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1.
Dr. Sherry Bursztajn Harvey B. Nudleman Stephen A. Berman 《Cell and tissue research》1987,248(3):535-540
Summary The removal of the acetylcholine receptors (AChRs) from the surface of muscle cells serves as an important mechanism in the regulation of the AChR turnover rate. Our previous studies have shown that cultured myotubes contain coated pits and vesicles bearing -bungarotoxin (BTX)-binding sites (Bursztajn 1984; Bursztajn and Fischbach 1984). In this study we have used BTX conjugated to horseradish peroxidase (HRP) and quantitative electron microscopy to determine the intracellular pathway(s) of acetylcholine receptors during the internalization process. To accomplish this, cultured rat myotubes were incubated with BTX-HRP at 4° C after which cells were washed and incubated at 37° C for 0 min to 2 h. After warming the cells, coated pits, coated vesicles and smooth membraned vesicles containing the peroxidase reaction product were present. A threefold increase in coated vesicles containing the reaction product was observed 1 min after warming the cells. The number of smooth-membraned vesicles remained constant at this time point. However, 5 to 15 min after warming the cells, a fivefold increase in the number of smooth membraned vesicles was observed. After 1 h at 37° C the reaction product was present in the lysosomal like bodies, but was not observed in the Golgi complex or the small coated vesicles associated with the Golgi complex. Our observations indicate that there is a size segregation between those coated vesicles containing BTX-HRP reaction product and those in which reaction product is absent. Our studies also suggest that within minutes of AChR internalization coated vesicles lose their coat and become smooth-membraned vesicles. 相似文献
2.
Cellular distribution of three mammalian Ca2+-binding proteins related to Torpedo calelectrin. 总被引:7,自引:3,他引:4
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M Geisow J Childs B Dash A Harris G Panayotou T Südhof J H Walker 《The EMBO journal》1984,3(12):2969-2974
Addition of Ca2+ to post-microsomal fractions of bovine adrenal or liver produced a sedimentable complex of membrane vesicles and cytoplasmic proteins. Proteins with apparent mol. wts. 70 000, 36 000 and 32 500 were solubilized from this complex by Ca2+ chelation. The 36 000 mol. wt. protein (p36) was immunoprecipitated by an antiserum specific for pp36, a major substrate for Rous sarcoma virus src-gene tyrosine kinase. This protein was present in many mesenchymal cells and associated with membrane cytoskeleton of bovine fibroblasts in a Ca2+-dependent manner. The 70 000 and 32 500 mol. wt. proteins were widely distributed in established cell lines, but were not clearly associated with cell organelles in tissue sections, nor retained in cytoskeleton preparations. On immunoblots p36 reacted strongly with antibodies produced against the electric fish protein Torpedo calelectrin and the similar Ca2+-binding properties and subunit mol. wts. of these proteins suggests that they might be functionally related. Since Torpedo calelectrin, p70, p36 and p32.5 were bound by lipid vesicles or microsomal membranes at micromolar free Ca2+ concentrations, regulated association with intrinsic membrane components may be involved in the functions of these widespread proteins. 相似文献
3.
Summary The surface-associated vesicles in retinal arterioles and venules were studied after fixation in glutaraldehyde-tannic acid or after intravitreal injection of peroxidase or lactoperoxidase. The vesicles were concentrated along the abluminal (basal) surface of the endothelial cells and along the plasma membranes of smooth muscle cells in arterioles and of pericytes in post-capillary venules. They were rarely encountered in the deeper regions of these cells. In perpendicular sections through the cell surface the majority of vesicles were in continuity with the plasma membrane whereas in tangential sections, they appeared to lie free in the cytoplasm. All such vesicles were labeled after exposure to tannic acid or to the heme-proteins. Peroxidase-reaction product was never seen in the lumen of the vessels. These observations suggest that the surface vesicles in endothelial cells, smooth muscle cells and pericytes are invaginations of the plasma membrane and are thus not involved in the transcytosis or endocytosis of proteins. The vesicles in the latter two cell types may be involved in some aspect of contractility rather than pinocytosis.Supported by grants EYO4831, Research to Prevent Blindness, Inc., and the Michigan Eye Bank 相似文献
4.
