Effects of hydroxyproline on the growth and cell-wall protein metabolism of excised root segments of Pisum sativum

Summary Hydroxyproline, in the presence of sucrose, enhanced the extension growth of excised 2–4 mm pea root segments in aseptic media. About 90% of protein-bound hydroxyproline in the pea root segments was confined to the cell-wall fraction where it occurred as trans-4-hydroxy-l-proline. The amounts of wall-bound hydroxyproline increased dramatically towards the cessation of extension growth, but when the segments were cultured in trans-hydroxyproline, this increase was considerably less.Externally supplied cis and trans-hydroxyproline inhibited the formation of protein-bound [¹⁴C]hydroxyproline from [¹⁴C]proline without affecting the total amount of [¹⁴C]proline incorporated into proteins. Studies with -dipyridyl showed that, although some of the externally supplied trans-[¹⁴C]hydroxyproline was incorporated directly into cell-wall proteins, most of it was first converted into proline which was then incorporated into proteins and subsequently reconverted, in part, into hydroxyproline. The effect of externally supplied hydroxyproline is discussed in relation to protein-bound proline hydroxylation.     

Metabolism ofl-proline toN-acetyl-d-glucosamine during germ tube formation ofCandida albicans

The metabolism ofl-proline toN-acetyl-d-glucosamine (GlcNAc) during germ tube formation ofCandida albicans (C. albicans) ATCC 1002 was studied. In uptake experiments, 6.9 nmol ofl-[¹⁴C]proline were taken up by 1×10⁶ cells during 3 h of incubation at 37°C. The percentage of germ tube formation was 94 under the same condition. The presence of GlcNAc reduced the uptake ofl-proline to 3.0 nmol. The percentage of germ tube formation was 95 in the presence and absence of GlcNAc. The [³H]GlcNAc uptake was 3.0 nmol and was constant whetherl-proline was present or not. After the preparation of a chitin fraction from germ tubes that were labeled withl-[¹⁴C]proline, the radioactivity froml-proline was detected in the glucosamine (GlcN) fraction by thin-layer chromatography (TLC). The metabolism ofl-proline to GlcNAc in chitin during germ tube formation was confirmed in this experiment.     

A sensitive method for the separation and quantitation of (14C)proline and (14C)hydroxyproline from the collagen of rat uterus

A specific and sensitive method is described for the isolation and quantitation of [¹⁴C]proline and [¹⁴C]hydroxyproline from uterine collagen of the immature rat. Selectivity is achieved in this isolation by using a protease-free bacterial collagenase. There is complete release of hydroxyproline from uterine protein if the latter is suspended by sonication prior to treatment with collagenase. There is a consistent recovery of [¹⁴C]proline and [¹⁴C]hydroxyproline when they are added to protein hydrolysates of uterus and then subjected to the procedures required for their isolation and quantitation. It is possible using this method to determine the incorporation of [¹⁴C]proline into collagen of the rat uterus and to quantitate its conversion to [¹⁴C]hydroxyproline. Coupled with the colorimetric methods for proline and hydroxyproline, it is also possible to determine their specific activity.     

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.     

Effect of L-azetidine 2-carboxylic acid on growth and proline metabolism in Escherichia coli

The effects of L-azetidine 2-carboxylic acid on growth and proline metabolism in a proline-requiring auxotroph of Escherichia coli are described. The homologue inhibited growth of the wild type and it, alone, did not substitute effectively for proline as a growth supplement for the mutant. In medium containing 0.05 mM proline, the addition of increasing amounts of homologue progressively inhibited growth of the wild type but stimulated growth of the mutant at homologue: proline ratios of 10 : 1 and 50 : 1. This suggested that the homologue exerted a “sparing effect” on proline in the mutant.The incorporation of L-[U-¹⁴C]proline and L-[³H]azetidine 2-carboxylic acid into hot trichloroacetic acid-insoluble material in the mutant was measured. Amino acid analysis of the insoluble material from cells incubated with radiolabeled proline alone revealed that proline was partially degraded and metabolized to other amino acids prior to incorporation into protein. The addition of unlabeled homologue to the incubation medium significantly reduced proline catabolism, suggesting that the homologue exerted a sparing effect on proline in this mutant. In medium containing unlabeled proline and radiolabeled L-azetidine 2-carboxylic acid, the homologuewas incorporated both intact and partially degraded prior to incorporation into protein. Alanine was the major L-azetidine 2-carboxylic acid catabolite.     

