Cellulose and 1,3-glucan synthesis during the early stages of wall regeneration in soybean protoplasts

Protoplasts isolated from cultured soybean cells (Glycine max (L.) Merr., cv. Mandarin) were used to study polysaccharide biosynthesis during the initial stages of cell wall-regeneration. Within minutes after the protoplasts were transferred to a wall-regeneration medium containing [¹⁴C]glucose, radioactivity was detected in a product which was chemically characterized as cellulose. The onset and accumulation of radioactivity into cellulose coincided with the appearance fibrils on the surface of protoplasts, as seen under the electron microscope. At these early stages, a variety of polysaccharide-containing polymers other than cellulose were also synthesized. Under conditions where the protoplasts were competent to synthesize cellulose from glucose, uridine diphosphate-[¹⁴C]glucose and guanosine diphosphate-[¹⁴C]glucose did not serve as effective substrates for cellulose synthesis. However, substantial amounts of label from uridine diphosphate glucose were incorporated into 1,3-glucan.Abbreviations ECM extracellular material - GLC gas liquid chromatography - GDP-glucose guanosine diphosphate glucose - UDP-glucose uridine diphosphate glucose - U enzyme units as defined by Sigma Chemical Corp., St. Louis, Mo., USA     

Role of cell-wall biogenesis in the initiation of auxin-mediated growth in coleoptiles of Zea mays L.

The involvement of cell-wall polymer synthesis in auxin-mediated elongation of coleoptile segments from Zea mays L. was investigated with particular regard to the growth-limiting outer epidermis. There was no effect of indole acetic acid (IAA) on the incorporation of labeled glucose into the major polysaccharide wall fractions (cellulose, hemicellulose) within the first 2 h of IAA-induced growth. 2,6-Dichlorobenzonitrile inhibited cellulose synthesis strongly but had no effect on IAA-induced segment elongation even after a pretreatment period of 24 h, indicating that the growth response is independent of the apposition of new cellulose microfibrils at the epidermal cell wall. The incorporation of labeled leucine into total and cell-wall protein of the epidermis was promoted by IAA during the first 30 min of IAA-induced growth. Inhibition of IAA-induced growth by protein and RNA-synthesis inhibitors (cycloheximide, cordycepin) was accompanied by an inhibition of leucine incorporation into the epidermal cell wall during the first 30 min of induced growth but had no effect on the concomitant incorporation of monosaccharide precursors into the cellulose or hemicellulose fractions of this wall. It is concluded that at least one of the epidermal cell-wall proteins fulfills the criteria for a growth-limiting protein induced by IAA at the onset of the growth response. In contrast, the synthesis of the polysaccharide wall fractions cellulose and hemicellulose, as well as their transport and integration into the growing epidermal wall, appears to be independent of growth-limiting protein and these processes are therefore no part of the mechanism of growth control by IAA.Abbreviations CHI cycloheximide - COR cordycepin - DCB 2,6-dichlorobenzonitrile - GLP growth-limiting protein(s) - IAA indole-3-acetic acid     

DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Enhancement of growth and polysaccharide production in suspension cultures of protocorm-like bodies from Dendrobium huoshanense by the addition of putrescine

Putrescine at 0.6 mM stimulated protocorm-like body growth and polysaccharide synthesis in suspension cultures of Dendrobium huoshanense. The specific growth rate of protocorm-like body increased from 0.047 to 0.056 day⁻¹, and the maximum dry weight and polysaccharide production reached 33.2 and 2.94 g l⁻¹, respectively, while they were 24.6 and 2.12 g l⁻¹, respectively, in the control. The administration of polyamine inhibitor, α-dl-difluoromethylarginine, at 1 mM, decreased protocorm-like body growth and polysaccharide production to 21.4 and 1.76 g l⁻¹, respectively.     

