Isolation and characterization of two forms of Met-tRNAf deacylase from rabbit reticulocyte ribosomes

We have separated and purified two forms of Met-tRNA_f deacylase (or two separate enzymes), an activity that mediates in part the suppression of polypeptide chain initiation that occurs in heme deficiency or with double-stranded RNA, 1000-fold from the 0.5 M KCl wash of rabbit reticulocyte ribosomes. Deacylase I is a minor activity with an S_20,w of 5.9, D_20,w of 4.9 and M_r of 110 000, while deacylase II is the major activity with an S_20,w of 3.3, D_20,w of 7.1 and M_r of 43 000. Both convert crude reticulocyte or pure yeast, wheat germ, and E. coli [³⁵S]Met-tRNA_f to [³⁵S]methionine and tRNA^Met_f and have no effect on reticulocyte [³⁵S]fMet-tRNA_f, [³H]Ala-tRNA or [³H]Lys-tRNA. However, while deacylase I has similar activity throughout the pH range of 6.1–8.1, deacylase II has a sharp pH optimum at 7.9 and is almost completely inactive at 6.1. In addition, deacylase II shows a much greater affinity for pure Met-tRNA_f than deacylase I (K_m of 1.5–3 nM vs. 100 nM), and, while deacylase II is selectively inhibited by tRNA^Met_f, deacylase I is inhibited similarly by any added tRNA.     

Use of an antibody to characterize and determine the role of the major Met-tRNAf deacylase from rabbit reticulocyte ribosomes

Inhibition of polypeptide chain initiation in rabbit reticulocyte lysate by phosphorylation of eukaryotic initiation factor-2(alpha) results, secondarily, in the enzymatic deacylation of Met-tRNAf on the 48 S initiation complexes that accumulate. We have prepared an antibody to a highly purified preparation of the major Met-tRNAf deacylase activity on rabbit reticulocyte ribosomes, termed deacylase II. Antibody, but not similarly purified normal IgG, completely neutralizes the activity of Met-tRNAf deacylase II and has no effect on Met-tRNAf deacylase I, a separate, minor, reticulocyte activity with the same substrate specificity but very different physical and enzymatic properties, strongly suggesting that deacylase I and II are distinct proteins. We partially purified Met-tRNAf deacylase activities from rabbit liver, myocardium and bone marrow ribosomes and found them to be similar to each other and to reticulocyte deacylase I in their enzymatic properties and insensitivity to anti-deacylase II, suggesting that deacylase I may be a general form of this enzyme, present in many cells, while deacylase II may be induced specifically during erythroid differentiation. Addition of the antibody to reticulocyte lysate incubated in the absence of hemin or presence of hemin plus 0.1 microgram/ml poly(I X C) did not reverse the inhibition of protein synthesis but did reduce the rate of turnover/utilization of Met-tRNAf and increase the level of Met-tRNAf bound to 48 S initiation complexes, demonstrating that the deacylase does not directly inhibit protein synthesis under these conditions but does mediate the deacylation, loss, and thus greater than expected turnover of Met-tRNAf in the 48 S complexes that accumulate.     

Dual effects of the ricin A chain on protein synthesis in rabbit reticulocyte lysate. Inhibition of initiation and translocation

Ricin A chain caused inhibition of protein synthesis by reticulocyte lysate with concomitant depurination of 28S rRNA. The partial reaction(s) of protein synthesis inhibited was investigated by following the appearance of [35S]methionine from initiator [35S]Met-tRNA into 40S ribosomal subunits, 80S monosomes and polysomes. Ricin A chain caused an accumulation of [35S]Met in monosomes which did not enter polysomes. In these respects the effects of the ricin A chain resembled those of diphtheria toxin, an inhibitor of elongation-factor-2-catalyzed translocation. This is consistent with the previously proposed site of action of ricin as an inhibitor of elongation. However, the inhibitory effects of the ricin A chain and diphtheria toxin are not equivalent because we observed that the rate of formation of the 80S initiation complex was reduced approximately sixfold with the ricin A chain relative to diphtheria toxin. Analysis of methionine-containing peptides bound to 80S monosomes in ricin-A-chain-inhibited and diphtheria-toxin-inhibited lysates, programmed with globin mRNA, revealed a predominance of Met-Val, suggesting that the elongation cycle is inhibited at the translocation step. Translocation was also implicated as the step blocked in both the ricin-A-chain-inhibited and diphtheria-toxin-inhibited lysates, by the finding that nascent peptide chains were unreactive towards puromycin. It is concluded that ricin-A-chain-modified ribosomes are deficient in two protein synthesis partial reactions: the formation of the 80S initiation complex during initiation and the translocation step of the elongation cycle.     

