears monomer-tetramer equilibrium at pH 5.5. The pH appears to be a necessary and sufficient factor determining the types of oligomers formed. Although Ca2+ did not change the type of oligomerization, it had a very significant effect on the values of the thermodynamic parameters characterizing the associations. The delta G0 values for a monomer-dimer equilibrium were -7 to -8 kcal/mol, while those for a monomer-tetramer equilibrium were -20 to -23 kcal/mol. At pH 5.5, the values of delta H0, delta S0, and delta C0p were large and negative in the absence of Ca2+ and large and positive in the presence of 35 mM Ca2+, implying markedly different reaction mechanisms. Extrapolation of the results to 37 degrees C and 1 mM chromogranin A suggests that chromogranin A is virtually 100% tetramer at pH 5.5 in the presence of 35 mM Ca2+ but is 96% dimer at pH 7.5 in the absence of Ca2+, the two conditions resembling those seen in vivo. These results suggest that chromogranin A is mostly dimer in the endoplasmic reticulum and cis-Golgi area and is essentially all tetramer in the vesicle. 相似文献
4.
Ca2(+)-induced conformational change and aggregation of chromogranin A 总被引:10,自引:0,他引:10
Chromogranin A, the most abundant protein in bovine adrenal chromaffin granules, bound calmodulin in a Ca2(+)-dependent manner, and the calmodulin-binding property was utilized to purify chromogranin A. Chromogranin A has been described in the past as a "random-coil polypeptide" with little alpha-helix or beta-sheet conformation. However, circular dichroism measurements with pure, native chromogranin A revealed relatively high alpha-helical contents (40% at the intravesicular pH of 5.5). Fluorescence studies confirmed previous observations that chromogranin A binds Ca2+ with low affinity. Considering the high concentration of Ca2+ in the secretory vesicle, the effect of Ca2+ on the secondary structure and self-association of chromogranin A was examined. Ca2+ induced a decrease of alpha-helicity of chromogranin A from 40 to 30% at pH 5.5. In contrast, at pH 7.5 the same amount of Ca2+ increased alpha-helicity of the protein from 25 to 40%. Boiling of the adrenal extract, a commonly used purification procedure for chromogranin A, resulted in the isolation of conformationally distinct chromogranin A molecule. Unlike secretory protein-I of the parathyroid gland (Gorr, S.-V., Dean, W. L., Radley, T. L., and Cohn, D. V. (1988) Bone Mineral 4, 17-25), chromogranin A aggregated rapidly in the presence of Ca2+. The extent and rate of aggregation were highly dependent on Ca2+ concentration. Although both the rate and extent of aggregation at pH 7.5 were much lower than those at pH 5.5, aggregation of chromogranin A proceeded at both pH's. In this respect, chromogranin A differs from human chromogranin C which was shown by Gerdes et al. (Gerdes, H.-H., Rosa, P., Phillips, E., Baeuerle, P. A., Frank, R., Argos, P., and Huttner, W. B. (1989) J. Biol. Chem. 264, 12009-12015) to aggregate at pH 5.2 but not at pH 7.4. 相似文献
5.
Inositol 1,4,5-trisphosphate-triggered Ca2+ release from bovine adrenal medullary secretory vesicles 总被引:3,自引:0,他引:3
The effect of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and calcium ionophore A23187 on Ca2+ release from bovine adrenal medullary secretory vesicles and microsomes was examined. Ins(1,4,5)P3 released 3.5 nmol of Ca2+/mg protein from secretory vesicles and 1.5 nmol of Ca2+/mg protein from microsomes as measured by a Ca2(+)-selective electrode. However, A23187 promoted Ca2+ uptake into vesicles while releasing Ca2+ from microsomes. Ins(1,4,5)P3-induced Ca2+ release from secretory vesicles was rapid, but the released Ca2+ was absorbed within 3 min during which the Ins(1,4,5)P3-releasable pools were refilled. The in situ calcium content of secretory vesicle measured by atomic absorption spectrometry was 112 +/- 6.3 nmol/mg protein indicating the potential importance of secretory vesicles as an intracellular Ca2+ store. The high Ca2(+)-buffering capacity of secretory vesicles is presumed to be due to the high Ca2(+)-binding capacity of chromogranin A, the major intravesicular protein, which has calsequestrin-like properties. 相似文献
6.
