Differential protein synthesis in response to sulphate and phosphate deprivation: Identification of possible components of plasma-membrane transport systems in cultured tomato roots

Isolated roots of Lycopersicon esculentum Mill., cultured in axenic conditions were starved of sulphate or phosphate, and uptake capacities for the respective oxyanion-transport systems were observed for several days after sulphate or phosphate withdrawal. Sulphate-uptake capacity of the intact roots, measured in a 20-min period, increased from a control level of 100 nmol · g^–1 · h^–1 to 1100 nmol · g^–1 · h^–1 in 10 d, and phosphate-uptake capacity increased from 500 to 1400 nmol · g^–1 · h^–1 over 4 d. Newly synthesised polypeptides of these root cultures were pulse-labelled in vivo for 2 h, by adding [³H]leucine to the culture medium. The tissue was immediately homogenised and soluble and membrane fractions were prepared. A highly purified plasma-membrane fraction was separated from the crude microsomal membrane fraction using an aqueous two-phase partitioning technique. All fractions were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. A 28-kilodalton (kDa) soluble polypeptide, and 36-, 43-, and 47-kDa plasma-membrane polypeptides were observed to have increased labelling after 4 d of sulphate deprivation. Longer periods resulted in additional polypeptides with increased [³H]leucine incorporation. The synthesis of a 25-kDa membrane polypeptide and a 65-kDa soluble polypeptide was increased after 4 d of phosphate deprivation. Two-dimensional electrophoresis afforded greater resolution of the plasmamembrane polypeptides, confirming increased synthesis of the 36-kDa polypeptide and the presence of the 28-kDa polypeptide in the plasma-membrane preparation from sulphate-starved roots. These polypeptides were also observed in protein-stained two-dimensional gels as low-abundant protein components of the plasmamembrane fraction. It is suggested that the 36-kDa polypeptide may be a component of the plasma-membrane sulphate-transport system and that the 25-kDa polypeptide may be a component of a phosphate-transport system.Abbreviations kDa kilodalton(s) - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - SDS Sodium dodecyl sulphate This work was supported by the Agricultural and Food Research Council via grants-in-aid to Long Ashton Research Station. We are also grateful for discussions with our colleagues D.T. Clarkson (LARS) and J.-C. Davidian (ENSA/INRA, Montpellier).     

Lysine metabolism in a barley mutant resistant to S(2-aminoethyl)cysteine

Lysine and S(2-aminoethyl)cysteine (AEC) metabolism were investigated in normal barley (Hordeum vulgare L. cv. Bomi) and a hemozygous recessive AEC-resistant mutant (R906). Feedback regulation of lysine and threonine synthesis from [¹⁴C] acetate was unimpaired in plants of the mutant 3 d after germination. Seeds of Bomi and R906 contained similar total amounts of lysine, threonine, methionine and isoleucine. Concentrations of these amino acids in the soluble fraction of plants grown 6 d without AEC were also similar. The concentration of AEC in R906 plants was less than in the parent variety when both were grown in the presence of 0.25 mM AEC for 6 d. The uptake of [³H]AEC and [³H]lysine by roots of R906 was, respectively, 33% and 32% of that by Bomi roots whereas the uptake of these compounds into the scutellum was the same in both the mutant and its parent. The uptake of [³H]leucine and its incorporation into proteins was also the same in Bomi and R906 plants. These results suggest that a transport system specific for lysine and AEC but not leucine is altered or lost in roots of the mutant R906. AEC is incorporated into protein and this could be the reason for inhibition of growth rather than action as a false-feedback inhibitor of lysine biosynthesis.Abbreviations AEC S(2-aminoethyl)cysteine - LYS lysine - THR threonine     

