The use of C14-labelled substrate in histochemical demonstration of different forms of phosphorylase

Active and total phosphorylase activity, using labelled C¹⁴-glucose-1-phosphate as the substrate, is demonstrated by histoautoradiographic method. This method can demonstrate the polysaccharide synthesizedin vitro by phosphorylase without intervention from the unlabelled pre-existing glycogen. C¹⁴-glucose can not replace C¹⁴-glucose-1-phosphate as substrate. The distribution of phosphorylase in tissue sections, except in cases of very low activity, is similar to that obtained by customary dilute Lugol's iodine staining method. The relative difference of intensity between active and total phosphorylase, as revealed by iodine staining, is also reflected by histoautoradiographic method. Histoautoradiographic method has several advantages over the iodine staining method. This method is more sensitive for demonstration of very low phosphorylase activity which may escape detection by iodine staining. Branching enzyme activity, especially when it favors synthesis of glycogen type of polysaccharide instead of amylopectin type, can be better detected by this method. Active phosphorylase substrate medium can be used to demonstrate this activity in plant tissues, where the presence of pre-existing starch often prohibits the use of iodine staining method. Stripping film method for autoradiography is recommended for the study of this enzyme activity.