The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low M concentrations of octanoyl-CoA. However, even a large excess (500 M) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low M concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above effect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolismin vivo.Abbreviations ACBP
acyl-CoA binding protein
- BSA
bovine serum albumin
- CPT
carnitine palmitoyltransferase
- CPT0
malonyl-CoA sensitive CPT of the outer mitochondrial membrane
- CPT
malonyl-CoA insensitive CPT of the inner mitochondrial membrane
- OG
octylglucoside
- OMV
outer membrane vesicles
- IMV
inner membrane vesicles
Affiliated to the Department of Experimental Medicine, University of Montreal 相似文献
5.
Yalcin Cetin 《Cell and tissue research》1990,259(2):313-319
Summary Previous immunochemical investigations have demonstrated various opioid peptides in the pancreas. However, controversies exist related to the cellular localization of these peptides in the endocrine pancreas. Therefore, the guinea pig endocrine pancreas was immunohistochemically investigated for the presence of opioid peptides derived from pro-dynorphin, pro-enkephalin or pro-opiomelanocortin. Immunoreactivities were demonstrated on serial semithin sections by the peroxidase anti-peroxidase technique. In routinely immunostained sections, immunoreactivities for dynorphin A and -neo-endorphin were localized in pancreatic enterochromaffin cells, but not in islet cells. Immunoreactivity for Met-enkephalin was confined exclusively to B-cells and was localized only in some secretory granules. However, pre-treatment of semi-thin sections with trypsin and carboxypeptidase B led to a marked increase of Met-enkephalin immunoreactivity in B-cells. In addition, immunoreactivities for Met-enkephalin-Arg-Gly-Leu and bovine adrenal medulla dodecapeptide could be demonstrated in B-and A-cells, and -endorphin immunoreactivity was localized in A-cells. In no case, however, were immunoreactivities detected for bovine adrenal medulla docosapeptide, peptide F, corticotropin, melanotropin or dynorphin 1–32. The immunohistochemical findings indicate that opioids of different peptide families are present in the guinea pig endocrine pancreas. Since several opioid peptides of the corresponding pro-hormones could be demonstrated in the reference organs but not in the pancreas, it is concluded that the biosynthetic pathways of the respective precursors are different from those in the adrenal medulla or in the pituitary. 相似文献
6.
Sonoko Furuya 《Cell and tissue research》1990,261(1):97-105
Summary The ultrastructure of diaphragmed fenestrae and the process of their de novo formation were examined in cultured endothelial cells cloned from fenestrated capillaries of bovine adrenal medulla. One clone frequently formed many diaphragmed fenestrae in highly attenuated regions of endothelium during 1–1.5 months of culture on reconstituted collagen gel. Stereo views of thick sections showed round or oval clusters of geometrically arranged fenestrae, each with a central knob. The number of diaphragmed fenestra per cluster was 50±13/m2. The inner diameter of the fenestrae was 50–60 nm. In the cells having small clusters of fenestrae, plasmalemmal vesicles, each having a thin diaphragm with a central knob, were accumulated (56±18/m2) and arranged geometrically, mostly on the basal plasmalemma. At the border between the cytoplasm and the eluster of fenestrae, plasmalemmal vesicles of the basal plasmalemma fused with the opposing apical plamalemma. A model for the process of fenestrae formation in vitro is proposed. 相似文献
7.
Priz.-Doz. Dr. K. Unsicker 《Cell and tissue research》1973,145(3):417-442
Summary According to their ultrastructure and histochemistry three types of efferent nerve fibers can be distinguished in the bird's adrenal gland. The main part is made up of cholinergic fibers recognizable by a positive reaction for acetylcholinesterase and two specific populations of granules within the synaptic ending. Synaptic vesicles measuring 300 to 500 Å in diameter and dense-cored vesicles with a diameter of about 1 000 Å are discernible.In the periphery of the gland cholinergic axons for the innervation of adrenal cells form large bundles surrounded by a perineural sheath. The bundles cross the capsule and are situated within the adrenal chromaffin cords or at their periphery. Finally small groups of fibers enter a group of chromaffin cells which are surrounded by a basal lamina and which consist of about a dozen or more cells producing adrenaline and noradrenaline. Synaptic endings occur, above all in passeriform species, in the center of a chromaffin cell complex. They are either attached to the innervated cells or their dendrite-like processes, or embedded into the cells, or connected to short spines of the innervated cells. Synaptic and dense-cored vesicles leave the bouton by exocytosis. One synaptic terminal may innervate up to three A- or NA-cells. The existence of different types of synapses for A- and NA-cells cannot be excluded.Supported by a grant from the Deutsche Forschungsgemeinschaft (Un 34/1). 相似文献
8.
