Glycogen extraction from skeletal muscle sarcoplasmic reticulum: structural and functional implications
In this investigation, skeletal muscle sarcoplasmic reticulum (SR) was purified from female Sprague Dawley rats (200-250 g). SR samples were subjected to two different biochemical glycogen-extraction protocols. The results suggest that both amylase and removal of EDTA (No-EDTA) from the homogenization and storage buffers reduced the amount of glycogen associated with the SR. Both of these treatments failed to impair SR calcium (Ca2+) handling when assayed under conditions where exogenous ATP was added and utilized for SR Ca2+ transport. In fact, these treatments seemed to cause a small increase in both SR Ca2+-uptake and release rates under these assay conditions. As expected, glycogen phosphorylase content was reduced as a result of glycogen extraction in the presence of amylase, however this was not the case for No-EDTA samples. Interestingly, many other proteins differed in content after glycogen extraction. These treatments resulted in a greater recovery of the sarco(endo)plasmic reticulum Ca2+ adenosine triphosphatase (SERCA) and a substantial loss of glycogen phosphorylase and glycogen debranching enzyme (AGL) in amylase-treated samples. Creatine kinase (CK) and pyruvate kinase (PK) contents were increased as a result of both glycogen-extraction conditions. It was imperative to consider these altered protein contents while analyzing the data and assessing the effects of glycogen extraction on SR Ca2+ handling.
After normalizing to SERCA content, only No-EDTA samples had higher adenosine triphosphate (ATP)-supported SR Ca2+-uptake rates compared to control samples. For endogenously synthesized ATP-supported SR Ca2+-uptake experiments, normalizing data to protein content (either CK and SERCA or PK and SERCA) revealed that amylase-treated samples had lower SR Ca2+-uptake rates, compared to control samples. Although not significant, SR Ca2+-uptake rates for No-EDTA samples were also lower than control samples. These data suggest that changes in endogenously supported SR Ca2+-uptake due to glycogen extraction affected the source of ATP synthesis (either PK or CK), the effectiveness of energy utilization for Ca2+ transport (SERCA), or altered the metabolic channeling properties.