MODIFICATION OF GLYCOGEN PHOSPHORYLASE B BY GLUTARALDEHYDE . PREPARATION AND ISOLATION OF ENZYME DERIVATIVES WITH ENHANCED STABILITY

Incubation of glycogen phosphorylase b with glutaraldehyde, a bifunctional protein reagent, resulted in inactivation of the enzyme. Approximately 60% inactivation could be observed after incubation of the enzyme with 0.05% reagent for 10 min. The partially inactivated enzyme showed multiple protein bands upon polyacrylamide gel electrophoresis. The various protein species were differentially susceptible to thermal denaturation. Heating at 50° for 5 hr resulted in the denaturation and precipitation of 90% of the modified enzyme. Gel electrophoretic analysis of the remaining protein showed only one major (80-90%) and one minor protein component. The minor protein component could be removed by gel filtration on a Sephadex G-200 column. This modified and heat-treated enzyme was considerably more resistant than native phosphorylase b toward heat, cold, or urea denaturation. Titration with trinitrobenzenesulfonic acid revealed that the stabilized enzyme contained 7-11 less free amino groups than native phosphorylase b. Amino acid analysis also indicated that the modified enzyme contained 10% less lysyl residues than native phosphorylase b. Titration of the enzyme with Ellman reagent suggested that sulfhydryl groups had not reacted in the modified enzyme. The specific activity of the modified and heat-treated phosphorylase b was approximately 65% that of the native enzyme. Although the affinities of this enzyme species toward glucose 1-phosphate and glycogen were the same as those of native phosphorylase b, the homotropic interaction of AMP which was observed with native enzyme could not be demonstrated with the stabilized phosphorylase b. Modification of phosphorylase b with monofunctional aldehyde was also investigated. Enzyme derivatives of these modifications were also more resistant to denaturation than native phosphorylase b. The relative stability of 1% butyraldehyde-modified and glutaraldehyde-stabilized phosphorylase b depended upon denaturation conditions. While butyraldehyde-modified enzyme showed a higher resistance toward heat denaturation (50-51°) the glutaraldehyde derivative appeared to be more stable upon exposure to cold or urea solutions. Kinetic studies indicated that homotropic interactions of adenosine monophosphate was enhanced in the 1% butyraldehydemodified phosphorylase b. © 1969, American Chemical Society. All rights reserved.