INFLUENCE OF MOLECULAR SIZE OF GLYCOGEN ON THE PHOSPHORYLASE REACTION

Earlier experiments in viva have revealed that after glucose-C4 administration to animals in acute experiments, glycogen, whether from liver or muscle, was non-uniformly labeled.Within individual molecules various tiers of glucose residues were unequally labeled (1,2).Glycogen molecules of differing size in a given polydisperse population were also noted to incorporate label to different degrees (3).In muscle, higher concentrations of isotope were found in the largest molecules, whereas in liver it was the fraction of glycogen containing the smallest molecules that was most heavily labeled.In an attempt to elucidate the basis for this metabolic inhomogeneity with respect to size, glycogen samples have been prepared from liver and muscle.These have been compared in regard to certain chemical properties and to their reactivities with muscle and liver phosphorylase in vitro. MethodsGlycogen samples were isolated from cold trichloroacetic acid extracts of tissues (TCA glycogen) and by hot aqueous KOH digestion (KOH glycogen) (4).Fractionation of TCA glycogen was accomplished by the method of differential centrifugation.It was found that KOH glycogen, which was of smaller average molecular size, could only be conveniently fractionated by the method of differential precipitation with ethanol (3).Estimation of molecular weights by light scattering methods and analysis of samples for radioactivity were carried out as previously described (3).Structural Studies of Glycogen-The periodate oxidation method for end group determination (5, 6) was modified for use with samples of 5 or 10 mg. of glycogen.Glycogen samples, dissolved in 0.2 ml. of 3 per cent NaCl, were allowed to react with 0.2 ml. of 0.3 N NaI04 at 0" in the dark.Excess periodate was destroyed by the addition of 2 drops of ethylene glycol.The reaction mixture was diluted with a little Con-free water, and the formic acid produced was measured by titration with 0.01 N Ba(OH)t by using a Scholander microburette (7).During the titration the solution was agitated by a stream of COz-free Nz, and a mixture of methyl red and methylene blue was used as indicator.