Regulation of mammalian protein synthesis in vivo. Protein synthesis in rat liver and kidney after the administration of sublethal doses of cyclohyximide.

Protein synthesis in vivo was studied at various times after the administration of sublethal doses of cycloheximide to rats. Cycloheximide caused an inhibition, followed by a dose-and time-dependent stimulation, of incorportation of labelled precursor into proteins of the liver and kidney. The stimulation of protein synthesis at 24h was not due to a change of precursor pool or the specific radioactivity of the precursor used. During the stimulatory period, leucine incorporation into various cellular protein fractions varied; incorporation into total nuclear protein was the most affected.     

An investigation of the location and possible compartmentation of the precursor pool of phenylalanine for protein synthesis in human granulocytes.

Evidence is presented that in human granulocytes the immediate precursor pool of phenylalanine for protein synthesis in vitro is intracellular significant compartmentation.     

Identification of the precursor protein for the heparan sulfate proteoglycan of human colon carcinoma cells and its post-translational modifications

Human colon carcinoma cells synthesize a high-molecular-weight heparan sulfate proteoglycan which is localized at the cell surface. In this study we have performed a series of immunoprecipitation and pulse-chase experiments associated with various pharmacological agents that interfere with the synthesis and post-translational modification of the proteoglycan. We demonstrate that colon carcinoma cells synthesize the heparan sulfate proteoglycan from a 400-kDa precursor protein that is immunologically related to the Engelbreth-Holm-Swarm (EHS) tumor cell proteoglycan. The cells contain a large pool of precursor protein with a half-life of about 75 min. Most of the precursor protein receives heparan sulfate side chains and is then transported to the cell surface and released into the medium. A portion of the precursor pool, however, does not receive heparan sulfate chains but is secreted into the medium. The glycosylation and subsequent secretion of the 400-kDa precursor protein was inhibited by NH4Cl and even more by monensin, indicating that the transit of precursor from the rough endoplasmic reticulum to the cell surface occurred through the Golgi complex and acidic compartments. The existence of a sizable pool of precursor protein was confirmed by additional experiments using cycloheximide and xyloside. These experiments showed that the half-life of the precursor protein was also 75 min and that stimulation of heparan sulfate synthesis by xyloside was greatly enhanced (about 12-fold) after new protein core synthesis was blocked by cycloheximide. Although the structural models proposed for the EHS and colon carcinoma heparan sulfate proteoglycans differ, the observation that they are derived from a precursor protein with dimensional and immunological similarities suggests that they may be genetically related.     

Germination of wheat embryos and the transport of amino acids into a protein synthesis precursor pool

Wheat (Triticum aestivum L. var. Lew) embryonic axes take up externally supplied radioactive amino acid (from a solution greater than 2 millimolar) such that the specific radioactivity of the total internal amino acid rapidly reaches that of the external solution. Nevertheless, incorporation of radioactive amino acid into protein increases steadily as the concentration of external amino acid is increased, indicating that the amino acid that is precursor to protein synthesis is not in equilibrium with the total internal amino acid pool. When the external source of amino acid is removed, incorporation of radiolabeled amino acid into protein continues at a rate comparable to that of embryos maintained in the radioactive solution. In explanation of these data, it is suggested that there are two separate cytoplasmic pools of amino acids, one a protein synthesis precursor pool, and the second, an expandable pool into which exogenous radioactive amino acids are taken up. The protein synthesis pool is fed at a limited rate from the expandable pool and at a far greater rate from an endogenous source. As a consequence, the specific activity of the amino acid that is the precursor for protein synthesis is considerably below that of the total internal pool and is determined by the rate of movement into the protein synthesis pool from the expanded radioactive cytoplasmic pool.

The rate of movement of amino acids from the expandable pool into the protein synthesis pool increases approximately 5-fold during the initial 4.5 hours of embryo germination. When this change is considered in analyzing the relative rates of protein synthesis, there is probably no more than a 2-fold increase in protein synthetic capacity between embryos germinated for 1.5 and 4.5 hours. The leveling off of the change in transport capacity after 4.5 hours suggests that the earlier increase in the rate of this process may be a necessary step before the embryos can begin to accelerate their growth rate.

