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1.
Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca2+. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca2+ induced by incubation with ionomycin.3. Elevated Ca2+ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca2+ stimulus. In the oocytes, cortical granule components necessary for Ca2+-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca2+ stimulus and downstream events. 相似文献
2.
One of the more striking morphological events during egg activation is exocytosis of the cortical granules. In the frog egg, the wave of cortical granule exocytosis takes about 100 sec to traverse the animal half, and travels slower in the vegetal half. We examined cortical granule exoctyosis during activation with respect to this animal/vegetal difference. In eggs which were acquiring the ability to be activated (recovering from CO2-intoxication or undergoing meiotic maturation), animal half cortical granules became capable of responding to activating stimuli prior to vegetal half ones. Since Ca2+ is involved in exocytosis, we examined the effect of Ca2+ on cortical granule breakdown in vitro. There was no difference in sensitivity to Ca2+ of cortical granules from immature vs. mature eggs, but animal half cortical granules were more sensistive to Ca2+ than vegetal half ones. Finally, we found that prick-activation of eggs at the vegetal pole was frequently unsuccessful but would occur when external Ca2+ was raised. These experiments show that there are regional differences in the frog egg with respect to cortical granule responsiveness, and they suggest that the differences are due to Ca2+ sensitivity. 相似文献
3.
Carl E. Creutz 《Biochemical and biophysical research communications》1981,103(4):1395-1400
Proteins from adrenal medullary cytosol that bind to chromaffin granule membranes in the presence of Ca2+ were isolated by affinity chromatography on granule membranes coupled to Sepharose 4B. Cytosol was applied to the affinity column in the presence of 2 mM free Ca2+. One group of proteins was eluted at 50 μM Ca2+ and had molecular weights of 60,000, 46,000, 36,000, 34,000, 32,000 and 26,000. At 0.1 μM Ca2+ additional proteins of molecular weights 70,000, 44,000 and 33,000 were eluted. Both groups of proteins aggregated isolated chromaffin granules in the presence of Ca2+. Since exocytosis involves cytosol-membrane interactions regulated by Ca2+, these proteins may have functional roles in this process. The term “chromobindins” is introduced to describe these proteins. 相似文献
4.
T. Senda Keisuke Yamashita Toshio Okabe Nakaba Sugimoto Morihiro Matsuda 《Cell and tissue research》1998,292(3):513-519
Quick-freeze deep-etch electron microscopy showed the presence of bridge-like structures between adjacent secretory granules in rat anterior pituitary secretory cells. These intergranular bridges were variable in length and thickness. The finest bridges were 7–8 nm in length, while the longest ones were as long as 80 nm. Annexin II, one of the Ca2+-dependent phospholipid-binding proteins, is known to interlink between two membranes and induce aggregation of liposomes and chromaffin granules under the presence of Ca2+. In anterior pituitary cells, annexin II was detected by immunoelectron microscopy at the contact sites of secretory granules with other granules. The anterior pituitary cells treated under the presence of extracellular Ca2+ with Clostridium perfringens enterotoxin which induces Ca2+ influx showed multigranular exocytosis, i.e., multiple fusions of secretory granules with each other and with the plasma membrane. The granule-granule fusion in progress could be captured by the quick-freeze deep-etch technique. The membranes of adjacent secretory granules were partially fused at their contact sites where intergranular strands were no longer seen, while there existed intergranular strands between unfused portions of the granule membranes. From these results, we consider that the intergranular bridges, some of which may be composed of annexin II, are involved in Ca2+-induced granule-granule fusion in anterior pituitary cells. 相似文献
5.
Actin cytoskeleton modulates calcium signaling during maturation of starfish oocytes 总被引:2,自引:0,他引:2
Kyozuka K Chun JT Puppo A Gragnaniello G Garante E Santella L 《Developmental biology》2008,320(2):426-435
Before successful fertilization can occur, oocytes must undergo meiotic maturation. In starfish, this can be achieved in vitro by applying 1-methyladenine (1-MA). The immediate response to 1-MA is the fast Ca2+ release in the cell cortex. Here, we show that this Ca2+ wave always initiates in the vegetal hemisphere and propagates through the cortex, which is the space immediately under the plasma membrane. We have observed that alteration of the cortical actin cytoskeleton by latrunculin-A and jasplakinolide can potently affect the Ca2+ waves triggered by 1-MA. This indicates that the cortical actin cytoskeleton modulates Ca2+ release during meiotic maturation. The Ca2+ wave was inhibited by the classical antagonists of the InsP3-linked Ca2+ signaling pathway, U73122 and heparin. To our surprise, however, these two inhibitors induced remarkable actin hyper-polymerization in the cell cortex, suggesting that their inhibitory effect on Ca2+ release may be attributed to the perturbation of the cortical actin cytoskeleton. In post-meiotic eggs, U73122 and jasplakinolide blocked the elevation of the vitelline layer by uncaged InsP3, despite the massive release of Ca2+, implying that exocytosis of the cortical granules requires not only a Ca2+ rise, but also regulation of the cortical actin cytoskeleton. Our results suggest that the cortical actin cytoskeleton of starfish oocytes plays critical roles both in generating Ca2+ signals and in regulating cortical granule exocytosis. 相似文献
6.
