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 onedimensional 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 ؎ The advent of proteomics, the experimental approach to assess global protein function, has introduced new techniques for studying important subproteomes of cellular proteins that operate in a coordinated fashion. One such subproteome is the entire complement of metabolic enzymes found in a selected muscle cell at any given time. Among these enzymes are those involved in glycolysis, a process by which ATP is generated during the enzymatic breakdown of glucose to pyruvate.Glycolysis is important in fast twitch muscles where ATP, produced at intermediate steps of the glycolytic pathway, fuels contraction and a variety of other ATP-dependent cellular processes. Information about the subcellular distribution, concentrations, and mutual interactions of the glycolytic enzymes and their metabolites is important for a complete understanding of metabolism in fast twitch muscles.In vertebrate fast twitch skeletal muscle fibers the glycolytic enzymes are regarded as soluble components of the fluid myoplasm because they are present in the supernatant fraction recovered after homogenization and centrifugation of the tissue sample (1). However, this assignment has often been a matter of dispute because the supernatant fraction may be only a rough approximation of the cytosol in situ (2-4). Not only are proteins in dilute slurries isolated under conditions far different from those in intact cells, additional information about their interactions may be lost using conventional methods of fractionation. Enzyme-enzyme interactions constitute an area of intense interest in structural genomics and enzymology (5, 6). Clearly studies in these areas would benefit from new approaches for isolation of proteins and determining their concentrations and stoichiometric relationships under conditions more closely approximating their native state. This information could help settle some controversial points debated over the past 50 years, such as whether large supramolecular complexes exist for glycolytic enzymes in the native cytosol (for a summary, see Ref. 5).Warmke et al. (7) pioneered a microvolumetric method of subcellular protein enrichment and fractionation in which cytosolic constituents of muscle were distinguished ...

Striated muscles across phyla share a highly conserved sarcomere design yet exhibit broad diversity in contractile velocity, force, power output, and efficiency. Insect asynchronous flight muscles are characterized by high-frequency contraction, endurance, and high-power output. These muscles have evolved an enhanced delayed force response to stretch that is largely responsible for their enhanced oscillatory work and power production. In this study we investigated the contribution of flightin to oscillatory work using sinusoidal analysis of fibers from three flightless mutants affecting flightin expression: 1) fln0, a flightin null mutant, 2) Mhc13, a myosin rod point mutant with reduced levels of flightin, and 3) Mhc6, a second myosin rod point mutant with reduced levels of phosphorylated flightin. Fibers from the three mutants show deficits in their passive and dynamic viscoelastic properties that are commensurate with their effect on flightin expression and result in a significant loss of oscillatory work and power. Passive tension and passive stiffness were significantly reduced in fln0 and Mhc13 but not in Mhc6. The dynamic viscous modulus was significantly reduced in the three mutants, whereas the dynamic elastic modulus was reduced in fln0 and Mhc13 but not in Mhc6. Tension generation under isometric conditions was not impaired in fln0. However, when subjected to sinusoidal length perturbations, work-absorbing processes dominated over work-producing processes, resulting in no net positive work output. We propose that flightin is a major contributor to myofilament stiffness and a key determinant of the enhanced delayed force response to stretch in Drosophila flight muscles.

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