Fibronectin synthesis during oogenesis and early development of the amphibian Pleurodeles waltlii

Fibronectin (FN) synthesis during oogenesis and early embryogenesis of the amphibian Pleurodeles waltlii was investigated. The isotopically labelled amino acids [3H]leucine and [35S]methionine were incubated with oocytes or microinjected into embryos. Newly synthesized FN was analysed by polyacrylamide gel electrophoresis, using the high resolution two-dimensional gel system described by O'Farrell. With this method and fluorography we demonstrate that FN synthesis begins during oogenesis. De novo synthesized FN appears during cleavage and gastrulation. Using actinomycin D we show the presence of maternal messenger RNA coding for FN. It is translated during the cleavage and gastrulation stages.     

Alanine and glutamine synthesis and release from skeletal muscle. II. The precursor role of amino acids in alanine and glutamine synthesis.

The synthesis and release of alanine and glutamine have been studied in the intact rat epitrochlaris skeletal muscle preparation. Aspartate, cysteine, leucine, valine, methionine, isoleucine, serine, theronine, and glycine increased significantly the formation and release of alanine from muscle. Cysteine, leucine, valine, methionine, isoleucine, tyrosine, lysine, and phenylalanine increased the rate of glutamine synthesis. Only ornithine, arginine, and tryptophan were without effect on the synthesis of either alanine or glutamine. Half-maximal stimulation of alanine and glutamine formation by added amino acids was observed with concentrations ranging between 0.5 and 1.0 mM. Increases in alanine and glutamine formation were not accompanied by changes in pyruvate production or glucose uptake. The progressive decline in alanine and glutamine synthesis noted on prolonged incubation was prevented by the addition of amino acids to the incubation medium. Stimulation of alanine synthesis by added amino acids was unaffected by inhibition of glycolysis with iodoacetate. Inhibition of alanine aminotransferase with aminooxyacetate significantly decreased alanine formation. Pyruvate and ammonium chloride did not increase further the rate of either alanine or glutamine formation above that produced by added amino acids. These data indicate that most amino acids are precursors for alanine and glutamine synthesis in skeletal muscle. A general mechanism is presented for the de novo formation of alanine from amino acids in skeletal muscle, and the importance of proteolysis for the supply of amino acid precursors for alanine and glutamine synthesis is discussed.     

Differential synthesis of polypeptides during morphogenesis of Mucor.

The extent of differential gene expression during morphogenesis of Mucor racemosus was investigated by two-dimensional polyacrylamide gel electrophoresis of neutral and acidic polypeptides. Cellular proteins were labeled with [35S]methionine in cells growing in either the yeast or hyphal form, or in yeast cells undergoing the transition of hyphae. The results showed that of the 400 to 500 polypeptides resolved by electrophoresis, relatively few were specific to one or the other morphological form. The major change in the patterns of proteins synthesized during morphogenesis was a change in rates of synthesis of individual polypeptides. Experiments in which morphogenesis was affected under aerobic or anaerobic conditions showed that the majority of changes in the protein patterns were associated with morphogenesis and were not a specific response to O2.     

Antibodies against the COOH-terminal undecapeptide of subunit II, but not those against the NH2-terminal decapeptide, immunoprecipitate the whole human cytochrome c oxidase complex

Antibodies against synthetic peptides derived from the DNA sequence of human cytochrome c oxidase subunit II (COII) have been tested for their capacity to immunoprecipitate the whole enzyme complex. Antibodies against the COOH-terminal undecapeptide of COII (anti-COII-C), when incubated with a Triton X-100 mitochondrial lysate from HeLa cells pulse-labeled with [35S]methionine under conditions selective for mitochondrial protein synthesis and chased for 18 h in unlabeled medium, precipitated the pulse-labeled three largest subunits (mitochondrially synthesized) of cytochrome c oxidase in proportions close to equimolarity. Antibodies against the NH2-terminal decapeptide of COII (anti-COII-N), although equally reactive as the anti-COII-C antibodies with the sodium dodecyl sulfate-solubilized COII, did not precipitate any of the three labeled subunits from the Triton X-100 mitochondrial lysate. In other experiments, all the 13 subunits which have been identified in the mammalian cytochrome c oxidase were immunoprecipitated from a Triton X-100 mitochondrial lysate of cells long-term labeled with [35S]methionine by anti-COII-C antibodies, but not by anti-COII-N antibodies. By contrast, in immunoblots of total mitochondrial proteins dissociated with sodium dodecyl sulfate, the anti-COII-C antibodies reacted specifically only with COII. These results strongly suggest that, in the native cytochrome c oxidase complex, the epitope recognized by the anti-COII-C antibodies is in the COII subunit and that, therefore, in such complex, the COOH-terminal peptide of COII is exposed to antibodies, whereas the NH2-terminal peptide is not accessible.     

