Ultrastructural localization of glycolytic enzymes on sarcoplasmic reticulum vesicles

We have previously obtained indirect evidence that sarcoplasmic reticulum (SR) vesicles from cardiac and skeletal muscle contain the complete chain of glycolytic enzymes from aldolase to pyruvate kinase. To investigate directly whether pyruvate kinase and other glycolytic enzymes are anatomically associated with the SR, electron microscopic immunogold labeling studies were carried out in isolated SR vesicles using specific primary antibodies against selected glycolytic enzymes and Ca²⁺-ATPase, and appropriate secondary antibodies labeled with 6-nm or 12-nm gold particles. Pyruvate kinase was broadly dispersed on the cytoplasmic side of the SR membrane of both cardiac and skeletal muscle vesicles. With 6-nm gold particles, the density of binding to pyruvate kinase was 2522 ± 445 and 4171 ± 1379 particles/μm² for cardiac and skeletal muscle SR, respectively. Binding densities to Ca²⁺-ATPase were similar (2550 ± 639 particles/μm² for cardiac SR, 3877 ± 408 particles/μm² for skeletal muscle 5R). Immunogold labeling of ultrathin sections indicated that pyruvate kinase was attached to the SR membrane and located immediately adjacent to the Ca²⁺- ATPase. Aldolase and glyceraldehyde phosphate dehydrogenase were also found to be attached to the cytoplasmic side of SR vesicles and located in close proximity to Ca²⁺-ATPase. These results provide the first ultrastructural evidence that glycolytic enzymes are anatomically associated with SR membranes and located near the SR Ca²⁺-ATPase. The results further support the hypothesis that ATP generated by SR-associated glycolytic enzymes is coupled to SR Ca²⁺ active transport.