Planta
Planta 143, 89 99 (1978)
9 by Springer-Verlag 1978
The Mode of Secretion of ~-Amylase in Barley Aleurone Layers Robert Locy* and Hans Kende MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA
Abstract. The involvement of the endomembrane system of barley ( H o r d e u m v u l g a r e L.) aleurone cells in the secretion of gibberellin-induced hydrolases has been investigated at the biochemical level. Our results show that at least 40 60% of the e-amylase activity in homogenates of aleurone layers occurs in a membrane-bound, latent form. The latent e-amylase can be assayed quantitatively following disruption of membranes by treatment with Triton X-100, ethanol, sonication, or osmotic shock and shear. The association of e-amylase with the membrane is not an artifact arising from homogenization of the tissue, and acid protease is also enriched in the same subcellular fraction as the a-amylase. The membrane fraction with which the e-amylase is associated has many properties of the endoplasmic reticulum (ER). When membranebound e-amylase is prepared in buffers containing 3 m M MgCI2 two fractions from a sucrose step gradient contain most of the e-amylase activity. These fractions are enriched in the E R marker enzyme, NADH-dependent cytochrome-c reductase, and show densities characteristic of smooth and rough ER during subsequent purification on continuous gradients. In step gradients prepared with ethylenediaminetetraacetic-acid-treated membranes, e-amylase activity is contained primarily in one fraction having the density of smooth ER. Electron microscopy of the purified fractions is consistent with e-amylase being Present address: Department of Horticultural Science, North Carolina State University, Raleigh, NC 27607, USA Abbreviations: CNTPE= N-carbobenzoxy-L-tyrosine p-nitrophenylester; Cyt oxidase=cytochrome oxidase; ER=endoplasmic reticulum; EDTA=ethylenediaminetetraacetic acid; GA3= gibberellic acid; IDPase=inosine diphosphatase; K+-ATPase= pH 6.5 K+-stimulated adenosine triphosphatase; MES=2-(Nmorpholino)ethanesulfonic acid; MOPS=3-(N-morpholino)propanesulfonie acid; NADH:Cyt c reductase=cyanide-insensitive NADH-linked cytochrome-creductase; RER =rough endoplasmic reticulum; Tris = tris-(hydroxymethyl)-aminomethane *
associated with smooth and rough ER. However, it has not been ruled out that the enzyme is also associated with plasma membrane, Golgi membranes, or tonoplast. Examination of the isoenzyme patterns of secreted, of total-homogenate and of membrane-associated e-amylases, as well as the results from pulsechase experiments using L-[3H]leucine for labeling of ~-amylase, are all consistent with the hypothesis that membrane-associated e-amylase is an intermediate in the secretory process.
Key words: Aleurone cells - e-Amylase - Gibberellin - H o r d e u m - Membrane fractionation - Secretion (enzymes).
Introduction In the cereal aleurone layer, e-amylase (Filner and Varner, 1967) and a protease (Jacobsen and Varner, 1967) were shown to be synthesized d e n o v o and secreted in response to GA 3. Ultrastructural studies (Jones, 1969a, b; Jones and Price, 1970; Vigil and Ruddat, 1973) indicated that R E R accumulated after treatment of aleurone layers with GA3, and the hypothesis was advanced that ER-derived vesicles are involved in the secretory process (Colborne et al., 1976; Jones, 1969b; Vigil and Ruddat, 1973). This hypothesis has been further examined in several laboratories. Jones (1972) and Chen and Jones (1974a, 1974b) were unable to find biochemical or autoradiographic evidence that secretion of e-amylase involved a membrane-bound intermediate. In contrast, Gibson and Paleg (1972, 1975) using wheat and Firn (1975) using barley were able to demonstrate that a substantial fraction (40-70%) of the e-amylase activity in an aleurone-layer homogenate was associated with a membrane. Our studies were carried
0032-0935/78/0143/0089/$ 02.20
90
out to help in clarifying this controversy and to expand our knowledge concerning the mode of enzyme secretion in plants.
