Preadaptation of protein synthesis in wheat seedlings to high temperature

The optimum temperature of protein synthesis in wheat seedlings (Triticum aestivum L.), measured as ¹⁴C-leucine incorporation, depends on the growing temperature. Plants grown at reduced temperature (4 C) reach their optimum at 27.5 C, whereas plants kept at 36 C have the highest rate of protein synthesis at 35 C. The transition is gradual. The activation energy of protein synthesis for seedlings grown at medium or reduced temperature is lower (about 11 kcal/mole), than for plants grown at higher temperatures (15 keal/mole). The decline of the rate of protein synthesis beyond the temperature optimum is also affected by the growth temperature; only plants kept at 30 or 36 C show a sharp decrease with increasing slope; plants kept at 4, 10, and 20 C exhibit a linear and comparatively moderate decline.     

Impairment of glycoprotein secretion by phenobarbital in rat liver slices

The effects of phenobarbital on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Phenobarbital (2 mM) decreased [¹⁴C]-glucosamine and [¹⁴C]leucine incorporation into liver proteins and markedly inhibited their incorporation into medium (secretory) proteins. This inhibitory effect of phenobarbital was dose dependent and not reversible under the conditions of this study. In the presence of cycloheximide, an inhibitor of peptide synthesis, phenobarbital still inhibited the release of glycoproteins into the medium; however, the specific activity of liver glycoproteins was increased. The effects of phenobarbital on hepatic macromolecular secretion, independent of its effects on synthesis, were determined by prelabeling proteins in a liver slice system with either [¹⁴C]leucine of [¹⁴C]glucosamine. When phenobarbital was present, the secretion of these prelabeled proteins into the medium was impaired. 12 h after intraperitoneal injections of phenobarbital, glycoprotein secretion was inhibited from liver slices prepared from the pretreated rats. This inhibition of secretion occurred even though protein synthesis was stimulated and intracellular glycosylations unaffected. The results of this study indicate that phenobarbital impairs the secretion of glycoproteins by the liver.     

Further studies of the transport of protein to nerve endings

Mice were injected intracerebrally with [l-¹⁴C]leucine, and the specific activities of subcellular fractions of brain and effractions of isolated nerve endings were determined. There was a progressive increase in the specific activity of protein associated with isolated nerve endings after incorporation of [l-¹⁴C]leucine into whole brain protein had terminated. Although, the incorporation of [¹⁴C]leucine into soluble protein of whole brain did not differ significantly in mice which were 3 months or 1-year old, the subsequent increase in specific activity of soluble protein isolated from nerve endings was significantly greater in the younger animals; 6-month-old mice were intermediate. Therefore, changes in some aspect of the transport of protein to nerve endings is altered even after sexual maturity. Anaesthetization with pentobarbitone during incorporation of [¹⁴C]leucine into protein, and inhibition of protein synthesis with acetoxycycloheximide after incorporation of [¹⁴C]leucine was complete, did not interfere with the subsequent appearance of radioactive protein at the nerve ending. Evidence is presented for the transport, from a proximal site of synthesis, of protein associated with particulate components of the nerve ending, including synaptic vesicles.     

Protein Synthesis in Bromegrass (Bromus inermis Leyss) Cultured Cells during the Induction of Frost Tolerance by Abscisic Acid or Low Temperature

Bromus inermis Leyss cell cultures treated with 75 micromolar abscisic acid (ABA) at both 23 and 3°C developed more freezing resistance than cells cultured at 3°C. Protein synthesis in cells induced to become freezing tolerant by ABA and low temperature was monitored by [¹⁴C]leucine incorporation. Protein synthesis continued at 3°C, but net cell growth was stopped. Most of the major proteins detected at 23°C were synthesized at 3°C. However, some proteins were synthesized only at low temperatures, whereas others were inhibited. ABA showed similar effects on protein synthesis at both 23 and 3°C. Comparative electrophoretic analysis of [¹⁴C]leucine labeled protein detected the synthesis of 19, 21 and 47 kilodalton proteins in less than 8 hours after exposure to exogenous ABA. Proteins in the 20 kilodalton range were also synthesized at 3°C. In addition, a 31 kilodalton protein band showed increased expression in freezing resistant ABA treated cultures after 36 hours growth at both 3 and 23°C. Quantitative analysis of [¹⁴C]leucine labeled polypeptides in two-dimensional gels confirmed the increased expression of the 31 kilodalton protein. Two-dimensional analysis also resolved a 72 kilodalton protein enriched in ABA treated cultures and identified three proteins (24.5, 47, and 48 kilodaltons) induced by low temperature growth.     