Synthesis of Proteins by Isolated Euglena gracilis Chloroplasts

Intact Euglena gracilis chloroplasts, which had been purified on gradients of silica sol, incorporated [³⁵S]methionine or [³H]leucine into soluble and membrane-bound products, using light as the only source of energy. The chloroplasts were osmotically shocked, fractionated on discontinuous gradients of sucrose, and the products of protein synthesis of the different fractions characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The soluble fraction resolved into three zones of radioactivity, the major one corresponding to the large subunit or ribulose diphosphate carboxylase. The thylakoid membrane fraction contained nine labeled polypeptides, the two most prominent in the region of 31 and 42 kilodaltons. The envelope fraction contained a major radioactive peak of about 48 kilodaltons and four other minor peaks. The patterns of protein synthesis by isolated Euglena chloroplasts are broadly similar to those observed with chloroplasts of spinach and pea.     

INCORPORATION IN VIVO OF RADIOACTIVE LEUCINE INTO NEURONAL AND GOAL NUCLEAR PROTEINS OF RAT BRAIN

Purified neuronal and glial nuclei were separated from rat brain cells. The fraction rich in neuronal nuclei contained 68 ± 9 per cent neuronal nuclei and the fraction rich in glial nuclei contained 89 ± 6 per cent glial nuclei. The fraction rich in neuronal nuclei isolated from cells of adult rat brain incorporated l -[4,5-³H]leucine into TCA-insoluble material at a rate comparable to those of the microsomal and the soluble fractions of the brain, and at a much higher rate than the fraction rich in glial nuclei. The proteins soluble in buffered-saline, the acid-soluble deoxyribonucleoproteins, and the residual proteins of the neuronal nuclei are apparently the proteins which account for the higher specific activity of neuronal proteins compared with glial nuclear proteins. In liver and kidney, the incorporation of [³H]leucine into nuclear proteins was lower than into other subcellular fractions from the same organs.     

Membrene turnover in imbibed and dormant embryos of the wild oat (Avena fatua L.)

A comparative study of protein synthesis has been carried out with embryos excised from dormant (D) and non-dormant (ND) caryopses of the wild oat. Although D embryos imbibed in water or ND embryos imbibed in abscisic acid do not germinate, they incorporate [¹⁴C]leucine into TCA-insoluble material for the first 48 h as readily as embryos that do germinate (ND embryos imbibed in water, or D embryos imbibed in gibberellic acid). Pulsechase experiments with [¹⁴]leucine show that in both D and ND embryos the proteins associated with the membranes undergo turnover. The rates of decay of incorporated radioactivity are similar in both dormant and germinating embryos up to 98 h following embryo excision. Fractionation of the membrane proteins in SDS-polyacrylamide gels indicates that the different polypeptides have different rates of turnover. It is concluded that membrane proteins in imbibed D embryos are in a state of constant turnover, and that this is a part of the replacement processes necessary to maintain the integrity of hydrated cells. The continuation of such synthetic events could account for long term survival of dormant Avena fatua in the imbibed state.Abbreviations CCRSE cytochrome relative stain equivalents - D dormant - ND nondormant - ABA abscisic acid - GA gibberellic acid GA₃     

Effect of experimental colchicine encephalopathy on brain protein synthesis and tubulin metabolism

Colchicine blocks axoplasmic flow and produces neurofibrillary degeneration. Brain slices from mice injected intracerebrally with colchicine incorporated more [¹⁴C]leucine into protein and had a decreased uptake of [¹⁴C]leucine into the perchloric acid-soluble pool than did their controls. Brain RNA content was decreased and free leucine increased by colchicine-induced encephalopathy. The specific activities of proteins from subcellular fractions of colchicine-injected brain were increased in the nuclear fraction, the 100,000-g supernatant, and its vinblastine-precipitable tubulin. The ratio of the specific activity of the crude mitochondrial fraction to that of the total homogenate was decreased, as would consistent with impaired movement of newly labeled protein into synaptosomes. Colchicine-injected brain extracts contained one or more cytosol fractions that stimulated ribosomal incorporation of [¹⁴C]leucine into protein in a cell-free system. Colchicine-binding-activity measurements indicated loss of soluble and particulate tubulin in colchicine-injected brains; the decrease of soluble tubulin was verified by its selective precipitation with vinblastine. Colchicine encephalopathy did not affect the rate of spontaneous breakdown of in vitro colchicine binding activity. Similarities of colchicine encephalopathy to the neuron's response to axonal damage suggest that colchicine-induced increase in protein synthesis may, in part, reflect a neuronal response to blockage of neuroplasmic transport.     