Material and Methods Preparation of Barley Aleurone Layers Barley (Hordeum vulgare L. cv. Himalaya, 1969 harvest, obtained from the Agronomy Department, Washington State University, Pullman, Wash., USA) half-seeds were prepared as described by Chrispeels and Varner (1967) and allowed to imbibe water for 3 5 d at 25 ~ C. Following imbibition, half-seeds, or aleurone layers isolated from half-seeds according to Phillips and Paleg (1972), were incubated as described by Johnson and Kende (1971) except that each flask contained up to 150 half-seeds or aleurone layers. In some experiments it was necessary to inactivate the secreted, ceil-wall-bound amylase. This was accomplished by a 10-min treatment of the aleurone layers with 0.001 N HC1 as described by Varner and Meuse (1972).
Preparation and Fractionation of Tissue Homogenates Aleurone layers were homogenized (30-60 layers) in 1.8 ml of a medium consisting of 0.4 M sucrose, 25 m M Tris-MES buffer (pH 7.5), 10 m M KC1, 1 m M EDTA, and either 0.1 or 3 m M MgC12. Homogenization was carried out for 5 min using a motor-driven chopper (Cunningham et al., 1966). In the initial studies the unfiltered homogenate was centrifuged at 1000 x g. The supernatant liquid was saved, the pellet was resuspended in the homogenization medium and recentrifuged, and this whole procedure repeated one more time. The three supernatant liquids were combined and applied to a Sepharose 4B column (Pharmacia Fine Chemicals, Piscataway, N.J., USA). In subsequent studies, the homogenate was filtered through Miracloth (Chicopee Mills, Milltown, N.J., USA) and centrifuged at 13,000xg for 15 rain. The supernatant liquid was applied directly to the Sepharose 4B column. Sepharose column chromatography of the centrifuged homogenate was performed as described by Firn (1975) except that the column dimensions were 2.5 cm (diameter) x 10 cm. The fractions eluting in the void volume from the Sepharose 4B column were further fractionated by sucrose density gradient ultracentrifugation. The pooled fractions were layered over sucrose step gradients consisting of 5 ml of 50% (w/w) sucrose (special enzyme grade; Schwartz-Mann, Orangeburg, N.Y., USA) overlayered by 5 ml each of 37, 25 and 20% sucrose. All sucrose solutions contained 25 mM Tris-MES buffer (pH 7.5), 10 m M KC1, 1 m M EDTA, and either 0.1 or 3 m M MgC12. The gradients were centrifuged at 25,000 rpm in an SW 27 rotor using a Beckman L-2 Ultracentrifuge (Beckman Instruments, Palo Alto, Cal., USA). The gradients were fractionated, and the absorbance at 280 nm was determined using an ISCO UA-5 absorbance monitor (Instrumentation Specialties Co., Lincoln, Neb., USA) The appropriate particulate fractions (see figure legends) were further fractionated by applying them to the top of linear, 20-50% sucrose gradients. These gradients were centrifuged as above for 4 h and fractionated as above.
Enzyme Assays e-Amylase was assayed according to Jones and Varner (1967), except that Triton X-100 (Research Products International Corp.,
R. Locy and H. Kende : Mode of Secretion of a-Amylase Elk Grove Village, Ill., USA) was added to all enzyme aliquots to a final concentration of 0.1% (v/v). One enzyme unit(E.U.) of a-amylase activity is the amount of enzyme that causes a change in absorbance at 620 um of 1 in 1 min. N A D H : Cyt c reductase, Cyt oxidase, IDPase, and K § -ATPase were assayed by the procedures of Hodges and Leonard (1974). Glucan synthetases I and II were assayed as described by Ray (1977). Protease activity was assayed spectrophotometrically using the synthetic protease substrate CTNPE. The reaction mixture contained 10 m M MOPS buffer (pH 6.9), 0.1 m M dithiothreitol, 10% acetonitrile, and 0.1 m M CTNPE. The amount of enzyme added was selected so that the reaction rate was linear and proportional to enzyme concentration. The reaction was initiated by the addition of the CTNPE, and the rate of the reaction was followed spectrophotometrically at 400 nm. All spectrophotometric determinations were made using a Gilford Model 240 spectrophotometer equipped with a Gilford model 6040 recorder (Gilford Instruments, Oberlin, O., USA).
Protein and RNA Determinations Protein was determined by the Folin phenol method (Lowry et al., 1951) using bovine serum albumin as a standard. RNA was determined by the orcinol reaction as described by Schneider (1957) using yeast R N A as a standard.