Translation of immobilized genetic information by yeast cell-free protein synthesizing system

A cell-free protein-synthesizing system for the purpose of specific genetic information translation was constructed: ribosome, t-RNA, and enzymes extracted from yeast cells were combined with an immobilized mRNA. The soluble fraction mixed with energy sources and amino acids was incubated with the immobilized mRNA such as poly(U), yeast mRNA, and myeloma mRNA to incorporate [(3)H]phenylalanine into polypeptides of de novo synthesis. By supplying amino acids to these protein-synthesizing systems, amino acid incorporation was ascertained. Lower efficiency of the incorporation in the immobilized system than that of the homogeneous system was mainly attributed to the heterogeneous reaction where the mass-transfer process is diffusion limited. Results obtained show the possibility of a system for specific translation of a desired genetic code.     

Developmental regulation of protein synthesis during Polysphondylium pallidum cyst germination: A two-dimensional gel electrophoresis study

Microcyst germination in Polysphondylium pallidum can be used as a model for studying gene expression because temporally regulated modulations in protein synthesis occur in this developmental pathway. Germinating cysts were labeled with [³⁵S]methionine for half-hourly periods during the synchronous germination sequence, and the proteins labeled in each period were resolved by two-dimensional polyacrylamide gel electrophoresis. Three major classes of proteins observed were distinguished by the time of onset and duration of their synthesis: (a) proteins made throughout germination; (b) proteins synthesized only during a portion of the germination pathway; and (c) polypeptides whose synthesis started at 1 or 1.5 h and then continued throughout germination.     

Protein synthesis in imbibing wheat embryos.

Polypeptides synthesized by imbibing wheat embryos have been compared with those made by cell-free extracts programmed with bulk poly(A)-rich RNA from dry wheat embryos. Newly synthesized polypeptides, labeled with [35S]methionine, were resolved by one-dimensional and two-dimensional electrophoresis and then records of the separations were prepared by fluorography. When programmed by bulk poly(A)-rich RNA from dry wheat embryos, a nuclease-treated rabbit reticulocyte lysate synthesizes an array of polypeptides which is broadly similar to that formed when a wheat germ extract is programmed with the same RNA. Polypeptides made in both homologous and heterologous cell-free systems, under the direction of bulk poly(A)-rich RNA from dry wheat embryos, are broadly similar to those formed during early (0--40 min) imbibition of dry wheat embryos. As imbibition progresses beyond 40 min, there are profound changes in the one-dimensional and two-dimensional electrophoretic distributions of newly made polypeptides present in the 23 000 x g supernatant fraction of cell-free homogenates; characteristically, low-molecular-weight and basic polypeptides comprise a diminishing proportion of the total polypeptides as imbibition progresses beyond 40 min. Ribosomal proteins are conspicuous among the proteins formed during early imbibition and especially prominent among the products formed when homologous cell-free polypeptide synthesis is programmed by bulk poly(A)-rich RNA from dry wheat embryos.     

Control of protein synthesis during blastocyst formation in the mouse.