Effects of fusicoccin and abscisic acid on glucose uptake into isolated beetroot protoplasts

Uptake of glucose, 3-O-methylglucose and sucrose into beetroot protoplasts is considerably stimulated by 10^–6M fusicoccin. This effect is decreased in the presence of 10mM Na⁺ or K⁺, 2 mM Mg²⁺ or Ca²⁺. Whereas fusicoccin causes no change in the pH-optimum of the sugar uptake (pH 5.0), the apparent K_m of this uptake which obeys a biphasic kinetics is decreased by the action of fusicoccin. In the protoplast suspension, fusicoccin induces an acidification which is suppressed by uncoupling agents. Correspondingly, uncouplers as well as vanadate and diethylstilbestrol markedly inhibit the effect of fusicoccin on sugar uptake. The present data support the view that glucose uptake into beetroot protoplasts depend on the proton-pumping activity of the plasmalemma-ATPase. cis–Abscisic acid diminishes significantly the fusicoccin-enhanced glucose uptake. By using a radioimmunoassay, the internal abscisic acid content of the protoplast was estimated to be in the range of 10^–6 M. Protoplasts isolated from bundle tissue contain twice as much abscisic acid as those derived from storage parenchyma. Because protoplasts from the bundle tissue were shown to take up sugars much faster than those from the storage cells, the observed effect of abscisic acid might reflect an involvement of this hormone in the regulation of carbohydrate partitioning in the beet.Abbreviations ABA cis–abscisic acid - bundle protoplast protoplasts isolated from the conducting tissue of beetroots - DES diethylstilbestrol - FC fusicoccin - 3-OMG 3-O-methylglucopyranose - PCMBS p–chloromercuribenzenesulfonic acid - storage protoplasts protoplasts isolated from storage parenchyma     

Aluminum Ions Induce Oat Protoplasts to Produce an Extracellular (1-->3)beta-d-Glucan

Aluminum chloride induced mesophyll protoplasts of oat (Avena sativa) to produce an extracellular polysaccharide (EPS). EPS induced by AlCl₃ appeared identical to that produced in response to the phytotoxin victorin (JD Walton, ED Earle [1985] Planta 165: 407-415). Al ions at 1 millimolar were toxic to protoplasts, but maximum EPS production occurred at a sublethal concentration of 200 micromolar, assayed at pH 6.0. As measured by incorporation of [¹⁴C]glucose, AlCl₃ stimulated EPS production 10- to 15- fold. Pretreatment of protoplasts with cycloheximide prevented EPS production but not cell death in response to AlCl₃, indicating that protein synthesis was necessary for EPS production but not for the phytotoxicity of Al ions. The trivalent salts of Y, Yb, Gd, and In also induced EPS production but those of Sc, Fe, Ga, Cr, and La did not. Mesophyll protoplasts from an acid-soil tolerant oat cultivar, Coker 83-23, produced less EPS in response to AlCl₃ than the acid-soil sensitive cultivar Fla 501. EPS was also produced by wheat (Triticum aestivum) and barley (Hordeum vulgare) protoplasts in response to AlCl₃. An Al-tolerant cultivar of wheat, Atlas, produced less EPS than an Al-sensitive cultivar, Scout, but an Al-tolerant cultivar of barley, Dayton, produced more than the Al-sensitive cultivar Kearney. Therefore, production of EPS by protoplasts in response to Al ions did not appear to be related to Al ion tolerance at the level of whole plants. EPS fluoresced in the presence of Calcofluor and Sirofluor and was degraded by purified laminarinase [(1→3)β-d-glucanase] but not pectinase (polygalacturonase). EPS was composed solely of glucose in 1→3 linkages; hence it is a (1→3)β-d-glucan (callose).     

Magnesium-Dependent stimulation of protein synthesis by the insulin mimic,pervanadate

