The role of cholesteryl 14-methylhexadecanoate in peptide elongation reactions

Hradec, J; Dusek, Z; Bermek, Engin; Matthaei, Heinrich

doi:10.1042/bj1230959

Cited by 36 publications

(52 citation statements)

References 23 publications

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“…Hradec and coworkers (13) first reported that EF-1 preparations contained a unique cholesterol ester (cholesterol-14-methylhexadecanoate) which was also present in a complex of aminoacyl-tRNA synthetases (14). A similar result on the presence of cholesterol in a eukaryote aminoacyl-tRNA synthetase preparation has been reported by Bandyopadhyay and Deutscher (16).…”

Section: Discussionsupporting

confidence: 54%

“…Cholesterol-14 methylhexadecanoate was not found in the EF-1 preparations from calf brain, and preliminary attempts on our -part to activate EF-1H preparations that have been extracted with organic solvents as described by Hradec et al. (13) have been unsuccessful. We do not know whether EF-1H merely represents a storage form of EF-1L, a high-molecular-weight regulatory complex, or an artifact of the isolation procedure.…”

Section: Discussionmentioning

confidence: 97%

See 1 more Smart Citation

Multiple Forms of Elongation Factor 1 from Calf Brain

Moon¹,

Redfield²,

Millard³

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

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Heavy and light forms of elongation factor 1 (EF-1) from calf brain have been partially purified. The heterogeneous heavy species (EF-1H) with molecular weights of 2.5 X 105 to over 1 X 106 appears to be a complex or aggregate of the light form of the enzyme (EF-IL); the latter has a molecular weight of between 50,000 and 60,000. EF-1H but not EF-IL, contains significant amounts of free and esterified cholesterol. Although EF-1 and EF-IL are both active in aminoacyl-tRNA binding to ribosomes, EF-IL reacts with GTP and aminoacyl-tRNA more efficiently than EF-1H.We have described studies on the interactions of elongation factor 1 (EF-1) from calf brain with guanosine nucleotides and aminoacyl-tRNA (1, 2). Evidence was first obtained with a nitrocellulose filter assay that an aminoacyl-tRNA EF-1 GTP ternary complex could be formed since EF-1 * GTP was retained on the filter in the absence but not in the presence of aminoacyl-tRNA (1). More recent experiments with Sephadex G-150 chromatography (2) provided further evidence for ternary complex formation. These results also suggested that different forms of EF-1 were involved in these interactions, and that a low-molecular-weight species of the enzyme appeared to be part of the ternary complex. The interaction of the ternary complex with calf-brain ribosomes has also been demonstrated (3), and data in support of the view that the ternary complex contains a form of EF-1 having a molecular weight of less than 100,000 have been obtained.Multiple forms of EF-1 have been demonstrated in other mammalian tissues (4, 5) and wheat (6), although their nature and function are not clear. The present study describes the partial purification of EF-1 from calf brain and the separation of heavy and light forms of the enzyme. The characteristics of these EF-1 species were examined. MATERIALS AND METHODSPurification of EF-1 from Calf Brain. A typical purification started with 10 calf brains. Each brain (about 450 g) was homogenized in a Waring Blender for 20 see in 100 ml of a buffer containing 50 mM Tris HCl (pH 7.4)-25 mM KCl-5 mM MgCl2-0.25 M sucrose. The homogenate was centrifuged for 10 min at 5000 rpm in a Sorvall GSA rotor and then for 30 min at 9000 rpm.(NH4)2SO4 Fractionation. 16.4 g of solid (NH4)2SO4 was added to each 100 ml of the post mitochondrial fraction. After 30 min the solution was centrifuged for 30 min at 9000 rpm in a Sorvall GSA rotor. The precipitate was discarded, and 21.4 g of solid (NH4)2SO4 was added to each 100 ml of the supernatant. After 30 min of stirring, the solution was centrifuged.The pellet was dissolved in a small volume of buffer containing 50 mM Tris* HCl (pH 7.4)-i mM dithiothreitol (buffer A) and dialyzed against the same buffer. After dialysis, the A280 of the sample was adjusted to 35-40 per ml. Calcium PhosphateStep. To the dialyzed (NH4)2SO4 fraction we added 0.7 volume of a 3% calcium phosphate suspension (Calbiochem. Corp.). After 1 hr of stirring, the suspension was centrifuged for 5 min at 5000 rpm in a Sorvall GSA rotor. The su...

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Section: Discussionsupporting

confidence: 54%

Section: Discussionmentioning

confidence: 97%

Multiple Forms of Elongation Factor 1 from Calf Brain

Moon¹,

Redfield²,

Millard³

et al. 1973

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

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“…Blood was withdrawn by cardiac puncture into bottles containing a solution of heparin (50 units/ml of blood) and was chilled immediately in ice. Polyribosomes from rat liver were isolated as described by Hradec et al (1971) and ribosomal subunits from rat liver as reported by Hradec et al (1974). Peptide-elongation factors were separated and purified by the method of Bermek & Matthaei (1970).…”

Section: Animalsmentioning

confidence: 99%

“…Assay of radioactivity Portions of incubation mixtures (75,u1) in which peptide-bond formation was assayed were applied to discs of Whatman GF 83 filters, and the filters were washed as described by Hradec et al (1971). Mixtures for the assay of aminoacyl-tRNA formation were treated in the same way, omitting only washing with hot trichloroacetic acid.…”

Section: Incubationsmentioning

confidence: 99%

All factors required for protein synthesis are retained on heparin bound to Sepharose

Hradec¹,

Dusek²

1978

Biochemical Journal

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1. Postmitochondrial supernatants ofrabbit reticulocyte lysates were chromatographed on heparin bound to Sepharose 4B, and the fraction retained on affinity columns was separated by subsequent gel filtration on Sepharose 4B into three fractions, two of them active in protein synthesis. 2. The heavier fraction sedimented at 40 S and contained more than 10% RNA. This consisted predominantly of a 12S component, with smaller amounts of the 9S and 4S RNA species. The lighter fraction (18-20S) was composed of proteins with less than 1 % RNA. 3. Different enzymic activities were associated with these fractions. 4. In the presence of both fractions, efficient translation took place on combined ribosomal subunits of rat liver with added cofactors. Globin messenger ribonucleoprotein stimulated this translation 5-6-fold. 5. Relatively large complexes of all factors required for protein synthesis are apparently isolated from reticulocytes by affinity chromatography on heparin-Sepharose 4B. Such complexes may occur naturally in the cytoplasm of mammalian cells.

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“…This stimulating effect on enzymes present in the soluble fraction of the cell has been investigated in our laboratory during the past years [2,31. Evidence has been obtained that the presence of this compound in the molecules of aminoacyl-tRNA synthetases [4] as well as in peptide elongation factors [5] may be essential for the normal activity of these enzymes.…”

Section: Introductionmentioning

confidence: 99%