Dissociative Mechanism of Thermal Denaturation of Rabbit Skeletal Muscle Glycogen Phosphorylase b

Abstract

The thermal stability of rabbit skeletal muscle glycogen phosphorylase b was characterized using enzymological inactivation studies, differential scanning calorimetry, and analytical ultracentrifugation. The results suggest that denaturation proceeds by the dissociative mechanism, i.e., it includes the step of reversible dissociation of the active dimer into inactive monomers and the following step of irreversible denaturation of the monomer. It was shown that glucose 1-phosphate (substrate), glucose (competitive inhibitor), AMP (allosteric activator), FMN, and glucose 6-phosphate (allosteric inhibitors) had a protective effect. Calorimetric study demonstrates that the cofactor of glycogen phosphorylase-pyridoxal 5'-phosphate-stabilizes the enzyme molecule. Partial reactivation of glycogen phosphorylase b preheated at 53 degrees C occurs after cooling of the enzyme solution to 30 degrees C. The fact that the rate of reactivation decreases with dilution of the enzyme solution indicates association of inactive monomers into active dimers during renaturation. The allosteric inhibitor FMN enhances the rate of phosphorylase b reactivation.

Kinetics of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscle: Mechanism of protective action of α‐crystallin

“…The sedimentation coefficients were corrected to solvent density and viscosity (208C, water) in the standard way as described earlier. 14 …”

“…There is a lag period on the kinetic curve. To explain the appearance of the lag period, we proposed the kinetic scheme 14 including the stage of the conformational transition in the dimeric form of Phb. The theory of the lag periods for thermoinactivation of oligomeric enzymes that allows the number of the conformational transitions preceding the stage of irreversible denaturation to be determined was elaborated by Bednarck et al 32 The fact that the inactivation rate increases with decreasing Phb concentration means that the stage of irreversible denaturation of monomers is preceded by the stage of reversible dissociation of dimer into monomers.…”

Thermal Dissection of Lentil Seedling Amine Oxidase Domains by Differential Scanning Calorimetry

“…Our experiments showed that in LSAO, T m and ÁH of the DSC profile are independent of the protein concentration. Based on reports of Takahashi and Sturtevant 18) and Kurganov et al 19) The independence of thermal parameters from protein concentration shows that oligomerization does not contribute to the thermal unfolding process. So in the thermal denaturation of LSAO dissociation to monomers does not play any role.…”

Dissociative mechanism for irreversible thermal denaturation of oligomeric proteins

“…The Phb molecule consists of two identical subunits with molecular masses of 97.4 kDa each. The data on analytical ultracentrifugation provide evidence of the dissociation of the dimer into monomers at elevated temperatures Kurganov et al 2000;Meremyanin et al 2008). …”

“…1 Kinetics of thermal inactivation of Phb from rabbit skeletal muscles at 53°C (0.08 M Hepes-NaOH, pH 6.8). The relative enzymatic activity A/A 0 versus time plot was constructed on the basis of the data represented in the work by Kurganov et al (2000). Enzyme concentration: 1 1.6 and 2 5.2 mg/mL function of protein concentration has the following form (Kurganov 1966):…”