Crystallized Enzymes from the Myogen of Rabbit Skeletal Muscle

Czok, R.; Bücher, Th.

doi:10.1016/s0065-3233(08)60311-3

Cited by 165 publications

(30 citation statements)

References 230 publications

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“…The physical effect that diminishes solubility at very low ionic strength is the removal of ions essential for satisfying the electrostatic requirements of the protein molecules. As these ions are removed, and in this region of low ionic strength cations are most important [23,24], the protein molecules seek to balance their electrostatic requirements through interactions among themselves. Thus they tend to aggregate and separate from solution.…”

Section: Crystallization Strategymentioning

confidence: 99%

Current approaches to macromolecular crystallization

McPherson

1990

European Journal of Biochemistry

370

248

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Given our current expertise, and the certain future developments in genetically altering organisms to produce proteins of modified structure and function, the concept of protein engineering is nearing reality. Similarly, our ability to describe and utilize protein structure and to define interactions with ligands has made possible the rational design of new drugs and pharmacological agents. Even in the absence of any intention toward applied use or value, the correlation of regulation, mechanism, and function of proteins with their detailed molecular structure has now become a primary concern of modern biochemistry and molecular biology.At the present time, there are numerous physical-chemical approaches that yield information regarding macromolecular structure. Some of these methods, such as NMR and molecular dynamics, are becoming increasingly valuable in defining detailed protein structure, particularly for lower-molecularmass proteins. There is, however, only one general technique that yields a detailed and precise description, in useful mathematical terms, of a macromolecule's structure, a description that can serve as a basis for drug design, and an intelligent guide for protein engineering. The method is X-ray diffraction analysis of single crystals of proteins, nucleic acids, and their complexes with one another and with conventional small molecules. Some inspirational examples of representative crystals are shown in Fig. 1 -3.In the past 20 years, the practice of X-ray crystallography has made enormous strides. Nearly all of the critical and timeconsuming components of the technique have been improved, accelerated, and refined. X-ray crystallography today is not simply an awesome method used by physical chemists to reveal the vast beauty of macromolecular architecture; it is a practical, reliable, and relatively rapid means to obtain straightforward answers to perplexing questions.X-ray diffraction data that once required years to obtain, can now be collected in a matter of weeks, even days in some cases. Computers of extraordinary speed and capacity are now common tools as are computer graphics systems of a versatility and cleverness that would have been unimaginable only a few years ago. Software, too, exists that is sophisticated yet friendly, flexible yet reliable, and readily available to anyone in need of it. The question, then, is where does the problem lie? What prevents the full utilization and exploitation of this enormously powerful approach.The answer, of course, is that for application of the method to a particular macromolecule, the protein or nucleic acid Correspondence to A. McPherson,

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Section: Crystallization Strategymentioning

confidence: 99%

Current approaches to macromolecular crystallization

McPherson

1990

European Journal of Biochemistry

370

248

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“…Rabbit skeletal muscle was studied because purified standards of all the glycolytic enzymes and other muscle proteins of interest were commercially available for this experimental animal. Rabbit skeletal muscle has also been widely used for histochemical localization studies (8,9) and enzymatic assays (10,11) (see also Table III of Ref. 9 and citations therein), allowing a basis for comparing results.…”

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confidence: 99%

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

Maughan

Henkin

Vigoreaux

2005

Molecular & Cellular Proteomics

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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 ...

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“…However, in Fig. 4(c (Czok & Biucher, 1960 buffer of the same pH, namely 7.7. The reported mobilities, which refer to migration of the centroid (Longsworth, 1943) of the descending boundary, clearly establish the equilibrium nature of the phosphate interaction with glyceraldehyde 3-phosphate dehydrogenase by virtue of the progressive change in mobility with increasing phosphate concentration.…”

Section: Resultsmentioning

confidence: 97%

Rabbit muscle myogen. Interactions with phosphate as the source of non-enantiography in moving-boundary electrophoresis

Lovell¹,

Winzor²

1976

Biochemical Journal

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Rabbit muscle myogen has been subjected to moving-boundary electrophoresis and velocity sedimentation in 0.0187M-potassium phosphate buffer, pH7.7, I=0.05. The ascending and descending electrophoretic patterns are sufficiently non-enantiographic to suggest the existence of rapid, reversible interactions in the myogen solutions. However, no evidence of pronounced macromolecular association was obtained in velocitysedimentation experiments. The source of the non-enantiography in electrophoresis has been traced to interactions ofphosphate with components ofmyogen, which should therefore be considered as a mixture, rather than a complex, of glycolytic enzymes.

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Crystallized Enzymes from the Myogen of Rabbit Skeletal Muscle

Cited by 165 publications

References 230 publications

Current approaches to macromolecular crystallization

Current approaches to macromolecular crystallization

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

Rabbit muscle myogen. Interactions with phosphate as the source of non-enantiography in moving-boundary electrophoresis

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