B. Suren Baliga

Distribution of ferritin mRNA and albumin mRNA between free and membrane-bound rat liver polysomes

Abstract Free and membrane-bound polyribosomes and their respective mRNAS were isolated from the livers of both normal rats and rats treated with a single large dose of iron a few hours before being killed. The membrane-bound and free polysomes were incubated in vitro with [ 3 H]leucine and a pH 5 enzyme preparation, and peptide chains of albumin and ferritin were identified by immunoprecipitation. Albumin peptide chains were almost completely confined to the bound ribosomes, whereas ferritin peptide chains were found three times more frequently on free ribosomes than on bound ribosomes. Messenger RNA from each class of ribosome was translated in a wheat germ system, and the products were isolated by immunoprecipitation. Albumin mRNA was restricted almost exclusively to membrane-bound ribosomes. However, ferritin mRNA was equally abundant in the total mRNA of bound and free ribosomes. This finding contrasts with the low capacity of the bound ribosomes to synthesize ferritin in vitro, and suggests that the bound ribosomes of liver contain non-translated ferritin mRNA. Brief treatment of the rats with iron caused a sharp increase in the amount of nascent ferritin chains and l -ferritin mRNA in the free liver ribosomes, but failed to change the nascent chains or ferritin mRNA content of the bound polyribosome fraction. Only an apoferritin subunit of 19 000 daltons was formed by the free and membrane-bound ferritin mRNA. This has significance for the published observation of several sizes of protein subunits in ferritin isolated from tissue ferritins. These may represent modifications of the peptide chain after translation or artifacts of isolation.

Mechanism of d-amphetamine inhibition of protein synthesis

At 1 h after intraperitoneal administration of D-amphetamine sulphate (15 mg/kg), rat brain polyribosomes show disaggregation accompanied by reduced capacity for in vitro peptide chain elongation. The direct action of amphetamine on cell-fine protein-synthesizing systems was therefore explored. When brain or liver polyribosomes from untreated rats were incubated with pH 5 enzyme, peptide chain elongation was not inhibited by the addition 4 mM amphetamine to the medium. On the other hand, an initiation-dependent system consisting of rat liver of brain mRNA and wheat germ S-30 fraction showed inhibition of [3H]leucine incorporation by 50% when 4 mM amphetamine were added. The metabolites of amphetamine, p-hydroxyamphetamine and p-hydroxynorephedrine, had no inhibitory action in either system, but the potent neurotoxin p-chloroamphetamine was a more powerful inhibitor of initiation than amphetamine. By using [3H]amphetamine, it was shown that amphetamine binds to the 80-S ribosomes of the wheat germ system. This binding depended on the presence in the system of natural liver or brain mRNA or several synthetic mRNAs, but was not promoted by polyuridylic acid as the messenger. Significantly, polyuridylic acid-dependent polyphenylalanine synthesis by the wheat germ system was not inhibited by amphetamine or p-chloroamphetamine. Therefore, it was concluded that amphetamine inhibits protein synthesis by interfering with initiation through a step related to formation of the mRNA ribosome complex.

Evidence of formation of a complex between GTP and elongation factor two and the binding of the complex to a specific site on mammalian ribosomes.

Abstract The function of EF 2 (transferase II) and GTP in translocation of peptidyl-tRNA by mammalian ribosomes has been explored. The formation of a complex between GTP and this factor was demonstrated by chromatography of EF 2 on Sephadex G-75 equilibrated with [ 3 H]GTP. Recovery of the complex on Millipore filters showed that the [ 3 H]GTP can be readily exchanged for unlabeled GTP, GDP or GDPCP, but not for ATP. Formation of the complex was not influenced by sulfhydryl reagents or cycloheximide, both of which affect translocation. Ribosomal binding of GTP in the presence of EF 2 was examined with ribosomes bearing peptidyl-tRNA at either the acceptor or donor sites, the location of the peptidyl-tRNA being confirmed by reaction with puromycin. EF 2-dependent binding of GTP occurred only when the peptidyl-tRNA was at the donor site. Release of the terminal phosphate of GTP during binding was also dependent on exposure of this specific binding site on the donor ribosomes, since it did not occur with acceptor ribosomes, but translocation of peptidyl-tRNA was not an obligatory requirement for GTP hydrolysis. It is concluded that free EF 2 first forms a complex with GTP that binds to the larger ribosomal subunit at a specific site which becomes occluded when peptidyl-tRNA is at the acceptor position on the ribosome and that hydrolysis of GTP on binding is also dependent on exposure of this site.