     

Effect of Loading Doses of l-Valine on Relative Contributions of Valine Derived from Protein Degradation and Plasma to the Precursor Pool for Protein Synthesis in Rat Brain

"Flooding" amino acid pools with high doses of labeled amino acids of low specific activity has been proposed to minimize the effects of recycling of amino acids derived from protein degradation on the specific activity of the amino acid precursor pool for protein synthesis. We have examined the influence of recycling on the precursor pool for protein synthesis under conditions in which plasma valine concentrations were normal (0.19 mM) and "flooded" (10-28 mM) by comparing the steady-state specific activity of the tRNA-bound valine with that of the plasma valine. Under normal and "flooding" conditions, the relative contributions of valine from protein degradation to the precursor pool were 63 and 26%, respectively; "flooding" with a plasma level of 28 mM raised the brain acid-soluble pool level to 3.1 mM but was no more effective in decreasing the relative contribution of valine from protein degradation to the precursor pool than "flooding" with a plasma level of 17 mM valine, which raised the brain acid-soluble level only to 2.3 mM. The results of these studies show that "flooding" amino acid pools does indeed reduce the effect of recycling on the precursor amino acid pool for protein synthesis, but it does not totally eliminate it.     

Brain Protein Synthesis in the Conscious Rat Using L-[35S]Methionine: Relationship of Methionine Specific Activity Between Plasma and Precursor Compartment and Evaluation of Methionine Metabolic Pathways

The method previously developed for the measurement of rates of methionine incorporation into brain proteins assumed that methionine derived from protein degradation did not recycle into the precursor pool for protein synthesis and that the metabolism of methionine via the transmethylation pathway was negligible. To evaluate the degree of recycling, we have compared, under steady-state conditions, the specific activity of L-[35S] methionine in the tRNA-bound pool to that of plasma. The relative contribution of methionine from protein degradation to the precursor pool was 26%. Under the same conditions, the relative rate of methionine flux into the transmethylation cycle was estimated to be 10% of the rate of methionine incorporation into brain proteins. These results indicate the following: (a) there is significant recycling of unlabeled methionine derived from protein degradation in brain; and (b) the metabolism of methionine is directed mainly towards protein synthesis. At normal plasma amino acid levels, methionine is the amino acid which, to date, presents the lowest degree of dilution in the precursor pool for protein synthesis. L-[35S]-Methionine, therefore, presents radiobiochemical properties required to measure, with minimal underestimation, rates of brain protein synthesis in vivo.     

Determination of Regional Rates of Cerebral Protein Synthesis Adjusted for Regional Differences in Recycling of Leucine Derived from Protein Degradation into the Precursor Pool in Conscious Adult Rats

The quantitative autoradiographic L-[1-14C]leucine method for the determination of regional rates of cerebral protein synthesis in vivo takes into account recycling of unlabeled leucine derived from protein degradation into the precursor pool for protein synthesis. We have evaluated the degree of recycling by measuring the ratio of the apparent steady-state leucine specific activity in the precursor amino acid pool (tRNA-bound leucine) to that in the arterial plasma. In the whole brain of the conscious rat this ratio (lambda WB) equals 0.58. The equivalent ratio for leucine in the acid-soluble pool in whole brain (psi WB) is 0.49. A first-degree polynomial equation for lambda WB as a function of psi WB was fitted from paired determinations. To determine the degree of recycling in local regions of the brain, we have measured in individual brain regions (i) psi i and calculated lambda i assuming that the fitted equation also applies to these localized regions. Our results indicate that the degree of recycling into the precursor pool does vary regionally; lambda i in the individual regions varies from 0.62 in the hypoglossal nucleus to 0.50 in the globus pallidus. Local rates of protein synthesis were then determined by the autoradiographic technique with regional corrections for recycling of unlabeled leucine. Rates of leucine incorporation into protein averaged 6.1 nmol/g of tissue/min in the brain as a whole, with the rates in gray matter about twice those in white matter.     

