Endogenously produced ATP via SR-associated glycolytic enzymes supported 15-fold more 45Ca transport than exogenously supplied ATP at low concentrations, indicating functional coupling.
To investigate whether the energy derived from glycolysis is functionally coupled to Ca2+ active transport in sarcoplasmic reticulum (SR), we determined whether glycolytic enzymes were associated with SR membranes and whether metabolism through these enzymes was capable of supporting 45Ca transport. Sealed right-side-out SR vesicles were isolated by step sucrose gradient from rabbit skeletal and cardiac muscle. Intravesicular 45Ca transport was measured after the addition of glycolytic substrates and cofactors specific for each of the glycolytic reactions being studied or after the addition of exogenous ATP and was expressed as transport sensitive to the specific Ca(2+)-ATPase inhibitor thapsigargin. We found that the entire chain of glycolytic enzymes from aldolase onward, including aldolase, GAPDH, phosphoglycerate kinase (PGK), phosphoglyceromutase, enolase, and pyruvate kinase (PK), was associated with SR vesicles from both cardiac and skeletal muscle. Iodoacetic acid, an inhibitor of GAPDH, eliminated 45Ca transport supported by fructose-1,6-diphosphate, the substrate for aldolase, but transport was completely restored by phosphoenolpyruvate (the substrate for PK), indicating that both of the ATP-producing glycolytic enzymes, GAPDH/PGK and PK, were associated with the SR and functionally capable of providing ATP for the Ca2+ pump. Addition of a soluble hexokinase ATP trap eliminated 45Ca transport fueled by exogenous ATP but had markedly less effect on 45Ca transport supported by endogenously produced ATP (via glycolysis). Similarly, at very low concentrations of ATP and ADP (10 to 50 nmol/L), ATP that was produced endogenously from ADP and phosphoenolpyruvate supported 15-fold more 45Ca transport than ATP that was supplied exogenously at the same concentration. These results are consistent with functional coupling of glycolytic ATP to Ca2+ transport and support the hypothesis that ATP generated by SR-associated glycolytic enzymes may play an important role in cellular Ca2+ homeostasis by driving the SR Ca2+ pump.
Xu et al. (Sat,) conducted a other in Basic science (rabbit skeletal and cardiac muscle). Glycolytic substrates and cofactors (endogenous ATP production) vs. Exogenous ATP was evaluated on Intravesicular 45Ca transport. Endogenously produced ATP via SR-associated glycolytic enzymes supported 15-fold more 45Ca transport than exogenously supplied ATP at low concentrations, indicating functional coupling.