Purification and properties of rat adrenal phenylethanolamine-N-methyl transferase☆

Abstract A procedure has been developed for the purification of phenylethanolamine- N methyl transferase (PNMT) (EC 2.1.1) from adrenal glands of rats. Ninety percent of the enzyme activity was in the 105,000 g supernatant fraction. After chromatography on Sephadex G-150 and DEAE-cellulose, the PNMT showed two molecular species but the same specific activity on polyacrylamide gel electrophoresis. The final product was enriched nearly 100-fold. The methylation reaction is linear with increasing enzyme concentration, and the enzyme pH optimum was 8.0. The enzyme is relatively stable at 40 °C, but activity is partially destroyed by incubation at 60 °C. Several substrates were tested: octopamine, norepinephrine, tyramine, phenylethanolamine. Greatest affinity was for octopamine. All these substrates and the methyl group donor, S -adenosylmethionine, were inhibitory at high concentrations. Preincubation of the enzyme with norepinephrine accelerated the initial rate of the methylation reaction, while preincubation with S -adenosylmethionine had no such effect. A specific antibody against this purified enzyme was prepared. This antibody inhibited the enzyme activity and also precipitated it. Various immunological studies using this antibody are described.

Influence of temperature and monovalent cations on reactivity of the donor and acceptor sites on mammalian ribosomes

Abstract Rat liver ribosomes were prepared with the peptidyl-tRNA either predominantly at the acceptor site (acceptor ribosomes) or at the donor site (donor ribosomes) and were washed free of elongation factors. When incubated at low concentrations of puromycin in the absence of monovalent salts, the donor ribosomes had little capacity to form a peptidyl derivative with puromycin at 0 °C but became reactive at 37 °C Acceptor ribosomes showed little reactivity towards puromycin at either temperature, thus indicating that they do not undergo significant translocation in media lacking monovalent cations. Incubation in the presence of increasing concentrations of NH4Cl raised the reactivity with puromycin of both donor and acceptor ribosomes to a maximum at 160 mM concentration. The acceptor ribosomes responded to NH4Cl proportionally more than the donor ribosomes which however still remained more active. This indicates that optimal levels of NH4Cl can cause the acceptor sites on some but not all acceptor ribosomes to become available to puromycin, presumably by translocation of peptidyl-tRNA from the acceptor site. The increase in reactivity of the fully translocated donor ribosomes shows that NH4Cl causes changes additional to translocation. Addition of increasing amounts of KCl to the medium also raised the capacity of donor and acceptor ribosomes to react with puromycin, but in this case reactivity of the acceptor ribosomes at 37 °C became indistinguishable from that of donor ribosomes in media containing more than 100 mM KCl, implying complete translocation above this salt concentration. The effect of temperature and salt concentration on binding of aminoacyl-tRNA to the acceptor site was also examined. Raising the concentrations of KCl from 50 to 150 mM increased binding of aminoacyl-tRNA to the acceptor ribosomes, but did not alter binding to donor ribosomes. Higher concentrations of KCl reduced the binding capacity of both types of ribosome. Binding of aminoacyl-tRNA to each ribosome type was temperature-dependent. It is concluded that temperature and the monovalent salt content of the medium play a part in non-enzymic translocation of peptidyl-tRNA from the acceptor to the donor site on mammalian ribosomes, and thus can change the capacity of a population containing acceptor ribosomes to react with puromycin and with aminoacyl-tRNA. In addition, temperature and salt concentration also affect reactivity of donor ribosomes with puromycin, thus indicating that factors other than translocation are involved in the promotion of puromycin peptide formation by monovalent salts. It is suggested that the use of authentic acceptor and donor ribosomes under defined conditions of salt and temperature can increase the information to be learned from reaction with puromycin.

Regulation of Protein Synthesis in Relation to Amino Acid Supply—A Review

Publisher Summary This chapter presents a review on regulation of protein synthesis in relation to amino acid supply. When amino acids or protein are administered to the fasting animal, the liver responds with a rapid but transient increase in protein synthesis, as evidenced by increased incorporation of labeled amino acids into liver protein and aggregation of ribosomes. This response seems to be determined by the free amino acid in the liver that is least abundant in relation to needs. Such a response occurs mainly in the synthesis of proteins retained within the cell, and is not necessarily shared by secreted proteins. Synthesis of serum albumin seems normally to be insensitive to amino acid supply. This suggests that the rate of albumin synthesis in the liver of the normal rat is regulated by the requirement to maintain the level of this protein in the serum, and only becomes sensitive to changes in amino acid supply when the rate of albumin production has fallen below a level adequate to maintain an optimum concentration in the serum.