Plasma membranes from chromaffin cells of bovine adrenal medullae and from chicken macrophages were isolated on a urografin density gradient, frozen and sectioned without previous chemical fixation. Their receptor binding sites were localized by specific labelling. The sections were then post-fixed in the presence of K2Cr2O7 to produce positive staining of the membrane proteins. Chromaffin cell membranes formed single vesicles. The nicotinic acetylcholine receptor (localized using a monoclonal antibody against its cholinergic binding site) was always found in patches on the surface of vesicles, whose profiles corresponded to thickened bilayers. Macrophage membrane vesicles were agglutinated. The mannose receptor (localized using the ligand, mannosylferritin) was randomly distributed within the electron-dense coat of the agglutinated vesicles or on electron-dense caps involved in agglutination. The binding sites of both receptors were intact, as revealed by their being recognized by a monoclonal antibody against their cholinergic binding sites and by the active binding of the mannosylated ligand which was inhibited by mannan. The distribution of the receptors on the vesicles reflected their distribution on the cell surface. 相似文献
9.
K. Ishimura T. Yoshinaga-Hirabayashi H. Fujita H. Ishii-Ohba H. Inano B. Tamaoki 《Histochemistry and cell biology》1988,89(1):35-39
Summary The localization of 3-hydroxysteroid dehydrogenase/isomerase (3-HSD) was studied in bovine adrenal glands by light as well as electron microscopic immunocytochemistry, using anti-bovine adrenal 3-HSD antibody. With light microscopy the cytoplasm of the glomerulosa cells was weakly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained though both the capsular connective tissue cells and the medullary cells were entirely negative for this reaction. Electron microscopic immunocytochemistry revealed that the positive reaction products for 3-HSD were present on the membrane of smooth endoplasmic reticulum of the cortical cells, especially that of the fasciculata and reticularis cells. Other cell organelles such as mitochondria and Golgi apparatus were entirely negative. The present results indicate that 3-HSD is present in the membrane of smooth endoplasmic reticulum of bovine adrenal cortical cells.Supported by grants from the Ministry of Education Science and Culture, Japan 相似文献
10.
E. Jost-Vu R. L. Hamilton C. A. Hornick J. D. Belcher R. J. Havel 《Histochemistry and cell biology》1986,85(6):457-466
Summary Plama lipoproteins (and other ligands) are endocytosed by hepatocytes and appear in multivesicular bodies (MVBs) in the Golgi-lysosome region of the cell prior to their degradation. We have isolated MVB fractions from livers of estradiol-treated rats, permitting studies of their properties (Hornick et al. 1985). Here we report our cytochemical studies of lysosomal enzyme activity in partially and highly purified MVB fractions and in MVBs in hepatocytes in situ.Only about 15% of partially or highly purified MVBs were positive for acid phosphatase and arylsulfatase, consistent with the prelysosomal nature of this compartment. Partially purified MVB fractions contained small round vesicles, 70–120 nm in diameter, which, stained intensely for these enzymes; occasionally these vesicles appeared to fuse with MVBs, suggesting that these structures are primary lysosomes. Such stained vesicles were rarely seen in highly purified MVB preparations. Acid phosphatase reaction product with cerium as capture reagent appeared as uniform precipitates surrounding endocytosed plasma lipoproteins in positively stained MVBs. Arylsulfatase reaction product, however, appeared as distinctive are or plaque-like deposits just inside the MVB-limiting membrane, often in continuity with intense reaction product contained in a fusing primary lysosome. Similar putative primary lysosomes were occasionally observed in isolated, intact Golgi fractions from the same livers. Similar histochemical reactivities of MVBs and putative primary lysosomes were observed in thin sections of hepatoyctes in situ.These observations support the conclusion that, in hepatocytes, MVBs represent the immediate prelysosomal compartment in the endocytic pathway of macromolecular catabolism, and suggest that MVBs are converted to secondary lysosomes by direct fusion with primary lysosomes arising from closely adjacent Golgi compartment.A portion of this work was published in abstract form in the Journal of Histochemistry and Cytochemistry, Vol. 34, page 120, 1986.This work was supported by U.S. Public Health Service Grant HL 14237 (Arteriosclerosis SCOR) 相似文献
11.