Effect of secretagogues on chromogranin A synthesis in bovine cultured chromaffin cells. Possible regulation by protein kinase C. 总被引:4,自引:1,他引:3
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Chromogranin A is a major component of storage granules in many different secretory cell types. After [35S]methionine labelling of proteins from cultured bovine chromaffin cells, chromogranin A was immunoprecipitated with specific antibodies, and the radioactivity incorporated into chromogranin A was determined and used as an index of its synthesis rate. Depolarization of cells with nicotine or high K+ evoked a Ca2+-dependent increase in chromogranin A synthesis, whereas muscarine, which does not evoke significant Ca2+ influx from bovine chromaffin cells, had no effect on chromogranin A synthesis. Forskolin, an activator of adenylate cyclase, affected neither the basal nor the nicotine-stimulated rate of chromogranin A synthesis. In contrast, 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, significantly enhanced the incorporation of radioactivity into chromogranin A. Sphingosine, an inhibitor of protein kinase C, abolished both nicotine-stimulated and TPA-induced chromogranin A synthesis. In addition, long-term treatment of chromaffin cells with TPA decreased protein kinase C activity and inhibited the nicotine-stimulated chromogranin A synthesis. These results suggest that protein kinase C may play an important role in the control of chromogranin A synthesis. 相似文献
7.
Secretion of sulfated and nonsulfated forms of parathyroid chromogranin A (secretory protein-I) 总被引:2,自引:0,他引:2
Chromogranin A (secretory protein-I) is an acidic, sulfated glycoprotein found in secretory granules of most endocrine cells but not in exocrine or epithelial cells. Parathyroid chromogranin A is sulfated on tyrosine residues, whereas adrenal chromogranin A appears to be sulfated mainly on oligosaccharide residues. Chromogranin B, on the other hand, is tyrosine-sulfated in the bovine adrenal whereas this protein is absent from the parathyroid. The role of this tissue- or species-specific sulfation of chromogranin is not known. Tyrosine sulfation is a common post-translational modification of proteins in the exocytotic pathway and has been suggested to play a role in the sorting or intracellular transport of secretory proteins. To test this, porcine parathyroid tissue slices were metabolically labeled with 35SO4 and [3H]Lys, and the tissue and incubation medium analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoprecipitation with chromogranin A-specific antiserum or by radioimmunoassay for parathormone. Secretion of total and 3H-labeled chromogranin A was about 3- and 7-fold higher, respectively, at 0.5 mM than at 3.0 mM Ca2+, and secretion of 35SO4-labeled chromogranin A was 67-fold higher. This indicates that either sulfated chromogranin A is directed primarily to the Ca2+-regulated pathway or that sulfation occurs following sorting to this pathway. Sodium chlorate (1-10 mM) inhibited sulfation in a dose-dependent manner by up to 95% but it had no effect on the onset or rate of chromogranin A secretion. These data indicate that regulated secretion of parathyroid chromogranin A does not require sulfation of tyrosine residues. 相似文献
8.
Chromogranins and secretogranins have traditionally been known as marker proteins of secretory granules that contain the highest concentrations of cellular calcium, reaching approximately 40 mM. In addition, chromogranin B was also shown to exist in the nucleus, localizing in the putative inositol 1,4,5-trisphosphate (IP3)-sensitive nucleoplasmic Ca2+ store vesicles. Chromogranins A (CGA) and B (CGB) are high-capacity, low-affinity Ca2+ binding proteins, binding 30-90 mol of Ca2+/mol with dissociation constants (Kd) of 1.5-4 mM. Yet the Ca2+-binding property of secretogranins has not been studied. Here, we show the localization of secretogranin II (SgII) in the nucleus, more specifically, in the IP3-sensitive nucleoplasmic Ca2+ store vesicles along with CGB and the IP3 receptors. We have also determined the Ca2+-binding property of SgII and found that SgII binds 61 mol of Ca2+/mol (910 nmol Ca2+/mg) with a Kd of 3.0 mM at the intragranular pH 5.5 and 30 mol of Ca2+/mol (440 nmol Ca2+/mg) with a Kd of 2.2 mM at a near-physiological pH 7.5. Chromogranin B also bound 50 mol of Ca2+/mol (670 nmol Ca2+/mg) with a Kd of 3.1 mM at pH 7.5. Given the high-capacity, low-affinity Ca2+-binding property of SgII and its presence in the IP3-sensitive nucleoplasmic Ca2+ store vesicles, these results suggest that SgII may function in the storage and control of Ca2+ in the nucleus through its interaction with CGB in the nucleoplasmic vesicles. 相似文献
9.