In order to evaluate the dependence of the embryo on new mRNA synthesis during the period leading to blastulation, quantitative and qualitative aspects of protein synthesis in developing mouse morulae were investigated using α-amanitin, an inhibitor of RNA polymerase II. Only 1 of 423 early morulae cultured for 27 hr in the presence of 11 μg/ml α-amanitin cavitated, although most progressed as far as fully compacted morulae. About two-thirds of the untreated embryos cavitated during the same period. Incorporation of [³⁵S]methionine into protein was measured at 3- or 4-hr intervals over a 24-hr period and showed a two- to fivefold increase in control embryos. This increase was blocked in the α-amanitin-treated group although initial levels of incorporation were maintained. Total uptake of the amino acid appeared to be unaffected by the inhibitor. RNA synthesis, as measured by [³H]uridine incorporation over the same period, was reduced by between 5 and 52%, and the preblastulation surge in RNA synthesis was also blocked by α-amanitin. Two-dimensional polyacrylamide gel electrophoresis of labeled polypeptides synthesized by the embryos after 24-hr incubation in the presence or absence of the inhibitor revealed three distinct classes of polypeptide. The majority of polypeptides continued to be synthesized in the presence of α-amanitin whereas a small number of polypeptides, the synthesis of which would normally have increased during the development of the morula to the blastocyst, were prevented from doing so. A few polypeptides which normally cease to be synthesized over this period continued to be synthesized in the presence of α-amanitin. It is concluded that, while most of the proteins detectable at the morula stage are synthesized on mRNA templates of relatively long translational life, the general surge in protein synthesis, including the increased synthesis of a few species of polypeptide, are dependent on continuous translational activity.     

Synthesis of an elastin component of molecular weight about 70,000 by polysomes from chick embryo aortas

Polysomes were isolated from aortas of 17-day-old chick embryos, and the synthesis of the nascent polypeptide chains was completed in vitro. When a mixture of a labeled amino acids found in elastin was used, the major radioactive product obtained was of molecular weight about 70,000 and was similar to elastin by several criteria. The 70,000 molecular weight product was extractable in propanol-butanol, it was not labeled with [³⁵S]methionine, and it was precipitated by antibodies against elastin. Polypeptides larger than 70,000 molecular weight were also synthesized but these larger polypeptides incorporated relatively small amounts of [¹⁴C]valine, and they appeared to represent proα chains of procollagen. The results suggest that the major gene product for elastin has a molecular weight of about 70,000.     

De novo synthesis of uracil nucleotides in mouse liver and intestine studied using [15N]alanine

The amount of newly synthesized uracil nucleotides in mouse liver and intestine was determined by analysis of 15N incorporation into the uracil nucleotide pool of these tissues after intraperitoneal infusion of 15N-labelled amino acids. The appearance of newly synthesized uracil nucleotides was linear with time, and essentially independent of the rate of infusion of L-[15N]alanine. Varying the amino acid used in the infusion could affect the enrichment in the uracil ring nitrogens, but had no significant effect on the calculated amount of de novo synthesis. These results demonstrate the utility of this method in measuring de novo uracil nucleotide synthesis in mouse liver and intestine in vivo. The method should be a valuable tool in the effort to understand the regulation and pharmacological manipulation of de novo uracil nucleotide synthesis.     

The pathway of triacylglycerol synthesis through phosphatidylcholine in Arabidopsis produces a bottleneck for the accumulation of unusual fatty acids in transgenic seeds

Engineering of oilseed plants to accumulate unusual fatty acids (FAs) in seed triacylglycerol (TAG) requires not only the biosynthetic enzymes for unusual FAs but also efficient utilization of the unusual FAs by the host-plant TAG biosynthetic pathways. Competing pathways of diacylglycerol (DAG) and subsequent TAG synthesis ultimately affect TAG FA composition. The membrane lipid phosphatidylcholine (PC) is the substrate for many FA-modifying enzymes (desaturases, hydroxylases, etc.) and DAG can be derived from PC for TAG synthesis. The relative proportion of PC-derived DAG versus de novo synthesized DAG utilized for TAG synthesis, and the ability of each pathway to utilize unusual FA substrates, are unknown for most oilseed plants, including Arabidopsis thaliana. Through metabolic labeling experiments we demonstrate that the relative flux of de novo DAG into the PC-derived DAG pathway versus direct conversion to TAG is ～14/1 in wild-type Arabidopsis. Expression of the Ricinus communis FA hydroxylase reduced the flux of de novo DAG into PC by ～70%. Synthesis of TAG directly from de novo DAG did not increase, resulting in lower total synthesis of labeled lipids. Hydroxy-FA containing de novo DAG was rapidly synthesized, but it was not efficiently accumulated or converted to PC and TAG, and appeared to be in a futile cycle of synthesis and degradation. However, FA hydroxylation on PC and conversion to DAG allowed some hydroxy-FA to accumulate in sn-2 TAG. Therefore, the flux of DAG through PC represents a major bottleneck for the accumulation of unusual FAs in TAG of transgenic Arabidopsis seeds.     

