Ultracentrifugal studies of the effect of molecular crowding by trimethylamine N-oxide on the self-association of muscle glycogen phosphorylase b.
The suitability of sedimentation equilibrium for characterizing the self-association of muscle glycogen phosphorylase b has been reappraised. Whereas sedimentation equilibrium distributions for phosphorylase b in 40 mM Hepes buffer (pH 6.8) supplemented with 1 mM AMP signify a lack of chemical equilibrium attainment, those in buffer supplemented additionally with potassium sulfate conform with the requirements of a dimerizing system in chemical as well as sedimentation equilibrium. Because the rate of attainment of chemical equilibrium under the former conditions is sufficiently slow to allow resolution of the dimeric and tetrameric enzyme species by sedimentation velocity, this procedure has been used to examine the effects of thermodynamic nonideality arising from molecular crowding by trimethylamine N-oxide on the self-association behaviour of phosphorylase b. In those terms the marginally enhanced extent of phosphorylase b self-association observed in the presence of high concentrations of the cosolute is taken to imply that the effects of thermodynamic nonideality on the dimer-tetramer equilibrium are being countered by those displacing the T<==>R isomerization equilibrium for dimer towards the smaller, nonassociating T state. Because the R state is the enzymically active form, an inhibitory effect is the predicted consequence of molecular crowding by high concentrations of unrelated solutes. Thermodynamic nonideality thus provides an alternative explanation for the inhibitory effects of high concentrations of glycerol, sucrose and ethylene glycol on phosphorylase b activity, phenomena that have been attributed to extremely weak interaction of these cryoprotectants with the T state of the enzyme.
Chebotareva
N A
NA
Centre for Macromolecular Hydrodynamics, University of Nottingham, Sutton Bonington, UK.
Harding
S E
SE
Winzor
D J
DJ
D000818:Animals; D003451:Cryoprotective Agents / Q000494:pharmacology; D019855:Ethylene Glycol / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008744:Methylamines / Q000737:chemistry*; D008956:Models, Chemical; D015233:Models, Statistical; D018482:Muscle, Skeletal / Q000737:chemistry*; D006005:Phosphorylases / Q000737:chemistry*; D011485:Protein Binding; D011817:Rabbits; D013395:Sucrose / Q000494:pharmacology; D013816:Thermodynamics; D014461:Ultracentrifugation
D003451:Cryoprotective Agents; D008744:Methylamines; D013395:Sucrose; D006005:Phosphorylases; D019855:Ethylene Glycol; C005855:trimethyloxamine; D005990:Glycerol