Incorporation Of 32P-phosphate Research Articles

Phosphorylation of Gα11Protein Contributes to Agonist-Induced Desensitization of 5-HT2AReceptor Signaling

Agonist treatment causes desensitization of many G protein-coupled receptor systems. Recent advances have delineated changes in receptors in the desensitization response; however, the role of G proteins remains unclear. We investigated the role of phosphorylation of Galpha q/11 proteins in agonist-induced desensitization of serotonin 2A (5-HT2A) receptors. In an embryonic rat cortical cell line (A1A1v), 24-h treatment with 100 nM (-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI), a 5-HT(2A/2C) receptor agonist, decreased DOI-stimulated inositol phosphate accumulation and increased the phosphorylation of Galpha q/11 proteins, as demonstrated by immunoprecipitation of Galpha q/11 and both incorporation of 32P phosphate and labeling with a S/T/Y phosphorylation-dependent antibody. Treatment with DOI for 30 min induced desensitization but did not increase phosphorylation of Galpha q/11 proteins, suggesting that different mechanisms are involved in desensitization after short- and long-term treatments. Mutation of S154A in a protein kinase C (PKC) and calcium/calmodulin dependent kinase (CaMK) consensus site in Galpha11 significantly reduced DOI-stimulated phosphorylation of Galpha11 and DOI-induced desensitization of 5-HT2A receptor signaling. Inhibition of PKC and CaMK attenuated phosphorylation of Galpha q/11 proteins and DOI-induced desensitization of 5-HT2A receptors. Expression of Galpha11 S154D, a phosphorylation mimic, reduced DOI-stimulated inositol phosphate accumulation. DOI treatment for 24 h also produced heterologous desensitization, as indicated by decreased bradykinin-stimulated inositol phosphate accumulation. These data suggest that phosphorylation of Galpha11 protein by PKC and CaMK contributes to agonist-induced homologous desensitization of 5-HT2A receptor signaling as well as heterologous desensitization. The phosphorylation of Galpha protein represents a novel mechanism involved in regulation of receptor signaling and agonist-induced desensitization of G protein-coupled receptors.

Circadian regulation of phospholipid metabolism in retinal photoreceptors and ganglion cells.

The neural retina is a key component of the vertebrate circadian system that is responsible for synchronizing the central circadian pacemaker to external light-dark (LD) cycles. The retina is itself rhythmic, showing circadian cycles in melatonin levels and gene expression. We assessed the in vivo incorporation of 32P-phosphate and 3H-glycerol into phospholipids of photoreceptor cells (PRCs) and retina ganglion cells (GCs) from chicks in constant illumination conditions (dark: DD or light: LL) over a 24-h period. Our findings showed that in DD there was a daily oscillation in 32P-labeling of total phospholipids synthesized in GCs and axonally transported to the brain. This metabolic fluctuation peaked during the subjective night (zeitgeber time [ZT] 20), persisted for several hours well into the subjective day and declined at subjective dusk (ZT 10-12). PRCs also exhibited an in vivo rhythm of 32P-phospholipid synthesis in DD. This rhythm peaked around ZT 22, continued a few hours into the day and declined by the end of subjective dusk. The major individual species labeled 1 h after 32P administration was phosphatidylinositol (PI) in both PRCs and GCs. Rhythmic phospholipid biosynthesis was also observed in DD after 3H-glycerol administration, with levels in GCs elevated from midday to early night. PRCs exhibited a similar rhythmic profile with the lowest levels of labeling during midnight. Phosphatidylcholine (PC) accounted for the individual species with the highest ratio of 3H-glycerol incorporation in both cell populations at all phases examined. By contrast, in LL the rhythm of 3H-glycerol labeling of phospholipids damped out in both cell layers. Our findings support the idea that, in constant darkness, the metabolism of retinal phospholipids, including their de novo biosynthesis, is regulated by an endogenous circadian clock.