Protein synthesis in a cell-free system prepared from human placenta. II. pH 5 enzyme inefficiency due to defects in tRNA charging with resulting loss of elongation factor 1.

Abstract Washed ribosomes and the pH 5 fraction of cell sap were isolated from human placenta and from rat liver and used for preparing a system for in vitro amino acid incorporation. In such a system, ribosomes prepared from human placenta and from rat liver showed the same capacity for amino acid incorporation, but the placental pH 5 fraction was one-third as effective as the pH 5 fraction of rat liver. In order to identify limiting factors in the placental pH 5 enzyme, its efficiency in attaching amino acids to tRNA and subsequently transferring amino acids from aminoacyl-tRNA to ribosomes was examined. Crude preparations of aminoacyl-tRNA synthetases were made from placenta and from rat liver and were compared for their capacity to charge rat liver tRNA with amino acids. The placental synthetase preparation was inferior in charging activity. In addition, tRNA isolated from full-term placenta had a reduced capacity for accepting amino acids in the presence of rat liver synthetase and especially in the presence of the placental synthetase preparation. Thus defective charging of tRNA contributes to the inefficiency of the placental pH 5 fraction in cell-free protein synthesis. The pH 5 fractions of human placenta and of rat liver were then compared for their ability to promote peptide chain elongation by transfer of amino acids from pre-formed aminoacyl-tRNA to nascent protein chains on rat liver ribosomes. The placental pH 5 fraction was much less effective in carrying out transfer, but could be fully corrected by adding elongation factor 1 to the preparation. The deficiency of elongation factor 1 in the placental pH 5 fraction was confirmed by comparing the ability of the pH 5 enzyme from placenta and from rat liver to stabilize aminoacyl-tRNA against spontaneous hydrolysis in vitro. Since only part of the elongation factor 1 of cell sap precipitates at pH 5, the amount of elongation factor 1 remaining in the supernatant fraction was assayed with stripped ribosomes. The supernatant fraction left after precipitation of the placental cell sap at pH 5 was found to have 70% of the elongation factor 1 content of the same fraction prepared from rat liver. Since the transfer capacity of the pH 5 enzyme fraction made from the placental cell sap was much more severely reduced, this suggested failure of elongation factor 1 to precipitate at pH 5 from placental cell sap. This was confirmed and shown to be due to the lack of charged tRNA in placental cell sap noted above. When rat liver tRNA was added to the placental cell sap before adjusting to pH 5, the precipitate obtained at pH 5 now contained more elongation factor 1, while the supernatant fraction had less elongation factor 1 activity. This effect was found to be specific for charged tRNA. A scheme is proposed whereby the amounts of certain cell sap factors may be coordinated in vivo in relation to the charging of tRNA.

Estimation of protein sulfhydryl groups with 5,5′-[35S] dithio-bis(2-nitrobenzoate)

Abstract 35 S-Labeled dithio-bis(2-nitrobenzoate) has been prepared and its usefulness for the measurement of protein sulfhydryl groups has been investigated. The technique is at least 20 times more sensitive than when dithio-bis(2-nitrobenzoate) is used colorimetrically. The adduct formed between the reagent and sulfhydryl groups is stable between pH 3–9 which should enable use of the reagent for the tagging of sulfhydryl-containing proteins in a wide variety of biochemical procedures. Removal of the adduct from a protein is achieved simply by treatment with a thiol reagent, with concomitant restoration of enzymic activity of the protein. The procedure has been successfully tested on alcohol dehydrogenase and mammalian ribosomes.

Control of RNA content of developing human placenta

Human placentae obtained early in pregnancy or at full term were examined for RNA content per cell, RNA polymerase types and activities, and chromatin template availability. The RNA:DNA ratio fell from 0.7 at 15 to 20 weeks to 0.4 at 40 weeks of pregnancy. Since RNase activities were similar at both times, the reduction in RNA content was attributed not to increased degradation, but to reduced synthesis. At both stages of pregnancy, about 55 to 60 per cent of the RNA polymerase activity in isolated placental nuclei was accounted for by RNA polymerase II, as judged by suppression of activity with alpha-amanitin and by separation of the extracted polymerases on DEAE-Sephadex. The relative roles of changes in polymerase activity and template availability were measured in nuclei from 20- and 40-week placentae. Nuclei showed 20 per cent greater polymerase activity in full-term than in early placentae, but the template availability of isolated chromatin for transcription by RNA polymerase II was 70 per cent less at full term. We conclude that the reduced amount of RNA per cell in the full-term placenta is due to reduced template availability that more than offsets the slight increase in polymerase activity.