Cold Inactivation of Glycogen Phosphorylase*

Glycogen phosphorylase b has been found more sensitive to storage at 0° than at 20° in glycerophosphate-cysteine buffer at pH 6.0. After storage at cold temperatures, enzymic activity is lost, inhomogeneous material can be detected in the ultracentrifuge, and a shift and increase of absorbance of enzyme-bound pyridoxal phosphate occur to shorter wavelengths. Inactivation may be reversed upon rewarming. While the initial rate of cold inactivation of phosphorylase b has been shown to follow first-order kinetics at all protein concentrations, the entire course of inactivation can be described by the first-order law only at low protein concentrations. Inactivation is effectively slowed by glycogen, pyridoxal phosphate, AMP, ATP, organic solvents, and buffer at pH 6.8, and is accelerated at pH 6.0 in the presence or cysteine and NaCl. An Arrhenius plot of phosphorylase b activities at pH 6.0 shows a marked discontinuity at approximately 13°, with activation energies of 17,000 and 46,000 calories for the upper and lower limbs, respectively. NaBH4-reduced phosphorylase b is also cold sensitive. Phosphorylase a, in contrast to phosphorylase b, is sensitive to cold only in solutions of high ionic strength. The present data suggest that the inactivation of phosphorylase results from a conformational change of enzyme structure induced by cold temperatures and is not caused by a loss of enzyme-bound pyridoxal phosphate.