Summary Plasma membranes from chromaffin cells of bovine adrenal medullae and from chicken macrophages were isolated on a urografin density gradient, frozen and sectioned without previous chemical fixation. Their receptor binding sites were localized by specific labelling. The sections were then post-fixed in the presence of K2Cr2O7 to produce positive staining of the membrane proteins. Chromaffin cell membranes fromed single vesicles. The nicotinic acetylcholine receptor (localized using a monoclonal antibody against its cholinergic binding site) was always found in patches on the surface of vesicles, whose profiles corresponded to thickened bilayers. Macrophage membrane vesicles were agglutinated. The mannose receptor (localized using the ligand, mannosylferritin) was randomly distributed within the electron-dense coat of the agglutinated vesicles or on electron-dense caps involved in agglutination. The binding sites of both receptors were intact, as revealed by their being recognized by a monoclonal antibody against their cholinergic binding sites and by the active binding of the mannosylated ligand which was inhibited by mannan. The distribution of the receptors on the vesicles reflected their distribution on the cell surface.Dedicated to Prof. Georg Maneke on the occasion of his 70th birthday 相似文献
12.
Glycoprotein III (GpIII) was purified from the soluble fraction of bovine chromaffin granules, the secretory vesicles of the adrenal medulla, by chromatography using wheat germ agglutinin-Sepharose followed by reverse-phase high performance liquid chromatography (HPLC). Characterization of this glycoprotein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse-phase HPLC, amino acid analysis and partial NH2-terminal sequence analysis indicated that GPIII was a disulfide-linked heterodimer with 37-kDa subunits. Analysis of in vitro translation products of adrenal medullary poly(A)+ RNA by immunoprecipitation using an anti-GpIII serum and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that both subunits are synthesized from a single precursor. Partial NH2-terminal sequence analysis allowed construction of oligonucleotides which were used as primers for a polymerase chain reaction to generate a GpIII-specific DNA probe. This probe was used to isolate a cDNA clone encoding the GpIII precursor from a bovine adrenal medullary cDNA library. The predicted amino acid sequence of GpIII has greater than 80% similarity to human serum protein-40,40, a protein implicated in the complement system, and to a major secretory product of Sertoli cells, glycoprotein 2, which is thought to play a role in spermatogenesis. Northern blot analysis confirmed that RNA encoding GpIII is also abundant in liver, testis, and brain. 相似文献
13.
Electron spin resonance studies of membrane proteins in erythrocytes in myotonic muscular dystrophy.
D A Butterfield A D Roses S H Appel D B Chesnut 《Archives of biochemistry and biophysics》1976,177(1):226-234
A goat antibody produced against bovine adrenal ferredoxin has been employed to establish immunochemically the involvement of adrenal ferredoxin in the cholesterol side-chain cleavage reaction catalyzed by mammalian adrenal mitochondria. When added to preparations of bovine adrenocortical mitochondria, this antibody was found to inhibit the conversion of cholesterol to pregnenolone and progesterone, the 11β-hydroxylation of deoxycorticosterone and the NADPH-dependent reduction of cytochrome c. These observations demonstrate that, similar to the NADPH-cytochrome c reductase and steroid 11β-hydroxylase reactions, adrenal ferredoxin is also required for the oxidative cleavage of the cholesterol side-chain catalyzed by bovine adrenocortical mitochondria.The goat antibody to bovine adrenal ferredoxin was also found to interact with the comparable iron-sulfur proteins present in mitochondria prepared from sheep, rat, mouse, cat, dog, guinea pig, rabbit, and human adrenals. The interaction of the antibody with these iron-sulfur proteins resulted in the inhibition of both the cholesterol side-chain cleavage and NADPH-cytochrome c reductase activities catalyzed by these adrenal mitochondria. The NADH-dependent reduction of cytochrome c catalyzed by mammalian adrenal mitochondria was not inhibited by the goat antibody to adrenal ferredoxin. These results demonstrate the immunochemical similarity existing among mammalian adrenal ferredoxins and their involvement in the adrenal cholesterol side-chain cleavage reaction. 相似文献
14.
