Autophosphorylation of glyceraldehydephosphate dehydrogenase and phosphorylation of protein from skeletal muscle...

Area of Science:

• Biochemistry
• Muscle Physiology

Background:

• Glyceraldehydephosphate dehydrogenase (GAPDH) is a key glycolytic enzyme.
• Phosphorylation can regulate enzyme activity and protein-protein interactions.

Purpose of the Study:

• To investigate the auto-phosphorylation of rabbit skeletal muscle GAPDH.
• To characterize the properties of the phosphoenzyme.
• To determine if the phosphoenzyme exhibits kinase activity.

Main Methods:

• Purification of glyceraldehydephosphate dehydrogenase from rabbit skeletal muscle.
• Enzyme kinetics studies to determine phosphorylation conditions (MgATP concentration).
• Stability assays under varying pH conditions.
• Hydrolysis studies using hydroxylamine.
• Investigating the effect of metabolites (NADH, NAD+, glyceraldehyde 3-phosphate) and calsequestrin on phosphorylation.
• Assessing the kinase activity of the phosphoenzyme on skeletal muscle proteins.

Main Results:

• Rabbit skeletal muscle GAPDH auto-phosphorylates with MgATP, with half-maximal phosphorylation at ~0.3 mM MgATP.
• Phosphorylation is independent of Ca2+ and results in a labile phosphoenzyme complex under alkaline conditions, stable in acid.
• The phosphoenzyme complex is a high-energy acyl phosphate, hydrolyzed by neutral hydroxylamine.
• Nicotinamide adenine dinucleotides (NADH, NAD+) and glyceraldehyde 3-phosphate reduce phosphorylation and can also dephosphorylate the enzyme.
• Calsequestrin inhibits GAPDH phosphorylation.
• The phosphoenzyme demonstrates kinase activity, phosphorylating Mr 80,000 and 72,000 proteins from skeletal muscle terminal cisternae/triad preparations, an activity enhanced by NADH.
• The phosphate transferred to protein substrates originates from the initial GAPDH phosphorylation.

Conclusions:

• Rabbit skeletal muscle GAPDH undergoes auto-phosphorylation, forming a high-energy acyl phosphate intermediate.
• The phosphoenzyme exhibits kinase activity, potentially linking glycolysis to muscle protein phosphorylation.
• Metabolites like NADH and NAD+ play regulatory roles in both the phosphorylation and dephosphorylation of GAPDH and subsequent protein phosphorylation.