Pool Radioactivity Research Articles

The scintillometric evaluation of DNA repair synthesis can be distorted by changes of thymidine pool radioactivity

The induction of DNA repair synthesis by UV radiation and methylmethane sulphonate (MMS) in mammalian cell lines of human (EUE, HeLa, FT, KB) and hamster (CHO, BHK) origin has been evaluated by means of autoradiography and the scintillometric procedure which implied the use of hydroxyurea (HU) to suppress DNA replication. While with UV radiation both methods produce concordant positive results, in the case of MMS the evidence of DNA repair synthesis obtained from the autoradiograms is occasionally accompanied by a lack of increase of DNA radioactivity in the treated cultures, as detected by scintillation counting. In such instances MMS is shown to reverse the enhancement of pool radioactivity in the cultures incubated with HU and even to reduce the radioactivity of thymidine pool below control values. By normalizing DNA radioactivities on the basis of pool variations, the discrepancy between autoradiography and scintillation counting is solved. The chromatographic analysis of thymidine pool components justifies the normalization procedure as it demonstrates that also in cultures treated with MMS or MMS + HU pool variations closely parallel the variations of thymidine triphosphate (dTTP) level. The normalization of DNA radioactivities based on the overall pool radioactivities gives an improved evaluation of the actual rate of DNA synthesis. It can be recommended for screening studies of DNA repair inducers because it allows one to correct false negative results without producing false positive data. Compared with the dTTP levels, overall pool radioactivities used as normalizing factors still produce an underestimate of DNA repair when high doses of MMS are applied to hamster cell cultures.

Scintillometric determination of DNA repair in human cell lines: A critical appraisal

The ability of a variety of chemical and physical agents to stimulate DNA repair synthesis in human cell cultures was tested by a simplified scintillometric procedure, with the use of hydroxyurea (HU) to suppress DNA replicative synthesis. After incubation with [ 3H]thymidine, the radioactivity incorporated in to DNA was determined in controls (C) and treated (T) cultures and in the corresponding HU series (C HU, T HU). The ratios T HU/C HU and T HU/T:C HU/C, indicating absolute and relative increases of DNA radioactivity, were calculated. When both ratios were significantly higher than 1, they were taken as indices of DNA repair stimulation, whereas, no stimulation in inferred when both of them are ⩽1. The scintillometric estimate of DNA repair was always in agreement with the autoradiographic observations, so that the procedure adopted can be used as a rapid test for screening investigations. Agents giving a relative but no an absolute increase of DNA radioactivity are generally not inducers of repair synthesis as estimated by autoradiography. However, the same scintillometric results are also occasionally observed with DNA repair inducers, such as methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), owing to alterations of thymidine pool radioactivity. These chemicals, besides affecting the levels of labelled precursors in the intracellular pool in the T series, differently modified the increase of pool radioactivity which is a regular effect of HU. With such chemicals, DNA repair synthesis can be detected only after normalization of th DNA radioactivity on the basis of pool alterations. The quantitative value of the autoradiographic estimate of DNA repair is also affected by the changes in the radioactivity of the thymidine pool although autoradiography retains its qualitative value. Dimethylnitrosamine, mitomycin C and potassium dichromate, described by other authors as inducers of DNA repair, also gave negative results by the scintillometric procedure after normalization of DNA radioactivities. However, in our hands, these agents were unable to stimulated repair synthesis, according to the results of autoradiography and isopynic centrifugation. The proposed scintillometric procedure is effective in indicating false negative inducers of DNA repair, not giving rise to false positives.

Studies on mannosyl carrier function of retinol and retinoic acid in epithelial and mesenchymal tissues.

