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Purification and physical properties of homogeneous initiation factor MP from rabbit reticulocytes
Abstract:
Initiation factor MP was purified 1570-fold with 67% recovery of total activity present in 0.5 M KCl extracts of rabbit reticulocyte ribosomes. Initiation factor MP forms a ternary complex with Met-tRNAf and GTP or a binary complex with Met-tRNAf alone, the details of which are presented in the accompanying paper (Safer, B., Adams, S. L., Anderson. W. F., and Merrick, W. C. (1975) J. Biol. Chem. 250, 9076-9082). Initiation factor MP was homogeneous by the following criteria: (a) electrophoresis as a single band in gels of 5, 6, 7, 8, 9, and 10% acrylamide; (b) equilibration as a single band during isoelectric focusing; (c) sedimentation as a single symmetrical boundary during sedimentation velocity experiments; (d) linear plots of sedimentation equilibrium data; (e) symmetrical absorbance (at 280 nm) and activity profiles during DEAE-cellulose and Sephadex G-200 chromatography, and (f) symmetrical distribution of initiation factor MP during sucrose density gradient band sedimentation. The molecular weight of the initiation factor MP monomer (0.2 mg/ml) by low speed sedimentation equilibrium was 90,800. Calculations based on the Stokes radius and sedimentation velocity show the existence of relatively stable 90,000-dalton monomers or 180,000-dalton dimers at low (0.1 mg/ml) and high (9.75 mg/ml) concentrations of initiation factor MP, respectively. Electrophoresis in sodium dodecyl sulfate gels indicates that initiation factor MP monomer is composed of two noncovalently linked subunits with molecular weights of 52,000 and 34,000. Despite a relatively normal amino acid composition and an isoelectric point of 6.4, initiation factor MP behaves as a basic protein, eluting from phosphocellulose at 650 mM KCl (pH 7.9). Both ternary complex formation and methionyl-puromycin synthesis co-purify, indicating that a single protein is required for both activities.
Insights
Purified initiation factor MP, a key protein in rabbit reticulocyte translation, forms complexes with Met-tRNAf and GTP. This factor, composed of two subunits, is essential for both ternary complex formation and methionyl-puromycin synthesis.
Area of Science:
- Molecular Biology
- Protein Biochemistry
- Translation Initiation
Background:
- Protein synthesis initiation is a critical regulatory step in gene expression.
- Rabbit reticulocyte lysates are a well-established system for studying eukaryotic translation.
- Specific initiation factors are required to assemble the ribosomal pre-initiation complex.
Purpose of the Study:
- To purify and characterize initiation factor MP from rabbit reticulocyte ribosomes.
- To investigate the complex formation properties of initiation factor MP.
- To determine the subunit composition and functional activities of initiation factor MP.
Main Methods:
- Multi-step protein purification including ion-exchange and size-exclusion chromatography.
- Analytical ultracentrifugation (sedimentation velocity and equilibrium) for molecular weight determination.
- Electrophoresis (native and SDS-PAGE) and isoelectric focusing for homogeneity and subunit analysis.
- Functional assays including ternary complex formation with Met-tRNAf and GTP, and methionyl-puromycin synthesis.
Main Results:
- Initiation factor MP was purified 1570-fold with 67% recovery.
- The factor demonstrated homogeneity through various biochemical and biophysical techniques.
- It forms a ternary complex with Met-tRNAf and GTP, and a binary complex with Met-tRNAf.
- The monomer molecular weight was determined to be 90,800, existing as monomers or dimers depending on concentration.
- SDS-PAGE revealed the monomer is composed of 52,000 and 34,000 dalton subunits.
- Both ternary complex formation and methionyl-puromycin synthesis activities co-purified, indicating a single protein responsible.
Conclusions:
- Initiation factor MP is a distinct protein essential for translation initiation in rabbit reticulocytes.
- The factor exhibits distinct oligomeric states and is composed of two non-covalently linked subunits.
- Its ability to form specific complexes and catalyze methionyl-puromycin synthesis highlights its crucial role in initiating protein synthesis.