Delineation of sodium-stimulated amino acid transport pathways in rabbit kidney brush border vesicles

Summary We have confirmed previous demonstrations of sodium gradient-stimulated transport ofl-alanine, phenylalanine, proline, and -alanine, and in addition demonstrated transport of N-methylamino-isobutyric acid (MeAIB) and lysine in isolated rabbit kidney brush border vesicles. In order to probe the multiplicity of transport pathways available to each of these¹⁴C-amino acids, we measured the ability of test amino acids to inhibit tracer uptake. To obtain a rough estimate of nonspecific effects, e.g., dissipation of the transmembrane sodium electrochemical potential gradient, we measured the ability ofd-glucose to inhibit tracer uptake.l-alanine and phenylalanine were completely mutually inhibitory. Roughly 75% of the¹⁴C-l-alanine uptake could be inhibited by proline and -alanine, while lysine and MeAIB were no more effective thand-glucose. Roughly 50% of the¹⁴C-phenylalanine uptake could be inhibited by proline and -alanine; lysine was as effective as proline and -alanine, and the effects of pairs of these amino acids at 50mm each were not cumulative. MeAIB was no more effective thand-glucose. We conclude that three pathways mediate the uptake of neutral,l, -amino acids. One system is inaccessible to lysine, proline, and -alanine. The second system carries a major fraction of thel-alanine flux; it is sensitive to proline and -alanine, but not to lysine. The third system carries half the¹⁴C-phenylalanine flux, and it is sensitive to proline, lysine, and -alanine. Since the neutral,l, -amino acid fluxes are insensitive to MeAIB, we conclude that they are not mediated by the classicalA system, and since all of thel-alanine flux is inhibited by phenylalanine, we conclude that it is not mediated by the classicalASC system.l-alanine and phenylalanine completely inhibit uptake of lysine. MeAIB is no more effective thand-glucose in inhibiting lysine uptake, while proline and -alanine appear to inhibit a component of the lysine flux. We conclude that the¹⁴C-lysine fluxes are mediated by two systems, one, shared with phenylalanine, which is inhibited by proline, -alanine, andl-alanine, and one which is inhibited byl-alanine and phenylalanine but inaccessible to proline, -alanine, and MeAIB. Fluxes of¹⁴C-proline and¹⁴C-MeAIB are completely inhibited byl-alanine, phenylalanine, proline, and MeAIB, but they are insensitive to lysine. Proline and MeAIB, as well as alanine and phenylalanine, but not lysine, inhibit¹⁴C--alanine uptake. However, -alanine inhibits only 38% of the¹⁴C-proline uptake and 57% of the MeAIB uptake. We conclude that two systems mediate uptake of proline and MeAIB, and that one of these systems also transports -alanine.     

Kynurenines Impair Energy Metabolism in Rat Cerebral Cortex

Growing evidence indicates that some metabolites derived from the kynurenine pathway, the major route of l-tryptophan catabolism, are involved in the neurotoxicity associated with several brain disorders, such as Huntington’s disease, Parkinson’s disease and Alzheimer’s disease, as well as in glutaryl-CoA dehydrogenase deficiency (GAI). Considering that the pathophysiology of the brain damage in these neurodegenerative disorders is not completely defined, in the present study, we investigated the in vitro effect of l-kynurenine (Kyn), kynurenic acid (KA), 3-hydroxykynurenine (3HK), 3-hydroxyanthranilic acid (3HA) and anthranilic acid (AA) on some parameters of energy metabolism, namely glucose uptake, ¹⁴CO₂ production from [U-¹⁴C] glucose, [1-¹⁴C] acetate and [1,5-¹⁴C] citrate, as well as on the activities of the respiratory chain complexes I–IV and Na⁺,K⁺-ATPase activity in cerebral cortex from 30-day-old rats. We observed that all compounds tested, except l-kynurenine, significantly increased glucose uptake and inhibited ¹⁴CO₂ production from [U-¹⁴C] glucose, [1-¹⁴C] acetate and [1,5-¹⁴C] citrate. In addition, the activities of complexes I, II and IV of the respiratory chain were significantly inhibited by 3HK, while 3HA inhibited complexes I and II activities and AA inhibited complexes I–III activities. Moreover, Na⁺,K⁺-ATPase activity was not modified by these kynurenines. Taken together, our present data provide evidence that various kynurenine intermediates provoke impairment of brain energy metabolism.     