The mannosyl derivative of phosphorylated vitamin A, mannosylretinylphosphate, is synthesized in vivo and in vitro by most epithelial tissues, including mouse epidermal cells. This compound is but one component of the complex biosynthetic machinery responsible for the assembly of glycoproteins in mammalian membranes; its specific role remains to be understood, even though it has been established that it functions as a carrier of mannose mostly in a direct transfer to protein. The system of five conjugated double bonds appears necessary for this function, since upon saturation the resulting perhydroretinylphosphate is incapable of acting in mannosyl transfer to endogenous microsomal proteins. Using vitamin A - deficient hamsters and rats, retinoic acid was shown to be as active as retinol in restoring the in vivo in corporation of mannose into glycoproteins to normal levels within a short time, in liver and tracheal tissues. Retinoic acid was also active in increasing adhesive properties of spontaneously transformed mouse dermal fibroblasts (3T12 cells) in a reversible manner. The free carboxyl group appeared to be a requirement for this activity inasmuch as the lactonized form of 11-hydroxyretinoic acid was inactive. Even though retinoic acid, as well as retinol, was able to enhance adhesion of 3T12 cells, only the cellular retinoic acid-binding protein was detected, suggesting that the cellular retinol-binding protein is not a requirement for this activity. A metabolite of retinoic acid, compound X, different from retinol, was found incorporated into mannosylretinoidphosphate (MXP) by 3T12 cells. This derivative constitutes up to 40% of the total radioactive pool of derivatives of retinoic acid after 48 hours of labeling. These data suggest that retinol, as mannosylretinylphosphate, and retinoic acid, as mannosylretinoidphosphate, function as mannosyl carriers in biologic membranes.

Photorespiration and Malate Formation in Grape Leaves

Summary Excised grape leaves were fed 14 CO 2 in a closed assimilation chamber for 10 and 30 min, respectively, and the incorporation of radioactive label into the watersoluble substances was determined. Under the experimental conditions glycine/serine and sucrose constituted the main radioactive pools, whilst only traces of label were detectable in glycolate. However, administration of glycolate oxidase inhibitors (HPMS or GBS) through the leaf petiole prior to 14 CO 2 labelling led to an accumulation of 14 C-glycolate, thus indicating that in grape leaves a considerable portion of fixed carbon is normally metabolized via the photorespiratory pathway. Environmental conditions which favour photorespiration caused enhanced incorporation of 14 CO 2 not only into glycolate pathway intermediates but also into glycerate and malate. This finding suggests a close precursor-product relationship between the relevant photorespiratory compounds on the one hand and glycolytic C 3 -compounds and malic acid on the other.

Bleomycin-induced pulmonary fibrosis. Effects of steroid on lung collagen metabolism.

Using bleomycin-induced pulmonary fibrosis as a model, we have set out to study collagen metabolism in the fibrotic process. As was previously shown, intratracheal administration of bleomycin in the rat caused increased deposition and net synthesis of collagen in the lung. This was accompanied by marked increases in the tissue-free proline pool size and less dramatic increases in the pool's radioactivity when lung mince was pulsed with radioactive proline to measure the net collagen synthesis. Using a technique based on the quantitation of the rate of release of hydroxyproline-containing peptides when lung homogenates were incubated in calcium-containing buffer, lung collagenolytic activity was markedly diminished as a result of bleomycin treatment. Concomitant treatment with the steroid methylprednisolone did not affect significantly this decrease in lung collagenolytic activity. Such steroid treatment, however, prevented the increase in bleomycin-induced lung collagen deposition and partially suppressed total lung collagen synthesis, without affecting the net rate of lung collagen synthesis expressed per mg of DNA. Steroid treatment also inhibited the marked increase in tissue-free proline pool size and radioactivity caused by bleomycin. The mechanism of amelioration of the fibrotic response by the steroid is discussed.

Topography of rhodopsin in retinal rod outer segment disk membranes. Photochemical labeling with 1-azidopyrene

1-Azido[3H]pyrene ([3H]AP) has been synthesized with high specific radioactivity (3 Ci/mmol) and used to photochemically label retinal rod outer segment disk membranes. The reagent reacts with rhodopsin and a Mr approximately 240000 protein as well as with membrane lipids. When [3H]AP-rhodopsin is digested with thermolysin in the disk membrane, both membrane-bound fragments of rhodopsin, F1 and F2, are found to contain [3H]AP. Reaction of the reagent appears to be restricted to the lipophilic surface of rhodopsin inasmuch as the presence of the nitrene scavenger glutathione in the aqueous medium does not significantly reduce 3H incorporation into rhodopsin. Labeled F1 and F2 were prepared, their cyanogen bromide peptides partially separated, and specific radioactivities determined. A factor of 4.4-fold in specific radioactivities of peptide pools was found, which suggests that some specificity has been shown in the reaction of [3H]AP toward different surfaces of rhodopsin.

Alternative metabolic fates of thymine nucleotides in human cells.