The nucleus also contains the inositol 1,4,5-trisphosphate receptor (IP3R)/Ca2+ channels in the nucleoplasm proper independent of the nuclear envelope or the cytoplasm. The nuclear IP3R/Ca2+ channels were shown to be present in small IP3-dependent nucleoplasmic Ca2+ store vesicles, yet no information is available regarding the IP3 sensitivity of nuclear IP3R/Ca2+ channels. Here, we show that nuclear IP3R/Ca2+ channels are 3-4-fold more sensitive to IP3 than cytoplasmic ones in both neuroendocrine PC12 cells and nonneuroendocrine NIH3T3 cells. Given the presence of phosphoinositides and phospholipase C and the importance of IP3-mediated Ca2+ signaling in the nucleus, the high IP3 sensitivity of nuclear IP3R/Ca2+ channels seemed to reflect the physiological needs of the nucleus to finely control the IP3-dependent Ca2+ concentrations. It was further shown that the IP3R/Ca2+ channels of secretory cells are 7-8-fold more sensitive to IP3 than those of nonsecretory cells. This difference appeared to result from the presence of secretory cell marker protein chromogranins (thus secretory granules) in secretory cells; expression of chromogranins in NIH3T3 cells increased the IP3 sensitivity of both nuclear and cytoplasmic IP3R/Ca2+ channels by approximately 4-6-fold. In contrast, suppression of chromogranin A expression in PC12 cells changed the EC50 of IP3 sensitivity for cytoplasmic IP3R/Ca2+ channels from 17 to 47 nM, whereas suppression of chromogranin B expression changed the EC50 of cytoplasmic IP3R/Ca2+ channels from 17 to 102 nM and the nuclear ones from 4.3 to 35 nM. Given that secretion is the major function of secretory cells and is under a tight control of intracellular Ca2+ concentrations, the high IP3 sensitivity appears to reflect the physiological roles of secretory cells. 相似文献
10.
A study of the effects of dihydropyridine Ca2+ channel modulators on the release of catecholamines from perfused rat adrenal glands, evoked by electrical stimulation of their splanchnic nerves, is presented. Electrically mediated secretory responses were compared to chemically mediated responses (exogenous acetylcholine, nicotine, or high K+). Intensities of stimuli were selected to produce quantitatively similar secretory responses (between 100 and 200 ng per stimulus). The main finding of the study is that responses to transmural stimulation (300 pulses at 1 or 10 Hz) and to acetylcholine were inhibited only partially (about 50%) by isradipine, an L-type Ca2+ channel blocker. In contrast, responses to high K+ (17.5 mM for 2 min) were highly sensitive to isradipine (IC50 = 8.2 nM). Responses to nicotine were also fully inhibited by this drug. Bay K 8644 (an L-type Ca2+ channel activator) potentiated mildly the secretory responses to electrical stimulation at 10 Hz and to acetylcholine, but increased threefold the responses to K+ and nicotine. It is, therefore, likely that responses mediated by high K+ or nicotinic receptors are triggered by external Ca2+ gaining access to the internal secretory machinery through L-type, dihydropyridine-sensitive voltage-dependent Ca2+ channels. However, in addition to nicotinic receptors, the physiological stimulation of adrenal medulla chromaffin cells through splanchnic nerves has other components, i.e., muscarinic receptor stimulation or the release of cotransmitters such as vasoactive intestinal polypeptide. The poorer sensitivity to dihydropyridines of secretory responses triggered by electrical stimulation of splanchnic nerve terminals or exogenous acetylcholine speaks in favor of alternative Ca2+ pathways, probably some dihydropyridine-resistant Ca2+ channels, in modulating the physiological adrenal catecholamine secretory process. 相似文献
11.
The (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase (Ca2+-transporting), EC 3.6.1.38) protein of rabbit skeletal sarcoplasmic reticulum (SR) rapidly incorporated 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-ATPase, activity was inhibited. The same pattern was found for modified intact SR and the Ca2+ uptake ability was inhibited. MgATP, CaATP and MgADP protected the Ca2+-ATPase activity concurrent with a decrease of about 1 mol of the NBD group per 10(5) g protein, but the Ca2+ uptake ability was not protected. Calcium alone had no effect on the modification. The modified ATPase protein or SR formed non-serial oligomers or aggregates, but the ATPase protein remained the predominant species present. In the presence of MgATP, oligomer formation was reduced partially but the major changes in the Ca2+-ATPase activity were due to the modification of the ATPase monomer. Thiolysis of the NBD-ATPase protein with dithiothreitol did not restore the Ca2+-ATPase activity, although more than 1 mol of the NBD group was removed from cysteine residues. Cysteine residues were modified in the NBD-ATPase protein or SR when the enzyme activity was inhibited. Trypsin digestion of NBD-SR or its ATPase protein released the A, B, A1, and A2 fragments. The A fragment and its subfragment A2 contained most of the label. Substrate MgATP protection studies showed that the A1 and A2 fragments were involved in maintaining the Ca2+-ATPase activity. Reagent-induced conformational changes of these fragments rather than direct active site group labeling accounted for the loss of ATPase activity. 相似文献
12.
