EFFECT OF EDTA ON HISTOCHEMICAL DEMONSTRATION OF PHOSPHORYLASE ACTIVITY

Effects of ethylenediaminetetraacetate on the phosphorylase activity were studied histochemically and biochemically; sections were incubated in a variety of media with or without EDTA, and stained by I 2 -KI solution and periodic acid-Schiff reagent for the demonstration of synthesized and total glycogen, respectively. Incubated sections and the substrate media used for the incubation were also assayed for glycogen by the anthrone method. Both qualitative and qualitative data showed that EDTA stimulated greatly the synthesis of glycogen from glucose 1-phosphate in sections, and markedly reduced the degradation of glycogen present in sections, which otherwise occurred following incubation in acetate buffer. Other chelating agents, such as 1,2-cyclohexane diamine tetraacetic acid, diethylene triamine pentaacetic acid, nitrilotriacetic acid and glycolether diamine tetraacetic acid, failed to replace for EDTA in the phosphorylase staining. On the other hand, only 1,2-cyclohexane diamine tetraacetic acid inhibited glycogen degradation similarly to EDTA. The stimulative effect of EDTA on the phosphorylase staining might be explained by inhibition of hepatic α-amylase and activation of phosphorylase. Celloidine coating and subsequent treatment of sections with cold 30% ethanol enhanced the phosphorylase staining. Addition of adenosine 5'-phosphoric acid to the substrate medium did not significantly stimulate the enzyme activity.