Purification of c~-Amylase a-Amylase was purified according to Rodaway and Kende (1978). For pulse-chase studies where purification of e-amylase was necessary, the same procedure was used, but the steps subsequent to glycogen-ethanol precipitation were omitted, c~-Amylase purified to this point gave a single band in SDS acrylamide gel electrophoresis.
Preparation of Radioactive c~-Amylase Isolated aleurone layers were incubated in 1 gM GA3 for 6 h as described above. Following this, the incubation medium was decanted and replaced with new medium containing 1 mCi L[3H]leucine (62Ci/mmol; Schwartz-Mann, Orangeburg, N.Y., USA). Incubation was continued for an additional 14 h, at which time the incubation medium was decanted and saved. The aleurone layers were homogenized in fresh incubation buffer using a mortar and pestle. The 3H-labeled c~-amylases from the incubation medium and from the homogenate were separately purified by the procedure of Rodaway and Kende (1978). All determinations of radioactivity were made by pipetting up to 1 ml of radioactive solution onto a tightly compressed pellet of Kimwipe (Kimberly-Clark Corp.,Neenah, Wis., USA) in a paper thimble. The Kimwipe was dried in an oven at 130~ for at least 1 h, and combusted in a Model 306 Packard sample oxidizer (Packard Instruments, Downers Grove, Ill., USA). The radioactivity in such samples was determined in a Packard model 3375 Scintillation Spectrometer.
Separation of c~-Amylase Isoenzymes a-Amylase isoenzymes were separated by electrophoresis on 7.5% acrylamide gels following essentially the procedure of Ingle (1968) with 10 m M Tris-glycine, pH 8.9, as running buffer. The electrophoresis of the protein (0.08~. 15 E.U./gel) was carried out on gels
R. Locy and H. Kende: Mode of Secretion of c~-Amylase of 6 mm diameter at 2.5 mAmp/gel using a Model 3371E LKB DC power supply (LKB Instruments, Rockville, Md., USA). The ~-amylase-containing areas of the gel were visualized by incubating the gel for 1 h at 37~ C in starch solution followed by staining with I2/KI solution. The starch solution and staining procedure were as described by Jacobsen et al. (1970).
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Planta 143, 89 99 (1978)
9 by Springer-Verlag 1978
The Mode of Secretion of ~-Amylase in Barley Aleurone Layers Robert Locy* and Hans Kende MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA
Abstract. The involvement of the endomembrane system of barley ( H o r d e u m v u l g a r e L.) aleurone cells in the secretion of gibberellin-induced hydrolases has been investigated at the biochemical level. Our results show that at least 40 60% of the e-amylase activity in homogenates of aleurone layers occurs in a membrane-bound, latent form. The latent e-amylase can be assayed quantitatively following disruption of membranes by treatment with Triton X-100, ethanol, sonication, or osmotic shock and shear. The association of e-amylase with the membrane is not an artifact arising from homogenization of the tissue, and acid protease is also enriched in the same subcellular fraction as the a-amylase. The membrane fraction with which the e-amylase is associated has many properties of the endoplasmic reticulum (ER). When membranebound e-amylase is prepared in buffers containing 3 m M MgCI2 two fractions from a sucrose step gradient contain most of the e-amylase activity. These fractions are enriched in the E R marker enzyme, NADH-dependent cytochrome-c reductase, and show densities characteristic of smooth and rough ER during subsequent purification on continuous gradients. In step gradients prepared with ethylenediaminetetraacetic-acid-treated membranes, e-amylase activity is contained primarily in one fraction having the density of smooth ER. Electron microscopy of the purified fractions is consistent with e-amylase being Present address: Department of Horticultural Science, North Carolina State University, Raleigh, NC 27607, USA Abbreviations: CNTPE= N-carbobenzoxy-L-tyrosine p-nitrophenylester; Cyt oxidase=cytochrome oxidase; ER=endoplasmic reticulum; EDTA=ethylenediaminetetraacetic acid; GA3= gibberellic acid; IDPase=inosine diphosphatase; K+-ATPase= pH 6.5 K+-stimulated adenosine triphosphatase; MES=2-(Nmorpholino)ethanesulfonic acid; MOPS=3-(N-morpholino)propanesulfonie acid; NADH:Cyt c reductase=cyanide-insensitive NADH-linked cytochrome-creductase; RER =rough endoplasmic reticulum; Tris = tris-(hydroxymethyl)-aminomethane *
associated with smooth and rough ER. However, it has not been ruled out that the enzyme is also associated with plasma membrane, Golgi membranes, or tonoplast. Examination of the isoenzyme patterns of secreted, of total-homogenate and of membrane-associated e-amylases, as well as the results from pulsechase experiments using L-[3H]leucine for labeling of ~-amylase, are all consistent with the hypothesis that membrane-associated e-amylase is an intermediate in the secretory process.