Calcium-binding proteins in the parathyroid gland. Detailed studies of parathyroid secretory protein 总被引:1,自引:0,他引:1
In order to identify calcium (Ca2+)-binding proteins in the parathyroid gland, we used electrophoretic blots of proteins separated by a two-dimensional nondenaturing/denaturing gel system and incubated them with 45Ca2+. Parathyroid secretory protein (PSP) and proteins with approximate molecular weights of 98,000, 88,000, 58,000, and 30,000 were noted to bind Ca2+ in cytosolic fractions from bovine parathyroid, adrenal, and pituitary glands. However, differences in the binding affinity and capacity of the various proteins were observed. PSP displayed a low affinity and high binding capacity for Ca2+. In the presence of 5 mM MgCl2 and 60 mM KCl, native PSP (immobilized on nitrocellulose filters) bound 7.5 mol of Ca2+/mol of protein monomer with an apparent Kd of 1.1 mM. Immunoblotting identified the association of PSP with parathyroid cell membranes in a Ca2+-dependent manner. This property, together with its heat stability, distinguished PSP from other cytosolic Ca2+-binding proteins which were identified. There was also evidence for a Ca2+-dependent protein-protein interaction (aggregation) of PSP present in a Nonidet P-40 extract of cell membranes. The high Ca2+ binding capacity of PSP and its Ca2+-dependent membrane association may be features that make PSP a potentially important protein in secretory cells. 相似文献
13.
Matrix free Ca2+ in isolated chromaffin vesicles 总被引:3,自引:0,他引:3
Isolated secretory vesicles from bovine adrenal medulla contain 80 nmol of Ca2+ and 25 nmol of Mg2+ per milligram of protein. As determined with a Ca2+-selective electrode, a further accumulation of about 160 nmol of Ca2+/mg of protein can be attained upon addition of the Ca2+ ionophore A23187. During this process protons are released from the vesicles, in exchange for Ca2+ ions, as indicated by the decrease of the pH in the incubation medium or the release of 9-aminoacridine previously taken up by the vesicles. Intravesicular Mg2+ is not released from the vesicles by A23187, as determined by atomic emission spectroscopy. In the presence of NH4Cl, which causes the collapse of the secretory vesicle transmembrane proton gradient (delta pH), Ca2+ uptake decreases. Under these conditions A23187-mediated influx of Ca2+ and efflux of H+ cease at Ca2+ concentrations of about 4 microM. Below this concentration Ca2+ is even released from the vesicles. At the Ca2+ concentration at which no net flux of ions occurs the intravesicular matrix free Ca2+ equals the extravesicular free Ca2+. In the absence of NH4Cl we determined an intravesicular pH of 6.2. Under these conditions the Ca2+ influx ceases around 0.15 microM. From this value and the known pH across the vesicular membrane an intravesicular matrix free Ca2+ concentration of about 24 microM was calculated. This is within the same order of magnitude as the concentration of free Ca2+ in the vesicles determined in the presence of NH4Cl.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
14.
1. Secretion from the mucous sublingual gland of the mouse has been investigated and compared with the serous parotid gland. The influence of acetylcholine, noradrenalin and adrenalin on the secretion of glycoproteins (e.g. mucins) and proteins (e.g. amylase) from these glands in vitro, and the involvement of cyclic AMP and Ca2+ has been studied. 2. Secretion from the parotid gland could be stimulated by both acetylcholine and the catecholamines. It appears that cyclic AMP plays an important role in the adrenergic secretory process, but not in the cholinergic-induced secretion. In the latter case, exogenous Ca2+ strongly increased the secretion. 3. Mucin secretion from the sublingual gland could be affected by acetylcholine in the presence of exogenous Ca2+. Noradrenalin and adrenalin induced only a slow mucin secretion and, for this secretory process, exogenous Ca2+ is also required. Though cyclic AMP is present in the sublingual gland, no influence on its level could be detected in this gland after stimulation of the adrenergic beta-receptor, whereas, in contrast to the parotid gland, dibutyryl cyclic AMP induced only a slow secretion. Because it was observed that the sublingual gland of the mouse is not innervated sympathetically, it seems reasonable to suppose that the catecholamines stimulate the mucin secretion from this gland via hormonal receptors and not via the adrenergic beta-receptor. 4. The protein secretion from the sublingual gland could be stimulated by both acetylcholine and the catecholamines. An involvement of cyclic AMP in this process was not observed. Addition of exogenous Ca2+ is less important, as was found for the mucin secretion. So it has been concluded that protein and mucin secretion from the sublingual gland are regulated via different pathways. 相似文献
15.