Interaction of [3H]gibberellin A1 with a sub-cellular fraction from lettuce (Lactuca sativa L.) hypocotyls

The relationship between protein synthesis and the incorporation of [³H]gibberellin A₁ ([³H]GA₁) into a 2,000xg pelletable (2KP) fraction from lettuce (Lactuca sativa L.) hypocotyl sections has been investigated. Concentrations of D-2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide (MDMP) between 10^-7 M and 10^-4 M caused increasing inhibition of growth, 2KP labelling and incorporation of [¹⁴C]leucine into soluble protein. Growth and 2KP radioactivity were highly correlated (r=0.996). Transfer to MDMP early or late in the course of GA response caused reductions in both growth and incorporation into the 2KP fraction. Exposure to the inhibitor had more effect at 4 h than at 20 h. The proportions of alkali-soluble and insoluble radioactivity in the 2KP fraction were also altered by this treatment. The implications of these findings are discussed.Abbreviations GA₁ gibberellin A₁ - MDMP D-2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide - 2KP a2,000xg pelletable fraction     

Heat shock response ofChlorella protothecoides during greening

Heterotrophically grown cells ofChlorella protothecoides were transferred to autotrophic medium and allowed to green at 25°C. The protein synthetic activity of the greening cells measured in terms of incorporation of [³5S]-methionine showed a maximum around 20 h of greening and thereafter started declining. Similarly, an analysis of densitometric tracings of the fluorographic profile of the polypeptides associated with both total cellular fraction and membrane fractions during different hours of greening revealed that maximum number of polypeptides were getting labelled around 20 h of greening. At 20 h of greening, the cells were shifted to 40°C and the effect of heat shock on protein synthesis was studied. The heat shock treatment caused a definite decrease in the incorporation of [³⁵S]-methionine into proteins. Due to heat shock, the synthesis of total soluble proteins was affected much more than that of the thylakoid membrane bound proteins. When the cells were transferred back to 25°C after a brief period of heat shock at 40°C, there was a considerable recovery in the protein synthesis and this recovery was found to be significant in the case of soluble proteins, while there was no such definite recovery in the synthesis of thylakoid membrane bound proteins.     

Components of fast and slow phases of axoplasmic flow

Abstract— Cat ventral roots removed at times from hours to days after injection of [³H]leucine into the seventh lumbar and first sacral spinal cord segments were homogenized and subjected to sub-cellular fractionation. The soluble fraction was further analysed for protein content by column chromatography with Sephadex. The fast phase of axoplasmic flow carries down labelled free leucine, polypeptide, and soluble protein. The slow phase of axoplasmic flow carries down labelled soluble protein. The sub-cellular components were labelled with the small particulate fraction showing a higher specific activity than the other sub-cellular fractions.     

Mitochondrial protein synthesis in chinese hamster cells (line CHO) synchronized by isoleucine deprivation

Mitochondrial protein synthesis was measured in line CHO cells after phases of the cell cycle were synchronized by isoleucine deprivation or mitotic selection. Maximum incorporation of [³H] leucine into mitochondrial polypeptides occurred within 2 hours after isoleucine was added to initiate G₁ traverse. In cells synchronized in G₁ by mitotic selection, the rate of mitochondrial protein synthesis was fairly constant throughout the cell cycle. SDS-polyacrylamide gel electrophoretic profiles of labeled mitochondrial polypeptides were similar in cells synchronized by either isoleucine deprivation or mitotic selection. Obvious changes in the distribution of polypeptides were not detected during various phases of the cell cycle. The increased rate of incorporation of [³H] leucine into mitochondrial polypeptides after reversal of G₁-arrest may indicate that mitochondrial protein synthesis and possibly mitochondrial biogenesis are synchronized in CHO cells deprived of isoleucine.     