Effect of antibody to the hemin-controlled translational repressor in rabbit reticulocyte lysate

We have examined the effect of the purified IgG from the serum of guinea pigs immunized with a highly purified preparation of rabbit reticulocyte, hemin-controlled translational repressor (HCR) on protein synthesis in the reticulocyte lysate. We have found that the anti-HCR (but not non-immune) IgG completely prevents or reverses the suppression of protein synthesis that occurs in hemin-deficient lysate, providing a direct and definitive demonstration that the inhibitory effect of hemin-deficiency is mediated solely by the activation of HCR. The anti-HCR IgG also prevents or reverses the phosphorylation of eIF-2 alpha and the reduced binding of Met-tRNAf to 40 S ribosomal subunits that accompanies the inhibition of protein synthesis in hemin-deficient lysate. In contrast, the anti-HCR IgG has no effect on the inhibition produced by low levels of double-stranded RNA (that is due to the activation of a separate protein kinase), but it does partly reverse inhibition due to oxidized glutathione, ethanol, and phosphatidylserine, indicating that the effect of these components is mediated, at least in part, by the activation of HCR. Finally, we have confirmed our earlier observation that an excess of proHCR, the inactive precursor of HCR, has little effect on the neutralization of HCR by limiting anti-HCR IgG, suggesting that the antigenic determinants on HCR are not exposed on ProHCR.     

Cation radius effects on cell-free translation in rabbit reticulocyte lysate

The effect of monovalent cation concentrations on the translation was examined in the rabbit reticulocyte cell-free system. The translation of standard reporter gene luciferase was studied using different concentrations of LiCl, NaCl, KCl, RbCl, CsCl, NH(4)Cl, and (CH(3))(4)NCl and the acetates of Na(+), K(+), and NH4(+). Only the salts of K(+), Rb(+), and NH4(+) and to some minor extent of Cs(+) significantly supported translation. Optimum concentrations were dependent on the cation used. Optimum concentrations ranged between 40 mM (NH(4)Ac), 80 mM (KCl, NH(4)Cl), and 100 mM (RbCl, KAc). The maximum efficiency of translation depends on the ionic radius of the cation used. KCl and RbCl were superior to all other salts tested in stimulating in vitro translation. The results were confirmed, using a second reporter system, M-hirudin. Here, however, broad optima were observed with RbCl being slightly superior to KCl in supporting translation.     

Effect of parvalbumin and S-100 protein on protein synthesis in rabbit reticulocyte lysate.

The effect of two high affinity Ca+ binding acidic proteins, parvalbumin and S-100 protein on protein synthesis in rabbit reticulocyte lysates (RRL), was investigated. Nuclease-treated RRL, supplemented with yeast mRNA, and 3H-leucine were incubated at 37 degrees C, and incorporation of 3H-leucine into protein was determined for 24 min. At 20 micrograms/100 microliters lysate concentration, both parvalbumin and S-100 protein caused a marked inhibition of protein synthesis compared with the control lysate. At a lower concentration parvalbumin was less inhibitory than histone H1; the effect of S-100 protein was not significant. The combined inhibitory effect of parvalbumin and H1 was not additive probably due to strong interaction between them as was evidenced by the enhanced absorbance of parvalbumin-H1 mixture. Spectrophotometric profiles of parvalbumin-tRNA mixture indicated that, unlike H1, parvalbumin did not inhibit protein synthesis by binding with nucleic acids. These results suggest an important role for parvalbumin in translational regulation.     

Regulation of eukaryotic initiation factor-2B activity by polyamines and amino acid starvation in rabbit reticulocyte lysate

