The existence of a hexameric intermediate with molten-globule-like properties in the thermal denaturation of bovine-liver glutamate dehydrogenase

Singh, Narinder; Liu, Zhongzheng; Fisher, Harvey F.

doi:10.1016/s0301-4622(96)02192-8

Cited by 18 publications

(19 citation statements)

References 26 publications

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“…Interaction of the enzyme with these ligands result in conformational changes in the allosteric protein [15,21]. The thermodynamics of formation of GDH-ligand complexes has been investigated, and a protective role for ADP, a positive effector for GDH, was reported [29]. In the present study, we have investigated the influence of some of the well-known allosteric effectors for the enzyme, both positive and negative, on its thermal aggregation.…”

Section: Influence Of Allosteric Effectors On Gdh Aggregationmentioning

confidence: 98%

“…It has been shown that cooperative binding of ligands such as NADPH and ADP affect thermal stability of the enzyme [25][26][27][28]. Activation energy for thermal denaturation of the GDH-ADP complex is substantially higher (about 40 kcal mol −1 ) than that of the native enzyme [29].…”

Section: Introductionmentioning

confidence: 99%

“…Various methods have been used to study denaturation of the enzyme: chemical [30], thermal [29], acid-induced [31], and mild denaturation with the use of a non-ionic detergent [32] have been reported. In the case of thermal denaturation of bovine GDH, as well as acid-induced denaturation of hyperthermophilic arhaeon Pyrococcus furiosus GDH [33], hexameric assembly in the molten globule state has been observed.…”

Section: Introductionmentioning

confidence: 99%

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Thermal aggregation of a model allosteric protein in different conformational states

Sabbaghian

Ebrahim‐Habibi

Nemat‐Gorgani

2009

International Journal of Biological Macromolecules

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Section: Influence Of Allosteric Effectors On Gdh Aggregationmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Thermal aggregation of a model allosteric protein in different conformational states

Sabbaghian

Ebrahim‐Habibi

Nemat‐Gorgani

2009

International Journal of Biological Macromolecules

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“…In most of the earlier studies, the molten globule state was obtained either as equilibrium or kinetic intermediate on the unfolding or refolding pathways of monomeric proteins. Recently Singh et al (44) reported an intermediate of the hexameric enzyme, glutamate dehydrogenase, with molten globule-like properties, based on differential scanning calorimetric studies. Our present study shows that ␣-crystallin, a multimeric protein, undergoes a transition at around 62°C in which the near UV-CD spectrum indicates loss of all its tertiary structure, but the far UV-CD spectrum shows an increase in the ellipticity.…”

Section: ␣-Crystallin Structural Changes and Chaperone-like Activitymentioning

confidence: 99%

Chaperone-like Activity and Temperature-induced Structural Changes of α-Crystallin

Raman

Rao

1997

Journal of Biological Chemistry

141

132

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␣-Crystallin is known to exhibit chaperone-like activity. We have studied its chaperone-like activity toward the aggregation of ␤ L -crystallin upon refolding of this protein from its unfolded state in guanidinium chloride. The chaperone-like activity of ␣-crystallin is less pronounced below 30°C and is enhanced above this temperature. The plot of percentage protection as a function of temperature shows two transitions; one at 30°C and another at around 55°C. We have performed steady state fluorescence, fluorescence polarization, fluorescence quenching, circular dichroism, sedimentation analysis, and gel filtration chromatography to probe the temperature-induced structural changes of ␣-crystallin. Our results show that at above 50°C, ␣-crystallin undergoes a transition to a multimeric molten globule-like state. Above 30°C, a minor but detectable perturbation in its tertiary structure occurs that might lead to the observed exposure of its hydrophobic surfaces. These results support our earlier hypothesis that ␣-crystallin prevents the aggregation of other proteins by providing appropriately placed hydrophobic surfaces; a structural transition above 30°C involving enhanced or reorganized hydrophobic surfaces of ␣-crystallin is important for its chaperone-like activity. It is possible that a structural alteration induced by temperature forms a part of the general mechanism of chaperone function, because they are required to function more effectively at nonpermissible temperatures.␣-Crystallin, a multimeric protein composed of both acidic (␣ A ) and basic (␣ B ) subunits with the molecular mass of approximately 20 kDa, is a major protein of the eye lens. It is also expressed in other tissues such as heart, kidney, brain, muscles, etc. (1-4) and under certain diseased conditions (5-8). It shares both structural and sequence homology with small heat shock proteins and behaves in several ways like small heat shock proteins (9 -12). Its expression can be induced by thermal (9) or hypertonic (13) stress. It was believed to play only a structural role in the formation of the transparent and highly refractive tissue of the eye lens. However, the discovery of its presence in non-lenticular tissues and its homology with small heat shock proteins suggests that it might have other functional properties.␣-Crystallin is shown to prevent the heat-induced aggregation of other crystallins and enzymes like a molecular chaperone (14). The chaperone-like property of this crystallin has been investigated by several workers in order to gain insight in both the mechanism of this property and its relevance to the eye lens transparency (15-30). ␣-Crystallin from the old human lenses (28) and from the selenite-induced cataract lenses of an animal model (29) are found to exhibit decreased chaperone-like activity. The chaperone-like activity of ␣-crystallin might be important in the formation and maintenance of the eye lens transparency, and the loss of transparency can be attributed to the loss of function of ␣-crystallin (17).Earlier we inv...

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“…Contains the mathematical solution of the glutamate dehydrogenase denaturation model proposed by Singh et al~1996!. …”

Section: Supplementary Materials In Electronic Appendixmentioning

confidence: 99%

Pressure‐induced thermostabilization of glutamate dehydrogenase from the hyperthermophile pyrococcus furiosus

et al. 1999

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In this paper, elevated pressures up to 750 atm~1 atm ϭ 101 kPa! were found to have a strong stabilizing effect on two extremely thermophilic glutamate dehydrogenases~GDHs!: the native enzyme from the hyperthermophile Pyrococcus furiosus~Pf !, and a recombinant GDH mutant containing an extra tetrapeptide at the C-terminus~rGDH t !. The presence of the tetrapeptide greatly destabilized the recombinant mutant at ambient pressure; however, the destabilizing effect was largely reversed by the application of pressure. Electron spin resonance~ESR! spectroscopy of a spin-label attached to the terminal cysteine of rGDH t revealed a high degree of mobility, suggesting that destabilization is due to weakened intersubunit ion-pair interactions induced by thermal fluctuations of the tetrapeptide. For both enzymes, the stabilizing effect of pressure increased with temperature as well as pressure, reaching 36-fold for rGDH t at 105 8C and 750 atm, the largest pressure-induced thermostabilization of an enzyme reported to date. Stabilization of both native GDH and rGDH t was also achieved by adding glycerol. Based on the kinetics of thermal inactivation and the known effects of glycerol on protein structure, a mechanism of pressure-induced thermostabilization is proposed.

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The existence of a hexameric intermediate with molten-globule-like properties in the thermal denaturation of bovine-liver glutamate dehydrogenase

Cited by 18 publications

References 26 publications

Thermal aggregation of a model allosteric protein in different conformational states

Thermal aggregation of a model allosteric protein in different conformational states

Chaperone-like Activity and Temperature-induced Structural Changes of α-Crystallin

Pressure‐induced thermostabilization of glutamate dehydrogenase from the hyperthermophile pyrococcus furiosus

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