Stimulatory effect of gamma-aminobutyric acid upon animo acid incorporation into protein by a ribosomal system from immature rat brain

Abstract—

• 1 GABAstimulated the incorporation of L-[U-¹⁴C]leucine, primarily into the particulate protein of a ribosomal system from immature rat brain, but not from immature rat liver.
• 2 The GABA effect required the presence of Na⁺ and occurred at GABA concentrations which are thought to be physiological (1–5 mM).
• 3 Of all other amino acids tested at tissue extract concentrations in the system, only glycine had a similar effect. No analogues of GABA tested had a significant stimulatory effect upon leucine incorporation into protein, with the exception of homocarnosine which was mildly stimulatory.
• 4 The effect of GABA upon the incorporation of L-[U-¹⁴C]leucine was examined in the presence of added amino acid substrates, both individually and as mixtures. Also, the incorporation of L-[U-¹⁴C]leucine was compared with incorporation of L-[U-¹⁴C]Iysine and L-[U-¹⁴C]phenylalanine. The results are discussed in terms of GABA interaction with activating, transfer and transport mechanisms of other amino acids, inhibition of proteinase activity, and the possibility that GABA is stimulating the synthesis or turnover of specific proteins in the brain ribosomal system.
• 5 The results illustrate the fact that studies of ‘protein synthesis’ in immature rat brain ribosomes, as measured by amino acid incorporation, will yield answers which depend heavily upon substrate conditions and upon the labelled amino acid used as the marker for protein synthesis or turnover.

     

Metabolic Requirement of Cucurbita pepo for Boron

Lateral roots of intact summer squash seedlings (Cucurbita pepo L.) were used to quantify the effects of boron deficiency on DNA synthesis, protein synthesis, and respiration. The temporal relationship between changes in these metabolic activities and the cessation of root elongation caused by boron deprivation was determined. Transferring 5-day-old squash seedlings to a hydroponic culture medium without boron for 6 hours resulted in a 62% reduction in net root elongation and a 30% decrease in the incorporation of [³H]thymidine into DNA by root tips (apical 5-millimeter segments). At this time, root tips from both boron-deficient and boron-sufficient plants exhibited nearly identical rates of incorporation of [¹⁴C]leucine into protein and respiration as measured by O₂ consumption. After an additional 6 hours of boron deprivation, root elongation had nearly ceased. Concomitantly, DNA synthesis in root apices was 66% less than in the boron-sufficient control plants and protein synthesis was reduced 43%. O₂ consumption remained the same for both treatments. The decline and eventual cessation of root elongation correlated temporally with the decrease in DNA synthesis, but preceded changes in protein synthesis and respiration. These results suggest that boron is required for continued DNA synthesis and cell division in root meristems.     

Phenotypical Temperature Adaptation of Protein Synthesis in Wheat Seedlings : QUALITATIVE ASPECTS. INVOLVEMENT OF AMINOACID:tRNA-LIGASES

Phenotypical temperature adaptation of protein synthesis in wheat (Triticum aestivum L.) seedlings is not affected by darkness (etiolation), by partial inhibition of protein biosynthesis (10(-3)m fluorophenylalanine), or by changing the amino acid precursor and the radioisotope ([(3)H]valine instead of [(14)C]leucine). The temperature coefficient (mu), as well as the optimum temperature of in vivo protein synthesis, increases with rising preadaptation temperature, as normally observed. Protein turnover studies revealed that only proteins with a short half-life time (t((1/2)) = 2 to 4 hours) are labeled to a measurable extent during the temperature adaptation experiments. A heat-labile protein has been detected and partially characterized by means of polyacrylamide gradient gels. Leucine:tRNA-ligase (EC 6.1 1.4) from heat-pretreated wheat seedlings exhibits enhanced thermal stability. In Arrhenius curves, the upper transition point shifts from 30 to 34 degrees C, depending on preadaptation temperature. Only the leucine:tRNA-ligase extracted from heat-adapted plants is stable when the enzyme extracts are subjected to a 34 degrees C heat treatment.     

