Amino acid synthesis from glucose-U-14C in Glossina morsitans

Amino acid synthesis from glucose-U-¹⁴C was investigated in 2 day post-emergent and pregnant females of Glossina morsitans. This insect can synthesize alanine, aspartic acid, cystine, glutamic acid, glycine, proline, and serine from glucose. Arginine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, taurine, threonine, and valine showed no radioactivity and hence may be classified as nutritionally indispensable amino acids. Although tyrosine and hydroxyproline were not synthesized from glucose, they are at least partially dispensable nutrients for this insect because their synthesis from phenylalanine has been demonstrated. After the labelled glucose injection the highest radioactivity was recovered in the proline fraction. This is probably related to its rôle as an important energy reserve for flight. The radioactive amino acids recovered from females and from their offspring following glucose-U-¹⁴C injection were similar to those recovered from younger females. Radioactivity was also detected in the expired CO₂ and the excreta. The amino acids alanine, arginine, cystine, glycine, histidine, leucine/isoleucine, lysine, methionine, proline, and valine were identified in the excreta, of which arginine and histidine were in the largest amounts. Only excreted alanine, glycine, and proline showed radioactivity.     

d-β-HYDROXYBUTYRATE: A MAJOR PRECURSOR OF AMINO ACIDS IN DEVELOPING RAT BRAIN

Abstract— D-β-hydroxybutyrate (β-OHB) was compared to glucose as a precursor for brain amino acids during rat development. In the first study [3-¹⁴C]β-OHB or [2-¹⁴C]glucose was injected subcu-taneously (01 μCi/g body wt) into suckling rats shortly after birth and at 6. 11, 13, 15 and 21 days of age. Blood and brain tissue were obtained 20 min later after decapitation. The specific activity of the labelled precursor in the blood and in the brain tissue was essentially the same for each respective age suggesting that the labelled precursor had equilibrated between the blood and brain pools before decapitation. [3-¹⁴C]β-OHB rapidly labelled brain amino acids at all ages whereas [2-¹⁴C]glucose did not prior to 15 days of age. These observations are consistent with a maturational delay in the flux of metabolites through glycolysis and into the tricarboxylic acid cycle. Brain glutamate, glutamine, asparate and GABA were more heavily labelled by [3-¹⁴C]β-OHB from birth-15 days of age whereas brain alanine was more heavily labelled by [2-¹⁴C]glucose at all ages of development. The relative specific activity of brain glutamine/glutamate was less than one at all ages for both labelled precursors suggesting that β-OHB and glucose are entering the‘large’glutamate compartment throughout development. In a second study, 6 and 15 day old rats were decapitated at 5 min intervals after injection of the labelled precursors to evaluate the flux of the [¹⁴C]label into brain metabolites. At 6 days of age, most of the brain acid soluble radioactivity was recovered in the glucose fraction of the [2-^,4C]glucose injected rats with 72, 74, 65 and 63% after 5, 10, 15 and 20 min. In contrast, the 6 day old rats injected with [3-¹⁴C]β-OHB accumulated much of the brain acid soluble radioactivity in the amino acid fraction with 22, 47, 57 and 54% after 5, 10, 15 and 20 min. At 15 days of age the transfer of the [¹⁴C]label from [2-¹⁴C]glucose into the brain amino acid fraction was more rapid with 29, 40, 45, 61 and 73% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. There was almost quantitative transfer of [¹⁴C]label into the brain amino acids of the 15-day-old [3-¹⁴C]β-OHB injected rats with 66, 89, 89, 89 and 90% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. The calculated half life for /?-OHB at 6 days was 19 8 min and at 15 days was 12-2 min. Surprisingly, the relative specific activity of brain GABA/glutamate was lower at 15 days of age in the [3-¹⁴C]β-OHB injected rats compared to the [2-¹⁴C]glucose injected rats despite a heavier labelling of brain glutamate in the [3-¹⁴C]β-OHB injected group. We interpreted these data to mean that β-OHB is a less effective precursor for the brain glutamate ‘subcompartment’ which is involved in the synthesis of GABA.     