Studies on the biochemistry and fine structure of silica shell formation in diatoms

In diatoms, the siliceous cell walls are enveloped by an organic component which includes 4-hydroxyproline and 3,4-dihydroxy-L-proline. The formation of these two amino acids were studied in Nitzschia angularis in Si-starvation synchrony. Both appear to arise from peptidyl proline. Its conversion to peptidyl hydroxyproline was shown in cell-free extracts and in kinetic studies using [¹⁴C]proline. Two lines of evidence indicate that dihydroxyproline does not arise from the further hydroxylation of peptidyl hydroxyproline: First, there was a lag of several minutes between the incorporation of [¹⁴C]proline into protein and the appearance therein of [¹⁴C]hydroxyproline but no such lag for the appearance of dihydroxyproline. Second, ,-dipyridyl blocked the formation of hydroxyproline, but not of dihydroxypyroline, from peptidyl proline. Cell walls made in the presence of dipyridyl differed little in overall chemical composition from walls made in its absence and were morphologically identical. [¹⁴C]dehydroproline was rapidly metabolized in the cells, with [¹⁴C]dihydroxyproline a prominent product. Studies of the conversion of [¹⁴C]proline to [¹⁴C]hydroxyproline at various stages of wall formation showed an increased synthesis of [¹⁴C]dihydroxyproline at the end of cell separation.     

Effect of beta-D-xylosides on collagen synthesis in cultured fibroblasts.

Cultured normal human skin fibroblasts were incubated with [¹⁴C]proline in the presence and absence of 1.0 mM p-nitrophenyl-β-D-xylose. Formation of non-dialyzable hydroxyproline was used as a measure of collagen synthesis. Although total [¹⁴C]proline incorporation was similar in the two cultures, [¹⁴C]hydroxyproline formation was significantly decreased in the β-xyloside-treated cultures. Increasing the period of incubation increased the radioactivity of the insoluble collagen fraction in untreated fibroblasts, however, in β-xyloside-treated cultures no such increase was observed. In contrast to the decreased production of collagen, growth of cells in the presence of the β-xyloside induced the synthesis of high levels of soluble glycosaminoglycans as measured by ³⁵SO₄ incorporation into isolated polysaccharide.     

Thyroid hormone inhibition of synaptosome amino acid uptake and protein synthesis.

Abstract— 3,3′,5-Triiodothyronine (T₃) inhibited L-[¹⁴C]leucine uptake into synaptosomes. Inhibition was competitive with a K_i of 3.1 × 10^?5m . Hofstee plot revealed an inverted hyperbolic curve suggestive of a two carrier or carrier plus diffusion mediated system for amino acid uptake. Both the carrier mediated and diffusional components were inhibited by thyroid analogues. l -Thyroxine and analogues inhibited the incorporation of l -[¹⁴C] leucine into cerebral synaptosome protein. At 50 μm , the triiodo-compounds were more inhibitory than tetraiodo->3,5-triiodo-l -thyronine >3,3′,5-triiodothyropro-pionic> l -thyroxine >3,5-diiodo-l -tyrosine. Thyroid analogue inhibition was not seen in liver or brain mitochondrial protein synthesis. 3,3′,5-Triiodothyronine had no effect on respiratory control or 2,4-DNP stimulated synaptosome respiration supported by malate plus pyruvate. Ouabain did not inhibit [¹⁴C]leucine uptake into adult synaptosomes. There was synergistic inhibition of synaptosome protein synthesis by thyroid analogues in the presence of 0.2 mm -ouabain. 3,3′,5-Triiodothyronine had no effect on synaptosome fraction ATPase or Na-K ATPase. Addition of T₃ induced further inhibition of synaptosome protein synthesis in the presence of either chloramphenicol (100μm ) or cycloheximide (50μg/ml). [¹⁴C]Glycine uptake and incorporation into synaptosome protein was inhibited by 3,3′,5-triiodothyronine. There was no inhibition of [¹⁴C]proline uptake or incorporation. The above evidence and kinetic data strongly favor a selective competitive block in amino acid transport at the synaptosome membrane leading to a decreased rate of protein synthesis.     