ENHANCED CEREBRAL PROTEIN SYNTHESIS IN DEVELOPING HYPOTHYROID RATS: EVIDENCE FOR DELAYED MATURATION

(1) Neonatal hypothyroidism resulted in a 40% increase in the incorporation of [¹⁴C]leucine into protein by cerebral cortical slices from 25-day-old rats. The uptake of the [¹⁴C]-labelled amino acid into the acid-soluble free amino acid pool was similar in hypothyroid and control groups which excluded the possibility that transport differences contributed to the observed differences in incorporation. (2) The conversion of [¹⁴C]leucine in the free amino acid pool to other metabolites was substantially greater in the hypothyroid state compared to euthyroid controls. (3) The correction of the incorporation data for radioactivity associated with [¹⁴C]leucine in the precursor pool, provided an estimate of cerebral protein synthetic rate which was markedly higher in thyroid hormone-deficient-rats compared to litter mate controls. (4) The administration of L-thyroxine to hypothyroid animals for two successive days essentially returned the accelerated metabolism of the precursor pool leucine to normal but failed to ameliorate the increased incorporation into protein. (5) Incubations conducted in the presence of high exogenous leucine levels, to eliminate possible differences in intracellular free amino acid pool size, provided additional evidence for an increased rate of cerebral protein synthesis in 25-day-old hypothyroid rats compared to controls. (6) The results are compatible with a retardation in the normal developmental decline in the rate of cerebral protein synthesis associated with hypothyroidism.     

Biosynthesis of thyroglobulin. Incorporation of [1-14C]galactose, [1-14C]mannose and [4,5-3H2]leucine into soluble proteins by rat thyroids in vitro

1. Rat thyroid lobes were incubated for various periods of time in Krebs–Ringer bicarbonate containing [³H]leucine and either [1-¹⁴C]galactose or [1-¹⁴C]mannose. Radioactivity in soluble proteins was determined after their separation by sucrose-gradient centrifugation. 2. The time-course of incorporation of label from [¹⁴C]-mannose into soluble thyroid proteins was parallel to that observed for [³H]leucine. There was a lag of at least 30min. before either label appeared in non-iodinated thyroglobulin (protein 17–18s). During this time both labels were detected in two fractions known to contain subunit precursors of thyroglobulin (fractions 12s and 3–8s). Radioactivity from double-labelled fractions 12s and 3–8s was transferred to protein 17–18s during subsequent incubation in an unlabelled medium. 3. In contrast, most of the [¹⁴C]galactose was immediately incorporated into protein 17–18s. 4. During the first hour of incubation, puromycin almost completely inhibited the incorporation of label from [³H]leucine and [¹⁴C]mannose into all protein fractions, but had little effect on the incorporation of [¹⁴C]galactose into protein 17–18s. 5. These results indicate that mannose is incorporated into the carbohydrate groups of protein 17–18s at an earlier stage in its formation than galactose. It is suggested that the synthesis of the carbohydrate groups of ghyroglobulin begins soon after formation of the polypeptide components, more than 30min. before these are aggregated to protein 17–18s; carbohydrate synthesis then proceeds in a stepwise manner, galactose being incorporated at about the time of aggregation of subunits to protein 17–18s. Most, if not all, the carbohydrate is added to thyroglobulin before it is iodinated.     

STUDIES ON AMINO ACID LEVELS AND PROTEIN METABOLISM IN THE BRAINS OF GALACTOSE-INTOXICATED CHICKS1

Abstract— Levels of free amino acids, profiles of polyribosomes, and rates of protein synthesis and degradation were examined in the brains of chicks fed toxic levels of galactose. The content of a number of amino acids were altered; alanine and leucine were most strikingly depressed, whereas levels of aspartate were elevated. Polyribosomal profiles were unaltered. There appeared to be no detrimental effect on protein synthesis as judged by in vivo incorporation of L-[U-¹⁴C]leucine and L-[guanidino-¹⁴C]arginine. Likewise, the half-lives of proteins, measured by the loss of L-[guanidino-¹⁴C]arginine, were similar in experimental and control groups. In contrast, initial rates of incorporation of [³H]glucosamine into glycoproteins were enhanced. The effect was greatest in the microsomal fraction and typically 50 per cent greater than controls. Levels of free glucosamine and protein-bound hexosamine were essentially unaltered in the galactose-fed chicks.     

Effects of temperature on L-leucine transport in toadfish liver in vivo

The effect of body temperature in the 4–30°C range on L-leucine uptake by toadfish liver in vivo was examined by means of a single-injection pulse technique. The ratio of [¹⁴C]leucine to [³H]mannitol or [³H]inulin in blood leaving the liver was measured as a function of time after hepatic portal vein injection. Recoveries of the two isotopes in liver and [¹⁴C]leucine incorporation into protein were determined.The Q₁₀ value for influx was 3.8, that for efflux 2.8. At all temperatures, the leucine influx was 8–10-times higher than its incorporation into protein. The directly energy-linked reactions appear to be the main site of increased temperature sensitivity at low temperatures.     