Ronald W. Holz 《Cellular and molecular neurobiology》1988,8(3):259-268
1. Calcium-dependent exocytosis of catecholamines from intact and digitonin-permeabilized bovine adrenal chromaffin cells was investigated. 2. 45Ca2+ uptake and secretion induced by nicotinic stimulation or depolarization in intact cells were closely correlated. The results provide strong support for Ca2+ entry being the trigger for exocytosis. 3. Experiments in which the H+ electrochemical gradient across the intracellular secretory granule (chromaffin granule) membrane was altered indicated that the gradient does not play an important role in exocytosis. 4. Ca2+ entry into the cells is associated with activation of phospholiphase C and a rapid translocation of protein kinase C to membranes. 5. The plasma membrane of chromaffin cells was rendered permeable to Ca2+, ATP, and proteins by the detergent digitonin without disruption of the intracellular secretory granules. In this system in which the intracellular milieu can be controlled, micromolar Ca2+ directly stimulated catecholamine secretion. 6. Treatment of the cells with phorbol esters and diglyceride, which activate protein kinase C, enhanced phosphorylation and subsequent Ca2+-dependent secretion in digitonin-treated cells. 7. Phorbol ester-induced secretion could be specifically inhibited by trypsin. The experiments indicate that protein kinase C modulates but is not necessary for Ca2+-dependent secretion. 相似文献
16.
H Sohma T Watanabe Y Kuroki H Yoshino N Matsushima M Yazawa T Akino 《Biochimica et biophysica acta》1992,1159(2):150-154
Calcium-dependent conformational changes of surfactant protein A (SP-A) and the collagenase resistant fragment (CRF) of SP-A were studied by measuring fluorescence spectra. The emission peaks of both SP-A and CRF in the absence of Ca2+ appeared at 343 nm when they were excited at 280 nm. In the presence of Ca2+, the peaks appeared at 340 nm and were accompanied by an increase in the fluorescence intensity. The magnitude of the fluorescence intensity change induced by Ca2+ was amplified by the addition of dithiothreitol (DTT) in both SP-A and CRF. The Ca2+ binding of CRF was measured by a flow dialysis method with 45CaCl2 in the Ca2+ concentration range where the Ca(2+)-induced fluorescence changes occurred. The maximum binding number of Ca2+ to CRF was about 2 mol per mol of CRF, and the value was independent of the presence of DTT. 相似文献
17.
S Cleary J K Phillips T-T Huynh K Pacak S Fliedner A G Elkahloun P Munson R A Worrell G Eisenhofer 《Hormones et métabolisme》2007,39(12):876-883
Chromogranin A (CGA) is a major secretory protein present in the soluble matrix of chromaffin granules of neuroendocrine cells and tumours, such as phaeochromocytomas. CGA has several functions, some of which may be involved in the distinct phenotypic differences of phaeochromocytomas in patients with von Hippel-Lindau (VHL) syndrome compared to multiple endocrine neoplasia type 2 (MEN 2). In this study, we therefore compared tumour and plasma levels of CGA in patients with phaeochromocytoma associated with the two syndromes. We show that phaeochromocytomas from MEN 2 patients express substantially more CGA than tumours from VHL patients at both the mRNA (3-fold greater) and protein (20-fold) level. We further show that relative to increases in plasma catecholamines, patients with phaeochromocytomas associated with MEN 2 have higher plasma concentrations of CGA than those with tumours in VHL syndrome. These data supplement other observations that phaeochromocytomas in VHL compared to MEN 2 patients express lower amounts of catecholamines and other chromaffin granule cargo, such as chromogranin B and neuropeptide Y. Possibly the differences in tumour CGA expression may contribute to differences in secretory vesicle formation and secretion in the two types of tumours. Alternatively the differences in expression in CGA and other secretory constituents may reflect downregulation of the entire regulated secretory pathway in VHL compared to MEN 2 tumours. 相似文献
18.
