title: The mode of secretion of α-amylase in barley aleurone layers.
abstract: The involvement of the endomenbrane system of barley (Hordeum vulgare 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 α-amylase activity in homogenates of aleurone layers occurs in a membrane-bound, latent form. The latent α-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 α-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 α-amylase. The membrane fraction with which the α-amylase is associated has many properties of the endoplasmic reticulum (ER). When membranebound α-amylase is prepared in buffers containing 3 mM MgCl2 two fractions from a sucrose step gradient contain most of the α-amylase activity. These fractions are enriched in the ER 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 ethylenediaminete-traacetic-acid-treated membranes, α-amylase activity is contained primarily in one fraction having the density of smooth ER. Electron microscopy of the purified fractions is consistent with α-amylase being 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 α-amylases, as well as the results from pulsechase experiments using L-[(3)H]leucine for labeling of α-amylase, are all consistent with the hypothesis that membrane-associated α-amylase is an intermediate in the secretory process.
authors.affiliation: MSU-DOE Plant Research Laboratory, Michigan State University, 48824, East Lansing, MI, USA.
authors.forename: R
authors.initials: R
authors.lastname: Locy
authors.forename: H
authors.initials: H
authors.lastname: Kende