Key words: Aleurone cells - e-Amylase - Gibberellin - H o r d e u m - Membrane fractionation - Secretion (enzymes).
Introduction In the cereal aleurone layer, e-amylase (Filner and Varner, 1967) and a protease (Jacobsen and Varner, 1967) were shown to be synthesized d e n o v o and secreted in response to GA 3. Ultrastructural studies (Jones, 1969a, b; Jones and Price, 1970; Vigil and Ruddat, 1973) indicated that R E R accumulated after treatment of aleurone layers with GA3, and the hypothesis was advanced that ER-derived vesicles are involved in the secretory process (Colborne et al., 1976; Jones, 1969b; Vigil and Ruddat, 1973). This hypothesis has been further examined in several laboratories. Jones (1972) and Chen and Jones (1974a, 1974b) were unable to find biochemical or autoradiographic evidence that secretion of e-amylase involved a membrane-bound intermediate. In contrast, Gibson and Paleg (1972, 1975) using wheat and Firn (1975) using barley were able to demonstrate that a substantial fraction (40-70%) of the e-amylase activity in an aleurone-layer homogenate was associated with a membrane. Our studies were carried
0032-0935/78/0143/0089/$ 02.20
90
out to help in clarifying this controversy and to expand our knowledge concerning the mode of enzyme secretion in plants.
Material and Methods Preparation of Barley Aleurone Layers Barley (Hordeum vulgare L. cv. Himalaya, 1969 harvest, obtained from the Agronomy Department, Washington State University, Pullman, Wash., USA) half-seeds were prepared as described by Chrispeels and Varner (1967) and allowed to imbibe water for 3 5 d at 25 ~ C. Following imbibition, half-seeds, or aleurone layers isolated from half-seeds according to Phillips and Paleg (1972), were incubated as described by Johnson and Kende (1971) except that each flask contained up to 150 half-seeds or aleurone layers. In some experiments it was necessary to inactivate the secreted, ceil-wall-bound amylase. This was accomplished by a 10-min treatment of the aleurone layers with 0.001 N HC1 as described by Varner and Meuse (1972).
Preparation and Fractionation of Tissue Homogenates Aleurone layers were homogenized (30-60 layers) in 1.8 ml of a medium consisting of 0.4 M sucrose, 25 m M Tris-MES buffer (pH 7.5), 10 m M KC1, 1 m M EDTA, and either 0.1 or 3 m M MgC12. Homogenization was carried out for 5 min using a motor-driven chopper (Cunningham et al., 1966). In the initial studies the unfiltered homogenate was centrifuged at 1000 x g. The supernatant liquid was saved, the pellet was resuspended in the homogenization medium and recentrifuged, and this whole procedure repeated one more time. The three supernatant liquids were combined and applied to a Sepharose 4B column (Pharmacia Fine Chemicals, Piscataway, N.J., USA). In subsequent studies, the homogenate was filtered through Miracloth (Chicopee Mills, Milltown, N.J., USA) and centrifuged at 13,000xg for 15 rain. The supernatant liquid was applied directly to the Sepharose 4B column. Sepharose column chromatography of the centrifuged homogenate was performed as described by Firn (1975) except that the column dimensions were 2.5 cm (diameter) x 10 cm. The fractions eluting in the void volume from the Sepharose 4B column were further fractionated by sucrose density gradient ultracentrifugation. The pooled fractions were layered over sucrose step gradients consisting of 5 ml of 50% (w/w) sucrose (special enzyme grade; Schwartz-Mann, Orangeburg, N.Y., USA) overlayered by 5 ml each of 37, 25 and 20% sucrose. All sucrose solutions contained 25 mM Tris-MES buffer (pH 7.5), 10 m M KC1, 1 m M EDTA, and either 0.1 or 3 m M MgC12. The gradients were centrifuged at 25,000 rpm in an SW 27 rotor using a Beckman L-2 Ultracentrifuge (Beckman Instruments, Palo Alto, Cal., USA). The gradients were fractionated, and the absorbance at 280 nm was determined using an ISCO UA-5 absorbance monitor (Instrumentation Specialties Co., Lincoln, Neb., USA) The appropriate particulate fractions (see figure legends) were further fractionated by applying them to the top of linear, 20-50% sucrose gradients. These gradients were centrifuged as above for 4 h and fractionated as above.
