Localization of phytase and acid phosphatase isoenzymes in aleurone layers of barley

Abstract

The localization of acid phosphatase (EC 3.1.3.2) in aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) grains was studied. Phosphatase (EC 3.1.3.26) activity, assayed with phytic acid as the substrate, is present in the dry grain at low leveis and increases during incubation in H2O at 25°C for three days. When aleurone layers are isolated from imbibed grain and incubated for 18 h in buffer with or without 50 μM gibberellic acid (GA3), the level of extractable phosphatase activity increases two‐ to threefold, and phosphatase is released into the medium. GA, promotes the release of phosphatase activity: aleurone layers incubated in GA, release twice as much phosphatase as layers incubated in buffer. Nine isoenzymes of phosphatase are found in aleurone layers of barley by non‐denaturing polyacrvlamide gel electropho‐resis. Six of these forms, isoenzymes 1,2,3,5,6 and 8, can be extracted from dry tissue, and after three days of imbibition in H2O an additional isoenzyme, isoenzyme 9, is found in aleurone extracts. When isolated aleurone layers are incubated for a further 22 h in buffer with or without GA3, isoenzyme 7 is found and yet another form, isoenzyme 4, is found in layers incubated in GA3. Eight isoenzymes are released from aleurone layers into the incubation medium. Isoenzymes 5 and 6 are released in buffer both with and without GA3, even when cycloheximide is present; cycloheximide inhibits the release of the other isoenzymes. Isoenzymes 1‐4, 7 and 8, on the other hand, are secreted into the incubation medium only when GA3, is present. Isoenzyme 9 is not released into the incubation medium. Acid phosphatase activity was localized in aleurone tissue using cytochemical, cell fractionation, and enzymatic methods. Cytochemical localization of ATPase (EC 3.6.1.8) in aleurone tissue showed the presence of enzyme activity in cell wall, protein bodies, endoplasmic reticulum, Golgi apparatus, and mitochondria. Analysis of organelle fractions isolated by density gradient centrifugation showed that the activity of acid phosphatase isoenzymes 1, 2 and 3 was prominently associated with the phytin globoid of protein bodies, and analysis of the activity released from the cell wall by enzymatic digestion showed that it was almost exclusively isoenzymes 5 and 6.