Purification and characterization of initiation factor IF-E2 from rabbit reticulocytes.

Initiation factor IF-E2 was isolated from rabbit reticulocytes and purified 120-fold to near homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose and phosphocellulose, and, when suitable, by sucrose density gradient centrifugation. The factor is a complex protein containing three nonidentical polypeptides of molecular weight 57,000, 52,000, and 36,000. It behaves as a complex throughout its purification and during polyacrylamide gel electrophoresis in nondenaturing buffer but its thress components are readily separated by electrophoresis in denaturing buffers. None of its components corresponds to any of the polypeptides of the other initiation factors or to any proteins of ribosomes washed in buffers containing a high salf concentration. A stoichiometric ratio of 1:1:1 was determined for the three polypeptides; based on the assumption of one copy each per complex, the calculated factor molecular weight is 145,000, a value in agreement with the measured value of 160,000. Initiation factor IF-E2 was radioactively labeled in vitro by reductive alkylation or by phosphorylation with a protein kinase also isolated from rabbit reticulocytes. Neither procedure causes a measurable change in the ability of the factor to form a ternary complex with GTP and the initiator methionyl-tRNA. 5'-Guanylyl-methylenediphosphonate may substitute for GTP, but only at relatively high concentrations. The binding of labeled initiation factor IF-E2 and methionyl-tRNA to the 40 S ribosomal subunit was studied by sucrose density gradient centrifugation. Appreciable binding of the factor is seen only when all three components of the ternary complex are included in the reaction mixture. The binding of either the factor or methionyl-tRNA was not stimulated by the addition of globin messenger RNA and initiation factor IF-E3. It was shown that all three polypeptide components of initiation factor IF-E2 are bound to these nascent initiation complexes.     

Purification and properties of a ribosomal casein kinase from rabbit reticulocytes.

A casein kinase was isolated and purifed from rabbit reticulocytes. About 90% of the enzyme activity co-sedimented with the ribosomal fraction, whereas about 10% of the enzyme activity was found in the ribosome-free supernatant. Both casein kinases (the ribosome-bound enzyme as well as the free enzyme) showed identical activity and the same molecular weight. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis a single band of about 70 000 mol.wt. was observed. Sucrose-gradient analysis, however, showed that the enzyme activity sedimented with a s20,w of approx. 7.5S. This observation suggested that the casein kinase is a dimer composed of subunits of identical molecular weight. The enzyme utilizes GTP as well as ATP as a phosphoryl donor. It preferentially phosphorylates acidic proteins, in particular the model substrates casein and phosvitin. Casein kinase is cyclic AMP-indepenoent. The Km values for ATP and GTP with phosvitin as a substrate were determined as 1.2 and 8.8 micrometer respectively.     

Purification of various forms of elongation factor 1 from rabbit reticulocytes

Previous studies have indicated that the high-molecular-weight form of elongation factor 1 (EF-1H) contained four subunits (α, β, γ, and δ). Using the conventional methods of gel-filtration and ion-exchange chromatography, various forms of elongation factor 1 (EF-1α, EF-βδ, EF-1βγδ) have been purified from rabbit reticulocyte lysate. The procedure described allows one to purify these factors from a single batch of lysate in sufficient amounts for physical and biochemical studies. EF-1α is a single polypeptide of M_r 52,000, and has an isoelectric point of 9.1. EF-1βδ and EF-1βγδ are composed of two and three nonidentical polypeptides, respectively, as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Both proteins can form stable aggregates in native conditions that can reach more than 2,000,000 Da. The isoelectric point for each polypeptide was determined; 5.8 for EF-1β, 5.5 for EF-1γ, and 4.8 for EF-1δ. The activity of both proteins was compared on a molecular basis by their ability to stimulate EF-1α in the poly(U)-directed synthesis of polyphenylalanine. On the basis of this assay EF-1βγδ is slightly more active than EF-1βδ. The similarity of the amino acid composition of EF-1γ and EF-1δ and the molar ratio of α:β:γ:δ in EF-1H of approximately 1:1:0.5:0.5 have led to the conclusion that EF-1δ is probably a breakdown product of EF-1γ, and that the native form of EF-1H probably contains only the α, β, and γ subunits.     

Purification and some properties of the histidyl-tRNA synthetase from the cytosol of rabbit reticulocytes.