Enzyme Assays e-Amylase was assayed according to Jones and Varner (1967), except that Triton X-100 (Research Products International Corp.,
R. Locy and H. Kende : Mode of Secretion of a-Amylase Elk Grove Village, Ill., USA) was added to all enzyme aliquots to a final concentration of 0.1% (v/v). One enzyme unit(E.U.) of a-amylase activity is the amount of enzyme that causes a change in absorbance at 620 um of 1 in 1 min. N A D H : Cyt c reductase, Cyt oxidase, IDPase, and K § -ATPase were assayed by the procedures of Hodges and Leonard (1974). Glucan synthetases I and II were assayed as described by Ray (1977). Protease activity was assayed spectrophotometrically using the synthetic protease substrate CTNPE. The reaction mixture contained 10 m M MOPS buffer (pH 6.9), 0.1 m M dithiothreitol, 10% acetonitrile, and 0.1 m M CTNPE. The amount of enzyme added was selected so that the reaction rate was linear and proportional to enzyme concentration. The reaction was initiated by the addition of the CTNPE, and the rate of the reaction was followed spectrophotometrically at 400 nm. All spectrophotometric determinations were made using a Gilford Model 240 spectrophotometer equipped with a Gilford model 6040 recorder (Gilford Instruments, Oberlin, O., USA).
Protein and RNA Determinations Protein was determined by the Folin phenol method (Lowry et al., 1951) using bovine serum albumin as a standard. RNA was determined by the orcinol reaction as described by Schneider (1957) using yeast R N A as a standard.
Purification of c~-Amylase a-Amylase was purified according to Rodaway and Kende (1978). For pulse-chase studies where purification of e-amylase was necessary, the same procedure was used, but the steps subsequent to glycogen-ethanol precipitation were omitted, c~-Amylase purified to this point gave a single band in SDS acrylamide gel electrophoresis.
Preparation of Radioactive c~-Amylase Isolated aleurone layers were incubated in 1 gM GA3 for 6 h as described above. Following this, the incubation medium was decanted and replaced with new medium containing 1 mCi L[3H]leucine (62Ci/mmol; Schwartz-Mann, Orangeburg, N.Y., USA). Incubation was continued for an additional 14 h, at which time the incubation medium was decanted and saved. The aleurone layers were homogenized in fresh incubation buffer using a mortar and pestle. The 3H-labeled c~-amylases from the incubation medium and from the homogenate were separately purified by the procedure of Rodaway and Kende (1978). All determinations of radioactivity were made by pipetting up to 1 ml of radioactive solution onto a tightly compressed pellet of Kimwipe (Kimberly-Clark Corp.,Neenah, Wis., USA) in a paper thimble. The Kimwipe was dried in an oven at 130~ for at least 1 h, and combusted in a Model 306 Packard sample oxidizer (Packard Instruments, Downers Grove, Ill., USA). The radioactivity in such samples was determined in a Packard model 3375 Scintillation Spectrometer.
Separation of c~-Amylase Isoenzymes a-Amylase isoenzymes were separated by electrophoresis on 7.5% acrylamide gels following essentially the procedure of Ingle (1968) with 10 m M Tris-glycine, pH 8.9, as running buffer. The electrophoresis of the protein (0.08~. 15 E.U./gel) was carried out on gels
R. Locy and H. Kende: Mode of Secretion of c~-Amylase of 6 mm diameter at 2.5 mAmp/gel using a Model 3371E LKB DC power supply (LKB Instruments, Rockville, Md., USA). The ~-amylase-containing areas of the gel were visualized by incubating the gel for 1 h at 37~ C in starch solution followed by staining with I2/KI solution. The starch solution and staining procedure were as described by Jacobsen et al. (1970).
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