The insulin mimic, peroxide of vanadate (pervanadate), stimulated ³⁵S-methionine incorporation into Xenopus oocyte protein in a Mg²⁺-dependent manner. Reducing the extracellular Mg²⁺ concentration from 1.0 to 0.1 mM decreased the pervanadate-stimulated component of incorporation by 35%; with 0.01 mM Mg²⁺ or lower, the pervanadate-stimulated component was abolished. In addition, reducing extracellular Mg²⁺ to 0.01 mM inhibited about 50% of the insulinstimulated component of methionine incorporation. Mg²⁺ depletion had no effects on incorporation in controls or when protein synthesis was stimulated by Zn²⁺ or bovine growth hormone. Thus, not all substances that stimulated protein synthesis showed a dependence on extracellular Mg²⁺. Reducing extracellular Ca²⁺ had no effects on methionine incorporation in control cells or in cells stimulated by pervanadate or insulin. When oocytes maintained in a paraffin oil medium were brought into contact with a 0.5 m?I droplet of buffer containing the Mg²⁺ indicator dye, mag-fura-2, and pervanadate, apparent droplet Mg²⁺ decreased rapidly, indicating net uptake by the cells. Insulin also caused a net uptake of Mg²⁺. In contrast, apparent extracellular Mg²⁺ was constant when cells were in contact with droplets containing no effectors. Together, these data indicate that extracellular Mg²⁺, but not Ca²⁺, is involved in the stimulation of protein synthesis by pervanadate, and to a lesser extent by insulin. Pervanadate appears to induce a net uptake of Mg²⁺, and this change in membrane transport may be an early event in signalling the increase in translation. © 1995 Wiley-Liss, Inc.     

Properties of phosphoenolpyruvate carboxylase in desalted extracts from isolated guard-cell protoplasts

Properties of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) obtained from isolated guard-cell protoplasts of Vicia faba L. were determined following rapidly desalting of the extract on a Sephadex G 25 column. The activity of PEP carboxylase was measured as a function of PEP and malate concentration, pH and K⁺ concentration within 2–3 min after homogenization of the guard-cell protoplasts. The activity of this enzyme was stimulated by PEP concentrations of 0.1 to 0.75 mM and by K⁺ ions (12 mM), but inhibited by PEP concentrations above 1 mM and by malate. Changes in the K_m(PEP) and V_max values with increasing malate concentrations (2.5 and 5 mM) indicate that the malate level, varying in relation to the physiological state of guard cells, plays an important role in regulating the properties of phosphoenolpyruvate carboxylase.Abbreviations CAM Crassulacean acid metabolism - GCP guard-cell protoplast - PEP phosphoenolpyruvate Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana

The electrical fusion of protoplasts has been studied in order to maximize the formation of heterokaryons for culture. Heterokaryons of Nicotiana tabacum L. mesophyll protoplasts and N. plumbaginifolia Viviani supension-cell protoplasts were identified in fixed and stained as well as living material; a quantitative fusion index was thereby developed. With this index the efficiencies of various electric fields and fusion-chamber designs have been determined. Optimal fusion was obtained with an alternating-current (AC) field of 150 V/cm and direct-current (DC) square-wave pulses of 1000 V/cm. A new, simple-to-use, largescale fusion chamber is described in which batches of up to 5·10⁵ protoplasts (0.5 ml of cells at 10⁶/ml) can be fused in 5–7 min with efficiencies approaching 40%. Half of the fusion products are heterokaryons, thus fusion is random. Of the fusion products, 60% are bi- or trinucleate. Using fusion procedures similar to those described here Bates and C. Hasenkampf (1985, Theor. Appl. Genet., in press) have recovered viable somatic hybrids which have been regenerated.Abbreviations AC alternating current - DC direct current - PEG polyethylene glycol     

Characterisation and bioactivity of protein-bound polysaccharides from submerged-culture fermentation of Coriolus versicolor Wr-74 and ATCC-20545 strains

The protein-bound polysaccharides of Coriolus versicolor (CPS) have been reported to stimulate overall immune functions against cancers and various infectious diseases by activating specific cell functions. A New Zealand isolate (Wr-74) and a patented strain (ATCC-20545) of C. versicolor were compared in this study. The fruit bodies of both strains were grown for visual verification. Both strains were grown in submerged-culture using an airlift fermentor with milk permeate as the base medium supplemented with glucose, yeast extract and salt. Metabolic profiles of both strains obtained over 7-day fermentation showed very similar trends in terms of biomass production (8.9–10.6 mg/ml), amounts of extracellular polysaccharide (EPS) from the culture medium (1150–1132 μg/ml), and intracellular polysaccharide (IPS) from the mycelium (80–100 μg/ml). Glucose was the dominant sugar in both EPS and IPS, and the polymers each consisted of three molecular weight fractions ranging from 2 × 10⁶ to 3 × 10³ Da. Both the EPS and IPS were able to significantly induce cytokine production (interleukin 12 and γ interferon) in murine splenocytes in vitro. Highest levels of interleukin 12 (291 pg/ml) and γ interferon (6,159 pg/ml) were obtained from samples containing Wr-74 IPS (0.06 μg/ml) and ATCC 20545 IPS (0.1 μg/ml), respectively. The results indicated that lower levels of EPS and IPS generally resulted in higher immune responses than did higher polymer concentrations.     

