Reevaluation of the "glycolytic complex" in muscle: a multitechnique approach using trout white muscle.
Preliminary characterization of the "glycolytic complex," formed in trout white muscle, revealed that phosphofructokinase (PFK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are bound to particulate matter largely by ionic interactions; increasing neutral salt or charged metabolite concentrations released bound PFK and GAPDH. GAPDH was consistently solubilized at lower salt concentrations, indicating that it is not bound as tightly as PFK, but both enzymes were readily solubilized at physiological concentrations of salts and metabolites. pH titrations indicated that PFK binding is dependent on group(s) with a pKa of 7.3 in 30 mM imidazole. PFK binding increased at lower pH values; at 150 mM KCl the apparent pKa value is 6.5. Experiments with polyethylene glycol 8000 (PEG), which is used to mimic the high in vivo protein concentrations under in vitro conditions, showed that the binding of PFK and GAPDH increased with increasing PEG concentrations. Interestingly, at 5% PEG, only the PFK binding response depended on the ionic composition of the medium--with increased binding occurring at the pH of the exhausted muscle and decreased binding at control pH values. These results suggested that only PFK reversibly bound to cellular structures in response to changing conditions and disagrees with previous studies showing binding of several glycolytic enzymes as measured using the dilution method (F. M. Clarke, F.D. Shaw, and D.J. Morton (1980) Biochem. J. 186, 105-109). In order to determine whether artifactual binding was measured by the dilution method, two new methodologies were employed to measure enzyme binding in vivo: (a) whole muscle slices were pressed to quickly extrude cellular juice, and (b) muscle strips were finely minced and centrifuged to liberate cytoplasmic contents. Both methods indicated that, under physiological conditions, up to 70% of the total cellular phosphofructokinase may be bound, but other glycolytic enzymes are bound to a lesser extent (10-30%). This result contrasts those obtained with the dilution method, and suggests that dilution of cellular contents may result in an overestimation of the percentage of enzyme associated with cellular structures; this is dramatically shown for glyceraldehyde-3-phosphate dehydrogenase. The viability of the glycolytic complex in trout white muscle is discussed in light of the decreased binding measured using these new methodologies.
Brooks
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SP
Institute of Biochemistry, Carleton University, Ottawa, Ontario, Canada.
Storey
K B
KB
D000205:Actomyosin / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D005993:Glycerolphosphate Dehydrogenase / Q000302:isolation & purification; D006019:Glycolysis; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D009132:Muscles / Q000201:enzymology* / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000302:isolation & purification; D005082:Physical Exertion; D011092:Polyethylene Glycols; D012995:Solubility; D014337:Trout
D011092:Polyethylene Glycols; D000205:Actomyosin; D005993:Glycerolphosphate Dehydrogenase; D010732:Phosphofructokinase-1