AMINO ACID INCORPORATION INTO PROTEIN IN NEURONAL CELL BODY AND NEUROPIL FRACTIONS IN VITRO

Abstract—

• 1 The incorporation of radioactivity from [³H]lysine into acid-insoluble material in vitro in mixed cell suspensions and isolated neuronal and neuropil fractions has been followed.
• 2 In the mixed cell suspension, incorporation was linear in fresh preparations for up to 60 min. In cold stored preparations, incorporation began to fall off after 30 min. Incorporation, at 4-11 pmol/mg protein/h, was intermediate between that in the tissue slice and in a cell-free preparation. Addition of a mixture of non-labelled amino acids at 1 mM produced a 30-40 per cent inhibition of incorporation. Molar rates of incorporation of glutamate and tryptophan into the mixed cell fractions were respectively 73 and 1-4 times that of lysine.
• 3 Only 8 per cent of the incorporated radioactivity could be recovered in soluble as opposed to particulate material. After hydrolysis of the protein, followed by paper chromatography and autoradiography, radioactivity was detected only in the position corresponding to lysine.
• 4 Incorporation in the separated cell fractions was not markedly reduced by the centri-fugation procedure. Incorporation into the neuronal fraction was 2-2-6 times that into the neuropil fraction, depending on the amino acid used. Incorporation into both was reduced by some 40 per cent by addition of an amino acid mixture.
• 5 Comparison of in vivo and in vitro data suggest that the differences in rate of incorporation are characteristic of neurons and neuropil in situ.

     

Asparagine biosynthesis by Oryza sativa seedlings

l-Aspartate-[U-¹⁴C] was quickly metabolized in rice seedlings into amino acids, organic acids and sugars. On feeding simultaneously with ammonium for 2 hr, about 1% of the total soluble radioactivity was recovered as asparagine. Major amino acids labelled were aspartate, glutamate, glutamine and alanine in both shoots and roots. On the other hand, on feeding l-aspartate-[U-¹⁴C] to rice seedlings precultured in an ammonium medium, asparagine accounted for 35% of the total soluble radioactivity in the roots. Different labelling patterns in amino acids from those of non-precultured tissues were observed, and the main amino acids labelled in this case were asparagine and γ-aminobutyrate in the roots; glutamate, asparagine and glutamine in the shoots. It was observed in the roots that this increase of asparagine labelling was associated with a decrease of label in glutamate.     

Axonal transport in nigro-neostriatal neurons during morphine tolerance development and abstinence in rats.

Axonal transport of [³H]protein in the nigro-neostriatal pathway in rats was examined during acute and chronic morphine administration and during morphine abstinence. Two days after a microinjection of [³H]lysine into the left substantia nigra zona compacta, more than 95% of the radioactivity present in the rat forebrain was protein-bound. Examination of frozen frontal brain sections revealed that 80–90% of the labelled protein of the injected side was located in brain areas traversed by the nigro-neostriatal pathway. As a positive control, intranigrally administered colchicine reduced the amount of [³H]protein transported after 5 days to the nucleus caudatus-putamen (neostriatum) to approx 18-26% of control. In animals rendered morphine-dependent by subcutaneous implantation of tablets containing 75 mg of morphine base, 27–86% more radioactivity accumulated in the neostriatum at 3, 4 and 5 days after [³H]lysine injection. In contrast, 23–48% less radioactivity was recovered in the neostriatal areas of animals withdrawing from morphine 24 h after [³H]lysine. Gel electrophoresis of soluble and particulate [³H]protein fractions from neostriatal tissues indicated that the gel patterns of radioactivity were not altered by chronic morphine administration. Neither morphine administration nor morphine abstinence altered the rate or amount of [³H]lysine incorporation into protein of the substantia nigra. These data demonstrate that chronic morphine administration was accompanied by a generalized increase in the amount of labelled protein transported to the neostriatum but the procedure was not sufficiently sensitive to detect a minor qualitative alteration of any particular protein(s). Furthermore, these data suggest that either the capacity or the rate of nigro-neostriatal protein transport may be increased during chronic morphine administration in the rat.     