Purification and properties of pyruvate dehydrogenase phosphatase from bovine heart and kidney 总被引:10,自引:0,他引:10
Pyruvate dehydrogenase phosphatase was purified to apparent homogeneity from bovine heart and kidney mitochondria. The phosphatase has a sedimentation coefficient (S20,w) of about 7.4 S and a molecular weight (Mr) of about 150 000 as determined by sedimentation equilibrium and by gel-permeation chromatography. The phosphatase consists of two subunits with molecular weights of about 97 000 and 50 000 as estimated by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Phosphatase activity resides in the Mr 50 000 subunit, which is sensitive to proteolysis. The phosphatase contains approximately 1 mol of flavin adenine dinucleotide (FAD) per mol of protein of Mr 150 000. FAD is apparently associated with the Mr 97 000 subunit. The function of this subunit remains to be established. The phosphatase binds 1 mol of Ca2+ per mol of enzyme of Mr 150 000 at pH 7.0, with a dissociation constant (Kd) of about 35 microM as determined by flow dialysis. Use of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate (EGTA) at pH 7.6 in conjunction with flow dialysis gave a Kd value for Ca2+ of about 8 microM. In the presence of both the phosphatase and the dihydrolipoyl transacetylase (E2) core of the pyruvate dehydrogenase complex, two equivalent and apparently non-interacting CA2+-binding sites were detected per unit of Mr 150 000, with a Kd value of about 24 microM in the absence and about 5 microM in the presence of EGTA. In the presence of 0.2 M KCl, which inhibits phosphatase activity about 95%, the phosphatase exhibited only one Ca2+-binding site, even in the presence of E2. The phosphatase apparently possesses an "intrinsic" Ca2+-binding site, and a second Ca2+-binding site is produced in the presence of E2. The second site is apparently altered by increasing the ionic strength. It is proposed that the second site may be at the interface between the phosphatase and E2, with Ca2+ acting as a bridging ligand for specific attachment of the phosphatase to E2. 相似文献
19.
S H Yoo 《Biochemistry》1992,31(26):6134-6140
Chromogranin A (CGA), the most abundant protein in bovine adrenal chromaffin granules, is a high-capacity, low-affinity Ca(2+)-binding protein found in most neuroendocrine cells, and binds calmodulin (CaM) in a Ca(2+)-dependent manner. The binding of chromogranin A to calmodulin was determined by measuring the intrinsic tryptophan fluorescence of chromogranin A in the presence and absence of Ca2+. Binding was specifically Ca(2+)-dependent; neither Mg2+ nor Mn2+ could substitute for Ca2+. Chelation of Ca2+ by EGTA completely eliminated the chromogranin A-calmodulin interaction. CaM binding was demonstrated by a synthetic CGA peptide representing residues 40-65. When the CGA peptide and CaM were mixed in the presence of 15 mM CaCl2, the intrinsic tryptophan fluorescence emission underwent a substantial blue-shift, shifting from 350 to 330 nm. Like the intact CGA, the peptide-CaM binding was specifically Ca(2+)-dependent, and neither Mg2+ nor Mn2+ could induce the binding. Calmodulin bound both to CGA and to the synthetic CGA peptide with a stoichiometry of one to one. The dissociation constants (Kd) determined by fluorometric titration were 13 nM for the peptide-CaM binding and 17 nM for intact CGA-CaM binding. The Kd values are comparable to those (approximately 10(-9) M) of other CaM-binding proteins and peptides, demonstrating a tight binding of CaM by CGA. The CaM-binding CGA residues 40-65 are 100% conserved among all the sequenced CGAs in contrast to 50-60% conservation found in the entire sequence, implying essential roles of this region. 相似文献
20.
The actions of the protonophore CCCP on intracellular Ca2+ regulation and exocytosis in chromaffin cells have been examined. Simultaneous fura-2 imaging and amperometry reveal that exposure to CCCP not only perturbs mitochondrial function but that it also alters vesicular storage of Ca2+ and catecholamines. By disrupting the pH gradient of the secretory vesicle membrane, the protonophore allows both Ca(2+) and catecholamine to leak into the cytosol. Unlike the high cytosolic Ca2+ concentrations resulting from mitochondrial membrane disruption, Ca2+ leakage from secretory vesicles may initiate exocytotic release. In conjunction with previous studies, this work reveals that catalytic and self-sustained vesicular Ca(2+) -induced exocytosis occurs with extended exposure to weak acid or base protonophores. 相似文献