Synexin protein is non-selective in its ability to increase Ca2+-dependent aggregation of biological and artificial membranes 总被引:2,自引:0,他引:2
Synexin, a soluble protein which increases the specificity of Ca2+ to aggregate isolated bovine chromaffin granules was prepared from bovine adrenal medullary tissue by the method of Creutz, Pazoles and Pollard (J. Biol. Chem. , 2858–2866, 1978). We also find that synexin increases both the initial rate and final amplitude of Ca2+-promoted aggregation of granule membranes. This effect is Ca2+-specific. However in contrast to Creutz , we find that synexin also potentiates aggregation of adrenal medulla and liver mitochondria and microsomes as well as phosphatidylserine vesicles. This lack of membrane specificity argues against the suggestion of Creutz that synexin specifically binds the granule to the plasma membrane prior to exocytosis . 相似文献
7.
8.
《生物化学与生物物理学报:生物膜》2001,1510(1-2):18-28
Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca2+-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-β-cyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-β-cyclodextrin complexes restored the Ca2+-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca2+, annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca2+ concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca2+ concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol. 相似文献
9.
Teresita Yap Nelson Mary Y. Lorenson Laurence S. Jacobs A. E. Boyd III 《Molecular and cellular biochemistry》1987,74(1):83-94
Calcium is necessary for secretion of pituitary hormones. Many of the biological effects of Ca2+ are mediated by the Ca2+-binding protein calmodulin (CaM), which interacts specifically with proteins regulated by the Ca2+-CaM complex. One of these proteins is myosin light chain kinase (MLCK), a Ca2+-calmodulin dependent enzyme that phosphorylates the regulatory light chains of myosin, and has been implicated in motile processes in both muscle and non-muscle tissues. We determined the content and distribution of CaM and CaM-binding proteins in bovine pituitary homogenates, and subcellular fractions including secretory granules and secretory granule membranes. CaM measured by radioimmunoassay was found in each fraction; although approximately one-half was in the cytosolic fraction, CaM was also associated with the plasma membrane and secretory granule fractions. CaM-binding proteins were identified by an 251-CaM gel overlay technique and quantitated by densitometric analysis of the autoradiograms. Pituitary homogenates contained nine major CaM-binding proteins of 146, 131, 90, 64, 58, 56, 52, 31 and 22 kilodaltons (kDa). Binding to all the bands was specific, Cat+-sensitive, and displaceable with excess unlabeled CaM. Severe heat treatment (100°C, 15 min), which results in a 75% reduction in phosphodiesterase activation by CaM, markedly decreased 251I-CaM binding to all protein bands. Secretory granule membranes showed enhancement for CaM-binding proteins with molecular weights of 184, 146, 131, 90, and 52000. A specific, affinity purified antibody to chicken gizzard MLCK bound to the 146 kDa band in homogenates, centrifugal subcellular fractions, and secretory granule membranes. No such binding was associated with the granule contents. The enrichment of MLCK and other CaM-binding proteins in pituitary secretory granule membranes suggests a possible role for CaM and/or CaM-binding proteins in granule membrane function and possibly exocytosis. 相似文献
10.