The histidyl-tRNA synthetase of rabbit reticulocyte cytosol has been purified 84 000-fold to apparent homogeneity with a specific activity of 687 nmol of histidyl-tRNA formed per min per mg of protein. Ten to 15% of the enzyme activity is sedimented with the ribosomes while the remainder is in the cytosol. The purified enzyme has a molecular weight of 122 000 as determined by sucrose density gradient centrifugation. Gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate suggests that it is composed of two similar subunits with a molecular weight of approximately 64 000. The enzyme has a magnesium optimum of 45 mM; however, this is reduced to 5 mM in the presence of an intracellular potassium concentration (160 nM). The enzyme acylates the two histidine tRNA isoacceptors of rabbit reticulocytes with similar Km values and at similar rates.     

Purification and properties of a homogeneous aryl sulfatase A from rabbit liver.

Aryl sulfatase A (aryl sulfate sulfohydrolase EC 3.1.6.1) has been purified > 10,000-fold from rabbit liver; by disc gel electrophoresis the enzyme appears homogeneous. Various properties of the enzyme have been determined and comparisons are made with other aryl sulfatases. Sodium dodecyl sulfate gel electrophoresis indicates that the enzyme is made up of monomers of molecular weight ～ 70,000. At pH 7.4 the enzyme exists as a dimer whereas a tetrameric form predominates at pH 4.8.The enzyme exhibits the anomalous kinetics often observed with aryl sulfatase A from mammalian tissues (the enzyme is modified to an inactive form while degrading substrate and the inactive form can be reactivated by sulfate ion). The enzyme activity has been studied under a variety of reaction conditions. Two pH optima are observed and neither enzyme concentration or changes in ionic strength appear to have an effect on the relative magnitudes of the optima. Aryl sulfatase A is competitively inhibited by potassium sulfate, potassium phosphate, and sodium sulfite (K_i = 2.9 × 10^?3 M, 3.4 × 10^?5 M, and 1.1 × 10^?6 M, respectively). Kinetic constants for some substituted phenyl sulfate esters have been determined. The variation in V is not consistent with a reaction mechanism involving a rate-limiting breakdown of a common intermediate.The inactive (modified) form of the enzyme has been isolated from reaction mixtures containing aryl sulfatase A and substrate. A procedure is presented for determining the relative amount of modified and native enzyme in these preparations. In the presence of substrate, sulfate displaces the equilibrium between native and modified enzyme in favor of native enzyme. In the absence of substrate neither sulfate or phosphate have an effect on the equilibrium. A study is made of the temperature dependence of the process in which the modified enzyme is converted back to native enzyme. The relatively small entropy of activation for the conversion of the modified to the native form (ΔS3 = ?8 cal/mole deg) does not seem to be consistent with a major modification of protein conformation.     

Purification and initial characterization of peptidyl-tRNA hydrolase from rabbit reticulocytes.

We have identified an activity in rabbit reticulocyte lysate as peptidyl-tRNA hydrolase, based upon its ability to hydrolyze native reticulocyte peptidyl-tRNA, isolated from polyribosomes, and N-acylaminoacyl-tRNA, and its inability to hydrolyze aminoacyl-tRNA, precisely the same substrate specificity previously reported for peptidyl-tRNA hydrolase from bacteria or yeast. The physiological role of the reticulocyte enzyme may be to hydrolyze and recycle peptidyl-tRNA that has dissociated prematurely from elongating ribosomes, as suggested for the bacterial and yeast enzymes, since reticulocyte peptidyl-tRNA hydrolase is completely incapable of hydrolyzing peptidyl-tRNA that is still bound to polyribosomes. We have purified reticulocyte peptidyl-tRNA hydrolase over 5,000-fold from the postribosomal supernatant with a yield of 14%. The purified product shows a 72-kDa band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis that has co-purified with enzyme activity and comprises about 90% of the total stained protein, strongly suggesting that the 72-kDa protein is the enzyme. Sucrose density gradient analysis indicates an apparent molecular mass for the native enzyme of 65 kDa, implying that it is a single polypeptide chain. The enzyme is almost completely inactive in the absence of a divalent cation: Mg2+ (1-2 mM) promotes activity best, Mn2+ is partly effective, and Ca2+ and spermidine are ineffective. The hydrolase shows a Km of 0.60 microM and Vmax of 7.1 nmol/min/mg with reticulocyte peptidyl-tRNA, a Km of 60 nM and Vmax of 14 nmol/min/mg with Escherichia coli fMet-tRNA(fMet), and a Km of 100 nM and Vmax of 2.2 nmol/min/mg with yeast N-acetyl-Phe-tRNA(Phe). The enzyme has a pH optimum of 7.0-7.25, it is inactivated by heat (60 degrees C for 5 min), and its activity is almost completely inhibited by pretreatment with N-ethylmaleimide or incubation with 20 mM phosphate. The fact that the enzyme hydrolyzes E. coli but not yeast or reticulocyte fMet-tRNA(fMet) may be explained, at least in part, by structural similarities between prokaryotic tRNA(fMet) and eukaryotic elongator tRNA that are not shared by eukaryotic tRNA(fMet).     