Variations in UDP-N-acetylglucosamine and UDP-N-acetylmuramyl-pentapeptide pools in Escherichia coli after inhibition of protein synthesis.

The pool levels of the nucleotide precursors of peptidoglycan were analyzed after inhibition of protein synthesis in various Escherichia coli strains. In all cases UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) cell pools increased upon treatment with chloramphenicol or tetracycline. Similar results were observed after the treatment of K-12 strains with valine. Since the intermediate nucleotide precursors did not accumulate after the arrest of protein synthesis and since a feedback mechanism was unlikely, the increases of the UDP-MurNAc-pentapeptide pool appeared as a consequence of that of the UDP-GlcNAc pool by the unrestricted functioning of the intermediate steps of the pathway. The highest increase (sixfold) of UDP-GlcNAc was observed with strain K-12 HfrH growing in minimal medium and treated with chloramphenicol. When a pair of isogenic Rel+ and Rel- strains were considered, both the UDP-GlcNAc and UDP-MurNAc-pentapeptide pools increased upon treatment with chloramphenicol or valine. However, the UDP-GlcNAc pool of the Rel+ strain was at a high natural level, which increased only moderately (20%) after the addition of valine. The increase of the UDP-GlcNAc pool after the various treatments could be due to an effect on some upstream step by an unknown mechanism. The possible correlations of the variations of the precursor pools with the rate of synthesis and extent of cross-linking of peptidoglycan were also considered.     

Homocysteine biosynthesis in green plants. Physiological importance of the transsulfuration pathway in Chlorella sorokiniana growing under steady state conditions with limiting sulfate

The physiological roles of the transsulfuration and direct sulfhydration pathways in Chlorella sorokiniana growing under steady state photoautotrophic conditions with limiting sulfate were studied by following the patterns of assimilation of 35SO4(2-) into sulfur amino acids. The labeling patterns expected of each pathway were defined by means of models based on the rates of net synthesis of the terminal pools of GSH, protein cysteine, and protein methionine. The labeling patterns observed are entirely consistent with the transsulfuration pathway and inconsistent with the direct sulfhydration pathway. By analysis of the amounts of radioactivity present in key intermediates at labeling times as short as 1 s, it was demonstrated that direct sulfhydration makes no detectable contribution to homocysteine biosynthesis, and if operative contributes no more than approximately 3% of the total homocysteine biosynthesized. From the combined determinations of the initial rates of labeling and net rates of synthesis of the various sulfur amino acids, a tentative working model is presented that summarizes our best current estimates of the major fluxes of sulfur in the experimental system. The labeling data further showed that soluble cysteine consists of at least two pools. One pool, termed "rapidly turning over" cysteine comprises less than 1% of the total soluble cysteine, and is the precursor of GSH, protein cysteine, and, almost certainly, cystathionine. The other pool, "slowly turning over" cysteine, appears to be in equilibrium with "rapidly turning over" cysteine, but not to be further metabolized.     

The role of different pathways of mevalonate synthesis in the regulation of sterol and bile acid synthesis in the mammalian liver

The role of various pathways of synthesis are considered for mevalonic acid, the first specific precursor of sterols, in the production of cholesterol and bile acids in the mammalian liver. It is emphasized that the mevalonate synthesis with participation of acetyl-CoA-carboxylase and hydroxymethylglutaryl-CoA-reductase not bound with the endoplasmic reticulum membranes results in formation of the pool of mevalonic acid and other precursors necessary mainly for the organism supply with bile acids under conditions of cholesterol synthesis inhibition.     

Determination of the Rates of Protein Synthesis and Degradation in Lemna minor

Attempts to measure the rates of synthesis and degradation of protein in plant tissues with isotopes are complicated by the presence of at least two pools of amino acids, only one of which contributes to the synthesis of protein. Direct measurement of the protein precursor pool is thus difficult. This paper shows that one solution to this problem is to assume that the amino-acyl transfer RNA is the strict precursor of protein amino acid. By using labeled methionine, the variation with time of the specific radioactivities of methionine bound to RNA and protein have been examined under two different growth conditions in Lemna minor. From these data rates of flux of methionine into and out of protein may be easily determined.     