Are Polyamines Transported in Etiolated Peas?

To investigate the possible transport of polyamines and their precursor amino acids, ¹⁴C-labeled putrescine, spermidine, arginine, or lysine were injected into cotyledons of 4-day etiolated pea (Pisum sativum L. cv Alaska) seedlings. After 4 hours the shoot, root, and cotyledons were homogenized and the extracted, dansylated polyamines separated by thin-layer chromatography. Little radioactivity was transported from the cotyledons when [¹⁴C]putrescine or [¹⁴C]spermidine were injected and of the radioactivity in the axis, none could be recovered as polyamines. Injection of [¹⁴C]arginine or [¹⁴C]lysine, on the other hand, led to a significant transport of radioactivity into the axis, of which a large fraction was present in the form of the diamines, putrescine or cadaverine, respectively. These results indicate that polyamines in the growing regions of etiolated pea seedlings probably arise from transport and conversion of amino acid precursors.     

Specific non-histone protein fractions associated with ‘active’ chromatin in immunized rats

Summary Non-histone proteins of normal, non-immunized rats and rats immunized with mouse spleen cells were labelled with three different amino acids: [³H]tryptophan, [³H]methionine and [³H]leucine. Chromatin was fractionated at increasing salt concentrations into three fractions: 0.35 M NaCl-soluble, 2 M NaCl-soluble and residual. Non-histone protein fractions F (M_r 12 000) and H (M_r 3 000) highly labelled with [³H]tryptophan, lower with [³H]methionine but not with [³H]leucine, were present mainly in the residual fraction. After DNAse II treatment non-histone protein fractions F and H disappeared in chromatin fractions and were present in Mg^2– soluble fractions which suggests that, similar to the fractions I (M_r below 3 000) and B (M_r 120 000) described previously (5), these fractions may be associated with active transcribed genes.     

A chemical and autoradiographic study of cellulose synthesis during the encystment of Acanthamoeba castellanii

When cells of Acanthamoeba castellanii are placed in a non-nutrient medium, they differentiate into cysts which possess cellulosic walls. In the present study, the source of the glucosyl unit for cyst wall cellulose was investigated by following the encystment of trophozoites grown in the presence of ¹⁴C-labeled fatty acids (uniformly labeled palmitate or oleate) or [3-³H]glucose. Cells were fractionated at the beginning and after 30 hr of encystment using a modified Schmidt-Tannhauser procedure. In cells grown on fatty acids, 90% of the labeled material was in the lipid fractions both before and after encystment with the total amount of label/cell changing very little. Both partial and complete acid hydrolysis of the glycogen of the acidsoluble fraction and the alkali-insoluble residue of the cyst wall indicated that the glucose of both fractions was not radioactive, although Acanthamoeba is known to have a functional glyoxylate pathway.Fractionation data of cells grown on [³H]glucose indicated a sevenfold increase in radioactivity in the wall insoluble fraction and a fivefold decrease in the acid-soluble fraction with the cpm/cell of the other fractions changing very little after 30 hr of encystment. Approximately 70% of the ³H-labeled material was recovered as glucose from the 30-hr wall insoluble fraction following complete acid hydrolysis. The specific radioactivity of glucose in the cyst wall insoluble fraction was the same as that of glycogen glucose isolated from the acid soluble fraction of trophozoites. Electron microscopic autoradiography showed that the majority of nonlipid radioactivity was due to glycogen in trophozoites. Autoradiograms failed to reveal Golgi bodies or any particular region of the cell as being the specialized site of cellulose synthesis. The results of the fractionation and autoradiographic studies are consistent with the concept that glycogen is a precursor of cyst wall cellulose, and that glucosyl units of glycogen and/or other glucose derivatives are converted to cellulose without significant dilution under the experimental conditions used.     

