A fractionated reticulocyte lysate retains high efficiency for protein synthesis.
The rabbit reticulocyte lysate is a highly efficient system for protein synthesis in vitro, and therefore has been used frequently in studies of translational control. However, when the lysate is fractionated into ribosomes and soluble proteins by centrifugation, there is a severe loss of activity, thereby making the system less suitable for identifying components involved in translational control. We have devised a new method of cell fractionation which employs rapid (20-min) centrifugation to pellet ribosomes. The post-ribosomal supernatant (S-100), high salt-washed ribosomes, and the high salt wash are all required in the reconstituted protein synthesis assay, and greater than 70% of the activity of the unfractionated lysate is attained. Proteins in the high salt wash have been further fractionated by ammonium sulfate precipitation, and the assay system has been used to measure the activities of eIF-4B and eIF-4F. This highly active fractionated lysate system should be useful in measuring the specific activities of translational components and may be appropriate for detecting changes due to covalent modifications such as phosphorylation.
Morley
S J
SJ
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616.
Hershey
J W
JW
D000818:Animals; D002498:Centrifugation; D004591:Electrophoresis, Polyacrylamide Gel; D039562:Eukaryotic Initiation Factor-4F; D039642:Eukaryotic Initiation Factors; D005786:Gene Expression Regulation; D066298:In Vitro Techniques; D008715:Methionine / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism
D039562:Eukaryotic Initiation Factor-4F; D039642:Eukaryotic Initiation Factors; D010448:Peptide Initiation Factors; C031799:eIF-4B; D008715:Methionine