Liposome-mediated delivery of DNA to carrot protoplasts

The encapsulation of DNA within liposomes and subsequent fusion of the liposomes with carrot (Daucus carota L.) protoplasts were examined to determine optimum conditions for effective liposome-mediated delivery of DNA to protoplasts. Escherichia coli [³H]DNA could be encapsulated with 50% efficiency using encapsulation volumes as low as 0.5 ml. Incorporation of liposome-encapsulated [³H]DNA by carrot protoplasts increased linearly for 2.5 h, and increasing the ratio of protoplasts to liposomes increased the total amount of radioactive label incorporated within the protoplasts. Liposome-mediated incorporation of [³H]DNA by protoplasts increased over a range of polyethylene glycol concentrations up to 20%, but Ca²⁺ did not increase liposome-mediated incorporation when present in the liposome-protoplast incubation mixture. Optimum incorporation was observed when the pH of the liposome-protoplast incubation medium was decreased to 4.8. Encapsulation experiments using DNA of the plasmid pBR322 indicated that an average of 200–1,000 intact copies of pBR322 were sequestered within each nucleus after liposome delivery.     

Molecular weight distribution of cellulose in primary cell walls

The distribution pattern of the degree of polymerization (DP) of cellulose present in the cell walls of mesophyll- and suspension-cultured cells of tobacco was compared to that of newly synthesized ¹⁴C-labeled cellulose from regenerating tobacco protoplasts and suspension-cultured cells. The cellulose was nitrated, and, after fractionation according to differences in solubility in acetone/water, the DP pattern of labeled or unlabeled cellulose nitrate was determined by viscosity measurements. A low (DP<500) and high DP-fraction (DP>2500) of cellulose were predominant in the cell walls of protoplasts, suspension — cultured cells, and mesophyll cells. The average DP of the high molecular weight fraction of cellulose in the cell walls of mesophyll was higher (DP4,000) than in protoplasts or suspension — cultured cells (DP 2,500-3,000). In all cell walls tested, minor amounts of cellulose molecules with a broad spectrum of a medium DP were present. Pulse — chase experiments with either protoplasts or suspension —cultured cells showed that a large proportion of the low and medium DP-cellulose are a separate class of structural components of the cellulose network. The results are discussed in relation to the organization of cellulose in the primary cell wall.Abbreviations DP degree of polymerisation - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid     

Determination of malate levels during the swelling of vacuoles isolated from guard-cell protoplasts

A method for the preparation of vacuoles from guard cells ofVicia faba L. is described. Vacuoles were released from guard-cell protoplasts by osmotic shock and purified on a Ficoll gradient. Contamination of the vacuoles was examined by assaying marker enzymes, such as fumarase, glucose-6-phosphate dehydrogenase, phosphofructokinase, acid phosphatase and mannosidase. Potassium ions in the incubation medium caused increases in the volume of the vacuoles by a factor of about 2.6, while the malate level remained unchanged. In contrast, malate synthesis was stimulated during the swelling phase when complete guard-cell protoplasts were exposed to K⁺. The possible role of K⁺ as an efficient osmotic effector is discussed.Abbreviations DEAE diethylaminoethyl - GCP guard-cell protoplast(s) - GCV guard-cell vacuoles(s) - MCP mesophyll cell protoplast(s) - MCV mesophyll cell vacuole(s)     

Effect of “osmotic stress” on protein and nucleic acid synthesis in isolated tobacco protoplasts