Specific protein synthesis in isolated epithelium of guinea-pig seminal vesicle. General features

Four intrinsic soluble proteins are synthesized and secreted by sexually mature guinea-pig seminal-vesicle mucosa, which comprises a monolayer of a homogeneous columnar epithelial cell. All four proteins can be extracted readily in 154mm-NaCl from the organ's luminal constituents in which they are present in high concentration. They are referred to as proteins 1, 2, 3 and 4 in order of their elution during DEAE-cellulose column chromatography. Specific primary antibodies were harvested from goats that had been inoculated with the purified vesicular proteins; secondary antibodies were obtained from a donkey inoculated with goat γ-globulins. Double-antibody-immunoprecipitation techniques were developed to precipitate the vesicular proteins. Thus proteins newly synthesized from ¹⁴C-labelled amino acids could be precipitated and the incorporated radioactivity assessed. Isolated seminal-vesicle mucosa, incubated in only a buffered salt solution containing glucose, readily synthesized the soluble secreted proteins from added [¹⁴C]lysine plus [¹⁴C]glycine, [¹⁴C]histidine plus [¹⁴C]glutamate, [¹⁴C]glutamine alone and [¹⁴C]arginine alone. The rates of incorporation (d.p.m./mg of total soluble protein) of labelled lysine and glycine and of labelled arginine were linear with time over 180min. With the other labelled precursors, rates diminished between 60 and 180min. Labelled protein could be detected after only 10–15min of incubation. Only 4–9% of the newly synthesized protein remained associated with the mucosa; the remainder was found in the cell-free incubation medium. The isolated seminal-vesicle mucosal preparation will provide a unique opportunity to study the synthesis and secretion of abundant cell-specific proteins by this androgen-dependent tissue.     

Incorporation of 3H-Gibberellin A20 in roots of G2 peas

Following application of ³H-Gibberellin A₂₀ (GA₂₀) to roots of G2 pea seedlings and homogenization of the roots, about 3% of the radioactivity in the tissue could be precipitated from a 30,000 × g supernatant with trichloroacetic acid (TCA) (soluble fraction) while about 5% of the radioactivity pelleted at 30,000 × g (particulate fraction). The radioactivity in the particulate fraction was soluble in sodium dodecyl sulfate (SDS), but was not dialyzable and was insoluble in ethanol. Electrophoresis of the soluble fraction gave only one band of radioactivity, while that of the particulate fraction gave multiple bands. Acid hydrolysis of the soluble fraction released radioactivity that ran coincident with acid-treated GA₂₀ on silicic-acid column chromatography. The particulate fraction gave numerous radioactive peaks following acid hydrolysis, two of which were coincident with GA₂₀ and GA₂₉ (hydroxylation product of GA₂₀) on silicic acid chromatography. Treatment of the particulate and soluble fractions with RNase, DNase, and proteases showed a significant solubilization of radioactivity only with the proteases, suggesting that the GA is bound to a proteinaceous macromolecule. Complete proteolytic hydrolyis followed by thin layer chromatography showed 65% of the radioactivity from the soluble fraction running separately from free GAs or the individual amino acids; the particulate fraction gave mainly (60%) free GAs on enzymatic hydrolysis and much smaller amounts (17%) in a position separate from that of the GAs or amino acids. Binding of ³H-GA to protease-sensitive material was obtained with biologically active ³H-GA₂₀ and ³H-GA₁.     

