α-Amylase Secretion by Single Barley Aleurone Layers

Abstract

The secretion of .alpha.-amylase from single isolated (H. vulgare L. cv Himalaya) aleurone layers was studied in an automated flow-through apparatus. The apparatus, consisting of a modified sample analyzer linked to a chart recorder, automatically samples the flow-through medium at 1-min intervals and assays for the presence of .alpha.-amylase. The release of .alpha.-amylase from aleurone layers begins after 5-6 h of exposure to GA and reaches a maximum rate after 10-12 h. The release of .alpha.-amylase shows a marked dependence on Ca2+ and in the absence of Ca2+ it is only 20% of that in the presence of 10 mM Ca2+. Withdrawal of Ca2+ from the flow-through medium results in the immediate cessation of enzyme release, and addition of Ca2+ causes immediate resumption of the release process. The effect of Ca2+ is concentration-dependent, being half-maximal at 1 mM Ca2+ and saturated at 10 mM Ca2+. Ruthenium red, which blocks Ca2+ but not Mg2+ efflux from barley aleurone layers, renders .alpha.-amylase release insensitive to Ca2+ withdrawal. Inhibitors of respiratory metabolism cause a burst of .alpha.-amylase release which lasts for 0.5-5 h. Following this phase of enhanced .alpha.-amylase release, the rate of release declines to zero. Pretreatment of aleurone layers with HCl prior to incubation in HCN also causes a burst of .alpha.-amylase release, indicating that the inhibitor is affecting the secretion of .alpha.-amylase and not its movement through the cell wall. The rapid inhibition of .alpha.-amylase release upon incubation of aleurone layers at low temperature (5.degree. C) or in 0.5 M mannitol also indicates that enzyme release is dependent on a metabolically linked process and is not diffusion-limited. Cytochemical observations show that, although the cell wall matrix of aleurone layers undergoes extensive digestion after GA treatment, the innermost part of the cell wall is not degraded and could influence enzyme release.