We recently reported that the translational control of protein synthesis by glucose 6-phosphate in gel-filtered, rabbit reticulocyte lysate is exerted on the activity of eukaryotic initiation factor (eIF)-2B, the factor that catalyzes the exchange of GTP for GDP bound to eIF-2, by a mechanism that is independent of the phosphorylation of eIF-2 (alpha subunit). We now demonstrate that two other conditions regulate the activity of eIF-2B in rabbit reticulocyte lysate: polyamines (spermidine and spermine) and amino acid deficiency. In the absence of added polyamines, protein synthesis in gel-filtered lysate is reduced to about 70% and eIF-2B activity to about 35% of optimal. The former is likely a result of the latter, since we find that reticulocyte lysate has about twice the eIF-2B necessary to recycle the eIF-2.GDP generated under conditions of optimal protein synthesis. In contrast, the reduction in eIF-2B activity (to about 50% of optimal) occurring in the absence of added amino acids in unfractionated or gel-filtered lysate is insufficient, by itself, to slow the rate of protein synthesis, and the inhibition of protein synthesis that does occur with amino acid deficiency is exerted on polypeptide chain elongation, not initiation. The reduction in eIF-2B activity occurring with amino acid deficiency cannot be reversed by adding more glucose 6-phosphate or polyamines nor can the reduced eIF-2B activity seen with polyamine deficiency be overcome by increasing the glucose 6-phosphate, suggesting that these three components regulate eIF-2B activity by different mechanisms.     

Poly(A) dependent translation in rabbit reticulocyte lysate

Poly(A) tail has been known to enhance mRNA translation in eukaryotic cells. However, the effect of poly(A) tail in vitro is rather small. Rabbit reticulocyte lysate (RRL) is widely used for studying translation in vitro. Translation in RRL is typically performed in nuclease-treated lysate in which most of the endogenous mRNA have been removed. In this condition, the difference in the translational efficiency between poly(A)⁺ and poly(A)⁻ mRNAs is about two-fold. We studied the effect of poly(A) tail on luciferase mRNA translation in nuclease uncreated reticulocyte lysate, in which endogenous globin mRNAs were actively translated. In the case of capped mRNAs. stimulation of translation by poly(A) addition was about 1.5- to 1.6-fold and the effect of the poly(A) length was small. However, in the case of uncapped mRNAs, the addition of poly(A) tail increased luciferase expression over 10-fold. The effect of the poly(A) tail was dependent on its length. The difference in the translational efficiency was not due to the change of mRNA stability. These data indicate that RRL has the potential to translate mRNA in a poly(A) dependent manner.     

Regulation of heme-controlled eukaryotic polypeptide chain initiation factor 2 alpha-subunit kinase of reticulocyte lysates.

We have obtained highly purified preparations of the heme-controlled eukaryotic initiation factor 2 alpha-subunit (eIF-2 alpha) kinase (HCI) from rabbit reticulocyte lysates containing five different polypeptides. One of these is a 87-kDa (p87) phosphopeptide which appears to show an autokinase activity. The controlled digestion with trypsin of HCI preparations leads to the suggestion that phosphorylation of p87 is not needed for kinase activity and, furthermore, that another 89-kDa polypeptide could be the kinase catalytic subunit. In agreement with this, monoclonal antibodies directed against p87 do not interfere with eIF-2 alpha kinase activity. Moreover, the anti-p87 antibodies and those directed against the mammalian 90-kDa heat shock protein recognize the same p87 polypeptide from rabbit reticulocyte lysates. Upon incubation of the HCI preparation with hemin (5-10 microM), the eIF-2 alpha kinase is converted into an inactive form and appears to become associated with related peptides forming high molecular weight complexes which can be reversibly activated by 2-mercaptoethanol. The maintenance of the integrity of the porphyrin ring is absolutely required for kinase inactivation and although the presence of metal ion is not essential, the iron and cobalt metalloporphyrins are more effective than protoporphyrin IX. The formation of the inactive form of HCI by hemin is prevented by either N-ethylmaleimide, monoclonal antibodies directed against p87, or phosphorylation of p87. The data strongly suggest that hemin regulates eIF-2 alpha kinase activity by promoting formation of the inactive dimer HCI.p87 via disulfide bonds and direct binding of hemin. A model of HCI regulation is discussed.     

In vitro synthesis of L-gulono-gamma-lactone oxidase by rabbit reticulocyte lysate

In vitro synthesis of L-gulono-gamma-lactone oxidase [L-gulono-gamma-lactone:oxygen 2-oxidoreductase, EC 1.1.3.8], one of the microsomal flavin enzymes, was performed with poly(A)+ RNA of rat liver using the rabbit reticulocyte lysate system in order to study the biosynthesis of the enzyme. The apparent molecular weight of the synthesized enzyme protein was almost the same as that of the purified L-gulono-gamma-lactone oxidase from rat liver. It was demonstrated that the enzyme protein was not detectable when guinea pig poly(A)+ RNA was used for the translation, indicating that the mRNA for the enzyme is absent in the guinea pig or, if it exists, is not translatable.     