High hydrostatic pressure applied between sperm attachment and the onset of cortical granule exocytosis will inhibit this exocytotic event in sea urchin eggs. Such pressure-treated zygotes, nevertheless, are activated and capable of development. Thus, this technique can be used as a tool to study the relationship between cortical granule breakdown and other fertilization-related responses. We have studied whether the exocytosis of cortical granules is necessary for proton efflux (acid release) to occur. Our results indicate that although Ca2+ is released while the eggs are under pressure (a prerequisite for the following events to take place), cortical granule exocytosis and acid release are pressure-sensitive and completely inhibited at pressures above 400 atm (6000 psi) and 275 atm (4000 psi), respectively. However, upon decompression, acid release is initiated which amounts to 65–70% of that seen in the unpressurized controls, suggesting that the efflux mechanism does not require cortical granule exocytosis and must result from some modification of the original plasma membrane of the egg. The remaining 30–35% of the acid release is related to cortical granule exocytosis, since it can be obtained upon induction of the cortical granule fusion 30 min later under atmospheric pressure. The initiation of acid release after decompression indicates that the efflux mechanism is not transiently turned on at fertilization, but undergoing long-term modification; the recovery of the ability to induce cortical granule fusion after fertilization under pressure suggests a refilling of cytoplasmic Ca2+ stores within this time course. 相似文献
11.
Zhe Xu Leah Lefevre Tom Ducibella Richard M. Schultz Gregory S. Kopf 《Developmental biology》1996,180(2):594
Results of numerous experiments indicate that the transient rise in intracellular Ca2+following sperm–egg fusion is essential for the subsequent events that constitute egg activation. Some events of egg activation, e.g., cortical granule exocytosis, however, appear more sensitive to intracellular Ca2+than other events, e.g., cell cycle resumption. To examine if specific events of egg activation have different thresholds for Ca2+, we manipulated buffered intracellular Ca2+concentrations by microinjecting Ca2+-BAPTA buffers and then examined the effect on the cortical granule exocytosis, recruitment of maternal mRNAs, and cell cycle resumption. We find that whereas cortical granule exocytosis occurs over a narrow threshold range of injected free Ca2+concentrations between 0.5 and 1.0 μM,recruitment of maternal mRNAs is only partially stimulated at injected free Ca2+concentrations of 2.5 μM,and no evidence for cell cycle resumption was observed (up to 2.5 μMCa2+). Although the Ca2+- and phospholipid-dependent protein kinase, protein kinase C, is implicated in aspects of egg activation, calmodulin is also a potential target for the transient increase in Ca2+that occurs following fertilization. Whereas incubation of eggs in the presence of the calmodulin antagonist W-7 followed by insemination does not block cortical granule exocytosis, cell cycle resumption, as assessed by the metaphase-to-anaphase transition, a decrease in histone H1 kinase activity and the time course for the emission of the second polar body are significantly delayed/inhibited. 相似文献
12.
Proteins of the chromaffin granule membrane were covalently labeled in situ with sulfhydryl-specific fluorophores. Using MIANS (maleimide iodoaminonaphthyl sulfonate) as the donor and fluorescein mercury acetate or fluorescein-5-maleimide as the acceptor, Förster fluorescence resonance energy transfer (FRET) could be employed to measure the degree of inter-membrane and intra-membrane protein-protein contact upon Ca2+-induced aggregation of the membranes. The four major findings were: (1) Raising the Ca2+ concentration to approx. 500 μM causes the proteins to aggregate in the plane of the membrane. This is demonstrated by Ca2+-induced increases in the fluorescence resonance energy transfer in double labeled membranes. This effect is not protein-concentration dependent and occurs at calcium concentrations too low for granule aggregation, implying intra-membrane protein clustering or patching. To our knowledge this is the first direct demonstration of the fluid mosaic nature of subcellular organelles. (2) If two sets of granules are labeled separately, Ca2+-induced aggregation brings at least 74% of the labeled proteins into close transmembrane proximity. This effect is also observed at 10–100-fold slower rates in the absence of calcium and can be greatly reduced by depleting the granule membrane of labeled peripheral proteins. It is enhanced if the granules are aggregated by Ca2+ or K+. We conclude that (some) peripheral proteins can transfer from one membrane surface to another. (3) Aggregation of separately labeled sets of membranes by Ca2+ also produces transmembrane energy transfer since: (a) the Km for Ca2+-induced quantum transfer is in the same range as the Km for aggregation; (b) the reaction is protein-concentration dependent; (c) reversal of aggregation also (partially) reverses donor quenching. (4) A kinetic analysis of the transmembrane effect shows it to be 5–10-fold slower than aggregation itself, supporting earlier suggestions (Haynes, D.H., Kolber, M. and Morris, S.J., (1979) J. Theor. Biol. 81, 713–743) that lipid and protein rearrangements are secondary to granule membrane aggregation. 相似文献
13.
Summary Cortical granules from fertilized eggs, oocytes and nurse cells of Ophryotrocha labronica have been analyzed for the presence of calcium using cryo-ultramicrotomy and X-ray microprobe analysis. All cortical granules showed a significant peak for calcium, but yolk granules were without calcium. These results support the hypothesis that the discharge of cortical granules shortly after fertilization is a self-propagating phenomenon involving the diffusion of Ca2+ from bursting granules. 相似文献
14.