Homologous and Heterologous Phosphorylation of the Vasopressin V1a Receptor

The vasopressin V1a receptor undergoes homologous and heterologous desensitizations which can be mimicked by activation of protein kinase C. This suggests that phosphorylation of the V1a receptor may be involved in the desensitization mechanisms. Such a phosphorylation was presently investigated in HEK 293 cells stably transfected with rat vasopressin V1a receptor. Metabolic labelling and immunoprecipitation of epitope-tagged V1a receptor evidenced a 52-kDa band and a 92-kDa band. Glycosidase treatments and immunoblotting experiments suggest that the 52-kDa band corresponds to an immature unprocessed receptor protein, whereas the 92-kDa band would correspond to a highly glycosylated form of the mature V1a receptor. Exposure of the cells to vasopressin induced a selective 32P phosphate incorporation in the 92-kDa form of the receptor. This homologous ligand-induced phosphorylation was dose dependent with maximal phosphate incorporation corresponding to four times the basal level. Stimulation of the endogenous phospholipase C-coupled m3 muscarinic receptor by carbachol-induced heterologous phosphorylation of the V1a receptor whose amplitude was half that of the homologous phosphorylation. This heterologous phosphorylation was associated with a reduced vasopressin-dependent increase in intracellular calcium.

Decreased phosphorylation of GAP-43/B-50 in striatal synaptic plasma membranes after circling motor activity.

The effects of spontaneous circling motor activity on the in vitro phosphorylation of the protein kinase C substrate GAP-43/B-50 was studied on striatal membranes of developing rats (30 days of age). At this time of postnatal development, permanent plastic changes in cholinergic and dopaminergic systems are produced by physiological motor activity. Exercised animals showed a significant reduction of 31% in the level of GAP-43/B-50 endogenous phosphorylation in the contralateral striatum respect to the ipsilateral side (P < 0.01), while control animals did not show asymmetric differences. Compared to controls, the contralateral striatum of exercised animals showed a 33% reduction in the incorporation of 32P-phosphate into GAP-43/B-50 30 minutes post-exercise (P < 0.01). This change in GAP-43/B-50 phosphorylation was correlated with the running speed developed by the animals (r:0.8986, P = 0.015). GAP-43/B-50 immunoblots revealed no changes in the amount of this protein in any group. Moreover, a significant variation of 25% (P < 0.05) in the PKC activity was seen between both exercised striata. Interhemispheric differences were not found in control animals. We conclude that endogenous phosphorylation of this protein is also altered by motor activity in the same period that permanent changes in striatal neuroreceptors are triggered after motor training.

Immunochemical characterization of the gap junction protein connexin45 in mouse kidney and transfected human HeLa cells

Antibodies to the gap junction protein connexin45 (Cx45) were obtained by immunizing rabbits with fusion protein consisting of glutathione S-transferase and 138 carboxy-terminal amino acids of mouse Cx45. As shown by immunoblotting and immunofluorescence, the affinity-purified antibodies recognized Cx45 protein in transfected human HeLa cells as well as in the kidney-derived human and hamster cell lines 293 and BHK21, respectively. In Cx45-transfected HeLa cells, this protein is phosphorylated as demonstrated by immunoprecipitation after metabolic labeling. The phosphate label could be removed by treatment with alkaline phosphatase. A weak phosphorylation of Cx45 protein was also detected in the cell lines 293 and BHK21. Treatment with dibutyryl cyclic adenosine- or guanosine monophosphate (cAMP, cGMP) did not alter the level of Cx45 phosphorylation, in either Cx45 transfectants or in 293 or BHK21 cells. The addition of the tumor-promoting agent phorbol 12-myristate 13-acetate (TPA) led to an increased 32P phosphate incorporation into the Cx45 protein in transfected cells. The Cx45 protein was found in homogenates of embryonic brain, kidney, and skin, as well as of adult lung. In kidney of four-day-old mice, Cx45 was detected in glomeruli and distal tubules, whereas connexin32 and -26 were coexpressed in proximal tubules. No connexin43 protein was detected in proximal tubules. No connexin43 protein was detected in renal tubules and glomeruli at this stage of development. Our results suggest that cells in proximal and distal tubules are interconnected by gap junction channels made of different connexin proteins. The Cx45 antibodies characterized in this paper should be useful for investigations of Cx45 in renal gap junctional communication.