Three types of experiments have been used to study the metabolism of thymine nucleotides by human cells. (1) Cells were labelled continuously with [3H]thymidine and the incorporation of label into DNA compared with the specific radioactivities of pools of individual thymine nucleotides separated by chromatography on polyethylene-imine-cellulose. (2) Cellular thymine nucleotides were labelled with [3H]thymidine at 13 degrees C, followed by incubation at 37 degrees C in unlabelled medium. Incorporation of label into DNA and loss of label from the nucleotide pools were monitored during the 'chase' period at 37 degrees C. (3) The experiments described in (2) above were repeated in the presence of the DNA-synthesis inhibitor cytosine arabinoside, in order to demonstrate more clearly and to quantify degradative pathways for thymine nucleotides. In phytohaemagglutinin-stimulated lymphocytes and in bone-marrow cells, only a proportion (25-60%) of labelled thymine nucleotide was incorporated into DNA, the rest being rapidly degraded and lost from the cell. In contrast, an established cell line (HPB-ALL) from a patient with acute lymphoblastic leukaemia of thymic origin incorporated 100% of its exogenously labelled thymine nucleotides into DNA. These results indicated that alternative metabolic routes are open to thymine nucleotides in human cells. In lymphocytes from patients with megaloblastic anaemia and in normal lymphocytes treated with methotrexate, the utilization of labelled thymine nucleotides for DNA synthesis was more efficient than in controls. These results offer an explanation for the observation of a normal pool of thymidine triphosphate in the cells of patients with untreated megaloblastic anaemia even though the amount of this compound available for DNA synthesis appears to be decreased.

Effect of age and dietary protein level on ovine nitrogen metabolism—II. Incorporation of [ 14C]valine into plasma proteins

Uptake from plasma and incorporation into plasma proteins of a pulse-dose of [ 14C]-valine were studied in 8-month and 9-yr old ewes fed corn-soy diets containing 9.6 and 19.9% crude protein in trials 1 and 2 respectively. Diets were fed at maintenance levels to 7 ewes of each age in each trial. In trial 1, total body plasma pool radioactivity of whole and deproteinized plasma decreased 68.6 and 98.9% in young ewes and 67.6 and 98.9% in old ewes over a 12 h period. In trial 2, decreases in radioactivity of these same pools were 55.1 and 98.6% for young ewes and 58.8 and 98.9% for old ewes. Total plasma protein ( TPP) and globulin ( G) concentrations were higher in old animals in both trials while albumin ( A) concentrations were comparable. In trial 1, changes in radioactivity of A and G pools from 2 to 12 h after dosing were 19.4% decrease and 41.3% increase for young ewes and 11.0 and 33.6% increase for old ewes. In trial 2, A radioactivity decreased 25.8% and G increased 40.1% for old ewes. Old ewes tended to have greater radioactivity than young ewes in teh G fraction in both trials. Old ewes consistently had a larger percent of TPP radioactivity in the G and a lower percent in the A fraction in trial 2. It was concluded that higher dietary protein intake resulted in increase incorporation or radioactivity into plasma proteins, and that animal age seemed to affect plasma protein concentrations and likely rate of incorporation of radioactivity into plasma proteins.

Metabolism of palmitate in perfused rat liver. Computer models of subcellular triacylglycerol metabolism.

1. In the preceding paper [Kondrup (1979) Biochem. J.184, 63-71] the separation of two major fractions of hepatic triacylglycerol was described. One fraction contained triacylglycerol from the endoplasmic reticulum and from the Golgi apparatus. The other fraction contained triacylglycerol from the cytoplasmic lipid droplets. In the present paper possible precursor-product relationships between the two fractions were investigated by means of computer models. 2. The fatty acids present in di- and tri-acylglycerol in the fractions isolated in the time studies were analysed by gas chromatography. From this analysis the relative specific radioactivities, and contents, of palmitate in acylglycerols in the two fractions at the various time points were calculated. 3. A computer was used to predict relative specific radioactivities of pools in defined models of hepatic triacylglycerol metabolism. The acceptability of the models was evaluated by comparing predicted with measured relative specific radioactivities. 4. It is suggested that triacylglycerol in cytoplasmic lipid droplets does not originate (a) directly from triacylglycerol in the endoplasmic reticulum, (b) from a sub-pool of it or (c) directly from non-esterified fatty acids entering the cell. Rather, it is formed from diacylglycerol (and acyl-CoA) in the endoplasmic reticulum. Diacylglycerol, on the other hand, is furnished in part by hydrolysis of triacylglycerol in the endoplasmic reticulum. 5. This suggestion is discussed in relation to previous models of hepatic fatty acid metabolism.