Effect of L-azetidine 2-carboxylic acid on growth and proline metabolism in Escherichia coli

The effects of L-azetidine 2-carboxylic acid on growth and proline metabolism in a proline-requiring auxotroph of Escherichia coli are described. The homologue inhibited growth of the wild type and it, alone, did not substitute effectively for proline as a growth supplement for the mutant. In medium containing 0.05 mM proline, the addition of increasing amounts of homologue progressively inhibited growth of the wild type but stimulated growth of the mutant at homologue: proline ratios of 10 : 1 and 50 : 1. This suggested that the homologue exerted a “sparing effect” on proline in the mutant.The incorporation of L-[U-¹⁴C]proline and L-[³H]azetidine 2-carboxylic acid into hot trichloroacetic acid-insoluble material in the mutant was measured. Amino acid analysis of the insoluble material from cells incubated with radiolabeled proline alone revealed that proline was partially degraded and metabolized to other amino acids prior to incorporation into protein. The addition of unlabeled homologue to the incubation medium significantly reduced proline catabolism, suggesting that the homologue exerted a sparing effect on proline in this mutant. In medium containing unlabeled proline and radiolabeled L-azetidine 2-carboxylic acid, the homologuewas incorporated both intact and partially degraded prior to incorporation into protein. Alanine was the major L-azetidine 2-carboxylic acid catabolite.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline nutrition and catabolism in <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ¹-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ¹-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this strain had l-proline dehydrogenase activity as detected both by reaction of Δ¹-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min⁻¹ mg⁻¹) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min⁻¹ mg⁻¹). A soluble fraction from this strain had Δ¹-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min⁻¹ mg⁻¹) as determined by the NAD⁺-dependent oxidation of dl-Δ¹-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.     

The convulsant drugs 3-mercaptopropionate and methionine sulfoximine inhibitl-glutamate andl-aspartate binding to a hydrophobic protein fraction from rat cerebral cortex

The action of the convulsant drugs, methionine sulfoximine (MSO), 3-mercaptopropionate (3-MP), megimide (MG), and allylglycine on the binding ofl-[¹⁴C]aspartate,l-[¹⁴C]glutamate and [¹⁴C]GABA to a hydrophobic protein fraction isolated from rat cerebral cortex was studied. Using the convulsant at 10^–4 M concentration and the radioactive ligands at 10⁶ M the binding ofl-[¹⁴C]glutamate was inhibited 60% by 3-MP and 40% by MSO, while MG and allylglycine had no effect. The binding ofl-[¹⁴C]aspartate was inhibited 55%, and 10–20% by 3-MP and MSO, respectively, while MG and allylglycine had not effect. None of the drugs mentioned, except for a minimal inhibition by MG, altered the binding of [¹⁴C]GABA. Neither MSO nor 3-MP affected the high-affinity sites forl-[¹⁴C]glutamate orl-[¹⁴C]aspartate, but they had a strong inhibitory action on the medium affinity site. These results are discussed in relation to the possible mechanism of action of these drugs onl-glutamate andl-aspartate receptors.     

In vitro cultures of Cinchona species

The uptake of l-[methylene-¹⁴C]-tryptophan from culture medium into root organs of Cinchona ledgeriana and the subsequent incorporation of the radiolabel into quinine and quinidine is reported. In addition, feeding unlabelled l-tryptophan at levels of 500mg/l to the cultures results in a 5-fold increase in the yields of both quinoline alkaloids.     

Substrate regulation of ascorbate transport activity in astrocytes

Astrocytes possess a concentrativel-ascorbate (vitamin C) uptake mechanism involving a Na⁺-dependentl-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellularl-ascorbate on the activity of this transport system. Initial rates ofl-ascorbate uptake were measured by incubating primary cultures of rat astrocytes withl-[¹⁴C]ascorbate for 1 min at 37°C. We observed that the apparent maximal rate of uptake (V _max) increased rapidly (<1 h) when cultured cells were deprived ofl-ascorbate. In contrast, there was no change in the apparent affinity of the transport system forl-[¹⁴C]ascorbate. The increase inV _max was reversed by addition ofl-ascorbate, but notD-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures withl-ascorbate did not affect uptake of 2-deoxy-D-[³H(G)]glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.     