Does protein synthesis decline in lithium-treated sea urchin embryos because RNA synthesis is inhibited?

Vegetalization of sea urchin embryos by Li⁺ is characterized by rates of protein synthesis which are normal during cleavage, and decline after hatching. This paper tests the hypothesis that Li⁺ interferes with RNA synthesis during cleavage, resulting in the decline in protein synthesis at hatching when newly synthesized mRNA becomes critical for further normal development. Treatment with Li⁺ does cause a decline in the incorporation of [³H]guanosine into RNA. However, this decline could be accounted for by reduced uptake of the labeled precursor with a concomitant reduction in precursor pool specific activity. Therefore, reduced protein synthesis after hatching in Li⁺-treated embryos cannot be accounted for by a comparable reduction in RNA synthesis.     

Accumulation of free amino acids in growing Xenopus laevis oocytes

The sizes of amino acid pools in growing Xenopus laevis oocytes have been measured. The total free amino acid content per oocyte increases nearly 25-fold during oocyte growth. Together, glutamic acid and aspartic acid account for approximately 59-75% of the total amino acid pool in Xenopus oocytes. On the other hand, methionine and cysteine are the least abundant of the amino acids detected, each accounting for less than 0.7% of the total pool in developing oocytes. It is argued that the acid-extractable amino acid pool represents the precursor pool used in protein synthesis.     

Tunicamycin inhibits proteoglycan synthesis in rat ovarian granulosa cells in culture

The effects of tunicamycin, an inhibitor of N-linked oligosaccharide biosynthesis, on the synthesis and turnover of proteoglycans were investigated in rat ovarian granulosa cell cultures. The synthesis of proteoglycans was inhibited (40% of the control at 1.6 micrograms/ml tunicamycin) disproportionately to that of general protein synthesis measured by [3H]serine incorporation (80% of control). Proteoglycans synthesized in the presence of tunicamycin lacked N-linked oligosaccharides but contained apparently normal O-linked oligosaccharides. The dermatan sulfate and heparan sulfate chains of the proteoglycans had the same hydrodynamic size as control when analyzed by Sepharose 6B chromatography. However, the disulfated disaccharide content of the dermatan sulfate chains was reduced by tunicamycin in a dose-dependent manner, implying that the N-linked oligosaccharides may be involved in the function of a sulfotransferase which is responsible for sulfation of the iduronic acid residues. When [35S]sulfate and [3H]glucosamine were used as labeling precursors, the ratio of 35S/3H in chondroitin 4-sulfate was reduced to approximately 50% of the control by tunicamycin, indicating that the drug reduced the supply of endogenous sugar to the UDP-N-acetylhexosamine pool. Neither transport of proteoglycans from Golgi to the cell surface nor their turnover from the cell surface (release into the medium, or internalization and subsequent intracellular degradation) was affected by the drug. Addition of mannose 6-phosphate to the culture medium did not alter the proteoglycan turnover. When granulosa cells were treated with cycloheximide, completion of proteoglycan diminished with a t1/2 of approximately 12 min, indicating the time required for depleting the core protein precursor pool. The glycosaminoglycan synthesizing capacity measured by the addition of p-nitrophenyl-beta-xyloside, however, lasted longer (t1/2 of approximately 40 min). Tunicamycin decreased the core protein precursor pool size in parallel to decreased proteoglycan synthesis, both of which were significantly greater than the inhibition of general protein synthesis. This suggests two possibilities: tunicamycin specifically inhibited the synthesis of proteoglycan core protein, or more likely a proportion of the synthesized core protein precursor (approximately 50%) did not become accessible for post-translational modifications, and was possibly routed for premature degradation.     

Kinetic analysis of amino acid pools and protein synthesis in amphibian oocytes and embryos.

