Rabbit muscle phosphorylase derivatives with oligosaccharides covalently bound to the glycogen storage site

• Published in: Biochemistry (Easton) 1982-06, Vol.21 (13), p.3043-3050
• Main Authors: Philip, George, Gringel, Gisela, Palm, Dieter
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; chemical modification; Crystallization; Kinetics; muscles; Muscles - enzymology; Oligosaccharides - pharmacology; phosphorylase; Phosphorylase a - isolation & purification; Phosphorylase a - metabolism; Phosphorylase b - isolation & purification; Phosphorylase b - metabolism; Phosphorylases - metabolism; Rabbits
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00256a002

Linear maltooligosaccharides, e.g., maltoheptaose or terminal 4-O-methylmaltoheptaose, activated by cyanogen bromide, react covalently with rabbit muscle phosphorylases b and a (EC 2.4.1.1). Site-specific modification prevents further binding to glycogen and shifts the phosphorylase a tetramer-dimer equilibrium in favor of the dimer. Use was made of these properties to separate by affinity chromatography and gel filtration phosphorylase a dimers with specifically bound oligosaccharide from unspecifically modified products. The phosphorylase a-maltoheptaose derivative carries one oligosaccharide residue per monomer and can be distinguished from the native enzyme by its electrophoretic mobility in polyacrylamide gels or by affinity electrophoresis. Phosphorylase a preparations with covalently bound maltooligosaccharides are enzymatically active in the presence of a primer and alpha-D-glucopyranose 1-phosphate (glucose-1-P). Methylation of the nonreducing chain terminus of the bound oligosaccharide has no effect on glycogen synthesis. These findings exclude the participation of bound oligosaccharides in chain elongation. Purified covalent phosphorylase a-maltoheptaose complexes are stable dimers. They are no longer activated by glycogen. The properties of covalently modified phosphorylase-oligosaccharides are consistent with and provide direct evidence for the existence of a glycogen storage site in rabbit muscle phosphorylases. Covalent occupation of the storage site renders the affinity of glucose-1-P to phosphorylase a independent of modulation by glycogen, supporting the assumption that the glycogen storage site is involved in interactions with the catalytic site.