Biosynthesis of the Snowdrop (Galanthus nivalis) Lectin in Ripening Ovaries

The biosynthesis and processing of the Galanthus nivalis agglutinin were studied in vivo in ripening snowdrop ovaries. Using labeling and pulse chase labeling experiments it could be demonstrated that the snowdrop lectin is synthesized as a precursor of relative molecular weight (M_r) 15,000 which is posttranslationally converted into the authentic lectin polypeptide of M_r 13,000 with a half-life of about 6 hours. Gel filtration of an extract of [³H]leucine labeled ovaries on Sepharose 4B showed that a significant portion of the newly synthesized lectin is associated with the particulate fraction. When the organellar fraction was fractionated on isopycnic sucrose gradients this lectin banded in the same density region as the endoplasmic reticulum (ER) marker enzyme NADH cytochrome c reductase. Both radioactivity in lectin and in enzyme activity shifted towards a higher density in the presence of 2 millimolar Mg-acetate indicating that the labeled lectin was associated with the rough ER. Labeled lectin could be chased from the ER with a half-life of 4 hours and then accumulated in the soluble fraction. Whereas the ER-associated lectin contains exclusively polypeptides of M_r 15,000 the soluble fraction contains both precursor molecules and mature lectin polypeptides. The snowdrop lectin in the ER is fully capable of binding immobilized mannose. It is associated into tetramers with an appropriate molecular weight of 60,000. These results indicate that newly synthesized snowdrop lectin is transiently associated with the ER before transport and processing.     

Occurrence and biosynthesis of catalase at different stages of seed maturation

It was to be shown whether during the biogenesis of microbodies some of their components were already present in the cell prior to the organelle's assembly. To this end, the occurrence and properties of catalase in soluble and particular fractions of ripening cucumber seeds were examined. Homogenates of seeds from ripening fruits were fractionated by isopycnic density gradient centrifugation, and thus catalase was found in three different fractions: as a soluble enzyme in the gradient supernatant, as a membrane fraction at density d=1.18 kg l^-1, and in association with microbodies. In the early steps of seed formation, catalase was detected at density d=1.18 kg l^-1 and in the gradient supernatant. At a later stage of seed maturation, however, catalase was primarily associated with microbodies which exhibited an equilibrium density of d=1.23 kg l^-1. M _r as well as subunit M _r of catalase were determined, and their close immunological relationship to leaf peroxisomal catalase and glyoxysomal catalase was demonstrated. Biosynthesis of catalase at different stages of seed maturation was investigated by in vivo labeling with l-[³⁵S]methionine, l-[¹⁴C]leucine and -[³H]aminolaevulinic acid. Electrophoretic analysis of de novo synthesized catalase subunits revealed the occurrence of a heavy form (M _r 57,500) in the soluble fraction; this form was preferentially labeled. A light form, M _r 53,500, was detected in microbodies and also in the soluble fraction. The findings lend support to the hypothesis that the rate of catalase synthesis is highest in an early stage of seed formation, when globulins have already been formed, but before de novo synthesis of malate synthase has commenced. Prior to microbody assembling, a cytoplasmic pool of catalase was labeled.Abbreviations EDTA Na₂-ethylenediaminotetraacetate - Hepes 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - M _r molecular weight     

Transport and metabolism of [3H]iso-pentenyladenine following application to the tips of intact and decapitated tap roots of Pinus pinea