A highly flexible tRNA acylation method for non-natural polypeptide synthesis

Here we describe a de novo tRNA acylation system, the flexizyme (Fx) system, for the preparation of acyl tRNAs with nearly unlimited selection of amino and hydroxy acids and tRNAs. The combination of the Fx system with an appropriate cell-free translation system allows us to readily perform mRNA-encoded synthesis of proteins and short polypeptides involving multiple non-natural amino acids.     

Hypoxic stress inhibits the appearance of wound-response proteins in potato tubers

Potato (Solanum tuberosum L.) tubers respond to environmental stresses by alterations of macromolecular synthesis. In an aerobic environment tubers respond rapidly to wounding by synthesizing a set of proteins, the most prominent of which display apparent molecular weights of 78, 48, 38, and 31 kilodaltons. These proteins become intensely labeled by [³⁵S]methionine within 2 hours of wounding. The 78 kilodalton polypeptide has been identified by immunoprecipitation as phenylalanine ammonia-lyase. By contrast, tubers incubated in hypoxic conditions for a period as short as 1.5 hours exhibit significantly reduced incorporation of amino acids such that newly synthesized polypeptides are not detected. However, a second set of proteins is synthesized by wounded tubers after prolonged incubation in a hypoxic environment. One peptide of this set is precipitated by an antibody directed against aldolase; several of these proteins may be enzymes of glycolysis necessary for anaerobic metabolism. The results indicate that there is a complex regulatory mechanism which allows mature potato tubers to respond to changes in the environment.     

Protein Synthesis by Bradyrhizobium japonicum Bacteroids Declines as a Function of Nodule Age

Isolated bacteroids of Bradyrhizobium japonicum accumulated exogenously supplied [(sup35)S]methionine or [(sup3)H]leucine and incorporated them into cytosolic proteins. The accumulation of these labeled amino acids was inhibited by azide. Only 3 to 6% of these accumulated amino acids were incorporated into protein. Protein synthesis was not stimulated by incubation of bacteroids in the presence of potassium salts, malate, or amino acids, but azide, chloramphenicol, and acridine did inhibit the process. No prominent differences were observed in autoradiograms after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of (sup35)S-labeled bacteroid proteins as a function of nodule age. The rates of protein synthesis and protein turnover declined during nodule development. Protein synthesis declined about 60% between 14 and 20 days after planting, which is the period of a rapid increase in acetylene reduction activity. This correlation suggests a metabolic mechanism by which significant amounts of cellular energy are diverted to the nitrogen fixation process.     

Biogenesis of mitochondria. Phospholipid synthesis in vitro by yeast mitochondrial and microsomal fractions

The ability in vitro of yeast mitochondrial and microsomal fractions to synthesize lipid de novo was measured. The major phospholipids synthesized from sn-[2-(3)H]glycerol 3-phosphate by the two microsomal fractions were phosphatidylserine, phosphatidylinositol and phosphatidic acid. The mitochondrial fraction, which had a higher specific activity for total glycerolipid synthesis, synthesized phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and phosphatidic acid, together with smaller amounts of neutral lipids and diphosphatidylglycerol. Phosphatidylcholine synthesis from both S-adenosyl[Me-(14)C]methionine and CDP-[Me-(14)C]choline appeared to be localized in the microsomal fraction.     

