Concentrations of Glycolytic Enzymes and Other Cytosolic Proteins in the Diffusible Fraction of a Vertebrate Muscle Proteome*

We used a novel microvolumetric technique based on protein diffusion to characterize the subproteome of muscle that consists of diffusible proteins, including those involved in cell metabolism. Muscle fiber segments were mechanically demembranated under mineral oil and transferred into drops of relaxing solution. After the fiber segment was depleted of diffusible proteins, the content of each drop and residual segment was analyzed by one-dimensional polyacrylamide gel electrophoresis. Proteins were identified through peptide mass fingerprinting and quantified using purified protein standards. Ten of the most abundant cytosolic proteins, distinguished by their ability to readily diffuse out of the skinned fiber, were glycolytic enzymes whose concentrations ranged from 2.6 ± 1.0 g liter⁻¹ (phosphoglucose isomerase) to 12.8 ± 1.1 g liter⁻¹ fiber volume (pyruvate kinase). The concentrations of the other five most abundant cytosolic proteins were as follows: glycogen phosphorylase, 6.0 ± 2.3 g liter⁻¹; phosphoglucose mutase, 2.2 ± 0.2 g liter⁻¹; adenylate kinase, 1.6 ± 1.3 g liter⁻¹; phosphocreatine kinase, 6.6 ± 2.6 g liter⁻¹; and parvalbumin, 0.7 ± 0.4 g liter⁻¹. Given the molecular weight and subunit number of each enzyme, the combined concentration of the 15 most abundant cytosolic proteins was 82.3 g liter⁻¹; the volume fraction was 0.093. The large volume fraction of diffusible proteins favors nonspecific interactions and associations, particularly if the glycolytic enzymes and diffusible phosphocreatine kinase are restricted to the I-band as previous studies suggest. The relative molar concentration of glycolytic enzymes is roughly consistent with a stoichiometry of 1:2 for enzymes catalyzing the hexose and triose sugar reactions, respectively, a stoichiometry that may favor metabolic channeling of intermediates during glycolysis. Our results indicate that subcellular fractionation of muscle proteins, in which cytosolic constituents are distinguished by their ability to diffuse readily from demembranated cells, is a promising microvolumetric technique that allows conclusions to be drawn about native protein-protein interactions based on concentration and stoichiometry.