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Ronald W. Holz - One of the best experts on this subject based on the ideXlab platform.
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localization of phosphatidylinositol 4 5 p2 important in exocytosis and a quantitative analysis of Chromaffin Granule motion adjacent to the plasma membrane
Annals of the New York Academy of Sciences, 2002Co-Authors: Ronald W. Holz, Daniel AxelrodAbstract:: A slow ATP-dependent priming step precedes a rapid, Ca2+-dependent triggering step in exocytosis in Chromaffin cells and in most, if not all, differentiated secretory cells. A major component of ATP-dependent secretion in permeabilized cells reflects the maintenance of the polyphosphoinositides, especially PtdIns-4,5-P2. Here we summarize recent experiments with PH-GFP (binds to PtdIns-4,5-P2) that indicate that PtdIns-4,5-P2 is localized primarily on the plasma membrane in Chromaffin cells, and that it is this pool that plays a role in exocytosis. It is demonstrated that transiently expressed PH-GFP inhibits secretion in subsequently permeabilized cells. Recent studies using total internal reflection fluorescent microscopy (TIRFM) to measure Chromaffin Granule motion adjacent to the plasma membrane are also summarized. The quantitative analysis indicates that Chromaffin Granule motion is highly restricted and suggests that Chromaffin Granules are caged or tethered immediately adjacent to the plasma membrane.
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evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory Granule membrane comparison of exocytosis of injected bovine Chromaffin Granule membranes and endogenous cortical Granules in xenopus laevis oocyt
Cellular and Molecular Neurobiology, 1994Co-Authors: Donalyn Scheuner, Ronald W. HolzAbstract:SUMMARY 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaflin Granule membranes and of endogenous cortical Granules to undergo exocytosis in Xenopus laevis oocytes and eggs in response to cytosolic Ca ~÷. Exocytosis of chromatfin Granule membranes was detected by the appearance of dopamine-/3-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.
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bovine Chromaffin Granule membranes undergo ca 2 regulated exocytosis in frog oocytes
Journal of Cell Biology, 1992Co-Authors: Donalyn Scheuner, Craig D Logsdon, Ronald W. HolzAbstract:We have devised a new method that permits the investigation of exogenous secretory vesicle function using frog oocytes and bovine Chromaffin Granules, the secretory vesicles from adrenal Chromaffin cells. Highly purified Chromaffin Granule membranes were injected into Xenopus laevis oocytes. Exocytosis was detected by the appearance of dopamine-beta-hydroxylase of the Chromaffin Granule membrane in the oocyte plasma membrane. The appearance of dopamine-beta-hydroxylase on the oocyte surface was strongly Ca(2+)-dependent and was stimulated by coinjection of the Chromaffin Granule membranes with InsP3 or Ca2+/EGTA buffer (18 microM free Ca2+) or by incubation of the injected oocytes in medium containing the Ca2+ ionophore ionomycin. Similar experiments were performed with a subcellular fraction from cultured Chromaffin cells enriched with [3H]norepinephrine-containing Chromaffin Granules. Because the release of [3H]norepinephrine was strongly correlated with the appearance of dopamine-beta-hydroxylase on the oocyte surface, it is likely that intact Chromaffin Granules and Chromaffin Granule membranes undergo exocytosis in the oocyte. Thus, the secretory vesicle membrane without normal vesicle contents is competent to undergo the sequence of events leading to exocytosis. Furthermore, the interchangeability of mammalian and amphibian components suggests substantial biochemical conservation of the regulated exocytotic pathway during the evolutionary progression from amphibians to mammals.
Carl E Creutz - One of the best experts on this subject based on the ideXlab platform.
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Current Protocols in Cell Biology - Isolation of Chromaffin Granules.
