Interaction of phosphorylase b with eosin. Influence of substrate and effectors on eosin-enzyme complexes

Abstract

The interactions of rabbit muscle glycogen phosphorylase b with Eosin (2',4',5',7'-tetrabromofluorescein) was studied. Eosin was found to be an effective inhibitor of the enzyme. The inhibition constants for the dye were estimated to be approx. 36 and 60 microM with respect to AMP and glucose 1-phosphate respectively. The binding of Eosin to phosphorylase b is accompanied by a red-shift of about 12 nm in the dye absorption-spectrum maximum, indicating low-polarity binding sites on the enzyme molecule for the dye. The absorbance in the difference absorption maximum at 537 nm was utilized to follow the conjugation of phosphorylase b with Eosin. Scatchard plots of the titration data revealed the existence of at least two classes of binding sites on the protein molecule for Eosin, and the dissociation constants measured in Tris/HCl buffer, pH 7.0 (IO.091), were 7.7 and 41.7 microM respectively. The influence of the substrates and effectors on Eosin-enzymes complexes was used to study the ligand-phosphorylase b interactions. IMP displaced the dye completely from the enzyme, indicating that there are two IMP-binding sites per phosphorylase b monomer. AMP binding to the enzyme with respect to Eosin concentration is of two types: a non-competitive one for the high-affinity site for AMP and a competitive one for the low-affinity site for the activator. The effects of glucose 6-phosphate, ATP, Pi and glycerol 2-phosphate in the system are in according dance with a partially competitive model. Glucoes 1-phosphate and UDP-glucose appear to affect only the high-affinity site for Eosin, whereas glucose and glycogen have no effect on Eosin-phosphorylase b complexes. Our results suggest that Eosin can be used as an efficient optical probe for studying the phosphorylase b system.

Properties of Small Molecules Affecting Insulin Receptor Function

“…The changes in the UV-vis spectrum accompanying binding of EO and ER to sIR and mIR are similar to those observed when these molecules bind to other proteins (52)(53)(54) with small variations of the wavelengths at the extrema. Although EO has increased quantum yield in solutions with low polarity (41), the quenching of ER observed in the presence of sIR may originate from a number of different mechanisms.…”

Regulation of Muscle Glycogen Phospfaorylase by Physiological Effectors

“…The fact that the Hill coefficient exceeds unity is indicative of the positive kinetic cooperativity between activator-binding sites in the dimeric molecule of phosphorylase b. Similar values for the Hill coefficient for glucose-I -P and AMP have been obtained also by Madsen and Shechosky (1967), Sealock and Graves (1967), Black and Wang (1968), and Oikonomakos et al (1979). Thus, the kinetics of rabbit muscle phosphorylase b is characterized by the positive homotropic cooperativity between the binding sites in the dimeric enzyme molecule.…”

Spectroscopic characterization of insulin and small molecule ligand binding to the insulin receptor

“…Fluorescein and derivatives have been shown to bind to a number of different proteins [5,6,13,14,29,33,42]. Furthermore, the changes in UV-vis and fluorescence spectra are of similar nature as those observed when EO and ER bind to other proteins [20,25,34,43].…”