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  • 1
    Electronic Resource
    Electronic Resource
    The role of the endoplasmic reticulum in the synthesis and transport of α-amylase in barley aleurone layers (1982)
    Springer
    Planta 156 (1982), S. 421-432 
    Details
    ISSN: 1432-2048
    Keywords: Aleurone ; α-Amylase ; Endoplasmic reticulum ; Gibberellin and enzyme formation ; Hordeum (α-amylase in aleurone)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The subcellular site of α-amylase (EC 1.6.2.1) synthesis and transport was studied in barley aleurone layers incubated in the presence or absence of gibberellic acid (GA3). Using [35S]methionine as a marker, the site of amino-acid incorporation into organelles isolated from aleurone layers incubated with and without GA3 was determined following purification by isopycnic sucrose-density-gradient centrifugation. Incorporation of radioactivity into trichloroacetic-acid-insoluble proteins was greatest in those fractions exhibiting activity of an endoplasmic reticulum (ER) marker enzyme. Further fractionation of densitygradient fractions by sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis showed that a major portion of the radioactivity in the ER fractions was present in a protein co-migrating with marker α-amylase. This protein was identified as authentic α-amylase by immunoadsorbent chromatography and affinity chromatography. The newly synthesized α-amylase associated with the ER was shown to be sequenstered within the lumen of the ER by experiments which showed that the enzyme was resistant to proteolytic degradation. The labelled α-amylase sequestered in the ER can be chased from this organelle when tissue is incubated in unlabelled methionine following a 1-h pulse of labelled methionine. The isoenzymic forms of α-amylase found in tissue homogenates and incubation media of aleurone layers incubated with and without GA3 were characterized after chromatography on diethylaminoethyl cellulose. In homogenates of GA3-treated aleurone layers, five peaks of α-amylase activity were detected, while in homogenates of aleurone layers incubated with-out GA3 only three peaks of activity were found. In incubation media, four isoenzymes were found after GA3 treatment and two were found after incubation without GA3. We conclude that at least five α-amylase isoenzymes are synthesized by the ER of barley aleurone layers and that this membrane system is involved in the sequestration and transport of four of these isoenzymes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00393313
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  • 2
    Electronic Resource
    Electronic Resource
    The isolation of endoplasmic reticulum from barley aleurone layers (1980)
    Springer
    Planta 150 (1980), S. 58-69 
    Details
    ISSN: 1432-2048
    Keywords: Aleurone ; α-Amylase ; Endoplasmic reticulum ; Hordeum ; Organelle isolation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Techniques for the isolation and purification of endoplasmic reticulum (ER) from aleurone layers of barley (Hordeum vulgare L.) were assessed. Neither differential centrifugation nor density gradient centrifugation of a homogenate separate the ER or other organelles of this tissue from the lipidcontaining spherosomes. Isopycnic sucrose gradient centrifugation of organelles first purified by molecular sieve chromatography on Sepharose 4B, however, results in separation of the organelles based on their differing buoyant densities. Manipulation of the magnesium concentration of the isolation media and density-gradient solutions affords isolation of ER at a density of 1.13–1.14 g cc-1 and 1.17–1.18 g cc-1. Electron microscopy shows that the membranes sedimenting at 1.13–1.14 g cc-1 are devoid of ribosomes and are characteristic of smooth ER, while those sedimenting at 1.17–1.18 g cc-1 are studded with ribosomes and have the features of rough ER. Endoplasmic reticulum isolated by isopycnic density gradient centrifugation can be further purified by rate-zonal centrifugation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00385616
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  • 3
    Electronic Resource
    Electronic Resource
    Quantitative and qualitative changes in the endoplasmic reticulum of barley aleurone layers (1980)
    Springer
    Planta 150 (1980), S. 70-81 
    Details
