The isolation and biological activity of multiple forms of aminoacyl transferase I of rat liver

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...

Section: Discussionmentioning

confidence: 96%

“…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.…”

mentioning

confidence: 99%

Multiple Forms of Elongation Factor 1 from Calf Brain

Moon¹,

Redfield²,

Millard³

et al. 1973

“…The enzyme responsible for the transfer of the y-phosphoryl of GTP to ribosomal protein was prepared from rat-liver cytosol by hydroxylapatite chromatography, followed by filtration through Sephadex G-200, using the protocol described by Schneir and Moldave (9) for the isolation of elongation factor 1 (EF-1). We refer to the activity as "protein kinase-GTP."…”

Section: F2p]atp and [Y-32p]gtp (4)mentioning

confidence: 99%

A Kinase That Transfers the γ-Phosphoryl Group of GTP to Proteins of Eukaryotic 40S Ribosomal Subunits

Ventimiglia

Wool

1974

An enzyme in rat-liver cytosol transferred the 7y-phosphoryl of GTP to serine and threonine residues of at least four proteins (S6, S10, S14 or S15, and S17) of the small (40S) subunit of rat-liver ribosomes. A number of nonribosomal proteins in the enzyme preparation were also phosphorylated; they were preferentially and tightly bound to the large subunit. The enzyme could be distinguished from protein kinase-ATP (which also phosphorylated ribosomal proteins) by a number of criteria: (1) GTP was the phosphoryl donor; (2) the pattern of phosphorylation of ribosomal proteins by the two enzymes was different; and (3) the protein kinase that used GTP as the phosphoryl donor was not stimulated by cyclic AMP (or by cyclic GMP).Eukaryotic ribosomal proteins are phosphorylated in vivo and in vitro by protein kinases bound to the particle or free in the cytoplasm (1-3); that reaction has been shown, or assumed, to result from the transfer of phosphate from ATP. We report now on a related but novel reaction. There is an enzyme ac--tivity in rat-liver cytosol that catalyzes the transfer of the yphosphoryl of GTP to several proteins of the 40S subunit of eukaryotic ribosomes. Preparation of Ribosomal Subunits. Liver ribosomes were isolated from male Sprague-Dawley rats that weighed 100-120 g (5, 6) and used to prepare ribosomal subunits (5, 7).Preparation of Enzymes. The enzyme responsible for the transfer of the y-phosphoryl of GTP to ribosomal protein was prepared from rat-liver cytosol by hydroxylapatite chromatography, followed by filtration through Sephadex G-200, using the protocol described by Schneir and Moldave (9) for the isolation of elongation factor 1 (EF-1). We refer to the activity as "protein kinase-GTP." A rat-liver protein kinase that transfers the y-phosphoryl group of ATP to serine and theonine in ribosomal proteins was prepared as described (3, 10); the enzyme is referred to as "protein kinase-

“…The difference in the elution patterns appears to be consistent with the known information on the structure of EF 1. The enzyme from liver has been reported to exist in multiple forms that have molecular weights ranging from 100,000 to greater than 300,000 (5). Reticulocyte EF 1 has a molecular weight of 186,000, and this protein has been dissociated into three subunits that were enzymatically inactive (6).…”

Section: Discussionmentioning

confidence: 99%

Interaction of Eukaryote Elongation Factor EF 1 with Guanosine Nucleotides and Aminoacyl-tRNA

Moon¹,

Redfield²,

Weissbach³

1972

Evidence for two species of elongation factor 1 (EF IA and EF 1B) from calf brain has been obtained by molecular sieve chromatography on Sephadex G-150.-A high molecular weight form, EF IAS interacts with GTP to form an EF 1A-GTP complex. GDP also reacts with EF 1, but unlike the reaction with-GTP, an EF 1B-GDP complex is formed that contains a lower molecular weight and labile species of EF 1. The results also indicate that EF IA-GTP reacts with aminoacyl-tRNA to form an aminoacyl-tRNA-EF IB-GTP complex. These results are discussed with regard to the role of EF 1 in aminoacyltRNA binding to ribosomes.We have recently demonstrated that elongation factor 1 (EF 1) from calf brain can interact with GDP and GTP to form complexes that are retained on nitrocellulose filters (1). The EF 1-GTP complex was not retained on the filter in the presence of Phe-tRNA, suggesting that a Phe-tRNA-EF 1-GTP complex was formed (1). Similar results have been obtained with the bacterial elongation factor Tu (EF Tu), i.e., a reaction of EF Tu-GTP with aminoacyl-tRNA to form a ternary complex, aminoacyl-tRNA-EF Tu-GTP (2).In the present experiments, evidence for the formation of a ternary complex with the eukaryote elongation factor was sought by chromatographic procedures. It has now been possible to obtain more evidence for the formation of a PhetRNA-EF 1-GTP complex by molecular sieve chromatography. In addition, evidence will be presented that GDP and GTP react differently with EF 1. These results are discussed with respect to the role of EF 1 in aminoacyl-tRNA binding to ribosomes. MATERIALS AND METHODSMost of the materials used were obtained from commercial sources. The preparation of calf-brain ribosomes and an outline of the purification procedure of EF 1 from calf brain have been reported (1). The EF 1 preparation bound 800 pmol of GTP per mg of protein, of which EF 1 accounted for 70% of the GTP binding based on the interaction with aminoacyl-tRNA, as assayed on a nitrocellulose filter (1).EF 1 was calculated to be about 10% pure, if a molecular weight of the protein of 2 X 105 is assumed. ,ug of poly(U), 7.5 ,ug of EF2 (3), 25 pmol of Phe-tRNA, 10 mM MgCl2,50 mM NH4Cl, 50 mM Tris HCl (pH 7.4), 1 mM DTT, 0.25 mM GTP, and 0.5 A20 of calf-brain ribosomes.The incubations were for 10 min at 370, and the reactions were terminated by the addition of 5 ml of 5% Cl3CCOOH. The acidified reaction mixtures were heated at 900 for 15 min and, after cooling, the incubations were filtered through nitrocellulose membranes (0.45 Mam, Millipore Corp.) as described (1); the membranes were assayed for radioactivity.All radioactive measurements were performed in a Beckman LS-100 scintillation counter with a counting fluid described by Bray (4). RESULTSThe initial purpose of these experiments was to separate an aminoacyl-tRNA-EF 1-GTP complex from unreacted aminoacyl-tRNA and GTP, so as to study the interaction of the ternary complex with mammalian ribosomes. A Sephadex G-150 column was considered ideal for this purpose, since preliminary ...