Recent work has demonstrated a role for Na+/Ca2+ exchange in potentiation of the Ca2+ entry elicited through the human platelet store-operated channel by controlling a Mn2+-impermeable Ca2+ entry pathway. Here we demonstrate that this involves control over the secretion of dense granules by a Na+/Ca2+ exchanger (NCX) and so autocrine signalling between platelets. NCX inhibition reduced dense granule secretion. The reduction in SOCE elicited by NCX inhibition could be reversed by the addition of uninhibited donor cells, their releasate alone, or exogenous ADP and 5-HT. The use of specific receptor antagonists indicated that ATP, ADP and 5-HT all played a role in NCX-dependent autocrine signalling between platelets following thapsigargin stimulation, by activating Mn2+-impermeable Ca2+ entry pathways. These data provide further insight into the mechanisms underlying the known interrelationship between platelet Ca2+ signalling and dense granule secretion, and suggest an important role for the NCX in potentiation of platelet activation via dense granule secretion and so autocrine signalling. Our results caution the interpretation of platelet Ca2+ signalling studies involving pharmacological or other manipulations that do not assess possible effects on NCX activity and dense granule secretion. 相似文献
15.
SantoDomingo J Fonteriz RI Lobatón CD Montero M Moreno A Alvarez J 《Cellular and molecular neurobiology》2010,30(8):1267-1274
We have investigated the dynamics of the free [Ca2+] inside the secretory granules of neurosecretory PC12 and INS1 cells using a low-Ca2+-affinity aequorin chimera fused to synaptobrevin-2. The steady-state secretory granule [Ca2+] ([Ca2+]SG] was around 20–40 μM in both cell types, about half the values previously found in chromaffin cells. Inhibition of SERCA-type
Ca2+ pumps with thapsigargin largely blocked Ca2+ uptake by the granules in Ca2+-depleted permeabilized cells, and the same effect was obtained when the perfusion medium lacked ATP. Consistently, the SERCA-type
Ca2+ pump inhibitor benzohydroquinone induced a rapid release of Ca2+ from the granules both in intact and permeabilized cells, suggesting that the continuous activity of SERCA-type Ca2+ pumps is essential to maintain the steady-state [Ca2+]SG. Both inositol 1,4,5-trisphosphate (InsP3) and caffeine produced a rapid Ca2+ release from the granules, suggesting the presence of InsP3 and ryanodine receptors in the granules. The response to high-K+ depolarization was different in both cell types, a decrease in [Ca2+]SG in PC12 cells and an increase in [Ca2+]SG in INS1 cells. The difference may rely on the heterogeneous response of different vesicle populations in each cell type.
Finally, increasing the glucose concentration triggered a decrease in [Ca2+]SG in INS1 cells. In conclusion, our data show that the secretory granules of PC12 and INS1 cells take up Ca2+ through SERCA-type Ca2+ pumps and can release it through InsP3 and ryanodine receptors, supporting the hypothesis that secretory granule Ca2+ may be released during cell stimulation and contribute to secretion. 相似文献
16.
Yoo SH 《Cell calcium》2011,50(2):175-183
The majority of secretory cell calcium is stored in secretory granules that serve as the major IP3-dependent intracellular Ca2+ store. Even in unicellular phytoplankton secretory granules are responsible for the IP3-induced Ca2+ release that triggers exocytosis. The number of secretory granules in the cell is directly related not only to the magnitude of IP3-induced Ca2+ release, which accounts for the majority of the IP3-induced cytoplasmic Ca2+ release in neuroendocrine cells, but also to the IP3 sensitivity of the cytoplasmic IP3 receptor (IP3R)/Ca2+ channels. Moreover, secretory granules contain the highest IP3R concentrations and the largest amounts of IP3Rs in any subcellular organelles in neuroendocrine cells. Secretory granules from phytoplankton to mammals contain large amounts of polyanionic molecules, chromogranins being the major molecules in mammals, in addition to acidic intragranular pH and high Ca2+ concentrations. The polyanionic molecules undergo pH- and Ca2+-dependent conformational changes that serve as a molecular basis for condensation-decondensation phase transitions of the intragranular matrix. Likewise, chromogranins undergo pH- and Ca2+-dependent conformational changes with increased exposure of the structure and increased interactions with Ca2+ and other granule components at acidic pH. The unique physico-chemical properties of polyanionic molecules appear to be at the center of biogenesis, and physiological functions of secretory granules in living organisms from primitive to advanced species. 相似文献
17.