[³H]iso-Pentenyladenine ([³H]iP) was fed for 24 h to the tips of intact and root tip-decapitated Pinus pinea seedlings. Twelve and 24 h after application to the roots of intact plants most of the applied radioactivity (±60%) was transported to the shoot. Root tip removal increased transport of the applied radioactivity to the shoot, but the overall pattern of distribution of radioactivity in the seedling did not change. Large amounts of radioactivity were recovered from the elongation zone of the root. Some radioactivity also accumulated in the older part of the root with well-developed lateral roots. When [³H]iP was applied one day after decapitation, no significant changes in the pattern of radioactivity distribution were found between the intact and decapitated root systems. However, when applied 7 days after decapitation there was a significant increase of radioactivity in the region of the root where lateral roots were emerging. HPLC separation of extracts from the different root sections showed that [³H]iP was extensively metabolized in the root. Six peaks of radioactivity, which co-chromatographed with authentic cytokinin standards, were detected.Abbreviations ABA abscisic acid - ADE adenine - IAA indole-acetic acid - iP iso-pentenyladenine - HPLC high performance liquid chromatography - [OG]DHZ O-glycosyldihydrozeatin - [9R-MP]DHZ ribosyldihydrozeatin monophosphate - [9G]iP iso-pentenyladenine-9-glucoside - [9R]Z ribosylzeatin - [9R]iP iso-pentenyladenosine - TLC thin layer chromatography     

The identification of polypeptides synthesised during the acquisition of teichoic acid synthetic activity in Bacillus licheniformis

An attempt has been made to identify proteins synthesised during induction of teichoic acid synthesis in Bacillus licheniformis ATCC 9945. The proteins are recognised as those produced on the change from teichuronic acid to teichoic acid synthesis that occurs after the transfer of the bacteria from phosphate-limited to phosphate-rich conditions. B. licheniformis was grown in phosphate-limiting conditions in the presence of threonine to stimulate threonine uptake. The bacteria were then transferred to phosphate-rich conditions and were pulsed-labelled with [¹⁴C]threonine during the change to teichoic acid synthesis. All of the proteins were extracted from the cells with sodium dodecyl sulphate and were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Radioactive polypeptides were identified by fluorography of the polyacrylamide gels. The radioactive polypeptides that were formed on change from teichuronic acid to teichoic acid synthesis were compared with the polypeptides present in a membrane sub-fraction that had high teichoic acid-synthesising activity. The labelling of nine polypeptides with [¹⁴C]threonine was dependent on new RNA synthesis. Of these nine polypeptides, five were also present in the membrane sub-fraction with the highest teichoic acid-synthesising activity.     

Phaseolus vulgaris phytohemagglutinin contains high-mannose and modified oligosaccharide chains

Phytohemagglutinin, the major lectin in the seeds of the common bean Phaseolus vulgaris L., was isolated by affinity chromatography from cotyledons of nearly mature seeds and from developing cotyledons labeled with [³H]glucosamine, [³H]mannose or [³H]fucose. The protein was subjected to exhaustive proteolysis and the carbohydrate composition of the resulting glycopeptides examined. Two classes of oligosaccharide side-chains were found. The sidechains of the first class are of the high-mannose type, containing two residues of N-acetylglucosamine and 8 or 9 mannose residues. The sidechains of the second class are of the modified type containing N-acetylglucosamine, mannose, fucose, xylose in molar ratios of 2:3.8:0.6:0.5. Two-dimensional gel electrophoresis shows that phytohemagglutinin can be fractionated into seven different glycosylated polypeptides, and that each one contains at least one modified oligosaccharide chain. The results indicate that most glycosylated polypeptides probably contain one chain of each class. The carbohydrate composition of the two types of chains is similar to that found in other plant glycoproteins, but this is the first report of a plant glycoprotein with both highmannose and modified oligosaccharides on the same polypeptide chain.Abbreviations endo H endo--N-acetylglucosaminidase H - GlcN glucosamine - GlcNAc N-acetylglucosamine - Man mannose - PHA phytohemagglutinin This work was done while A.V. was on leave from the Istituto Biosintesi Vegetali, C.N.R., via Bassini 15, I-20133 Milano, Italy     

