Affinity Labeling of Muscle Phosphorylase b with α-Cyclodextrin-Dialdehyde

Abstract

Rabbit muscle phosphorylase b was modified with a substrate analog, the 2', 3'-dialdehyde derivative of α-cyclodextrin (dial-α-CD). Although the inhibition of phosphorylase b by α-, β-, and γ-cyclodextrins gave rather high Ki values (10-25 ITIM), the dial-CD gave much smaller Ki values of 1.2-3.5mM. Moreover, the latter inhibition was time-dependent and accelerated by higher pHs and higher concentrations of dial-CD. Incorporation of the dial-CD into the enzyme was proportional to the loss of enzyme activity and became stationary at about 1 mol of dial-CD bound to a mol of enzyme subunit. Modification was greatly suppressed by the presence of substrate glycogen. Glucose 1-phosphate was not effective. The dial-CD-modified phosphorylase b was purified by Sephadex G-75 and Con A-Sepharose column chromatography. The modified enzyme gave a single band of activity having a K_app of 6.3% glycogen on affinity gel electrophoresis, which showed that the modified enzyme had a very low affinity for glycogen. Comparison of Km values of the native and modified phosphorylase b showed that the Km values for the glucan substrates increased 8 to 11-fold in the modified enzyme. These results suggested that the glycogen storage site of muscle phosphorylase b might be modified with dial-CD and the modified enzyme significantly decreased in the affinity for the substrate glycogen.