Mechanism of Thermal Aggregation of Rabbit Muscle Glyceraldehyde-3-phosphate Dehydrogenase

Abstract

Thermal denaturation and aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been studied using differential scanning calorimetry (DSC), dynamic light scattering (DLS), and analytical ultracentrifugation. The maximum of the protein thermal transition (T(m)) increased with increasing the protein concentration, suggesting that the denaturation process involves the stage of reversible dissociation of the enzyme tetramer into the oligomeric forms of lesser size. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. The DLS data support the mechanism of protein aggregation that involves a stage of the formation of the start aggregates followed by their sticking together. The hydrodynamic radius of the start aggregates remained constant in the temperature interval from 37 to 55 degrees C and was independent of the protein concentration (R(h,0) approximately 21 nm; 10 mM sodium phosphate, pH 7.5). A strict correlation between thermal aggregation of GAPDH registered by the increase in the light scattering intensity and protein denaturation characterized by DSC has been proved.

Oxidation of an Exposed Methionine Instigates the Aggregation of Glyceraldehyde-3-phosphate Dehydrogenase

“…There are numerous reports investigating the mechanism of GAPDH aggregation (11,13,22,23,(43)(44)(45)(46)(47) with several studies stipulating an essential role for intermolecular disulfide bond formation (11,13,22,23). These models of GAPDH aggregation are supported by the observation that insoluble disulfide-cross-linked forms of GAPDH have been found in vivo (11)(12)(13)18).…”

CP12 from Chlamydomonas reinhardtii, a Permanent Specific “Chaperone-like” Protein of Glyceraldehyde-3-phosphate Dehydrogenase

“…if it could prevent aggregation of its partner, GAPDH, an enzyme well known for its tendency to aggregate (41)(42)(43)(44) and consequently a substrate commonly used in chaperone studies (45,46).…”

Estudo da estabilidade térmica da hemoglobina extracelular gigante de Glossoscolex paulistus (HbGp): efeitos do estado de oxidação do ferro do grupo heme, pH e presença de surfactante

“…al. [31], e requer que no início do processo de agregação o sistema seja monodisperso, e com comportamento linear em função do tempo.…”

“…Na equação acima o parâmetro D * h,0 corresponde ao diâmetro hidrodinâmico da proteína no início do processo de agregação, t 0 é o tempo requerido para que o processo de agregação seja iniciado ou "lag period", e t 2D é o tempo necessário para que o valor de D h seja duplicado [31]. As análises usando a equação 14 são permitidas apenas na faixa de tempo que o sistema permanece monodisperso durante o processo de agregação [31].…”

“…As análises usando a equação 14 são permitidas apenas na faixa de tempo que o sistema permanece monodisperso durante o processo de agregação [31]. Os ajustes das curvas de cinética de agregação usando a equação 14 foram realizados usando o programa Origin 8.0Pro da Microcal (USA).…”