Immunocytochemical localization of secretory proteins in bovine pancreatic exocrine cells 总被引:1,自引:0,他引:1
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The bovine exocrine pancreatic cell produces a variety of enzymes and proenzymes for export. Biochemical studies by Greene L.J., C.H. Hirs, and G.E. Palade (J. Biol. Chem. 1963. 238:2054) have shown that the mass proportions of several of these proteins in resting pancreatic juice and zymogen granule fractions are identical. In this study we have used immunocytochemical techniques at the electron microscope level to determine whether regional differences exist in the bovine gland with regard to production of individual secretory proteins and whether specialization of product handling occurs at the subcellular level. The technique used is a modification of one previously reported (McLean, J.D., and S.J. Singer. 1970. Proc. Natl. Acad. Sci U.S.A. 69:1771) in which immunocytochemical reagents are applied to thin sections of bovine serum albumin-imbedded tissue and zymogen granule fractions. A double antibody technique was used in which the first step consisted of rabbit F(ab')2 antibovine secretory protein and the detection step consisted of sheep (F(ab')2 antirabbit F(ab')2 conjugated to ferritin. The results showed that all exocrine cells in the gland, and all zymogen granules and Golgi cisternae in each cell, were qualitatively alike with regard to their content of secretory proteins examined (trypsinogen, chymotrypsinogen A, carboxypeptidase A, RNase, and DNase). The data suggest that these secretory proteins are transported through the cisternae of the Golgi complex where they are intermixed before copackaging in zymogen granules; passage through the Golgi complex is apparently obligatory for these (and likely all) secretory proteins, and is independent of extent of glycosylation, e.g., trypsinogen, a nonglycoprotein vs. DNase, a glycoprotein. 相似文献
15.
Summary Whether both casein and noncasein (serum or whey) proteins of milk are contained within the same secretory vesicles of milk secreting mammary epithelial cells was explored. Antibodies to a major casein and to -lactalbumin of rat milk were localized in thin sections with colloidal gold-conjugated second antibodies. Antibodies to the casein component bound to an antigen present within lumina of Golgi apparatus cisternae and within secretory vesicles. This antigen was also recognized in structures within secretory vesicles and within alveolar lumina which were ultrastructurally identified as casein micelles. Antigens recognized by antibodies to -lactalbumin also were present in Golgi apparatus cisternae and within secretory vesicles. Both anti-casein and anti--lactalbumin antibodies recognized antigens within the same secretory vesicles. These observations show that one major noncasein protein of rat's milk is present in casein-containing secretory vesicles. 相似文献
16.
Anterograde and retrograde traffic between the rough endoplasmic reticulum and the Golgi complex 总被引:17,自引:7,他引:10
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《The Journal of cell biology》1995,131(6):1387-1401
The transfer of newly synthesized membrane proteins moving from the rough endoplasmic reticulum (RER) to the Golgi complex has been studied by electron microscopy in HEp-2 cells transfected with cDNAs for chimeric proteins. These proteins consist of a reporter enzyme, horseradish peroxidase (HRP), anchored to the transmembrane domains of two integral membrane proteins, the transferrin receptor and sialyl- transferase. The chimeras are distributed throughout the nuclear envelope, RER, vesicular tubular clusters (VTCs) and a network of tubules in the cis-Golgi area. At 20 degrees C tubules containing chimera connect the RER to the VTCs and to the cis-Golgi network. On transfer to 37 degrees C in the presence of dithiothreitol (DTT), the chimeras are seen to move from the RER and through the Golgi stack. With this temperature shift the direct connections with the RER are lost and free vesicles form; some of these vesicles contain HRP reaction product which is much more concentrated than in the adjacent RER while others lack reaction product entirely. In cells expressing SSHRPKDEL, DAB reaction product remains distributed throughout the RER, the VTCs, and the cis-Golgi network for prolonged periods in the presence of DTT and almost all of the vesicles which form at 37 degrees C are DAB-positive. Together these observations demonstrate that all three chimeras are transported from the RER to the cis-Golgi in free, 40-60-nm vesicles at 37 degrees C. They also suggest that the retrograde traffic which carries SSHRPKDEL back to the RER is probably mediated by vesicles with a similar morphology but which, in cells expressing membrane-anchored chimeras, lack detectable reaction product. 相似文献
17.
