Initiation of Protein Synthesis in Eukaryotes. Binding to Sepharose-heparin and Partial Purification of Initiation Factors from Krebs II Ascites Cells

Mast, C. van der; Thomas, Adri A. M.; Goumans, Hans; Amesz, Hans; Voorma, Harry O.

doi:10.1111/j.1432-1033.1977.tb11548.x

Cited by 48 publications

(10 citation statements)

References 24 publications

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“…[10]; reticulocyte lysate by Schreier and Staehelin [8] with a few modifications [11]; tRNA from rat liver, rabbit liver or rabbit reticulocytes by Rogg et al [12] and [3H]methionyltRNA by Stanley [13]. Crude initiation factors were precipitated with ammonium sulphate from the high salt ribosomal wash according to Schreier and Staehelin [14]: fraction A (0--40% saturation of ammonium sulphate); fraction B (40--50%) and fraction C (50--70%).…”

Section: Methodsmentioning

confidence: 99%

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Purification of free eukaryotic initiation factors eIF-4A and eIF-4D on cibacron blue F3G-A

Mast

Voorma

1980

Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein

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SummaryThe eukaryotic initiation factors eIF-4A and eIF-4D are almost exclusively found in the supernatant of a reticulocyte lysate. The basic steps of general purification schemes failed because these factors do not bind to Sepharoseheparin or, in the case of eIF-4A, to phosphocellulose and a new procedure had to be devised. Cibacron Blue F3G-A proved to be a successful alternative in the purification of these factors from the excessive load of proteins found in the supernatant.

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Section: Methodsmentioning

confidence: 99%

“…Affinity chromatography on Sepharose 6B-heparin was performed as reported previously [ 11 ]. Volumes of gel used in the purification steps to be described refer to the volume that the gel occupies in a column under normal running conditions.…”

Section: Methodsmentioning

confidence: 99%

Purification of free eukaryotic initiation factors eIF-4A and eIF-4D on cibacron blue F3G-A

Mast

Voorma

1980

Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein

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“…The following components were prepared as described : ribosomal subunits by Schreier and Staehelin [15], pH-5 enzymes by Falvey and Staehelin [16], hemoglobin 9-S mRNA by Krystosek et al [17], reticulocyte lysate by Schreier and Staehelin [15] with a few modifications [14], tRNA (from rabbit liver, rabbit reticulocytes or rat liver) by Rogg et al [18] and [3H]methionyl-tRNA~e' by Stanley [19]. Crude fractions of initiation factors were obtained by precipitation of the high salt ribosomal wash with ammonium sulphate as described by Schreier and Stae-helin [20] : fraction A (0 -40 % saturation), fraction B (40 -50 %) and fraction C (50 -70 %).…”

Section: Methodsmentioning

confidence: 99%

“…elF-I, eIF-2, elF-3, eIF-4A, eIF-4B, eIF-4C, eIF-4D and eIF-5 are eukaryotic initiation factors, see Anderson et al (1977) FEBS Lett. 76, 1-10 and [14].…”

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A Comparison of the Initiation Factors of Eukaryotic Protein Synthesis from Ribosomes and from the Postribosomal Supernatant

Thomas

Goumans

Amesz

et al. 1979

European Journal of Biochemistry

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A purification procedure is described for the initiation factors of protein synthesis from rabbit reticulocytes : (a) from the ribosomal wash and (b) from the postribosomal supernatant.A comparison is made between these preparations with respect to yield and specific activity. eIF-4A and eIF-4D occur mainly in the postribosomal supernatant; eIF-2, eIF-4C and eIF-5 are more evenly divided over both fractions, whereas eIF-1, eIF-3 and eIF-4B are found almost exclusively in the ribosomal wash. No significant difference in specific activity could be detected when factors from both sources were compared, with a possible exception of eIF-4A and eIF-4D.Several laboratories have reported the purification of initiation factors of protein synthesis from rabbit reticulocytes [l-71. As a first step the preparation of a crude ribosomal wash with high salt concentration was described. This procedure is based on the assumption that initiation factors are bound to ribosomes and are not present in the postribosomal supernatant. However, after completing the initiation sequence all of the initiation factors are released from the initiation complex into the cytoplasm, after which a new round of initiation is started [8 -101. Therefore, although detailed knowledge about the rate of protein synthesis and, more specifically, about the rate of attachment of each initiation factor to the ribosomes is lacking in vivo, the possibility arises that a certain amount of initiation factors exists free in the cytoplasm. Thus, the presence of initiation factors in the postribosomal supernatant might provide some information about their function in the process of initiation of protein synthesis.Another point of interest involves the question of whether supernatant factors differ from ribosomebound factors with respect to specific and total activity. It is possible that a pool of (inactivated?) factors exists, waiting for a signal to take part in another round of initiation. It has been shown that such a situation occurs in the case of certain mRNAs ~ Abbreviations. elF-I, eIF-2, elF-3, eIF-4A, eIF-4B, eIF-4C, eIF-4D and eIF-5 are eukaryotic initiation factors, see Anderson et al. (1977) FEBS Lett. 76, 1-10 and [14].De-finition. A260 unit; the quantity of material in 1 ml of a SOlution with an absorbance of 1 at 260 nm (2-cm path length).[ 1 1 -131. Therefore, useful information may be obtained by comparison of the specific activities and amounts of each initiation factor found in the postribosomal supernatant and on the ribosomes.We reported a procedure for the isolation of initiation factors from a crude lysate [14], in which an affinity chromatography step was used, i.e. binding of initiation factors to Sepharose-heparin. This paper describes a method to isolate initiation factors from the postribosomal supernatant utilizing chromatography on Sepharose-heparin at an early stage. Yield and specific activity of factors thus obtained were compared to those of factors isolated from the ribosomes via conventional methods. MATERIALS AND METHODS Mat...

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The Initiation Factors

Hershey

1982

Protein Biosynthesis in Eukaryotes

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Initiation of Protein Synthesis in Eukaryotes. Binding to Sepharose-heparin and Partial Purification of Initiation Factors from Krebs II Ascites Cells

Cited by 48 publications

References 24 publications

Purification of free eukaryotic initiation factors eIF-4A and eIF-4D on cibacron blue F3G-A

Purification of free eukaryotic initiation factors eIF-4A and eIF-4D on cibacron blue F3G-A

A Comparison of the Initiation Factors of Eukaryotic Protein Synthesis from Ribosomes and from the Postribosomal Supernatant

The Initiation Factors

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