The incorporation of labeled precursors into RNAs and proteins of isolated tobacco (Nicotiana tabacum L.) leaf protoplasts decreases with increasing osmotic pressure in the incubation medium. The incorporation of precursors into RNA and proteins is linear for 15–18 h after the isolation of the protoplasts, irrespective of the osmolarity of the culture media. The uptake of precursors is also affected by the osmolarity of the medium. However, the osmotic stress-induced inhibition of incorporation of precursors into RNA and proteins is also apparent if the differences in uptake are taken into consideration in the calculation. Incorporation of ³²P into TMV-RNA is also inhibited by osmotic stress. As assayed by the double labeling ratio technique, osmotic stress has less unequivocal effect on TMV protein synthesis.Abbreviations PP protoplast - RNase ribonuclease - rRNA ribosomal ribonucleic acid - SDS sodium dodecyl sulfate - SSC 0.1 M Na-acetate in 0.15 M NaCl - TCA trichloroacetic acid - TMV tobacco mosaic virus     

Cell wall regeneration and galactomannan biosynthesis in protoplasts from carob

Protoplast isolation from endosperms of developing carob (Ceratonia siliqua L.) seeds is reported for the first time. These protoplasts regenerated cell walls within 12 h. In order to assess their potential for galactomannan biosynthesis, the incorporation of radioactivity in the regenerated cell wall polysaccharides (CWP) and extracellular polysaccharides (ECP), after feeding these protoplasts with D-[U-¹⁴C]glucose or D-[U-¹⁴C]mannose was studied. The pattern of the radioactive label distribution in the neutral sugars of the trifluoroacetic acid (TFA) hydrolysate of CWP was different from that of the ECP. In the TFA hydrolysis products of the CWP, immediately after protoplast isolation, the greatest level of radioactivity (approximately 90%) was detected in glucose, galactose and mannose. After 2 days protoplast culture, the label in mannose increased. In contrast, immediately after protoplast isolation, approximately 90% of radioactivity of the ECP was detected in galactose and mannose. However, during culture, the radioactivity incorporation in mannose dropped to one third, while that in galactose and arabinose increased significantly. Hydrolysis of the CWP and ECP with -galactosidase and endo--mannanase confirmed that, at least part of mannose and galactose belonged to galactomannan molecules. These results were compared with those obtained upon feeding developing endosperm tissue with D-[U-¹⁴C]mannose. From our results we concluded that protoplasts from endosperm tissues of developing carob seeds, retained the ability of their original explant to synthesize galactomannan, making protoplasts candidates for the study of galactomannan biosynthesis.     

Stabilization of cortical microtubules by the cell wall in cultured tobacco cells

Cortical microtubules (MTs) in protoplasts prepared from tobacco (Nicotiana tabacum L.) BY-2 cells were found to be sensitive to cold. However, as the protoplasts regenerated cell walls they became resistant to cold, indicating that the cell wall stabilizes cortical MTs against the effects of cold. Since poly-l-lysine was found to stabilize MTs in protoplasts, we examined extensin, an important polycationic component of the cell wall, and found it also to be effective in stabilizing the MTs of protoplasts. Both extensin isolated from culture filtrates of tobacco BY-2 cells and extensin isolated in a similar way from cultures of tobacco XD-6S cells rendered the cortical MTs in protoplasts resistant to cold. Extensin at 0.1 mg·ml⁻¹ was as effective as the cell wall in this respect. It is probable that extensin in the cell wall plays an important role in stabilizing cortical MTs in tobacco BY-2 cells.     

Valine resistant plants derived from mutated haploid and diploid protoplasts of Nicotiana sylvestris and N. tabacum

Summary Protoplasts were derived from haploid and diploid Nicotiana sylvestris and N. tabacum. Exposure of the protoplasts to mutagenic doses of ultraviolet (U.V.) radiation prior to two selection rounds in the presence of 4 mM (or 5 mM) and 8 mM of valine, respectively, was required to obtain cell lines with persistent valine resistance. Such lines were obtained from haploid and diploid N. sylvestris protoplasts as well as from haploid protoplasts of N. tabacum but not from (1.8 × 10⁷) diploid N. tabacum protoplasts. The ratio between number of verified valine-resistant cell lines and the initial number of U.V. exposed protoplasts enabled the estimation of the following order of mutation frequency: haploid N. sylvestris > haploid N. tabacum > diploid N. sylvestris. Plants which retained the valine resistance and transmitted it to their sexual progeny were derived from the resistant cell lines.     