Protein synthesis in rabbit reticulocytes: control of protein synthesis initiation by a met-tRNA Met f deacylase and peptide chain initiation factors

The 0.5M KCl wash of rabbit reticulocyte ribosomes (I fraction) catalyzes the deacylation of Met-tRNA_f^Met. Upon DEAE-cellulose column chromatography, the deacylase activity elutes with the 0.1M KCl wash of the column (f1) and is well-resolved from the peptide chain initiation factors (1–3). The deacylase activity is specific for Met-tRNA_f^Met (retic., $\text{E.}$$\text{coli}$). Other aminoacyl tRNAs tested including fMet-tRNA_f^Met (retic., $\text{E.}$$\text{coli}$), Phe-tRNA ($\text{E.}$$\text{coli}$), Val-tRNA (retic.), and Arg-tRNA (retic.) are completely resistant to the action of the deacylase. In the presence of the peptide chain initiation factor (IF1) and GTP, retic. Met-tRNA_f^Met forms the initiation complex Met-tRNA_f^Met:IF1:GTP (2), and in this ternary complex Met-tRNA_f^Met is not degraded by the deacylase. $\text{E.}$$\text{coli}$ Met-tRNA_f^Met binds to IF1 independent of GTP, and in this complex, this Met-tRNA_f^Met is degraded by the deacylase.Prior incubation of f1 with Met-tRNA_f^Met (retic.) strongly inhibited protein synthesis initiation, presumably due to deacylation of the initiator tRNA. This inhibition by f1 was completely prevented when Met-tRNA_f^Met (retic.) was pre-incubated with peptide chain initiation factors.     

Temperature sensitivity of protein synthesis initiation in the reticulocyte lysate system. Reduced formation of the 40 S ribosomal subunit - Met-tRNAf complex at an elevated temperature.

Analysis of protein synthesis in the rabbit reticulocyte lysate system revealed the existence of a temperature-sensitive step in chain initiation which became irreversibly inactivated in the incubation at 42 degrees C. This inactivation of initiation was accompanied by a marked reduction in formation of the 40 S ribosomal subunit - Met-tRNAf complex. Decreased protein synthesis and a decrease in formation of the 40 S complex were also evident in unfortified lysates which had been prewarmed at 42 degrees C prior to protein synthesis. Hemin did not protect such lysates. The addition of supernatant fraction of a fresh lysate did not promote recovery of the reduced protein synthesis by such prewarmed lysates. Moreover, the addition of supernatant fraction prewarmed at 42 degrees C in the presence of added hemin caused little inhibition of protein synthesis by fresh lysate. The results indicate that the supernatant fraction is not involved in the inactivation.     

Quantitation and localization of globin messenger RNA in rabbit reticulocyte lysate.

A sensitive and quantitative method is described for the determination of globin mRNA distribution in rabbit reticulocyte lysate. The method uses high resolution sucrose density gradient centrifugation followed by [5'-3H]polyuridylate hybridization to poly(A)-mRNA in gradient fractions. Polyadenylate, purified globin mRNA, and ribonuclease-treated lysate are used to standardize the hybridization assay. It is demonstrated that changes of mRNA and ribosomal distribution do not affect quantitation of the total mRNA localization and Met-tRNAf which suggest that the monitoring of Met-tRNAf binding alone may not be sufficient to assess the mechanisms of control which affect the initiation of protein synthesis.     

Purification of two high molecular weight proteases from rabbit reticulocyte lysate

We have purified two high molecular weight proteases approximately 400-fold from rabbit reticulocyte lysate. Both enzymes hydrolyze 125I-alpha-casein and 4-methylcoumaryl-7-amide peptides with tyrosine, phenylalanine, or arginine at the P1 position. Both are inhibited by hemin, thiol reagents, chymostatin, and leupeptin. They differ, however, by other criteria. Degradation of 125I-lysozyme-ubiquitin conjugates and succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide by the larger 26 S protease is stimulated by ATP. Based on sedimentation, gel filtration, and nondenaturing polyacrylamide gel electrophoresis, the ATP-dependent protease has a molecular weight of 1,000,000 +/- 100,000 and is a multisubunit complex. The smaller 20 S protease has a molecular weight of 700,000 +/- 20,000 and is composed of 8-10 separate subunits with Mr values between 21,000 and 32,000. It does not require nucleotides for degradation of protein or peptide substrates. This smaller enzyme is similar, if not identical, to the "multicatalytic proteinase complex" first described by Wilk and Orlowski (Wilk, S., and Orlowski, M. (1983) J. Neurochem. 40, 842-849).