Rates of protein synthesis have been measured in Rana pipiens oocytes and embryos and in Xenopus oocytes from the incorporation kinetics of two different concentrations of amino acid. This method does not require an independent measurement of the amino acid pools, since the pool size can be calculated directly from incorporation data. The effects of the concentration and diffusion of injected amino acid on the calculated values for amino acid pool size and flow rate are discussed. When the endogenous amino acid pool is appreciably expanded by the injected amino acid, the total amino acid pool in the oocytes or embryos may be considered as the precursor pool for protein synthesis. Under these circumstances, compartmentation of amino acids does not affect the results, except when lysine is used as tracer. The rates of protein synthesis in ovarian oocytes of Rana pipiens and Xenopus laevis are 18 and 50–54 ng/hr, respectively. In Rana pipiens, the rate increases 70% during maturation and another 50% before the two-cell stage. Finally, the rate approximately doubles between the two-cell and blastula stages.     

Amino acid analogues: uptake, pool formation and incorporation of phenylalanine and two halogenated derivatives in cultured mammalian cells.

HeLa cells take up Phe and two of its ring halogenated derivatives (pFPhe and pClPhe) with rpaidity, concentrating them against the external medium both at 4 and 37 degrees C. The majority of amino acid (greater than 90%) is accumulated without energy expenditures at 4 degrees C, and can be quickly discharged by normal cell washing procedures in saline. At 37 degrees C the freely-diffusible (FDP) pool is accompanied by another which develops more slowly and cannot diffuse out freely during washings with saline but is extractable with trichloracetic acid (the slowly-diffusible pool, SDP, or more conventionally, the acid-soluble pool). Both of the analogues produced larger pools of the latter type than Phe itself from external concentrations ranging from 10(-5) to 10(-3) M. The incorporation of pFPhe into proteins over these same concentrations ranged from 30 to 90--95% of Phe incorporation, whereas pClPhe showed negligible incorporation. From these and similar analyses it can be concluded that amino acid pools form largely independently of protein synthesis, but bear a close relationship with the external amino acid concentration. The fraction of total uptake into cellular pools entering the SDP was relatively constant over a wide range of external concentrations. pFPhe incorporation into cellular proteins produced the same labelling distribution of Phe. It appears to ener all proteins, the vast majority of which have similar half-lives and turnover rates to Phe proteins. In competition, little or no interference was experienced between the analogue and Phe in uptake and pool formation until excessive amounts of one or the other were present (50--100x). By contrast, incorporation of pFPhe into protein was markedly reduced by the presence of Phe. However, the development of normal or large pools of pFPhe or Phe in cells prior to 3H-Phe incorporation did not affect the linear incorporation pattern of the radioisotope into protein. The relationship of pools to protein synthesis is discussed, and it is concluded that, although the SDP could contain potential precursor molecules for protein synthesis, it does not usually act as the direct supplier of amino acid for protein synthesis. Alternative explanations for precursor supply are discussed.     

Inactive and protein precursor pools of amino acids in the soybean hypocotyl

There are at least 2 amino pools for leucine and for valine in the soybean hypocotyl, a small protein precursor pool and a large inactive pool. The precursor pool decreased in size during incubation of excised hypocotyls presumably because the cotyledonary sources of amino acids had been removed. The precursor pool was subject to expansion by supplying the amino acid externally at high concentrations. After the transfer of tissue to unsupplemented media, the expanded pool was rapidly depleted.     

Kinetics of the appearance of nuclear estrogen binding sites in chicken liver

A single injection of estradiol to immature male chickens results in a very rapid increase in the number of both soluble and tightly bound nuclear estrogen binding sites. After an intermediate levelling off, a second longer lasting increase in binding sites is observed. Whereas the first rapid increase seems to be independent of RNA and protein synthesis, the second one obviously requires intact RNA and protein synthesis. From these results we conclude that there is a pool of cytoplasmic estrogen binding sites, which supplies the precursor for the nuclear binding sites. After this pool is exhausted, it is replenished by new synthesis of binding sites. However, although several laboratories have attempted to demonstrate directly the existence of such a cytoplasmic estrogen receptor in the chicken liver, all have so far been unsuccessful.     

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [¹³C₆]lysine or [²H₂]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.