THE INCORPORATION OF RADIOACTIVE AMINO ACIDS INTO BRAIN SUBCELLULAR PROTEINS DURING TRAINING

—Total proteins, free amino acids, tritiated water and subcellular proteins of mouse brain were examined for changes in radioactivity during operant conditioning after subcutaneous administration of labelled amino acids. The conditioning was based on appetitive learning, using sweetened milk as a reward. During training and incorporation for 20-30 min, both [³H]leucine and [1-¹⁴C]leucine underwent a significant increase in catabolism, resulting in a decreased radioactivity in the free amino acids. [2-²H]Methionine underwent a rapid loss of isotope, so that 90% of the radioactivity was in the form of tritiated water at the end of training, and this phenomenon masked any possible effect of training. The brain uptake of [³⁵S]methionine increased during the training, resulting in an increased radioactivity in the proteins. Uptake of [³H]lysine increased slightly during training only after 1 h incorporation and not after 20 or 30 min, as judged from a time course of radioactivity in the free amino acids. Incorporation into nuclear proteins increased selectively during 20 min, and into nuclear and cytosol proteins after 60 min incorporations. It is concluded that changes in the observed rate of incorporation of a precursor into brain subcellular proteins under the influence of behaviour might be the result of changes in precursor catabolism or uptake, or both, and that each amino acid behaves in a different way. Even the same amino acid gives different results depending on the isotope and its position in the amino acid.     

AXOPLASMIC TRANSPORT IN THE OPTIC NERVE AND TRACT OF THE RABBIT

The axonal transport of labelled proteins was studied in the optic system of adult rabbits after an intraocular injection of [³H]Ieucine. It was demonstrated that the precursor was incorporated into protein, which was transported along the axons of the retinal ganglion cells. Intraocularly injected puromycin inhibited protein synthesis in the retina and markedly inhibited the appearance of labelled protein in the optic nerve and tract. It was further demonstrated by intracisternal injection of [³H]leucine that an intraocular injection of puromycin did not affect the local protein synthesis in the optic nerve and tract. Cell fractionation studies of the optic nerve and tract showed that the rapidly migrating component, previously described as moving at an average rate of 110-150 mm/day, was largely associated with the microsomal fraction. About 40 per cent of the total protein-bound radioactivity in this component was found in the microsomal fraction and about 15 per cent was recovered in the soluble protein fraction. Most of the labelled material moving at a rate of 1-5-2 mm/day was soluble protein. The specific radioactivity of this component was about ten times greater than that of the fast one. In the slow component about 50 per cent of the radioactivity was found in the soluble protein fraction and about 10 per cent of the radioactivity was recovered in the microsomal fraction. Radioautography demonstrated incorporated label in the neuropil structures in the lateral geniculate body as early as 4-8 hr after intraocular injection. The labelling of the neuropil increased markedly during the first week, and could be observed after 3 weeks.     

EFFECT OF ACTINOMYCIN-D ON LABELLED MATERIAL IN THE RETINA AND OPTIC TECTUM OF GOLDFISH AFTER INTRAOCULAR INJECTION OF TRITIATED RNA PRECURSORS

—After injection of [³H]guanosine or [³H]uridine into the eye of goldfish, labelled acid-soluble radioactivity and RNA appeared in the contralateral optic tectum. When 0·1 μg actinomycin-D was injected into the eye 4 h before the precursor, the labelled RNA in the retina by 18 h after the injection was only 23 per cent of normal, but the acid-soluble radioactivity in the retina and the small amount of labelled acid-soluble material conveyed to the tectum were not significantly affected; by 15–20 days after the injection the acid-soluble radioactivity in the retina was reduced and the amount of labelled material conveyed to the tectum, including both RNA and acid-soluble fractions, was less than normal. When the actinomycin was injected at various times before or after the precursor and measurements were made 6 days later, it was found that the amount of labelled RNA conveyed to the tectum was maximally decreased if the inhibitor was given simultaneously with or up to 4 h before the precursor, whereas the amount of RNA was normal if the incorporation of the precursor had been allowed to proceed for 12 h before the inhibitor was given. This result would be consistent with the view that much of the RNA conveyed to the tectum had been synthesized in the retina within 12 h of the injection of the precursor, and had then presumably been axonally transported in the optic nerve to the tectum. However, since the acid-soluble material conveyed to the tectum was also reduced as a result of the actinomycin treatment, the results of these experiments with actinomycin do not unequivocally rule out the possibility that the RNA appearing in the tectum had been locally synthesized from the axonally transported acid-soluble material. In the retina, both the labelled RNA and acid-soluble fractions were reduced, to about 15 and 60 per cent of normal, respectively, without any relationship to the time between the injection of inhibitor and precursor. The discrepancy between the effects of the labelling of the retina and the labelling of material conveyed to the tectum could be correlated with the fact that the actinomycin caused severe damage to the retinal receptor cells, while leaving the ganglion cells relatively intact. The more pronounced effect of actinomycin on the receptor cells could in turn be correlated with the fact that these cells had a higher rate of RNA synthesis than the ganglion cells. This was demonstrated autoradiographically by the higher rate of incorporation of [³H]uridine into the receptor cells. Intracranial injection of actinomycin did not affect significantly the amount of labelled RNA conveyed to the tectum, which would argue against the local synthesis of this RNA. It is not certain, however, that the actinomycin penetrated deeply enough into the tectum to be effective.     