Characterization and autophosphorylation of Ca 2+/calmodulin-dependent protein kinase in the postsynaptic density of the rat forebrain

The enzymatic and regulatory properties of Ca 2+/calmodulin-dependent protein kinase in the postsynaptic density (mPSDp CaM kinase) of the rat forebrain was compared with those of soluble Ca 2+/calmodulin-dependent protein kinase II (CaM kinase II). mPSDp CaM kinase was different from soluble CaM kinase II in terms of substrate specificity, regulatory consequences and sites of autophosphorylation. Both soluble and PSD kinases generated Ca 2+-independent activity by autophosphorylation and Ca 2+-independent activity almost reached the maximum during the first minute of autophosphorylation. Ca 2+-independent activity of mPSDp CaM kinase was more stable than that of the soluble kinase under autophosphorylating conditions. Autophosphorylation of the kinases decreased the mobility of the kinases on SDS-polyacrylamide gels. The mobility shift and determination of 32P phosphate incorporation into the kinases demonstrated that there were three species in mPSDp CaM kinase α isoform: two active forms with and without the mobility shift (about 22 and 19%, respectively), and an inactive form (about 59%). However, there was only one species in the soluble kinase α isoform, which was active. The maximum incorporation of 32P phosphate into mPSDp CaM kinase α isoform was less than that of the soluble kinase. Tryptic peptide analysis indicated that the phosphorylation sites of mPSDp CaM kinase α isoform differed from those of the soluble kinase.

Measurement of relative amounts of phospho- and dephospho-B-50(GAP-43) peptides by fast atom bombardment-mass spectrometry

The biological role of phosphoproteins depends upon their degree of phosphorylation in vivo. Methods currently available to measure the degree of phosphorylation of a protein involve indirect procedures to detect the 32P-phosphate incorporation. We report here a direct method to measure relative amounts of phospho- and dephospho-forms of peptides based upon a mass spectrometric technique. The intensities of the molecular ions corresponding to the two forms of the peptides are proportional to their relative amounts. This is demonstrated for a peptide fragment of the protein B-5O(GAP-43) and for kemptide, respectively substrates for protein kinases C and A, and demonstrates the applicability of fast atom bombardment-mass spectrometry to quantitate peptides bearing post-translational modifications.

Phosphorylation ofEscherichia coli NADP+-specific glutamate dehydrogenase

Glutamate dehydrogenase fromEscherichia coli is phosphorylated in vitro in an ATP-dependent enzymatic reaction. The phosphorylated protein, when exposed to acid conditions, releases the phosphate; this implies that the phosphorylation site is not on a serine, tyrosine, or threonine residue(s). Treatment of glutamate dehydrogenase with diethyl pyrocarbonate, a highly specific histidine-modifying reagent, blocks incorporation of32P-phosphate from [γ-32P]ATP into the enzyme, suggestive that the phosphorylation site is a histidine residue(s). The phosphorylated glutamate dehydrogenase was identified on the basis of its comigration with highly purified glutamate dehydrogenase, isolated fromE. coli, on denaturing, nondenaturing, and isoelectric focusing polyacrylamide gels and by sequence analysis.

Tetradecanoyl phorbol-13-acetate counteracts the responsiveness of cultured thyroid cells to thyrotropin.

We have studied the effects of TPA on the metabolism of porcine thyroid cells cultured for 1-4 days in the absence (control cells) and in the presence of 0.1 mU/ml TSH (TSH cells). The phospholipid turnover, evaluated after a 2 hr incorporation of 32P-phosphate into phospholipids, is markedly modified by the presence of TPA (1.5 microM, 2 hr) in the incubation medium of control and TSH treated cells. The total incorporation is 3-4 times higher than untreated cells, the labelling of phosphatidylinositol (PI) is slightly decreased or unchanged whereas that of phosphatidylcholine (PC) is strongly increased. The increased labelling of PI, promoted by an acute TSH treatment is counteracted by TPA. This TPA effect is not observed when prelabelled cells are challenged for 5 min with the drug. A similar effect is observed when 10 nM TPA is added in the culture medium for 20 hr. The addition of TPA does not affect significantly the protein iodine content in 3 or 4 days control cells incubated for 45 min or 2 hr with 125I-iodine, but dramatically decreases the very high iodination rate of TSH cells. We have tested the TPA effect on the cyclic AMP accumulation for the last 5 min of a 2 hr incubation. TPA inhibits by about 50-80% the stimulation evoked by TSH and only by 10% that evoked by forskolin (0.1 mM). These results suggest a possible link between the PC turnover and the adenylate cyclase responsiveness to TSH and the iodination rate.

