Phosphorylation-induced conformational changes in the phosphorylaseab hybrid as revealed by resolution of pyridoxal 5′-phosphate with imidazole citrate and cysteine
Abstract:The accessibility of pyridoxal 5'-phosphates of the phosphorylase ab hybrid to resolution by imidazole citrate and cysteine was studied and compared with that of the b and a forms. Promotion of resolution of phosphorylated forms by raising the temperature or in the presence of glycogen indicates that the resistance of phosphorylase a and ab to resolution at 0 degrees C is due rather to their tetrameric state than their phosphorylation-related active conformation. The pattern of resolution of the ab hybrid was … Show more
“…An intermediate protein exhibiting significant GP activity was collected as Fraction X. Although some previous studies adopted an instantaneous increase in the eluent glycerophosphate concentration [e.g., jumping from 30 to 200 mM (Vereb et al 1987 , 1992 )] to eluate GP ab from the anion-exchange column, we found that this practice caused co-elution of GP ab and GP a (data not shown). Fig.…”
Section: Resultsmentioning
confidence: 70%
“…This is one of the major reasons for the stagnation of GP ab research. Previously, some dedicated researchers prepared GP ab in vitro by the partial phosphorylation of GP b using PhK as the catalyst (Vereb et al 1987 , 1992 ; Harris and Graves 1990 ). Notably, in the PhK-catalyzed GP b phosphorylation reaction, production of GP a (di-phosphorylated form, secondary product) is inevitable even at an early stage of the reaction, because the phosphorylation rate toward GP ab (mono-phosphorylated form, primary product) is faster than that toward GP b (non-phosphorylated form, original substrate) (Harris and Graves 1990 ).…”
Section: Resultsmentioning
confidence: 99%
“…GP ab has been usually identified through a radioactive labeling method using [γ- 32 P]ATP as the substate (Vereb et al 1987 , 1992 ; Harris and Graves 1990 ). However, the use of radioactive materials significantly hinders routine performance in GP ab research because researchers must follow strict rules to order, store, use, and dispose of these materials.…”
Section: Resultsmentioning
confidence: 99%
“…To focus on the catalytic site activity, PA-0, comprising the minimum essential dextrin structure for GP, was used as the assay substrate (Fig. 2 ); in contrast, macromolecular glycogen was used in the previous GP ab studies (Vereb et al 1987 , 1992 ; Harris and Graves 1990 ; Burkhardt and Wegener 1994 ). The results are summarized in Fig.…”
Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits, each activated by phosphorylation of the serine-14 residue. GP exists in three interconvertible forms, namely GPa (di-phosphorylated form), GPab (mono-phosphorylated form), and GPb (non-phosphorylated form); however, information on GPab remains scarce. Given the prevailing view that the two GP subunits collaboratively determine their catalytic characteristics, it is essential to conduct GPab characterization to gain a comprehensive understanding of glycogenolysis regulation. Thus, in the present study, we prepared rabbit muscle GPab from GPb, using phosphorylase kinase as the catalyst, and identified it using a nonradioactive phosphate-affinity gel electrophoresis method. Compared with the half-half GPa/GPb mixture, the as-prepared GPab showed a unique AMP-binding affinity. To further investigate the intersubunit communication in GP, its catalytic site was probed using pyridylaminated-maltohexaose (a maltooligosaccharide-based substrate comprising the essential dextrin structure for GP; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (substrate analogs lacking part of the essential dextrin structure). By comparing the initial reaction rates toward the PA-0 derivative (Vderivative) and PA-0 (VPA-0), we demonstrated that the Vderivative/VPA-0 ratio for GPab was significantly different from that for the half-half GPa/GPb mixture. This result indicates that the interaction between the two GP subunits significantly influences substrate recognition at the catalytic sites, thereby providing GPab its unique substrate recognition profile.
“…An intermediate protein exhibiting significant GP activity was collected as Fraction X. Although some previous studies adopted an instantaneous increase in the eluent glycerophosphate concentration [e.g., jumping from 30 to 200 mM (Vereb et al 1987 , 1992 )] to eluate GP ab from the anion-exchange column, we found that this practice caused co-elution of GP ab and GP a (data not shown). Fig.…”
Section: Resultsmentioning
confidence: 70%
“…This is one of the major reasons for the stagnation of GP ab research. Previously, some dedicated researchers prepared GP ab in vitro by the partial phosphorylation of GP b using PhK as the catalyst (Vereb et al 1987 , 1992 ; Harris and Graves 1990 ). Notably, in the PhK-catalyzed GP b phosphorylation reaction, production of GP a (di-phosphorylated form, secondary product) is inevitable even at an early stage of the reaction, because the phosphorylation rate toward GP ab (mono-phosphorylated form, primary product) is faster than that toward GP b (non-phosphorylated form, original substrate) (Harris and Graves 1990 ).…”
Section: Resultsmentioning
confidence: 99%
“…GP ab has been usually identified through a radioactive labeling method using [γ- 32 P]ATP as the substate (Vereb et al 1987 , 1992 ; Harris and Graves 1990 ). However, the use of radioactive materials significantly hinders routine performance in GP ab research because researchers must follow strict rules to order, store, use, and dispose of these materials.…”
Section: Resultsmentioning
confidence: 99%
“…To focus on the catalytic site activity, PA-0, comprising the minimum essential dextrin structure for GP, was used as the assay substrate (Fig. 2 ); in contrast, macromolecular glycogen was used in the previous GP ab studies (Vereb et al 1987 , 1992 ; Harris and Graves 1990 ; Burkhardt and Wegener 1994 ). The results are summarized in Fig.…”
Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits, each activated by phosphorylation of the serine-14 residue. GP exists in three interconvertible forms, namely GPa (di-phosphorylated form), GPab (mono-phosphorylated form), and GPb (non-phosphorylated form); however, information on GPab remains scarce. Given the prevailing view that the two GP subunits collaboratively determine their catalytic characteristics, it is essential to conduct GPab characterization to gain a comprehensive understanding of glycogenolysis regulation. Thus, in the present study, we prepared rabbit muscle GPab from GPb, using phosphorylase kinase as the catalyst, and identified it using a nonradioactive phosphate-affinity gel electrophoresis method. Compared with the half-half GPa/GPb mixture, the as-prepared GPab showed a unique AMP-binding affinity. To further investigate the intersubunit communication in GP, its catalytic site was probed using pyridylaminated-maltohexaose (a maltooligosaccharide-based substrate comprising the essential dextrin structure for GP; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (substrate analogs lacking part of the essential dextrin structure). By comparing the initial reaction rates toward the PA-0 derivative (Vderivative) and PA-0 (VPA-0), we demonstrated that the Vderivative/VPA-0 ratio for GPab was significantly different from that for the half-half GPa/GPb mixture. This result indicates that the interaction between the two GP subunits significantly influences substrate recognition at the catalytic sites, thereby providing GPab its unique substrate recognition profile.
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