Globin biosynthesis in sickle cell, Hb SC, and Hb C diseases

The wide range of globin synthesis ratios reported in patients with sickle cell disease casts doubt on whether the presence of genes for alpha- or beta-thalassemia in combination with Hb S can be detected by globin synthesis studies. We have studied globin synthesis in 20 patients with Hb SS who had a mean betaA/alpha ratio of 1.05+/-0.04, similar to that of 28 control children. In nine of these patients the percentage of newly synthesized radioactive alpha-chains in dimer or monomer forms was 16.3%+/-1.3, also similar to the control subjects. The remainder of alpha-chain was in hemoglobin tetramer. In nine patients with Hb SC, the (non-alpha)/alpha ratio was 0.97+/-0.04, and the free alpha-chain pool radioactivity in four patients was 14.1%+/-4.2. In three patients with Hb CC, betac/alpha ratios were 0.99, 1.07, and 1.10. These results indicate that globin synthesis ratios and alpha-chain radioactivity in the free alpha-chain pool of peripheral blood of patients with Hb SS, Hb SC, and Hb CC have narrow ranges, close to those of nonthalassemic controls. The data provide a basis for detecting syndromes with Hb S or Hb C associated with alpha- or beta-thalassemia. This precise differentiation is important for clinical studies of severity in sickle cell disease and for genetic counseling.

Alteration of the pattern of hippocampal nerve cell RNA labelling during training in rats

RNA synthesis in the CA3 region of brain hippocampus was studied in rats trained to a handedness reversal task. The newly synthesized RNA, labelled by intraventricular injection of radioactive orotic acid, was extracted and analyzed by micromethods. The result was the following. In the trained animals the incorporation of the labelled precursor into RNA, corrected by the pool radioactivity, was almost double in comparison with the controls. In the trained animals there was a stimulation of the incorporation in the high molecular weight (> 18S) RNA region and in two specific low molecular weight regions: 8–9 and 16–17S. Taking in account previous results of other authors, these data appear to imply a stimulation of brain RNA synthesis as a result of training.

Completely isolated molluscan neurons. An ultrastructural study

The neurons of the mollucs Lymnaea and Helix isolated by fermentative digestion followed by mechanical treatment do not differ ultrastructurally from intact ones. These cells have sufficient metabolic reserves and incorporate into RNA 8% of the total radioactive pool, even more than neurons in ganglia under equal conditions. Neuronal damage can occur, mainly during the pipetting, and this is usually expressed in vacuolization of the cytoplasm. It is important to note that alterations in cell ultrastructure develop earlier than changes in the membrane electrical properties. The surface of the isolated neurons is enlarged two-fold due to the infoldings of the cell membrane. So, the specific resistance of soma membrane of these neurons was calculated as 78 ± 13kΩ · sq. cm. On the surface of isolated neurons scraps of glial and neuronal processes not connected with their own cell bodies, and as a consequence not powerful, are sometimes found. Some endings of the neuronal processes on the surface of isolated neurons are ultrastructurally similar to the axo-somatic synapses.

METABOLISM AND MODE OF ACTION OF ANDROGENS IN TARGET TISSUES OF MALE RATS

ABSTRACT In order to get more information on the mode of action of anti-androgens, two series with low but biologically active doses of cyproterone acetate were started. In the first experiments 12 μg of [3H] cyproterone acetate was injected intravenously into adult rats castrated 3 days before treatment. Thirty min after injection the radioactivity uptake in the target organs and other tissues was measured. The metabolites were separated by thin layer chromatography. A large pool of radioactivity could be shown in the liver. Thin layer chromatography revealed that in this pool cyproterone acetate had been converted by more than 80% to one metabolite. In blood plasma, too, the metabolite accounted for the major part of radioactivity. When compared to skeletal muscle, the prostate, seminal vesicles, and m. bulbocavernosus and m. levator ani accumulated more radioactivity. Within 30 min unchanged cyproterone acetate was retained selectively thus showing its relative high affinity to target organs. In a second experimental series, adult castrated male rats were given 10 μg of cyproterone acetate intravenously 30 min before the injection of [3H] testosterone or [3H]5α-dihydrotestosterone. Under this condition androgen uptake in target tissues was reduced to about 70 % of the control values. The data parallel the results of in vivo studies on cytosol receptor displacement of androgens by cyproterone acetate. In agreement with previous investigators no significant influence of the anti-androgen on androgen metabolism was observed. The importance of the findings concerning the mode of anti-androgen action is discussed.

