Radioautographic visualization of β-glucans formed by pea membranes from UDP-glucose

Abstract

Radioautographic experiments were carried out using pea stem slices to determine the site of glucose incorporation from UDP-glucose. Cut or damaged pea stem cells were the only cells to incorporate [3H]glucose from UDP-[3H]glucose. The product formed at 20 μM UDP-glucose was observed in electron microscope thin sections in patches on the plasma membrane and the cell wall. The product formed at 5 mM UDP-glucose occurred in fibrillar bundles that stretched between the plasma membrane and the cell wall. This periplasmic material fluoresced when stained with aniline blue. Experiments in which slices were subjected to sequential incubations in radioactive 5 mM UDP-glucose followed by unlabelled 5 mM UDP-glucose, or incubations in the reverse order, indicated that incorporation of [3H]glucose into products insoluble in chloroform:methanol:water or hot water occurs at the plasma membrane, and radioactivity is displaced from the membrane by subsequent incubations. A similar experiment, in which slices were first incubated in radioactive 20 μM UDP-glucose followed by unlabelled 5 mM UDP-glucose, indicated that the synthesis of fibrillar material from 5 mM UDP-glucose displaces the labelled product that had been formed from 20 μM UDP-glucose. It is concluded that only cut or damaged pea stem cells utilize UDP-glucose and the plasma membrane enzymes that incorporate [3H]glucose from 20 μM or 5 mM UDP-[3H]glucose are in close physical proximity.