In Vitro Protein Synthesis by Plastids of Phaseolus vulgaris. I. Localization of Activity in the Chloroplasts of a Chloroplast Containing Fraction from Developing Leaves

The incorporation of uniformly labeled leucine-¹⁴C into protein by a chloroplast containing fraction from developing primary leaves of bean is reported. Chloroplasts, obtained from week old plants grown in darkness, and then illuminated with white light for 12 hours, were shown to be the principal sites of incorporating activity. Incorporation may continue for 2 hours. Rates of up to 50 μμmole leucine incorporated per mg protein per hour are observed when a 1 hour assay period is used. Incorporation is only partially sensitive to ribonuclease.     

The hormonal control of protein N-glycosylation in the developing rabbit mammary gland and its effect upon transferrin synthesis and secretion

Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [³H]leucine or [³H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [³H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+ prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [³H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [³H]mannose and [¹⁴C]leucine confirmed this. The decreased incorporation of [³H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does.     

Incorporation of fucose and leucine into PNS myelin proteins in nerves undergoing early Wallerian degeneration

The simultaneous incorporation of [³H]fucose and [1-¹⁴C]leucine into normal rat sciatic nerve was examined using an in vitro incubation model. A linear rate of protein precursor uptake was found in purified myelin protein over 1/2–6 hr of incubation utilizing a supplemented medium containing amino acids. This model was then used to examine myelin protein synthesis in nerves undergoing degeneration at 1–4 days following a crush injury. Data showed a statistically significant decrease in the ratio of fucose to leucine at 2, 3, and 4 days of degeneration, which was the consequence of a significant increase in leucine uptake. These results, plus substantial protein recovery in axotomized nerves, are indicative of active synthesis of proteins that purify with myelin during early Wallerian degeneration.     

Expression of the mitochondrial genome in HeLa cells. XXI. Mitochondrial protein synthesis during the cell cycle

Chloramphenicol sensitive [³H]leucine incorporation into protein (due to mitochondrial protein synthesis) in synchronized HeLa cells has been found to continue throughout interphase, its rate per cell approximately doubling from the G₁ to the G₂ phase. This increase in the rate of [³H]leucine incorporation during the cycle does not seem to parallel closely the increase in cell mass. In fact, the observations made on cultures incubated at 34.5 °C, where the G₁ and S phases are better resolved than at 37 °C, indicate that the rate remains constant during the G₁ phase, and starts to accelerate with the onset of nuclear DNA synthesis. Correspondingly, on a per unit mass basis, there appears to be a slight decline in the rate of [³H]leucine incorporation into protein during the G₁ phase, which is compensated by an increase in the early S phase. No significant variations were observed in the mitochondrial leucine pool labeling during the cell cycle; therefore, the observed pattern of [³H]leucine incorporation into protein should reflect fairly accurately the behavior of mitochondrial protein synthesis. Evidence has been obtained indicating a depression in the rate of incorporation of [³H]leucine into protein in mitochondria of mitotic cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the products of mitochondrial protein synthesis has not revealed any differences in the size distribution of the proteins synthesized in the various portions of the cell cycle.     

A possible mechanism of inhibition of protein synthesis by dimethylnitrosamine

1. The incorporation of [¹⁴C]leucine into liver proteins of rats was measured in vivo at various times after treatment of the animals with dimethylnitrosamine and was correlated with the state of the liver ribosomal aggregates. Inhibition of incorporation ran parallel with breakdown of the aggregates. 2. Inhibition of leucine incorporation into protein and breakdown of ribosomal aggregates were not preceded by inhibition of incorporation of [¹⁴C]orotate into nuclear RNA of the liver. 3. Evidence was obtained of methylation of nuclear RNA in the livers of rats treated with [¹⁴C]dimethylnitrosamine. 4. Zonal centrifugation analysis of radioactive, nuclear, ribosomal and transfer RNA from livers of rats treated with [¹⁴C]dimethylnitrosamine revealed labelling of all centrifugal fractions to about the same extent. 5. It is suggested that methylation of messenger RNA might occur in the livers of dimethylnitrosamine-treated rats and the possible relation of this to inhibition of hepatic protein synthesis is discussed.     