Current protocols in pharmacology, 2020Co-Authors: Carl E CreutzAbstract:Adrenal medullary Chromaffin Granules (dense core secretory vesicles) have been a valuable model system for the study of the proteins and membrane components involved in the process of exocytosis. Because of the abundance of Chromaffin Granules in a readily available tissue source, bovine adrenal medullae, and their unique sedimentation properties, it is possible to obtain large quantities of highly purified Granules and Granule membranes in a short period of time. Two protocols are presented here for the isolation of Chromaffin Granules: a basic protocol based on differential centrifugation in an iso-osmotic medium that yields intact Chromaffin Granules, and an alternate protocol based on sedimentation through a density step gradient that provides a greater yield of more highly purified Chromaffin Granules. Since in the latter case the Granules cannot be returned to a medium of physiological osmolarity without lysis after purification on the step gradient, the alternate protocol is more useful to obtain the Granule membranes or contents for further study. Curr. Protoc. Cell Biol. 48:3.39.1-3.39.10. © 2010 by John Wiley & Sons, Inc. Keywords: Chromaffin Granule; Chromaffin Granule membrane; secretory vesicle; dense core vesicle; adrenal medulla; catecholamine; chromogranin; exocytosis; cytochrome b562; dopamine-β-hydroxylase; bovine
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identification of the major Chromaffin Granule binding protein chromobindin a as the cytosolic chaperonin cct chaperonin containing tcp 1
Journal of Biological Chemistry, 1994Co-Authors: Carl E Creutz, A Liou, S L Snyder, A Brownawell, K WillisonAbstract:Abstract Chromobindin A is a multisubunit complex ATPase that binds to Chromaffin Granule membranes in a calcium-dependent manner and requires ATP for release from the membrane (Martin, W. H., and Creutz, C. E. (1987) J. Biol. Chem. 262, 2803-2810). Here we report that the seven previously characterized subunits of chromobindin A cross react with antisera specific to subunits of CCT, the chaperonin containing TCP-1 (Kubota, H., Hynes, G., Carne, A., Ashworth, A., and Willison, K. (1994) Curr. Biol. 4, 89-99). The chromobindin A subunits previously called chromobindins 12, 13, 14, 15, 16, 18, and 19 cross-react specifically with subunits beta, delta, theta, alpha, zeta, xi, and gamma, respectively, of CCT. Additional similarities in subunit molecular weights, isoelectric points, and the morphologies of the two protein complexes as determined by electron microscopy support identification of chromobindin A as an adrenal medullary form of CCT. The chromobindin A/CCT complex was found to bind at least 7-fold more efficiently to affinity columns of Chromaffin Granule membranes than of adrenal medullary cytosol proteins, suggesting a specific interaction occurs between the complex and membrane components. The results indicate that the previously described characteristics of chromobindin A are likely to be relevant to the functions of CCT and suggest that the adrenal medullary form of CCT may play a role in the activities of secretory vesicle membranes.
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annexin Chromaffin Granule membrane interactions a comparative study of synexin p32 and p67
Biochimica et Biophysica Acta, 1990Co-Authors: William J Zaks, Carl E CreutzAbstract:The Chromaffin Granule membrane binding and aggregating properties of three annexins, synexin, p32 and p67, have been studied and compared. Each protein was activated to bind and aggregate membranes with a biphasic Ca2+ dependence, with one phase titrating between pCa 5.0–3.5 and the second at higher levels of calcium (pCa ; Sr2+ >; Ba2+. The proteins appeared to bind to distinct but overlapping populations of receptor sites, and did so in a manner displaying positive cooperativity at the higher Ca2+ levels. The maximal efficacy of the proteins as membrane aggregators differed with synexin being 1–2-fold more efficacious than p32, which in turn was 7-fold more efficacious than p67. In combination, p67 was an effective inhibitor of Granule aggregation induced by synexin or p32, while p32 was able to both promote and inhibit synexin-induced Granule aggregation in a manner which varied with synexin concentration. The complexity of these annexin-membrane interactions may be a reflection of the multidomain structure of the annexins and may have implications for the differential functions of these proteins in cells.
Shyamali D Wimalasena - One of the best experts on this subject based on the ideXlab platform.
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kinetic evidence for channeling of dopamine between monoamine transporter and membranous dopamine β monooxygenase in Chromaffin Granule ghosts
Journal of Biological Chemistry, 2004Co-Authors: Shyamali D Wimalasena, Kandatege WimalasenaAbstract:Abstract The nature of coupling between the uptake and dopamine-β-monooxygenase (DβM) catalyzed hydroxylation of dopamine (DA) was studied in bovine Chromaffin Granule ghosts. Initial rate and transient kinetics of DA uptake and conversion were determined under a variety of conditions. The uptake kinetics of DA, norepinephrine (NE), and epinephrine demonstrate that DA is a better substrate than NE and epinephrine under optimal uptake conditions. The transient kinetics of DA accumulation and NE production under both optimal uptake and uptake and conversion conditions were zero-order with no detectable lag or burst periods. The mathematical analyses of the data show that a normal sequential uptake followed by the conversion process could not explain the observed kinetics, under any condition. On the other hand, all experimental data are in agreement with a mechanism in which DA is efficiently channeled from the vesicular monoamine transporter to membranous DβM for hydroxylation, prior to the release into the bulk medium of the ghost interior. The slow accumulation of DA under optimal conversion conditions appears to be caused by the slow leakage of DA from the channeling pathway to the ghost interior. Because DβM activity in intact Granules is equally distributed between soluble and membranous forms of DβM, if an efficient channeling mechanism is operative in vivo, soluble DβM may not have access to the substrate, making the catalytic activity of soluble DβM physiologically insignificant, which is consistent with the increasing experimental evidence that membranous DβM may be the physiologically functional form.
