Alpha-amylase secretion by single barley aleurone layers.

The secretion of alpha-amylase from single isolated (Hordeum 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 minute intervals and assays for the presence of alpha-amylase. The release of alpha-amylase from aleurone layers begins after 5 to 6 hours of exposure to gibberellic acid and reaches a maximum rate after 10 to 12 hours. The release of alpha-amylase shows a marked dependence on Ca(2+), and in the absence of Ca(2+) it is only 20% of that in the presence of 10 millimolar Ca(2+). Withdrawal of Ca(2+) from the flow-through medium results in the immediate cessation of enzyme release and addition of Ca(2+) causes immediate resumption of the release process. The effect of Ca(2+) is concentration-dependent, being half-maximal at 1 millimolar Ca(2+) and saturated at 10 millimolar Ca(2+). Ruthenium red, which blocks Ca(2+) but not Mg(2+) efflux from barley aleurone layers, renders alpha-amylase release insensitive to Ca(2+) withdrawal. Inhibitors of respiratory metabolism cause a burst of alpha-amylase release which lasts for 0.5 to 5 hours. 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 degrees C) or in 0.5 molar mannitol also indicates that enzyme release is dependent on a metabolically linked process and is not diffusion-limited. This conclusion is supported by cytochemical observations which show that, although the cell wall matrix of aleurone layers undergoes extensive digestion after gibberellin treatment, the innermost part of the cell wall is not degraded and could influence enzyme release.