13C NMR studies of the fluxes in the central metabolism of Corynebacterium glutamicum during growth and overproduction of amino acids in batch cultures

The carbon flux distribution in the central metabolism of Corynebacterium glutamicum was studied in batch cultures using [1-¹³C]- and [6-¹³C]glucose as substrate during exponential growth as well as during overproduction of l-lysine and l-glutamate. Using the ¹³C NMR data in conjunction with stoichiometric metabolite balances, molar fluxes were quantified and normalised to the glucose uptake rate, which was set to 100. The normalised molar flux via the hexose monophosphate pathway was 40 during exponential growth, whereas it was only 17 during l-glutamate production. During l-lysine production, the normalised hexose monophosphate pathway flux was elevated to 47. Thus, the carbon flux via this pathway correlated with the NADPH demand for bacterial growth and l-lysine overproduction. The normalised molar flux in the tricarboxylic acid cycle at the level of 2-oxoglutarate dehydrogenase was 100 during exponential growth and 103 during l-lysine secretion. During l-glutamate formation, the normalised flux through the tricarboxylic acid cycle was reduced to 60. In contrast to earlier NMR studies with C. glutamicum, no significant activity of the glyoxylate pathway could be detected. All experiments indicated a strong in vivo flux from oxaloacetate back to phosphoenolpyruvate and/or pyruvate, which might be due to phosphoenolpyruvate carboxykinase activity in C. glutamicum.     

Manipulation,by nutrient limitation,of the biosynthetic activity of immobilized cells of Capsicum frutescens Mill. cv. annuum

The relationship between the synthesis and accumulation of protein and capsaicin was investigated in cultured cells of Capsicum frutescens Mill. cv. annuum immobilized in reticulate polyurethane. Cells were cultured in media containing reduced concentrations of essential nutrients, in an attempt to manipulate the rates of protein synthesis. Cells cultured in the absence of orthophosphate for 7 d demonstrated no reduction in the incorporation of l-[U-¹⁴C]phenylalanine into soluble protein or an increase in incorporation into capsaicin, compared with controls supplied with orthophosphate. By day 15 of culture, however, a differential incorporation of label was observed. Over a 21-d culture period the intracellular phosphate did not completely disappear. Cells cultured in the absence of nitrate and phosphate combined, however, exhibited some reduction in incorporation of [¹⁴C]phenylalanine into protein and an increased incorporation into capsaicin after 7 d of culture, but the differences were greater at day 15, when increases in the total capsaicin content of the cultures were apparent. There was observed a relationship between the intracellular nitrate concentration, the culture growth index, and the incorporation of [¹⁴C]phenylalanine into soluble protein — each of these factors was inversely related to the incorporation of label into capsaicin and the total capsaicin content of the cultures.Abbreviations HPLC high-performance liquid chromatography - Phe phenylalanine     

Microbial Production of Abscisic Acid by Botrytis cinerea

The effect of oryzalexin D, which has been isolated as a group of novel phytoalexins of rice plant, on DNA, RNA, protein, lipid and chitin biosyntheses, respiration and cell membrane permeability was investigated in Pyricularia oryzae. The concentration for 50% inhibition (ED₅₀) by oryzalexin D of the mycelial growth of P. oryzae was 230 ppm. At this concentration, oryzalexin D inhibited equally the incorporation of [2–¹⁴C]thymidine, [2–¹⁴C]uridine, l-[U-¹⁴C]amino acid mixture, l-[methyl-¹⁴C]methionine and d-[l-¹⁴C]glucosamine into DNA, RNA, protein, lipid and chitin in intact cells, but did not inhibit these systems in a homogenate of the mycelia of P. oryzae. Oryzalexin D scarcely inhibited the respiration of the homogenate and mitochondria at ED₅₀. On the other hand, oryzalexin D at ED₅₀ caused leakage of potassium and inhibited the uptake of glutamate by mycelial cells of P. oryzae. These results suggest that interference with the cell membrane function is responsible for the primary mode of action.of oryzalexin D against P. oryzae.     

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.     

Induction of swelling in pea internode tissue by ethylene

Ethylene inhibits polar cell expansion of etiolated pea (Pisum sativum) internode tissue and results in a radial cell expansion, swelling. A special gas-tight growth chamber was developed to monitor continuously the induction of cell expansion inhibition of excised internode segments in the presence of ethylene. Following a complex induction period which lasts about 3 hours, a low, but steady-state growth rate is achieved. Ethylene removal experiments indicate that the gas induces an irreversible change in the cell expansion mode. If the tissue was transferred to an ethylene-free chamber after the steady-state rate had been achieved, this rate continued in the absence of the gas. If the gas was removed during an intermediate phase, that intermediate rate of growth continued after removal. In other experiments, segments were incubated with intact apical hooks and the induction period was much shorter than with isolated segments. Changes in imino acid metabolism have been correlated with the induction of swelling. Although total proline and hydroxyproline levels were not affected by ethylene treatment, incorporation of [¹⁴C]proline into a wall-associated protein was inhibited during the period when swelling occurred. These results suggest that ethylene is affecting proline pool sizes.