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the reduction of membrane bound dopamine β monooxygenase in resealed Chromaffin Granule ghosts is intragranular ascorbic acid a mediator for extragranular reducing equivalents
Journal of Biological Chemistry, 1995Co-Authors: Kandatege Wimalasena, Shyamali D WimalasenaAbstract:Abstract The role of internal and external reductants in the dopamine β-monooxygenase (DβM)-catalyzed conversion of dopamine to norepinephrine has been investigated in resealed Chromaffin Granule ghosts. The rate of norepinephrine production was not affected by the exclusion of internal ascorbate. The omission of ascorbate from the external medium drastically reduced the norepinephrine production without affecting the net rate of dopamine uptake. In the presence of the external reductant, the internal ascorbate levels were constant throughout the incubation period. The rate of norepinephrine production was not affected when ghosts were resealed to contain the DβM reduction site inhibitor, imino-D-glucoascorbate. Ghosts incubated with external imino-D-glucoascorbate reduced the norepinephrine production. The weak DβM reductant, 6-amino-L-ascorbic acid, was found to be a good external reductant for Granule ghosts. The outcome of the above experiments was not altered when dopamine was replaced with the reductively inactive DβM substrate, tyramine. These results and the known topology of membrane-bound DβM disfavor the direct reduction of the enzyme by the external reductant. Our observations are consistent with the hypothesis that external ascorbate is the sole source of reducing equivalents for DβM monooxygenation and that internal soluble ascorbate (or dopamine) may not directly reduce or mediate the reduction of membrane-bound DβM in resealed Granule ghosts.
Kandatege Wimalasena - One of the best experts on this subject based on the ideXlab platform.
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kinetic evidence for channeling of dopamine between monoamine transporter and membranous dopamine β monooxygenase in Chromaffin Granule ghosts
Journal of Biological Chemistry, 2004Co-Authors: Shyamali D Wimalasena, Kandatege WimalasenaAbstract:Abstract The nature of coupling between the uptake and dopamine-β-monooxygenase (DβM) catalyzed hydroxylation of dopamine (DA) was studied in bovine Chromaffin Granule ghosts. Initial rate and transient kinetics of DA uptake and conversion were determined under a variety of conditions. The uptake kinetics of DA, norepinephrine (NE), and epinephrine demonstrate that DA is a better substrate than NE and epinephrine under optimal uptake conditions. The transient kinetics of DA accumulation and NE production under both optimal uptake and uptake and conversion conditions were zero-order with no detectable lag or burst periods. The mathematical analyses of the data show that a normal sequential uptake followed by the conversion process could not explain the observed kinetics, under any condition. On the other hand, all experimental data are in agreement with a mechanism in which DA is efficiently channeled from the vesicular monoamine transporter to membranous DβM for hydroxylation, prior to the release into the bulk medium of the ghost interior. The slow accumulation of DA under optimal conversion conditions appears to be caused by the slow leakage of DA from the channeling pathway to the ghost interior. Because DβM activity in intact Granules is equally distributed between soluble and membranous forms of DβM, if an efficient channeling mechanism is operative in vivo, soluble DβM may not have access to the substrate, making the catalytic activity of soluble DβM physiologically insignificant, which is consistent with the increasing experimental evidence that membranous DβM may be the physiologically functional form.