Polypeptide pattern and enzymic character of vacuoles isolated from barley mesophyll protoplasts

Intact chloroplasts and vacuoles were isolated from mesophyll protoplasts of barley. The chloroplasts occupied about 15% of the cellular volume and contained 75% of the protein, whereas the vacuoles occupied about 80% of the volume and contained less than 4% of total cellular protein. Contamination of the vacuolar fraction by foreign protein is included in these values. Chlorophyll was absent from the vacuolar fraction, but less than 1% of several extra-vacuolar marker proteins were still present. The vacuoles contained hydrolytic enzymes. Several of them (-mannosidase, -galactosidase, N-acetylglucosaminidase) were soluble, whereas part of the activity of others semimented with the tonoplasts during centrifugation. Attached proteins could be released from the membranes during freezing in the presence of NaCl. One-dimensional gel electrophoretic separation of soluble vacuolar proteins under non-denaturing conditions yielded more than 10 protein bands. A comparative analysis was performed of thylakoids and vacuoles which were subfractionated into tonoplasts and soluble vacuolar constituents. Sodium dodecyl sulfate gel electrophoresis separated about 15 polypeptides of the soluble fraction which reacted with silver reagent. The tonoplast fraction yielded about 20 bands. A similar number of bands was observed when vacuoles incubated with the ¹⁴C-labelled SH-reagent N-ethylmaleimide were analysed for radioactive polypeptides. Silverstaining of the polypeptides and their SH-content did not correlate. Several polypeptides of the vacuolar fraction had molecular weights very similar to the molecular weights of known chloroplast proteins. However, with the exception of the two subunits of ribulose-1,5-bisphosphate carboxylase, contamination of the vacuolar fraction by chloroplast proteins could be ruled out as a possible cause of the close correspondence. The lipophilic carboxylic-group reagent N,N-dicyclohexylcarbodiimide ([¹⁴C]DCCD) reacted with several polypeptides of thylakoids and tonoplasts. However, the labelling patterns were different. The most heavily labelled polypeptide of thylakoids was the 8-kDa polypeptide of the basal part of the coupling factor CF₀. Tonoplast polypeptides heavily labelled with [¹⁴C]DCCD had molecular weights of 24, 28, and 56 kDa. The vacuolar 8-kDa polypeptide remained unlabelled.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - IA iodoacetamide - NEM N-ethylmaleimide - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate     

Relative Stability of Membrane Proteins in Escherichia coli

The relative stability of membrane proteins in Escherichia coli was investigated to determine whether these proteins are degraded at heterogeneous rates and, if so, whether the degradative rates are correlated with the sizes or charges of the proteins. Cells growing in a glucose-limited chemostat with a generation time of 15 h were labeled with [¹⁴C]leucine. After allowing 24 h for turnover of ¹⁴C-labeled proteins, the cells were labeled for 15 min with [³H]leucine. By this protocol, the rapidly degraded proteins have a high ratio of ³H to ¹⁴C, whereas the stable proteins have a lower ratio. The total cell envelope fraction was collected by differential centrifugation, and the proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The relative ratio for each protein was determined by dividing its ³H/¹⁴C ratio by the ³H/¹⁴C ratio of the total membrane fraction. Although most of the 125 membrane proteins had relative ratios close to the average for the total membrane fraction, 19 varied significantly from this value. These differences were also observed when the order of addition of [¹⁴C]leucine and [³H]leucine was reversed. In control cultures labeled simultaneously with both isotopes, the relative ratios of these 19 proteins were similar to that of the total membrane fraction. Thirteen of these proteins had low relative ratios, which suggested that they were more stable than the average protein. An experiment in which the normal labeling procedure was followed by a 60-min chase period in the presence of excess unlabeled leucine suggested that the low relative ratios of 3 of these 13 proteins may be due to a slow post-translational modification step. Six membrane proteins had high relative ratios, which indicated that they were degraded rapidly. In contrast to the relationships found for soluble proteins in mammalian cells, there were no strong correlations between the degradative rates and either the isoelectric points or the molecular weights of membrane proteins in E. coli.     