Some properties of the lipoxygenase from rabbit reticulocytes.

A lipoxygenase was enriched from the stoma-free supernatant of rabbit reticulocytes. The enzyme causes drastic deterioration of mitochondrial membranes. The release of matrix enzymes is paralleled by formation of products of lipid peroxidation. The enzyme reacts with isolated phospholipds and free cis-unsaturated fatty acids. Some properties were determined: molecular weight, isoelectric point, temperature and pH-dependence and Km value for linoleic acid. The enzyme is inhibited by reaction products and a variety of inhibitors, especially antioxidants and chelating agents.     

Purification and phosphorylation of initiation factor eIF-2 from rabbit skeletal muscle

Core particle DNA unfolding and refolding are followed by stopped-flow circular dichroism technique. When core particles are dissociated in the stopped-flow cuvette, the high CD deviation corresponding to the dissociated state is reached in the first millisecond, which means that the dissociation process is completed within the dead time of the apparatus which is ～1 ms. The same conclusion can be drawn when core particles are reassociated, since the low CD value, typical of the associated state, is immediately reached. Similarly histone release from chromatin is a very fast process. We also include some points of discussion about core particle assembly process.     

Further characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Immunochemical characterization and phosphorylation by casein kinase II

Eukaryotic initiation factor (eIF)-5, isolated from rabbit reticulocyte lysates, is a monomeric protein of Mr = 58,000-62,000. Immunochemical methods were employed to identify eIF-5 in crude cell lysates. Antisera against purified denatured eIF-5 were prepared in rabbits and characterized by immunoblotting and immunoprecipitation techniques using native and denatured eIF-5 as antigens. Monospecific antibodies to denatured eIF-5 were affinity-purified using eIF-5 blotted onto aminophenylthioether paper. Rabbit reticulocytes, HeLa cells and mouse L cells were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate. The denatured proteins were analyzed by polyacrylamide gel electrophoresis followed by immunoblotting with anti-eIF-5 antibodies. With each lysate, one major immunoreactive polypeptide was observed whose molecular weight corresponded to that of purified eIF-5 (Mr = 58,000-62,000). No degradation products or precursor forms of molecular weight higher than 62,000 were detected in any lysate. These results indicate that isolated eIF-5 is the same size as that found in crude lysates. Additional characterization of eIF-5 indicates that purified eIF-5 can be phosphorylated at serine residues in vitro by casein kinase II. Furthermore, in vitro phosphorylated eIF-5 retains full biological activity in catalyzing the joining of 60 S ribosomal subunits to a preformed 40 S ribosomal initiation complex to form an 80 S initiation complex. Based on its specific activity, we demonstrate that 1 pmol of rabbit reticulocyte eIF-5 mediates the formation of approximately 180 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.     

Crosslinking of eukaryotic initiation factor eIF3 to the 40S ribosomal subunit from rabbit reticulocytes

Complexes of purified 40S ribosomal subunits and initiation factor 3 from rabbit reticulocytes were crosslinked using the reversible protein crosslinking reagent, 2-iminothiolane, under conditions shown previously to lead to the formation of dimers between 40S proteins but not higher multimers. The activity of both the 40S subunits and initiation factor 3 was maintained. Protein crosslinked to the factor was purified by sucrose density gradient centrifugation following nuclease digestion of the ribosomal subunit: alternatively, the total protein was extracted from 40S: factor complexes. The protein obtained by either method was analyzed by two-dimensional diagonal polyacrylamide/sodium dodecyl sulfate gel electrophoresis. Ribosomal proteins were found in multimeric complexes of high molecular weight due to their crosslinking to components of eIF3. Identification of the ribosomal proteins appearing below the diagonal was accomplished by elution, radioiodination, two-dimensional polyacrylamide/urea gel electrophoresis, and radioautography. Proteins S2, S3, S3a, S4, S5, S6, S8, S9, S11, S12, S14, S15, S16, S19, S24, S25, and S26 were identified. Because many of the proteins in this group form crosslinked dimers with each other, it was impossible to distinguish proteins directly crosslinked to eIF3 from those crosslinked indirectly through one bridging protein. The results nonetheless imply that the 40S ribosomal proteins identified are at or near the binding site for initiation factor 3.     

Characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Evidence that the initiation factor is a monomeric protein of Mr of about 58,000-62,000

Eukaryotic initiation factor 5 (eIF-5) has been purified from the ribosomal salt-wash proteins of rabbit reticulocyte lysates. The purified factor migrates as a single polypeptide upon sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of about 58,000-62,000. In contrast, less pure preparations of reticulocyte eIF-5 behave in gel filtration columns and in glycerol gradient centrifugation in buffers containing 75-100 mM KCl as a protein of apparent Mr = 140,000-160,000. Presumably, this is due to association of the factor with other proteins, since eIF-5 activity present in such preparations can also be shown by (a) glycerol gradient centrifugation in buffers containing 500 mM KCl or (b) gel electrophoresis under denaturing conditions, to be associated with a 58,000-62,000-dalton protein. Furthermore, eIF-5 purified from rabbit reticulocyte lysates in the absence or presence of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity. It can be calculated that 1 pmol of the purified eIF-5 catalyzes the formation of nearly 50 pmol of 80 S initiation complex under in vitro initiation reaction conditions. Because of the highly catalytic activity of eIF-5 in initiation reactions, the presence of even low levels of eIF-5 in eIF-2 preparations causes hydrolysis of GTP bound to the 40 S initiation complex. This results in destabilization of Met-tRNA(f) bound to the 40 S complex in sucrose gradient centrifugation.     

Polypeptide chain initiation in eukaryotes. IV. Purification and properties of supernatant initiation factor from Artemia salina embryos

The eukaryotic supernatant initiation factor, described in earlier publications from this laboratory, has been isolated and purified over 3000-fold, to about 70 to 80% purity, from extracts of embryos of the brine shrimp Artemia salina. The native protein appears to consist of two equal subunits, each weighing approximately 74,000 daltons. Like the bacterial initiation factor IF2, its prokaryotic counterpart, the Artemia factor promotes the AUG-dependent binding of fMet-tRNA, or the poly (U)-dependent binding of N-acetyl-Phe-tRNA, to the small ribosomal subunit. However, unlike IF2, the reaction is GTP-independent and the factor functions catalytically for one molecule may promote the binding of up to 12 molecules of fMet-tRNA to 40 s subunits at 0 °C.     

Purification and properties of rabbit reticulocyte elongation factor 1.

Elongation factor 1 has been purified about 100-fold from the lysate of rabbit reticulocytes. The native enzyme is highly asymmetric ($\text{f}\text{f}{}_0\text{= 1.53}$) and has a molecular weight of 450,000. Polyacrylamide-gel electrophoresis in sodium dodecyl sulfate shows two major bands with molecular weights of about 53,000 and 50,000. Partially purified phospholipase C and AB preparations and elastase cause dissociation of the aggregate form of the enzyme to an active form which has a molecular weight of about 50,000. The effect of these phospholipase preparations is unexplained since rabbit reticulocyte elongation factor 1 contains little or no phospholipid. A protease contamination has been considered but no evidence of protease activity has been detected in the phospholipase preparations. In aminoacyl-tRNA binding, elongation factor 1 appears to show very little, if any, turnover. However, in the presence of elongation factor 2, under conditions where polymerization occurs, elongation factor 1 functions catalytically.     

