Localization of Carbohydrate Metabolizing Enzymes in Guard Cells of Commelina communis

Abstract

The localization of enzymes involved in the flow of carbon into and out of starch was determined in guard cells of Commelina communis. The guard cell chloroplasts were separated from the rest of the cellular components by a modification of published microfuge methods. The enzymes of interest were then assayed in the supernatant and chloroplast fractions. The chloroplast yield averaged 75% with 10% cytoplasmic contamination. The enzymes involved in starch biosynthesis, ADPglucose pyrophosphorylase, starch synthase, and branching enzyme, are located exclusively in the chloroplast fraction. The enzymes involved in starch degradation show a more complex distribution. Phosphorylase is located in both the supernatant and chloroplast fraction, 50% in each fraction. Most of the amylase and debranching enzyme activity is present in the supernatant (70%) fraction. The majority of the rest of the enzymes involved in the degradation of starch to malate and synthesis of starch from a hexose precursor were also investigated. All of the enzymes were present in the chloroplast except for hexokinase and phosphofructokinase. The inability to assay these enzymes could possibly have been due to the lack of or low activity of the enzymes or to nonoptimal assay conditions.