HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: M500053-MCP200v1 4/10/1541    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Maughan, D. W. Articles by Vigoreaux, J. O. Search for Related Content PubMed PubMed Citation Articles by Maughan, D. W. Articles by Vigoreaux, J. O. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 4:1541-1549, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Research

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

David W. Maughan^,,¶, Josh A. Henkin^,,|| and Jim O. Vigoreaux^,,||

From the Department of Molecular Physiology and Biophysics, University of Vermont College of Medicine and the ^|| Department of Biology and the Cell and Molecular Biology Program, University of Vermont, Burlington, Vermont 05405

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.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. St-Jean, T. Izard, and J. Sygusch A Hydrophobic Pocket in the Active Site of Glycolytic Aldolase Mediates Interactions with Wiskott-Aldrich Syndrome Protein J. Biol. Chem., May 11, 2007; 282(19): 14309 - 14315. [Abstract] [Full Text] [PDF]

				R. M. Murphy, E. Verburg, and G. D. Lamb Ca2+ activation of diffusible and bound pools of {micro}-calpain in rat skeletal muscle J. Physiol., October 15, 2006; 576(2): 595 - 612. [Abstract] [Full Text] [PDF]