Arginine and lysine product inhibition of bovine adrenomedullary carboxypeptidase H, a prohormone processing enzyme

Carboxypeptidase H (CPH) is one of the later enzymes in the cascade of proteolytic steps required for the posttranslational processing of peptide hormone precursors, including processing of proenkephalin. In this study, CPH activity in the soluble and membrane fractions of enkephalin-containing bovine chromaffin granules was competitively inhibited by its products arginine and lysine. Ki values for arginine and lysine were 4.6 +/- 1.3 and 7.6 +/- 1.9 mM, respectively, indicating that arginine was a more effective inhibitor than lysine. Other amino acids (at 10 mM) had no effect. The in vivo intragranular concentrations of lysine and arginine are similar to the measured Ki values, indicating that product inhibition of CPH by basic amino acids may occur in vivo.     

Metabolism of [2-14C] acetate to amino acids and proteins in segments of bean seedling roots

Summary ¹⁴C from [2-¹⁴C] acetate was found to be incorporated into soluble and protein amino acids in substantial amounts by bean root apices. The ¹⁴C was spread through a wide range of amino acids in both these fractions. Glutamic acid was found to be heavily labelled with ¹⁴C in both soluble and protein amino acid fractions. The data are discussed in relation to present ideas on transport and utilization of amino acids in root systems.     

Human–Human Hybridomas from Lung Cancer Patients Secrete Anti-DNA Antibodies

The effects of modification of the arginine/lysine ratio of dietary protein on the cholesterol kinetics were studied in male rats. Single amino acids (lysine to soybean protein and arginine to casein) were added to approximate the arginine/lysine ratio in different proteins. After acclimation to these diets for 30 days, rats were administered intravenous [¹⁴C]cholesterol and oral [³H]cholesterol. Analysis of the die-away curve of [¹⁴C]cholesterol showed an apparent independence of cholesterol kinetics to the dietary manipulations, but there was a moderate reduction of the size of the slowly exchangeable pool and of the biliary concentration of cholesterol when lysine was added to soybean protein. Addition of amino acids neither influenced cholesterol absorption nor the fecal excretion of the radioactivities from labeled cholesterol. The results indicate that manipulating the arginine/lysine ratio of dietary protein by adding single amino acids is not necessarily effective in ameliorating cholesterol metabolism in rats, although the arginine addition caused a significant reduction of serum cholesterol and triglyceride.     

A method for the estimation of amino acid radioactivity in biological samples

A method for quantitative estimation of total radioactivity present in the free amino acid fraction of tissue samples has been described. Samples deproteinized with cold acetone were extracted, in acidic medium, with ethyl (peroxide free); after centrifugation, the aqueous phase was used for amino acid derivatization at 40°C for 15 h with 1-flouro-2,4-dinitrobenzene in bicarbonate-buffered medium. Aliquots of the derivatized samples were acidified and extracted twice again with ethyl ether. The combined organic phases were placed in glass scintilation vials, dried, and used for the determination of its radiactivity, corresponding to the radioactivity present in the free amino acid fraction of the sample. Deproteinized samples of rat blood plasma, as well as hen egg white and yolk were tested after addition of known quantities of ¹⁴C-labelled amino acids or glucose, for validation of the method. No glucose radioactivity was found in any of the extracted samples. All radioactivity added to the samples in the form of ¹⁴C-labelled alanine, glutamic acid, leucine and phenylalanine was quantitatively recovered in the derivatized fraction; only a fraction of arginine radioactivity was recovered.     