    ISSN: 1432-2048
    Keywords: Aleurone ; α-Amylase ; Endoplasmic reticulum ; Gibberellin ; Hordeum ; Organelle isolation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Changes in the level of the endoplasmicreticulum (ER) marker enzyme cytochrome-c reductase (EC 1.6.2.1) were followed with time of imbibition of de-embryonated half-seeds of barley (Hordeum vulgare L.) and the subsequent incubation of their aleurone layers in gibberellic acid (GA3) and H2O. During imbibition there is an increase in the level of cytochrome-c-reductase activity and in the amount of 280-nm absorbance associated with this enzyme. When aleurone layers are incubated for a further 42 h in water, there is a doubling of the cytochrome-c-reductase activity. In GA3, the activity of cytochrome-c reductase reaches a maximum at 24 h of incubation and thereafter falls to below 70% of its level at the beginning of the incubation period. Changes in the cytochrome-c-reductase activity correlate with changes in the fine structure of the aleurone cell. The ER isolated in low Mg2+ from aleurone layers incubated in buffer for up to 18 h has buoyant density of 1.13–1.14 g cc-1 while that from layers incubated in GA3 for 7.5–18 h has a density of 1.11–1.12 g cc-1. The α-amylase (EC3.2.1.1) isolated with the organelle fraction by Sepharose gel filtration is associated with the ER on isopycnic and rate-zonal density gradients, and its activity can be enhanced by Triton X-100. The soluble α-amylase fraction from Separose-4B columns, on the other hand, is not Triton-activated but is acid-labile. Acid phosphatase (EC3.1.3.2) is distributed in at least three peaks on isopycnic gradients. In low Mg2+ the second peak of activity has a density of 1.12 g cc-1 in GA3-treated tissue and 1.13–1.14 g cc-1 in H2O-treated tissue. With high-Mg2+ buffers, this peak of phosphatase activity disappears. Acid-phosphatase activity is not enhanced by Triton X-100 nor is it acid-labile.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00385617
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    Paper (German National Licenses)
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  • 4
    Electronic Resource
    Electronic Resource
    Calcium regulation of the secretion of α-amylase isoenzymes and other proteins from barley aleurone layers (1983)
    Springer
    Planta 158 (1983), S. 1-9 
    Details
    ISSN: 1432-2048
    Keywords: Aleurone ; α-Amylase ; Calcium and enzyme secretion ; Enzyme secretion ; Gibberellin ; Hordeum (enzyme secretion)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effect of calcium on the secretion of α-amylase (EC 3.2.1.1) and other hydrolases from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was studied. Withdrawal of Ca2+ from the incubation medium of aleurone layers preincubated in 5 μM gibberellic acid (GA3) and 5 mM CaCl2 results in a 70–80% reduction in the secretion of α-amylase activity to the incubation medium. Agar-gel electrophoresis shows that the reduction in α-amylase activity following Ca2+ withdrawal is correlated with the disappearance of group B isoenzymes from the incubation medium. The secretion of isoenzymes of group A is unaffected by Ca2+. The addition of Ca2+ stimulates the secretion of group-B isoenzymes but has no measurable effect on either the α-amylase activity or the isoenzyme pattern of aleurone-layer extracts. Pulse-labelling experiments with [35S]methionine show that Ca2+ withdrawal results in a reduction in the secretion of labelled polypeptides into the incubation medium. Immunochemical studies also show that, in the absence of Ca2+, α-amylase isoenzymes of group B are not secreted into the incubation medium. In addition to its effect on α-amylase, Ca2+ influences the secretion of other proteins including several acid hydrolases. The secretion of these other proteins shows the same dependence on Ca2+ concentration as does that of α-amylase. Other cations can promote the secretion of α-amylase to less and varying extents. Strontium is 85% as effective as Ca2+ while Ba2+ is only 10% as effective. We conclude that Ca2+ regulates the secretion of enzymes and other proteins from the aleurone layer of barley.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00395396
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    Paper (German National Licenses)
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