Pressor-type vasopressin receptors in the adrenal cortex: properties of binding, effects on phosphoinositide metabolism and aldosterone secretion.

Specific, high affinity sites that bound tritium-labeled arginine-vasopressin (3H-AVP) were detected in a crude membrane fraction of rat adrenal capsules (chiefly zona glomerulosa). Binding displacement experiments with peptide analogs of AVP suggested that the binding site is a pressor (V1) type receptor for AVP. When added to dispersed rat adrenal glomerulosa cells, vasopressin (10(-8)-10(-6)M) stimulated the incorporation of 32P-phosphate into phosphatidylinositol, and the effect was blocked by the AVP receptor antagonist peptide d(CH2)5Tyr(Me)AVP. Vasopressin also increased the breakdown of phosphatidylinositol-4,5-bisphosphate within 1 min after its addition to the incubation medium. Superfused zona glomerulosa cells responded to AVP (10(-8)-(-6)M) by increasing their aldosterone production. The response could be blocked by the antagonist peptide. These data show that functionally active V1 receptors are present in rat glomerulosa cells, and suggest that vasopressin may regulate the function of the adrenal glomerulosa.

Phorbol myristate acetate stimulates formation of phosphatidyl inositol 4-phosphate and phosphatidyl inositol 4,5-bisphosphate in human platelets

The tumor-promoting phorbol ester PMA increased the incorporation of 32P-phosphate into PIP (150%) and PIP2 (50%) in human platelets over the same range of concentrations that stimulate protein kinase C activity (i.e. 1–10 ng/ml). PMA also increased the total content of PIP (2.5-fold) and PIP2 (1.5-fold). The increase in 32P-PIP and 32P-PIP2 was 50% completed at 2 min after 10 ng/ml PMA, and was maximal by 20 min. The increase in PIP and PIP2 was accompanied by a fall of 32P-PI and PI mass over the same time period and concentration range of PMA, but no 32P-PA was fourmed, indicating that phosphoinositide hydrolysis by phospholipase C was not stimulated. Inhibition of phospholipase C activity by increasing platelet cyclic AMP did not duplicate the effects of PMA. We conclude that PMA may directly affect inositol lipid kinases and/or phosphatases, or that PMA stimulation of protein kinase C provides feedback regulation of the enzymes that determine the levels of polyphosphoinositides involved in transmembrane stimulus-response coupling.

Phosphate depletion decreases mitogen-mediated stimulation of phospholipid synthesis in human peripheral lymphocytes

Concanavalin A-mediated stimulation of 32P-phosphate incorporation into phospholipids of human peripheral lymphocytes is comparatively studied in normal and phosphate-depleted media. In the phosphate-depleted medium, 2 hours after the start of cell activation, the stimulation sharply decreases for phosphatidylinositol (6.5-fold) and for phosphatidylcholine (in the latter case, the stimulation is even replaced by a slight inhibition of the incorporation). These results must be related to the rate-limiting effect of inorganic phosphate on ATP formation and thus on phospholipid synthesis, an effect which may be particularly pronounced when there is both phosphate depletion and cell activation.

Changes in phospholipid metabolism of SV3T3 transformed fibroblasts restricted by plating and cultivation at low serum concentration and subsequently re-stimulated by serum

We have studied 32P-phosphate incorporation, using 1 hour pulses, into phospholipids of SV3T3 transformed fibroblasts plated and cultured in a 1% serum medium. Two successive periods were observed: On the first 2 days after plating, cells grow exponentially with a lowered rate of multiplication. The percentages of incorporation of 32P-phosphate into the different phospholipids are then unchanged, but the rate of incorporation is decreased. On the 4th day, cells are almost completely arrested, and the percentages of incorporation into the different phospholipids are then sharply altered. If, during the first period, cells are subsequently re-stimulated by a medium containing 10% serum, i.e. by simply increasing the rate of exponential growth, it can be observed a response of phospholipid metabolism (for instance the “PI response”) similar to that which was previously reported as characteristic of untransformed fibroblasts.

Is phosphatidic acid a calcium ionophore under neurohumoral control?