Incorporation of Uracil by Synchronous Chlorella

AbstractLight‐dark synchronized Chlorella fusca was used to study the incorporation of radioactive uracil into the trichloroacetic acid insoluble fraction.In autospores the incorporation measured in darkened cells usually started immediately after uraci addition, and the time course showed three distinct linear phases with abrupt transitions between them. Chromatographic analyses of the radioactive pool components showed that the total amounts of monophosphate, diphosphate and triphosphate nucleotides of uracil did not limit the incorporation and thus did not cause the abrupt rate shifts.In autospores, 5, 9 and 14 h old cells, the initial incorporation rate increased with increasing initial uracil concentration to reach a constant value above 0.5 μ.M. Addition of glucose to the cells increased the incorporation rate over the whole uracil concentration range tested with autospores, but did not influence the rates of the other cells.The incorporation rate varied during the synchronous cycle in a manner which closely resembled the pattern for uptake and degradation rates, with the most pronounced similarity from 9 h onwards.

Turnover of ribosomal RNA in the submandibular gland of normal and isoproterenol-treated rats

From the decay in specific activity following the administration of 3H-cytidine and [ 3H-methyl]-methionine the half-life of ribosomal RNA in rat submandibular gland was determined. With [ 3H-methyl]methionine a half-life of 12 days of ribosomal RNA was obtained. This may be considered the minimal value since a shorter half-life, 7 days, was measured with 3H-cytidine as precursor. The difference in half-lives with the two methods of labeling is due to the presence of heterogeneous RNA sedimenting with the ribosomal RNA and labeled by 3H-cytidine but not methylated. An attempt was made to measure the half-life of ribosomal RNA in the submandibular gland during an accelerated hypertrophic and hyperplastic growth of the gland produced by the repeated administration of isoproterenol. When correction was made for the increase in RNA and ribosomal RNA that occurs during the enlargement of the gland the specific activity of ribosomal RNA increased instead of declining. During the experimental enlargement of the salivary gland the persistence of a radioactive pool excludes the possibility of measuring the turnover rate of ribosomal RNA accurately with methods presently available.

Fixative-soluble tritium pool size after tritiated thymidine injection into mice with skin stimulated by plucking.

Summary.— The size of the fixative-soluble tritium pool in skin has been studied in the hair growth cycle induced by plucking. The size of the pool appears to increase dramatically immediately after plucking and remains fairly high during the early phase of the growth cycle. In the middle of the cycle there is a second increase in size. Part of this radioactive pool is thought to be 3HTdR, or a substance capable of labelling S phase cells. The pool persists for several days. The thymidine incorporation quotient (T.I.Q.) shows much less scatter than the fresh or fixed tissue values or their difference. The T.I.Q. also changes during hair growth. The implications of these findings for cell kinetic studies using tritiated thyminine and autoradiography are discussed.

Inhibition of macromolecular synthesis in isolated mitochondria by phenylalanine and tryptophan

1. At 2 mg/ml phenylalanine (tryptophan) inhibited rat liver mitochondrial synthesis of DNA 28% (0%), RNA 13% (0%), glycoprotein 19% (22%) and protein 65% (50%). 2. At 2 mg/ml phenylalanine (tryptophan) inhibited rat cerebral cortex mitochondrial protein synthesis 81% (53%) but had no inhibitory effect on RNA, DNA, or glycoprotein synthesis. 3. Inhibition of the mitochondrial protein synthesis by 2 mg/ml phenylalanine or tryptophan was rapid, more effective with the L-isomers, and reversible. 4. The inhibition of protein synthesis by L-phenylalanine and L-tryptophan in the mitochondria was not related to the radioactive pool of amino acid ([ 3H]leucine). 5. Charging of tRNA by [ 3H]leucine in both the rat liver and cerebral cortex mitochondria was greatly decreased in the presence of 2 mg/ml of phenylalanine or tryptophan.

Synthesis of radioactive acetylcholine from ( 3 H)choline and its release from cerebral cortex slices in vitro.