Are sucrosyl-oligosaccharides synthesized in mesophyll protoplasts of mature leaves of Cucumis melo?

Biosynthesis of sucrosyl-oligosaccharides (raffinose, stachyose) was traced in source leaves of Cucumis melo after ¹⁴C-photoassimilation. The main carbon compound exported was ¹⁴C-labeled stachyose. No oligosaccharide synthesis was detected in young, importing leaves. Mesophyll protoplasts, isolated from mature leaves which had previously photosynthesized ¹⁴CO₂, did not contain ¹⁴C-oligosaccharides but contained [¹⁴C]-sucrose and ¹⁴C-hexoses. Isolated minor-vein-enriched fractions from the same leaves, however, showed nearly 30% of the ¹⁴C of the neutral fraction to be in oligosaccharides. Isolated, viable mesophyll protoplasts incubated with NaH¹⁴CO₃ also failed to incorporate radioactivity into oligosaccharides, although sucrose and galactinol synthesis was unimpaired. Galactinolsynthase activity in leaf extracts and in mesophyll protoplasts was 16.8 mol·h^-1·mg^-1 protein and 13.8 mol·h^-1·mg^-1 protein, respectively. Galactosyltransferase (EC 2.4.1.67), which synthesizes stachyose from raffinose and galactinol, had an activity of 50 nmol·h^-1·mg^-1 protein in leaf extracts and was also present in the minor-vein-enriched fraction, but could not be detected in mesophyll protoplast lysates. The results indicate that mesophyll cells may not be the site of stachyose synthesis although precursor compounds like sucrose and galactinol are synthesized there.Abbreviation HPLC high-performance liquid chromatography     

Isolation of functional extensor and flexor protoplasts fromPhaseolus coccineus L. pulvini: potassium induced swelling

Methods are described for the isolation of functional protoplasts from secondary pulvinus tissue (flexor and extensor) and from leaf mesophyll tissue of primary leaves ofPhaseolus coccineus L. Integrity of the protoplasts was shown by vital staining and their ability to evolve oxygen in the light. Extensor-cell protoplasts increased their volume for up to 60% upon addition of 10 mM KCl. This K⁺-induced swelling was accompanied by increased rates of proton extrusion.     

Glucocorticoids inhibit precursor incorporation into protein in splenic lymphocytes by stimulating protein degradation and expanding intracellular amino acid pools

In the presence of tracer concentrations of extracellular leucine (5 μM), treatment of rat splenic lymphocyte suspensions in vitro with 1 μM dexamethasone for 2.5–4 h caused a 30–35% inhibition of [³H]leucine incorporation into protein. As the extracellular leucine concentration was raised to 5 mM, this inhibition was progressively reduced to 0–12%. This phenomenon correlated with a marked dependence on extracellular leucine concentration of the dexamethasone-dependent enlargement of free intracellular leucine pools in splenic lymphocytes: a 123% increase in pool size with tracer extracellular leucine; a 10% increase with 5 mM leucine. Varying extracellular leucine had no effect on: (1) nuclear [³H]dexamethasone binding by the cells; (2) the concentration of dexamethasone needed for half-maximal inhibition of [³H]leucine incorporation; (3) the time course of onset and maximal expression of the hormonal inhibition of [³H]leucine incorporation; or (4) the magnitude of dexamethasone-dependent inhibition of [³H]uridine incorporation into RNA by these cells. There was no detectable effect of dexamethasone on uptake and retention of [³H]leucine by the cells, regardless of the extracellular leucine concentration. Treatment of splenic lymphocytes for 4 h in vitro with 1 μM dexamethasone caused a small shift of ribosomes from larger aggregate polysomes to smaller forms. Thus, glucocorticoid-induced inhibition of amino acid incorporation in splenic lymphocytes is a multicomponent response, of which an actual decrease in protein synthesis is only a small part. Enlargement of free intracellular amino acid pools, probably resulting from increased protein degradation, is the major contributing factor to the hormonal inhibition of amino acid incorporation.