There has recently been renewed interest in a previously observed phenomenon1 whereby hormones and neurotransmitters alter the rate of incorporation of 32P-phosphate into phospholipids, specifically, phosphatidylinositol (PI) and phosphatidic acid (PA)2. This has arisen from a theory formulated by Michell and coworkers which postulates that this ‘phospholipid effect’ is in some way intimately involved in the mechanism by which certain neurotransmitters and hormones activate membrane Ca gates2–5. However, although Michell's observations are consistent with a phospholipid involvement probably somewhere between receptor occupation and Ca gating, they do not suggest what the specific role of the phospholipids in the Ca gating mechanism might be. Recently, Salmon and Honeyman suggested6 that the critical event may be the net formation of PA following the net breakdown of PI which occurs in pancreas7, platelets8 and smooth muscle6,9. PA has been shown to behave as a Ca ionophore in a Pressman chamber10, and thus it is possible that an increase in PA concentration in cellular membranes might increase the Ca permeability of those membranes (see also ref. 5). In the rat parotid, receptors associated with Ca gating (muscarinic, α-adrenergic and substance P) show a phospholipid effect whereas β-adrenergic receptors (which act on adenylate cyclase) do not11,12. The phospholipid effect is Ca independent, and is not produced by the divalent cationophore A23187 (refs 11,12). Also, an easily quantifiable response of this tissue (K efflux measured as release of 86Rb) is absolutely dependent on the concentration of external Ca (ref. 13) and, by inference, on the magnitude of Ca influx14. We report here evidence suggesting that PA, which is formed during the reaction sequence of the phospholipid effect, may directly mediate the inward movement of Ca that results from activation of surface membrane receptors.

Two-dimensional gel analysis of cyclic amp effects in cultured s49 mouse lymphoma cells: Protein modifications, inductions and repressions

In this study, we used two-dimensional gel electrophoresis to analyze the responses of cultured S49 mouse lymphoma cells to incubation with analogs or inducers of cyclic AMP (cAMP). Putative phosphorylations were detected by charge alterations in proteins labeled with 35S-methionine and, in some cases, confirmed by labeling with 32P-phosphate. We assessed the relative stabilities of proteins affected by cAMP, the periods of susceptibility of proteins to cAMP-dependent modification and any cAMP-mediated changes in protein synthesis or stability. Five proteins (of about 650 resolved) behave as expected for “orthodox” substrates of a cAMP-activated protein kinase: both newly synthesized and prelabeled forms of these proteins are subject to modification; this modification involves an acidic charge shift of about one unit; and cAMP-mediated conversion of these proteins to their modified forms is virtually complete. The acidic forms of at least three of these proteins also exhibit cAMP-mediated increases in 32P-phosphate incorporation. Each protein comprised less than approximately 0.005% of cellular protein. Under basal conditions they appear to be phosphorylated to an extent about 20–30% of that found in fully stimulated cells. Nine proteins show cAMP-dependent changes in rates of synthesis with six inductions and three repressions. Most of these changes are of a magnitude of about 3 to 5 fold, and reach their maximal extents after about 4–5 hr of exposure to dibutyryl cAMP. In addition to the phosphorylations, inductions and repressions mentioned above, approximately 12 other reproducible cAMP-dependent changes in protein patterns are observed. Mutant cell lines deficient in catalytic activity of cAMP-dependent protein kinase show none of the changes in protein pattern attributable to cAMP.

Activation, after medium replacement, of 32P-phosphate incorporation into phospholipids of succinyl-concanavalin A arrested fibroblasts

When succinyl-concanavalin A arrested fibroblasts are incubated in culture medium lacking succinyl-concanavalin A, the cell cycle is triggered, and S phase occurs 12–14 hours later. In pulse experiments, 32P-phosphate incorporation into cell phospholipids is shown to fastly increase. When succinyl-concanavalin A arrested cells are incubated in succinyl-concanavalin A containing culture medium, the cells remain arrested, and yet an initial and transient activation of 32P-phosphate incorporation into phospholipids is observed.

Decreased incorporation of phosphate and amino acids into low-solubility, nuclear proteins in rapidly growing hepatomas

1. 1. The incorporation of 32P-phosphate and 3H-amino acids was studied in nuclear protein fractions of normal and neoplastic liver. 2. 2. The most marked change in hepatomas was a decreased incorporation of isotope into a low solubility fraction of non-histone proteins in rapidly growing tumors. 3. 3. The phosphate content of this fraction was lower in Morris hepatomas 9618A 2 and 7777 than host livers. 4. 4. Significant decreases were not seen in slowly growing hepatomas and regenerating rat liver.