Effect of 2-deoxy-d-Glucose on Aminoacids Metabolism in Rats’ Cerebral Cortex Slices

We studied the effect of different concentrations of 2-deoxy-d-glucose on the l-[U-¹⁴C]leucine, l-[1-¹⁴C]leucine and [1-¹⁴C]glycine metabolism in slices of cerebral cortex of 10-day-old rats. 2-deoxy-d-glucose since 0.5 mM concentration has inhibited significantly the protein synthesis from l-[U-¹⁴C]leucine and from [1-¹⁴C]glycine in relation to the medium containing only Krebs Ringer bicarbonate. Potassium 8.0 mM in incubation medium did not stimulate the protein synthesis compared to the medium containing 2.7 mM, and at 50 mM diminishes more than 2.5 times the protein synthesis compared to the other concentration. Only at the concentration of 5.0 mM, 2-deoxy-d-glucose inhibited the CO₂ production and lipid synthesis from l-[U-¹⁴C] leucine. This compound did not inhibit either CO₂ production, or lipid synthesis from [1-¹⁴C]glycine. Lactate at 10 mM and glucose 5.0 mM did not revert the inhibitory effect of 2-deoxy-d-glucose on the protein synthesis from l-[U-¹⁴C]leucine. 2-deoxy-d-glucose at 2.0 mM did not show any effect either on CO₂ production, or on lipid synthesis from l-[U-¹⁴C]lactate 10 mM and glucose 5.0 mM.     

THE EFFECT OF MORPHINE ADMINISTRATION ON THE INCORPORATION OF [14C]LEUCINE INTO PROTEIN IN CELL-FREE SYSTEMS FROM RAT BRAIN AND LIVER

—Ribosomes isolated from the brains of rats treated with morphine in vivo were less active in promoting the incorporation of [¹⁴C]leucine into protein than ribosomes isolated from untreated rats. This inhibitory phenomenon was studied in relation to dose of morphine, time after drug administration and the pharmacological responses of hypothermia and analgesia. The inhibition of [¹⁴C]leucine incorporation into brain proteins in vitro was transient after a single injection of morphine and dose-dependent, and related to the hypothermic response, but not prevented by keeping the rats at an ambient temperature which prevented hypothermia. The incorporation of [¹⁴C]leucine into protein by liver ribosomes was also inhibited in preparations from morphine treated rats.     

METHYL MERCURY INHIBITION OF SYNAPTOSOME AND BRAIN SLICE PROTEIN SYNTHESIS: IN VIVO AND IN VITRO STUDIES

Subacute methyl mercury (MeHg) intoxication was induced in adult rats following the daily intragastric administration of 1 mg MeHg/100 g body weight. Decreased [¹⁴C]leucine incorporation into cerebral and cerebellar slice protein was found. Weight loss occurred during the latent and neurotoxic phases but pair feeding did not reveal a significant defect in amino acid incorporation into slice protein. There was no decline in synaptosome protein synthesis in vitro during the latent phase but a significant decline in cerebellar and cerebral synaptosome synthesis was found during the neurotoxic phase. MeHg in vitro inhibited cerebral slice and synaptosome protein synthesis at half maximal concentrations of 7.5 and 12.5 μM respectively. Inhibition of synthesis in synaptosomes was non-competitive with K₁ of 4 × 10^?6M. MeHg had no effect on [¹⁴C]leucine or [¹⁴C]proline uptake into synaptosomes. There was no significant inhibition of synaptosome basal ATPase or Na + K ATPase at concentrations of MeHg (12 μM) giving half maximal inhibition of protein synthesis. No preferential inhibition of the chloramphenicol (55S) or cycloheximide sensitive components of synaptosome fraction protein synthesis was found, suggesting that the inhibition is common to both mitochondrial and extramitochondrial protein synthesizing systems. Addition of nucleotides and/or atractylate failed to influence protein synthesis and did not reverse the MeHg inhibition. Mannitol, as a replacement for the predominant cation species of the incubation medium, gave 40% inhibition of protein synthesis in the control but protected against further inhibition by MeHg.     

Effects of cycloheximide,d-threo-chloramphenicol,erythromycin and actinomycin D on De-novo synthesis of cytoplasmic and mitochondrial proteins in the cotyledons of germinating pea seeds

Summary Inhibitors of, and radioactive substrates for, protein synthesis were introduced into germinating pea (Pisum sativum L.) seeds, and protein synthesis was allowed to proceed in vivo. Subsequent analyses of subcellular fractions showed the following: Cycloheximide strongly inhibited the incorporation of [¹⁴C]leucine into both mitochondrial and cytoplasmic proteins. d-Threo-chloramphenicol and erythromycin did not affect cytoplasmic protein synthesis, but partially inhibited mitochondrial protein synthesis. These results suggest that most of the new mitochondrial proteins were originally synthesized in the cytoplasm. Actinomycin D did not appreciably affect the initial incorporation of [¹⁴C]leucine into either mitochondrial or cytoplasmic proteins, suggesting that information (mRNA) concerning the initially synthesized proteins may be present in the quiescent seeds. The lack of appreciable incorporation of [³H]thymidine into mitochondrial DNA supported our previons report that mitochondria may not be synthesized de novo in pea cotyledons.