Changes in fungi with age

Summary The uptake and incorporation of l-phenylalanine and l-leucine into various cell fractions of Rhizoctonia solani and Sclerotium bataticola decreased with cell age. As would be expected, most of the radioactivity from the amino acids was distributed in the soluble and protein fractions. Ratios of the radioactivity in the soluble fraction to that in the protein fraction indicated that in R. solani the mechanism for protein synthesis decreased with age. However, in S. bataticola, the decrease in protein synthesis with age may be due to a decrease in permeability to the amino acids.     

An investigation of acid-soluble nuclear proteins of human leucocytes in relation to fraction RP2-L, a component of neoplastic cells

1. The claim that tumour cells contain a specific nuclear protein was investigated. The presence of this component was confirmed in Walker tumour cells by the chromatography on CM-cellulose of nuclear proteins labelled with [¹⁴C]lysine. This protein was studied further in a number of human leucocyte cells. 2. The labelling of leucocyte nuclear proteins with [¹⁴C]lysine was attempted during incubation and culture in vitro. Incorporation of the label into acid-soluble nuclear proteins was highest in normal lymphocytes cultured with phytohaemagglutinin, followed by chronic-myeloid-leukaemic leucocytes and mixed samples of normal leucocytes incubated in plasma. Little incorporation was seen in similar extracts of chronic-lymphatic or normal leucocytes. 3. Lymphocytes were the only cells that gave nuclear extracts with amino acid analysis similar to that of unfractionated histones. 4. Little of the [¹⁴C]lysine in nuclear extracts of incubated leucocytes proved to be of chromosomal origin. No evidence was found of an RP2-L component in the highly labelled nuclear extracts of phytohaemagglutinin-treated lymphocytes until after 6 days of culture with [¹⁴C]lysine. This component was soluble in saline. 5. Evidence is presented that fraction RP2-L is a non-histone protein constituent of cell nuclei whose labelling with [¹⁴C]lysine may be dependent on the metabolic state of the cell. Thus this component is not specific to the neoplastic state.     

RAPIDLY LABELLING AND EXPORTED NEURONAL PROTEIN; [3H]FUCOSE AS A PRECURSOR, AND EFFECTS OF CYCLOHEXIMIDE AND COLCHICINE

Abstract— The characteristics of a rapidly labelled and rapidly transported neuronal perikaryal protein fraction (Rose & Sinha . 1974a) were investigated in three experiments. (1) The kinetics of labelling of neuronal cell body and neuropil fractions from [³H]fucose were followed and shown to be similar to those from [³H]lysine, the label first appearing in the neuronal fraction and then being exported. The neuronal/neuropil incorporation ratio fell from 1.37 at 1 h to 0.77 at 4 h. (2) When cycloheximide (5 mg/kg) was injected intraperitoneally 15 min after [³H]lysine, incorporation into neuronal protein was inhibited to a greater extent (85%) than into neuropil (60%). (3) Colchicine was injected at a dose (40 μg/kg) sufficient to prevent accumulation of radioactively labelled protein into synaptosomes but insufficient to affect total incorporation of precursor into protein. [³H]Lysine was injected 1 h after colchicine and neurons and neuropil fractions made 1 h and 4 h later; colchicine inhibited the export of labelled protein from the neuronal perikaryon and its accumulation in the neuropil. We conclude that the rapidly labelled neuronal protein is partially glycoprotein in character and may be normally transported from the cell body by way of the axonal/(dendritic?) flow mechanism.