Summary The fate of lectin labeled internalized plasma membrane in the ascites tumor form of the Chang rat hepatoma growing under in vivo and in vitro conditions was investigated cytochemically. Ascites cells were incubated in Concanavalin A (Con A) and horseradish peroxidase (PO), either with or without prior glutaraldehyde fixation and subsequently treated with 3,3-diaminobenzidine. In cells fixed before Con-A-PO labeling the reaction product was localized as a continuous and even layer upon the external surface of the plasma membrane. If unfixed cells were treated with Con A, coupled with PO at 4°C and reinbated in phosphate buffered saline at 37°C for varying periods of time, the Con-A-PO layer was of irregular thickness. In as little as 15 min of reincubation endocytotic vesicles containing PO positive material were closely associated with GERL components of the Golgi Apparatus. Localization of acid phosphatase (ACPase) within GERL vesicles, similar in size and location to those containing Con-A-PO reaction product, indicates that the Con-A-PO labeled vesicles may be a component of the Golgi apparatus in hepatoma cells.Supported by NIH Grant CA 16663. 相似文献
18.
Secretory Proteins from Adrenal Medullary Cells Are Carboxyl-Methylated In Vivo and Released Under Their Methylated Form by Acetylcholine 总被引:1,自引:0,他引:1
The carboxyl methylation of secretory proteins in vivo was investigated in bovine adrenal medullary cells in culture. Chromogranin A, the major intragranular secretory protein in adrenal medullary cells, and other secretory proteins were found to be carboxyl-methylated within secretory vesicles. The in vivo labeling pattern using [methyl-3H]methionine and the in vitro labeling pattern using S-adenosyl-[methyl-14C]methionine of intravesicular secretory proteins were similar. The detection of methylated chromogranin A in mature secretory vesicles required 3-6 h, a time consistent with the synthesis and storage of secretory proteins in this tissue. Carboxyl-methylated chromogranin A was secreted from medullary cells by exocytosis via activation of nicotinic cholinergic receptor and recovered still under the methylated form in the incubation medium. Since protein-carboxyl-methylase is cytosolic, these results suggest that methylation of secretory proteins is a cotranslational phenomenon. 相似文献
19.
R. H. Ashley D. M. Brown D. K. Apps J. H. Phillips 《European biophysics journal : EBJ》1994,23(4):263-275
A K+ channel was incorporated into voltage-clamped planar lipid bilayers from bovine chromaffin granules and resealed granule membranes (ghosts). It was not incorporated from plasma membrane-rich fractions from the adrenal medulla. The channel had a conductance of 400 pS in symmetric 450 mM KCI, with the permeability sequence K+ > Rb+ > Cs+ > Na+ > Li+, and was insensitive to both Ca2+ and charybdotoxin. It exhibited complex gating kinetics, consistent with the presence of multiple open and closed states, and its gating was voltage-dependent. The channels appeared to incorporate into bilayers with the same orientation, and were blocked from one side (the side of vesicle addition) by 0.2-1 mM TEA'. The block was slightly voltage-dependent. Acidification of resealed granule membranes in response to external ATP (which activated the vacuolartype ATPase) was significantly reduced in the presence of 1 mM intralumenal TEACI (with 9 mM KCl), and parallel measurements with the potential-sensitive dye Oxonol V showed that such vesicles tended to develop higher internal-positive membrane potentials than control vesicles containing only 10 mM KCI. 1 mM TEA+ had no effect on proton-pumping activity when applied externally, and did not directly affect either the proton-pumping or ATP hydrolytic activity of the partially-purified ATPase. These results suggest that chromaffin granule membranes contain a TEA+-sensitive K+ channel which may have a role in regulating the vesicle membrane potential.
Correspondence to: R. H. Ashley 相似文献
20.
《The International journal of biochemistry》1984,16(5):489-495
- 1.1. Ferredoxin reductase and ferredoxin were purified from the bovine corpus luteum and their properties compared to the corresponding adrenal proteins.
- 2.2. The luteal and adrenal proteins had similar absorbance spectra and molecular weights.
- 3.3. Evidence was obtained from spectrophotometric titrations for formation of 1:1 complexes between luteal ferredoxin reductase and ferredoxin and between ferredoxin and cytochrome P-450scc.
- 4.4. Adrenal ferredoxin reductase and ferredoxin were equally as effective as luteal ferredoxin reductase and ferredoxin in supporting cholesterol side-chain cleavage by luteal cytochrome P-450scc.