Interaction of flavin mononucleotide with dimeric and tetrameric forms of muscle phosphorylase β

Ultracentrifugal studies of the effect of molecular crowding by trimethylamine N‐oxide on the self‐association of muscle glycogen phosphorylase b

“…1, which comprises two clearly resolved boundaries, the sedimentation coefficients of which (after correction to s 20,w ) coincide with the respective values of 8.7 S and 13.4 S for dimer and tetramer. Interpretation of the areas of these two g(s*) peaks in terms of Eqn (5) yields a dimerization constant, K 2,4 , of 3.0 Â 10 4 m 21 for phosphorylase b in the present Hepes/AMP environment, a value which is in reasonable agreement with that of 2.4 Â 10 4 m 21 deduced previously by sedimentation velocity under similar (but not identical) conditions [4]. This evaluation of an equilibrium constant from the size of boundaries formed by resolving the mixture into its separate species is incompatible with the concept of any chemical interconversion to restore equilibrium during the course of the experiment.…”

“…Clearly, these results defy description in terms of a common ordinate intercept, C 2 (r F ), and a common slope, 2C 4 (r F ), the behaviour of a dimer±tetramer system in association equilibrium. Although the inverse dependence of the ordinate intercept upon rotor speed would be consistent with the concept of a pressuredependent chemical equilibrium, this possibility has been precluded in a previous investigation [4]. It is therefore concluded that sedimentation equilibrium patterns for phosphorylase b under the present conditions should be considered in terms of a noninteracting mixture of dimer and tetramer, rather than that of a dimer±tetramer system in reversible association equilibrium.…”

“…Allosteric activation of the enzyme by AMP induces the formation of tetramers, which are also favoured by a decrease in temperature or the presence of sulfate ions [2±5]. On the basis that adoption of the R state is a prerequisite for tetramer formation [3,6±8], the position of the dimer±tetramer equilibrium provides a means of monitoring effector-induced transitions between the inactive (T state) and active (R state) conformations of the enzyme subunits [4,9,10]. The present investigation was initiated with a view to determining the likely consequences of molecular crowding by high concentrations of inert solutes, which also have the potential to favour protein selfassociation [11±13] and hence to act as nonspecific effectors of the phosphorylase reaction.…”

“…Whereas initial characterization of the dimer±tetramer equilibrium entailed sedimentation velocity studies of glycogen phosphorylase b [4], the use of sedimentation equilibrium has been reported subsequently [17]. In this investigation, the results were analyzed on the basis that chemical equilibrium prevailed throughout the sedimentation equilibrium distributions, the usual assumption in studies of proteins for which the various oligomeric states are in rapid association equilibrium.…”

Sedimentation analysis of muscle glycogen phosphorylase b entrapped in hydrated reversed micelles of aerosol OT in octane

“…Allosteric activator AMP provokes the association of the enzyme with formation of tetramers (13)(14)(15)(16)(17)(18). The portion of the tetrameric form increases with the increase in protein concentration and the decrease of temperature (18).…”