Use of an antibody probe to study regulation of glycogen phosphorylase by its NH2-terminal region.

Antibodies that are specific for the NHz-terminal region of rabbit muscle glycogen phosphorylase were isolated. Studies, using synthetic peptides representing different segments of the NHz-terminal region of muscle phosphorylase, indicated the antibodies are highly specific for the first 4 NHz-terminal residues of the enzyme. The molecular weight of the complex formed between dimeric phosphorylase and the antibodies estimated by gel filtration suggests that only 1 molecule of antibody binds per dimer of phosphorylase. The antibodies were strongly inhibitory to both phosphorylase kinase and phosphorylase phosphatase. Apparent binding constants for glucose l-phosphate and AMP and inhibition by compounds that bind at or near the glucose l-phosphate and AMP sites were not affected by the antibodies. The apparent K,,, for the high molecular weight substrate, glycogen, was lowered 2-fold by the presence of the antibodies. The primary binding site for maltoheptaose, and presumably for glycogen, recently has been shown to be a site separate from the active site (Kasvinsky, P. J., Madsen, N. B., Fletterick, R. J., and Sygusch, J. (1978) J. Biol. Chem 253, 1290-1296). The improved binding affinity for glycogen, induced by the antibodies, is consistent with regulation of this glycogen site by the NHz-terminal region. The binding of the antibodies to phosphorylase b completely stabilized the enzyme to loss of its cofactor, pyridoxal 5’-phosphate, under conditions in which the cofactor is normally completely resolved. Because the antibodies did not affect the apparent binding affinities for compounds (glucose, glucose l-phosphate, and caffeine) that bind in the same hydrophobic active site crevice as pyridoxal phosphate, the suggestion is made that the dramatic effect of the antibodies on the pyridoxal5’-phosphate site is quite specific. The specific antibodies against muscle phosphorylase were able to bind to the liver isozyme of phosphorylase. When antibodies were bound to the liver isozyme, the apparent affinity (K,,,) for glucose l-phosphate was improved by 4.1-fold at saturating AMP. At concentrations of glucose l-phosphate lower than the Km, the antibodies increased enzyme activity by more than lofold. The conclusion is made that the structural character of the NH&erminal regions of liver and muscle