Robert A. Ronzio Kathryn E. Kronquist Douglas S. Lewis Raymond J. Macdonald Sharon H. Mohrlok John J. ODonnell 《生物化学与生物物理学报:生物膜》1978,508(1):65-84
Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5–10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50–100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and . The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes < smooth microsomes < zymogen granules.Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptide was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [3H]glucosamine or [3H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules.Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB3H4. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior. 相似文献
18.
The inositol 1,4,5-trisphosphate (IP3)-mediated intracellular Ca2+ releases in secretory cells play vital roles in controlling not only the intracellular Ca2+ concentrations but also the Ca2+-dependent exocytotic processes. Of intracellular organelles that release Ca2+ in response to IP3, secretory granules stand out as the most prominent organelle and are responsible for the majority of IP3-dependent Ca2+ releases in the cytoplasm of chromaffin cells. Bovine chromaffin granules were the first granules that demonstrated the IP3-mediated Ca2+ release as well as the presence of the IP3 receptor (IP3R) in granule membranes. Secretory granules contain all three (type 1, 2, and 3) IP3R isoforms, and 58–69% of total cellular IP3R isoforms are expressed in bovine chromaffin granules. Moreover, secretory granules contain large amounts (2–4 mM) of chromogranins
and secretogranins; chromogranins A and B, and secretogranin II being the major species. Chromogranins A and B, and secretogranin
II are high-capacity, low-affinity Ca2+ binding proteins, binding 30–93 mol of Ca2+/mol of protein with dissociation constants of 1.5–4.0 mM. Due to this high Ca2+ storage properties of chromogranins secretory granules contain ~40 mM Ca2+. Furthermore, chromogranins A and B directly interact with the IP3Rs and modulate the IP3R/Ca2+ channels, i.e., increasing the open probability and the mean open time of the channels 8- to 16-fold and 9- to 42-fold, respectively.
Coupled chromogranins change the IP3R/Ca2+ channels to a more ordered, release-ready state, whereby making the IP3R/Ca2+ channels significantly more sensitive to IP3. 相似文献
19.
The sea urchin fertilization envelope (FE) is an extraembryonic coat which develops from the egg vitelline envelope (VE) and the secreted paracrystalline protein fraction of the cortical granules at fertilization. The FE undergoes further developmental changes postinsemination which are characterized by changes in envelope permeability, solubility in reducing and denaturing solvents, and morphology. We have developed a procedure to uncouple cortical granule exocytosis from assembly of the paracrystalline protein fraction onto the VE template. Egg suspensions were inseminated in normal seawater and diluted into Ca2+- and Mg2+-free seawater at 15 sec postinsemination. Phase-contrast and electron microscopic observations showed that the embryos formed a normally elevated, extremely thin envelope through which the cortical granule exudate permeated. Secretion studies showed that eggs which were diluted into divalent ion-free seawater postinsemination secreted as much protein into the surrounding seawater as eggs which had their VEs removed prior to the experiment. We have termed the envelope elevated in divalent ion-free seawater the VE1 and we believe that it is the VE structural component of the FE based on its thickness and morphology. VE1s were isolated by gentle physical means and the preparations appeared to be greater than 80% pure based on radioactive mixing experiments and on malate dehydrogenase and glucose-6-phosphate dehydrogenase marker studies. VE1s were at least 80% soluble based on extraction of radioiodinated preparations with reducing and denaturing solvents. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of VE1s showed eight major polypeptides which ranged from 30,500 to 270,000 in molecular weight. 相似文献
20.
Mica Ohara-Imaizumi Yoko Nakamichi Takashi Sakurai 《Biochemical and biophysical research communications》2009,385(3):291-295
We simultaneously analyzed insulin granule fusion with insulin fused to green fluorescent protein and the subplasma membrane Ca2+ concentration ([Ca2+]PM) with the Ca2+ indicator Fura Red in rat β cells by dual-color total internal reflection fluorescence microscopy. We found that rapid and marked elevation in [Ca2+]PM caused insulin granule fusion mostly from previously docked granules during the high KCl-evoked release and high glucose-evoked first phase release. In contrast, the slow and sustained elevation in [Ca2+]PM induced fusion from newcomers translocated from the internal pool during the low KCl-evoked release and glucose-evoked second phase release. These data suggest that the pattern of the [Ca2+]PM rise directly determines the types of fusing granules. 相似文献