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

• Published in: Molecular & cellular proteomics Vol. 4; no. 10; pp. 1541 - 1549
• Main Authors: Maughan, David W, Henkin, Josh A, Vigoreaux, Jim O
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 01-10-2005
• Subjects: Animals; Chemical Fractionation; Cytosol - enzymology; Cytosol - metabolism; Diffusion; Glycolysis; Male; Muscles - cytology; Muscles - enzymology; Muscles - metabolism; Proteome - metabolism; Psoas Muscles - enzymology; Psoas Muscles - metabolism; Rabbits
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.M500053-MCP200

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.