Abstract— Guinea pig cerebral cortex slices were incubated for 60 min in a medium containing [3H]choline with or without the addition of 33 mM‐KCl for the last 30 min. KC1 caused the release into the medium of large amounts of both bioassayable and radioactive ACh, while at the same time their concentrations in the tissue decreased. The specific activity (d.p.m./pmol) of the ACh released by KC1 was greater than that released in control incubations, indicating that it comes from a newly synthesized, more radioactive store. The amounts of [3H]choline, [3H]ACh and the specific activity of tissue acetylcholine reached a plateau in the tissue 30 min after the addition of isotope. However isotopic equilibrium was not achieved because the specific activity of the ACh released, with or without KC1 in the subsequent 30 min, was less than the specific activity of the ACh remaining in the tissue. This implies the existence of a pool of ACh in the tissue which is turning over very slowly or is being synthesized from a less radioactive pool of choline. This pool of ACh does not contribute substantially to that released by KC1. Levorphanol at 10−3 M, as well as the analgesically inactive stereoisomer, dextrorphan, blocked the KCl‐stimulated release of both bioassayable and radioactive ACh. These drugs demonstrate the coupling of synthesis and release of ACh in cerebral cortex slices.

Arginine Transport and Metabolism in Osmotically Shocked and Unshocked Cells of Escherichia coli W

Abstract Exogenously derived arginine is extensively metabolized by early stationary phase cells of Escherichia coli W. This metabolism is substantial even during exposure to the substrate for only 15 sec at room temperature; at 300 sec only about 15% of the intracellular pool radioactivity is found in arginine. The major route of exogenous arginine metabolism is via an amineoxyacetic acid-inhibitable arginine decarboxylase which leads to the production and accumulation of large amounts of agmatine and putrescine. Much smaller amounts of ornithine, citrulline, proline, and glutamate also accumulate intracellularly. Cold osmotic shock treatment of the cells decreases the relative extent of arginine metabolism via the decarboxylase and increases metabolism via the other pathway (pathways). The net result is that proportionately more arginine is found in shocked cell pools than in the corresponding control cells. In the presence of 4 x 10-3 m amineoxyacetic acid the bulk of the pool radioactivity is found in arginine. Thus, under these conditions the pool radioactivity is a measure of arginine transport. Cold osmotic shock treatment reduces the amount of arginine-derived radioactivity associated with the cell in the presence and absence of 4 x 10-1 m amineoxyacetic acid. The shock treatment also reduces the capacity of the cell to transport exogenous lysine and leucine but not proline. In the presence of amineoxyacetic acid, l-lysine is a poor inhibitor of arginine transport, while l-canavanine, which itself is not well accumulated by these cells, is a modest inhibitor. l-Arginine is a good inhibitor of lysine transport while l-canavanine is a modest inhibitor. These data support the thesis that arginine and lysine are not transported by identical systems in this organism. Since lysine previously has been found to inhibit arginine uptake, these observations further support the conclusion that arginine transport cannot be reliably studied in E. coli without utilizing inhibitors such as amineoxyacetic acid which greatly attenuate the extensive metabolism of this amino acid.

Stimulation of Arginine Transport in Osmotically Shocked Escherichia coli W Cells by Purified Arginine-binding Protein Fractions

Abstract Cold osmotic shock treatment of early stationary phase Escherichia coli W cells reduced the capacity for arginine uptake and accumulation by about 25%. DEAE-cellulose chromatography of the shock fluid separated four protein-containing fractions having relatively specific arginine-binding activity. The reduced transport activity of shocked cells was partially restored by two of these purified protein fractions (I and III). The degree of restoration was linear between 5 and 10 µg per ml for Fraction I; above 10 µg per ml there was a marked reduction in the extent of stimulation. For Fraction III restoration of the initial rate (15 sec) was a curvilinear function of the protein concentration from 5 to 40 µg per ml. The degree of restoration of the steady state level of uptake (5 min) hardly changed within this protein concentration range. Fractions I and III have identical dissociation constants for arginine, the values ranging from 0.5 to 1 x 10-6 m. 14C-Arginine was not metabolized by concentrated crude shock protein. The material bound by this protein solution and by purified Fractions I and III was found to have the same Rf value as free arginine. Analysis of the intracellular pool radioactivity after exposure of shocked cells to 14C-arginine and to 14C-arginine plus optimal concentrations of Fractions I and III (in the presence of 4 x 10-3 m amineoxyacetic acid) showed that the bulk of the radioactivity (∼90%) remained in arginine, thereby showing that these proteins stimulate the arginine accumulation process. The proteins are specific in binding and stimulating the transport of arginine. They bound little or no lysine and canavanine and did not stimulate the transport of lysine in shocked cells. These data suggest that cold osmotic shock treatment of E. coli W releases proteins which may participate in the accumulation of arginine by these cells, and that these proteins are not capable of functioning in the accumulation of lysine.