Pyoverdine-mediated iron transport Fate of iron and ligand inPseudomonas aeruginosa

Summary Incubated in the presence of [⁵⁵Fe]ferri[¹⁴C]pyoverdine, iron-poorPseudomonas aeruginosa accumulated more⁵⁵Fe than¹⁴C over a 60-min period. Distribution studies showed (a) more¹⁴C than⁵⁵Fe in the soluble fraction during the first 20 min, (b) approximately 60% of the⁵⁵Fe associated with the membranes at 60 min, and (c) approximately 85% of the¹⁴C in the soluble fraction at 60 min. Cells osmotically shocked after incubating with [⁵⁵Fe]ferri[¹⁴C]pyoverdine for 60 min released⁵⁵Fe but not¹⁴C, suggesting separation of metal and ligand in the periplasmic space. Whereas the mechanism of dissociation of iron and ligand is not known, the decrease in transport observed in the presence of dipyridyl suggests involvement of reduction in this process. Transport of iron was energized by the proton motive force instead of by intracellular levels of ATP. The hydrogen ion gradient was the major driving force of transport. Cyanide-poisoned cells accumulated more¹⁴C than⁵⁵Fe over 60 min. Here, iron accumulated in the soluble fraction instead of on the membranes.     

What is pea legumin — Is it glycosylated?

Since there is some question as to whether or not legumin is glycosylated, this storage protein was isolated by various procedures from developing cotyledons of Pisum sativum L. supplied with [¹⁴C]-labeled glucosamine and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Legumin isolated by the classical method of Danielsson [(1949) Biochem. J. 44, 387–400] a procedure in which globulins extracted with a buffered salt solution are precipitated with ammonium sulfate (70% saturation) and legumin separated from vicilin by isoelectric precipitation, was labeled. The glucosamine incorporated into legumin was associated with low-molecular-weight polypeptides. In contrast, legumin isolated by the method of Casey [(1979) Biochem. J. 177, 509–520], a procedure where legumin is prepared by zonal isoelectric precipitation from globulins precipitated with 40–70% ammonium sulfate, was not labeled. However, the globulin fraction precipitated with 40% ammonium sulfate was labeled and the radioactive glucosamine was associated with low-molecular-weight polypeptides. Legumin isolated from protein bodies [Thomson et al. (1978) Aust. J. Plant Physiol. 5, 263–279] was not extensively labeled. However, the saltinsoluble fraction of protein body extracts was labeled and the radioactivity was associated with low-molecular-weight polypeptides. These results indicate that protein bodies contain a glycoprotein of low-molecular-weight that co-purifies with legumin isolated by the method of Danielsson but that is discarded when isolation methods developed more recently are used.     

The relationship between DNA synthesis and the synthesis of nuclear proteins in rat brain during development

—The incorporation of [³H]thymidine into nuclear DNA of rat brain progressively increased from birth until the 8th postnatal day and it was lowest in the adult brain. When isolated nuclei from brain cells were separated into a neuronal- and a glial-rich fraction (composed of glial and neuroblast nuclei in young animals), the specific radioactivity of the DNA was higher in the glial-rich fraction at all ages investigated. The incorporation of [³H]leucine into proteins of rat brain was considerably higher in the 8-than in the 1-day-old rat. The greatest difference in the rate of protein synthesis between 8- and 1-day-old brain occurred in the nuclear proteins, especially those associated with DNA. There was an accumulation of protein and RNA in nuclei from brain cells from birth to the 8th postnatal day. The increased content of proteins occurred primarily in the fraction soluble in buffered saline (nuclear sap).