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the reduction of membrane bound dopamine β monooxygenase in resealed Chromaffin Granule ghosts is intragranular ascorbic acid a mediator for extragranular reducing equivalents
Journal of Biological Chemistry, 1995Co-Authors: Kandatege Wimalasena, Shyamali D WimalasenaAbstract:Abstract The role of internal and external reductants in the dopamine β-monooxygenase (DβM)-catalyzed conversion of dopamine to norepinephrine has been investigated in resealed Chromaffin Granule ghosts. The rate of norepinephrine production was not affected by the exclusion of internal ascorbate. The omission of ascorbate from the external medium drastically reduced the norepinephrine production without affecting the net rate of dopamine uptake. In the presence of the external reductant, the internal ascorbate levels were constant throughout the incubation period. The rate of norepinephrine production was not affected when ghosts were resealed to contain the DβM reduction site inhibitor, imino-D-glucoascorbate. Ghosts incubated with external imino-D-glucoascorbate reduced the norepinephrine production. The weak DβM reductant, 6-amino-L-ascorbic acid, was found to be a good external reductant for Granule ghosts. The outcome of the above experiments was not altered when dopamine was replaced with the reductively inactive DβM substrate, tyramine. These results and the known topology of membrane-bound DβM disfavor the direct reduction of the enzyme by the external reductant. Our observations are consistent with the hypothesis that external ascorbate is the sole source of reducing equivalents for DβM monooxygenation and that internal soluble ascorbate (or dopamine) may not directly reduce or mediate the reduction of membrane-bound DβM in resealed Granule ghosts.
Vivian Hook - One of the best experts on this subject based on the ideXlab platform.
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characteristics of the Chromaffin Granule aspartic proteinase involved in proenkephalin processing
Journal of Neurochemistry, 2002Co-Authors: Anahit V Azaryan, Martin R Schiller, Liane M Mendemueller, Vivian HookAbstract:Proteolytic processing of neuropeptide precursors is required for production of active neurotransmitters and hormones. In this study, a Chromaffin Granule (CG) aspartic proteinase of 70 kDa was found to contribute to enkephalin precursor cleaving activity, as assayed with recombinant ([ 35 S]Met)preproenkephalin. The 70-kDa CG aspartic proteinase was purified by concanavalin A-Sepharose, Sephacryl S-200, and pepstatin A agarose affinity chromatography. The proteinase showed optimal activity at pH 5.5. It was potently inhibited by pepstatin A, a selective aspartic proteinase inhibitor, but not by inhibitors of serine, cysteine, or metalloproteinases. Lack of inhibition by Val-D-Leu-Pro-Phe-Val-D-Leu-an inhibitor of pepsin, cathepsin D, and cathepsin E-distinguishes the CG aspartic proteinase from classical members of the aspartic proteinase family. The CG aspartic proteinase cleaved recombinant proenkephalin between the Lys 172 -Arg 173 pair located at the COOH-terminus of (Met)enkephalin-Arg 6 -Gly 7 -Leu 8 , as assessed by peptide microsequencing. The importance of full-length prohormone as substrate was demonstrated by the enzyme's ability to hydrolyze 35 S-labeled proenkephalin and proopiomelanocortin and its inability to cleave tri- and tetrapeptide substrates containing dibasic or monobasic cleavage sites. In this study, results provide evidence for the role of an aspartic proteinase in proenkephalin and prohormone processing.
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Chromaffin Granule aspartic proteinase processes recombinant proopiomelanocortin pomc
Biochemical and Biophysical Research Communications, 1995Co-Authors: Anahit V Azaryan, Martin R Schiller, Vivian HookAbstract:Abstract Our search for proteases responsible for proenkephalin (PE) processing in adrenal medulla led to the isolation of a 70 kDa aspartic proteinase that cleaves PE between the basic residues of the Lys-Arg processing site (1). Studies in pituitary have also identified a similar aspartic proteinase that processes POMC (2,3). To compare the Chromaffin Granule (CG) 70 kDa aspartic proteinase with that in pituitary, processing of recombinant POMC by the CG enzyme was examined. POMC was expressed in the T7 expression system in E. coli , and purified to homogeneity. The CG 70 kDa aspartic proteinase converted POMC to 27 and 22 kDa bands that were detected by anti-N-POMC immunoblots, and to 26, 22, and 14 kDa bands that were immunoreactive with anti-β-lipotropin. POMC products represented by these bands indicate appropriate POMC processing by the CG 70 kDa aspartic proteinase. These results, combined with the similar biochemical properties of these two enzymes, suggest that the CG 70 kDa aspartic proteinase resembles the POMC-converting enzyme (PCE), an aspartic proteinase in pituitary (2,3).