Optimization of protein synthesis in isolated higher plant chloroplasts. Identification of paused translation intermediates

Protein synthesis in isolated, intact pea chloroplasts was optimized and compared to translation within chloroplasts in vivo. Many polypeptides labeled with [35S]methionine in isolated intact chloroplasts did not comigrate with polypeptides which were labeled within chloroplasts in vivo. Antibodies to the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (EC 4.1.1.39) immunoprecipitated [35S]-labeled large subunit plus several lower-molecular-mass translation products of isolated chloroplasts. The lower-molecular-mass soluble translation products synthesized in pulse-labeled chloroplasts were converted into full-length large-subunit polypeptides during a subsequent chase period. This result suggests that many of the polypeptides observed in pulse-labeled chloroplasts are incomplete translation products which are the result of ribosome pausing at discrete points along chloroplast mRNAs. The pulse-chase technique was used to follow synthesis of the 34.5-kDa precursor of the psb A gene product and its processing to the mature 32-kDa polypeptide in isolated chloroplasts. Chloroplast translation profiles obtained using the pulse-chase assay were very similar to translation profiles obtained in vivo thus extending the utility of protein synthesis in isolated chloroplasts.     

Influence of Auxin and Gibberellin on in Vivo Protein Synthesis during Early Pea Fruit Growth

Developing pea fruits (Pisum sativum L.) offer a unique opportunity to study growth and development in a tissue that is responsive to both gibberellins (GAs) and auxin (4-chloroindole-3-acetic acid[4-CI-IAA]). To begin a molecular analysis of the interaction of GAs and auxins in pea fruit development, in vivo labeling with [35S]methionine coupled with two-dimensional gel electrophoresis were used to characterize de novo synthesis of proteins during gibberellic acid (GA3)-, 4-CI-indoleacetic acid-, and seed-induced pea pericarp growth. The most significant and reproducible polypeptide changes were observed between molecular weights of 20 and 60. Comparing about 250 de novo synthesized proteins revealed that seed removal changed the pattern substantially. We identified one class of polypeptides that was uniquely seed induced and five classes that were affected by hormone treatment. The latter included 4-CI-IAA-induced, GA3-induced, GA3- and 4-CI-IAA-induced, 4-CI-IAA-repressed, and GA3- and 4-CI-IAA-repressed polypeptides. Similar patterns of protein expression were associated with both hormone treatments; however, changes unique to GA3 or 4-CI-IAA treatment also indicate that the effects of GA3 and 4-CI-IAA on this process are not equivalent. In general, application of 4-CI-IAA plus GA3 replaced the seed effects on pericarp protein synthesis, supporting our hypothesis that both hormones are involved in pea pericarp development.     

Fish oil fatty acids impair VLDL assembly and/or secretion by cultured rat hepatocytes

We determined the effect of the two major fish oil fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on VLDL assembly and secretion by cultured rat hepatocytes. The incorporation of [3H]glycerol into total triglyceride (cell plus media) was stimulated eight-fold when hepatocytes were incubated for 2 h with 1 mM EPA, DHA, or oleic acid (OA), suggesting that fish oil fatty acids stimulate hepatic triglyceride synthesis to an extent similar to OA. In contrast, mass quantitation of secreted triglyceride showed impaired triglyceride secretion with EPA and DHA compared to OA. During a 42-h time course, cells stimulated with EPA and DHA progressively accumulated triglyceride compared to cells stimulated with OA. To determine whether fish oil fatty acids impair very low density lipoprotein (VLDL) secretion, cells were labeled with [35S]methionine and the secretion of de novo synthesized apoB was measured. Compared to OA, EPA and DHA significantly impaired the secretion of both molecular weight forms of apoB. The cellular content of apoB was not altered by any of the fatty acids. The concordant decrease in the secretion of both triglyceride and apoB suggests that fish oil fatty acids impair VLDL assembly and/or secretion.     

Electrophoretic analysis of soluble proteins specifically synthesized under phosphate deficiency in the mycelia ofPholiota nameko

Mycelial soluble proteins ofPholiota nameko labeled in vivo during the Pi-supplied (P⁺) and the Pi-depleted (P⁻) cultures were separated by SDS-polyacrylamide gel electrophoresis and two-dimensional polyacrylamide gel electrophoresis, and visualized by fluorography. A comparison of protein profiles from the P⁺ and P⁻ cultures showed that Pi deficiency induces the synthesis of 15 polypeptides and an increase in the relative amount of 29 polypeptides. These result suggests that many proteins may be specifically synthesized de novo under Pi deficiency as part of the adaptive mechanism for this condition.