The initiation of haemoglobin synthesis in rabbit reticulocytes

1. The incorporation of labelled valine by rabbit reticulocytes into the N-terminal position of nascent haemoglobin was investigated by deaminating the nascent peptides with nitrous acid and isolating labelled alpha-hydroxyisovaleric acid and valine after acid hydrolysis. 2. The amount of radioactivity in alpha-hydroxyisovaleric acid relative to that in valine indicated the presence of 12.3% N-terminal valine having a free amino group. This high value suggests that most if not all nascent peptides contain valine in the N-terminal position. 3. Cell-free preparations containing reticulocyte ribosomes and pH5 enzymes incorporated alpha-hydroxy-[(14)C]isovaleryl-tRNA (where tRNA refers to transfer RNA), which was obtained by deamination of [(14)C]valyl-tRNA from yeast or liver with nitrous acid, into both soluble and nascent protein. 4. When the soluble protein was chromatographed on CM-cellulose, radioactivity was found to be associated with both the alpha-and beta-globin chains. 5. The kinetics of hydrolysis of [(14)C]valine, was also investigated. Most of the material was hydrolysed rapidly at pH10, but a minor component that was relatively stable appeared to be present to the extent of about 10% of the total valyl-tRNA. Valine was, however, the only hydrolysis product detected by paper chromatography. 6. It is concluded that chain initiation in haemoglobin synthesis involves valine as the N-terminal amino acid and that the amino group of nascent protein is probably not substituted.     

The isolation and characterization from rabbit reticulocytes of two forms of eukaryotic initiation factor 2 having different beta-polypeptides

We have isolated from the high salt wash of rabbit reticulocyte ribosomes two forms of the polypeptide chain initiation factor 2 (eIF-2) which differ with respect to their beta-subunit, GDP content, and sensitivity to Mg2+ in ternary (eIF-2 X GTP X Met-tRNAf) and binary (eIF-2 X GDP) complex formation. The form of eIF-2 eluting first from a cation exchange (Mono S, Pharmacia) column has a beta-subunit of lower molecular weight (eIF-2(beta L] and a more acidic pI value than the form eluting at a higher salt concentration (eIF-2(beta H]. These two forms of eIF-2 beta-polypeptides are also detected in reticulocyte lysates when the proteins are resolved by two-dimensional isoelectric focusing-dodecyl sulfate polyacrylamide gel electrophoresis followed by immunoblotting. The peptide mapping of the isolated beta-subunits after limited proteolysis by papain, pancreatic protease, alpha-chymotrypsin, or Staphylococcus aureus V8 protease further demonstrates that the two forms of beta-subunits are not the product of a non-specific proteolytic action that occurred during the purification procedure, but rather reflects the existence in vivo of both forms of eIF-2. The GDP content of eIF-2(beta L) and eIF-2(beta H) is approximately 0.85 and 0.22 mol of GDP/mol of eIF-2, respectively. The KD for GDP of eIF-2(beta L) was lower (2.2 X 10(-9) M) than that of eIF-2(beta H) (6.0 X 10(-8) M). In the presence of 1 mM Mg2+, the activities of eIF-2(beta L) and eIF-2(beta H) in forming a binary and a ternary complex are inhibited 90 and 25%, respectively. The extent of Mg2+ inhibition and its reversal by the guanine nucleotide exchange factor is directly proportional to the amount of GDP bound to eIF-2. No inhibition by Mg2+ is observed when eIF-2-bound GDP is removed by alkaline phosphatase. In the presence of the guanine nucleotide exchange factor, both forms of eIF-2 are equally active in ternary complex formation, and the complex formed is quantitatively transferred to 40 S ribosomal subunits.     

Purification and properties of a methionine formylmethionyl-tRNA-binding initiation factor from pig liver

(i) A factor, EIF-2, that binds methionyl-tRNA_f^Met in the presence of GTP has been isolated from pig liver. (ii) Dodecylsulfate-gel electrophoresis and sedimentation equilibrium centrifugation indicate that the factor has a molecular weight of 122,000 and that it consists of three unequal subunits. (iii) The apparent K_D for binding of methionyl-tRNA_f^Met varies with factor concentration. GTP participates in the binding with a K_D of 0.5 μm. β,γ-Methylene-guanosine triphosphate supports 40% of the binding observed with GTP. GDP is a competitive inhibitor with a K_i of 0.2 μm. The optimal, free Mg²⁺ concentration is approximately 50 μm. GTP and Mg²⁺ stabilize the factor against thermal inactivation and inactivation by N-ethyl maleimide. (iv) The factor is required for the formation of a sucrose gradient-stable complex between methionyl-tRNA_f^Met and the 40S ribosomal subunit. The presence of template is not necessary, but poly(A,U,G) increases the binding observed 1.5-fold. (v) The factor markedly stimulates synthesis in a reconstituted protein-synthesizing system with globin messenger RNA as template.