Quantitative histochemical assessment of the heterogeneity of glycogen phosphorylase activity in liver parenchyma of fasted rats using the semipermeable membrane technique and the PAS reaction.
Glycogen phosphorylase (EC 2.4.1.1) has been demonstrated in sections of liver from rats starved for 24 h. The method is based on the measurement of the amount of glycogen formed after incubation in a gelled medium containing glucose 1-phosphate as substrate, using the semipermeable membrane technique. Glycogen was demonstrated with the periodic acid-Schiff (PAS) reaction. Phosphorylase activity appeared to be highest in periportal areas. The optimum substrate concentration for revealing activity of the enzyme was 60-120 mM. After incubation in the absence of substrate, the staining intensity, as measured cytophotometrically as the mean integrated absorbance at 560 nm, was similar to that of an unincubated section. p-Chloromercuribenzoate, a non-specific inhibitor of glycogen phosphorylase activity, reduced the formation of final reaction product attributable to phosphorylase activity completely. The Michaelis constants (KM) of the enzyme in periportal and pericentral areas differed. This was probably due to the presence of the a form only in periportal areas and of the a and b forms in pericentral areas. The mean integrated absorbances in both the periportal and pericentral areas increased linearly with incubation time (4-16 min). A linear relationship was also found with section thickness (4-10 micron). The total activity of glycogen phosphorylase in the periportal areas was double the pericentral activity. It is concluded that the semipermeable membrane technique, combined with the PAS reaction for glycogen, can be used as a valid method for the demonstration and quantification of glycogen phosphorylase activity in livers from starved rats.
D000818:Animals; D005215:Fasting; D006651:Histocytochemistry / Q000379:methods; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008297:Male; D006005:Phosphorylases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013194:Staining and Labeling
D006005:Phosphorylases