Cyclic Amp-Dependent Protein Kinase in Rat Adrenal Mitochondrial Fraction

Although it has been proposed that cyclic AMP is an intracellular mediator in the stimulatory effect of ACTH on steroidogenesis, and that the action of cyclic AMP is exerted through the activation of protein kinase, it still remains to be resolved whether cyclic AMP-dependent protein kinases present in adrenocortical cells bear any direct or indirect relationship to steroidogenesis. My previous observation that cyclic AMP acts directly on rat adrenal mitochondrial fraction to stimulate its steroidogenesis suggests that cyclic AMP-dependent protein kinase might be present in mitochondria per se. The present studies were undertaken to examine whether cyclic AMP-dependent protein kinase activity and the existence of its substrate could be demonstrated in rat adrenal mitochondrial fraction. Adrenal subcellular fractions were obtained from male Sprague-Dawley rats pretreated with dexamethasone. Protein kinase activity was determined in a 0.2 ml reaction mixture containing 4.1 mM phosphate buffer (pH 6.5), 10 mM magnesium acetate, 0.3 mM EGTA, 10 mM sodium fluoride, histone (0.2 mg/tube), ATP-γ-32P (2.5 μM, 0.2~3 μCi/tube) and subcellular fraction, with or without cyclic AMP. Incubation was carried out at 30°C for 5 min., and trichloroacetic acid-precipitable radioactive phosphate was assayed. Cyclic AMP-dependent protein kinase activity was detected in the mitochondrial fraction as well as in soluble and microsomal fractions. The possibility of contamination of the mitochondrial fraction by other fractions was checked by comparing enzyme activities of protein kinase and various marker enzymes, 21α-hydroxylase, glucose-6-phosphate dehydrogenase, β-glucuronidase and 11β-hydroxylase, in each fraction. Contamination of microsomal and soluble fractions was found negligible, although the lysosomal enzyme activity was to some extent detectable in the mitochondrial fraction. In order to examine the ability of mitochondrial proteins to serve as substrates for mitochondrial protein kinase, mitochondrial fraction was incubated with ATP-γ-32P and Mg++ without histone. 32P-phosphate incorporation into the mitochondrial protein was clearly demonstrated. When phosphorylated mitochondrial proteins were separated by SDS-polyacrylamide gel electrophoresis, several radioactive peaks with a predominant peak were observed. Cyclic AMP stimulated incorporation of phosphate into several protein bands on the gel without significant effect on the major peak. These findings suggest that cyclic AMP-dependent protein kinase is present in adrenal mitochondria, and that a small but significant amount of mitochondrial protein undergoes phosphorylation by endogenous protein kinase in response to cyclic AMP. Therefore, cyclic AMP, by acting directly on adrenal mitochondria, might regulate its metabolic processes including steroidogenesis, and thus might act as the intracellular messenger of ACTH action.

Sphingomyelin and other phospholipid metabolism in the rabbit atheromatous and normal aorta.

Hypercholesterolemia induces atheroma formation, and the concentration of sphingomyelin is increased compared with that in the normal aortic intima-media. The aortic intima-media sphingomyelin concentration appears to increase exponentially with time on the cholesterol diet. Moreover, the entry rate of serum sphingomyelin into the aortic wall also appears to increase exponentially with time on the cholesterol diet and with the extent of aortic exposure to hypercholesterolemia. 32P-Phosphate incorporation into sphingomyelin and other phospholipids in the perfused rabbit aorta does not increase with atheromatosis, but the rate of sphingomyelin entry increases 27-fold during approximately the same period of atheroma formation and can account for all of the increase in sphingomyelin concentration in the intima-media.

Effects of X-Irradiation on DNA Precursor Metabolism and Deoxyribonucleoside Triphosphate Pools in Chinese Hamster Cells

The effect of x-irradiation on DNA precursor metabolism and deoxyribonucleoside triphosphate acid-soluble pools was examined. Pulse-labeling experiments indicated that the rates of incorporation into DNA of thymidine and the ribonucleosides and deoxyribonucleosides of adenine and guanine were similarly reduced in a biphasic manner with increasing doses of x-irradiation. The incorporation rate of cytidine into DNA was reduced after irradiation, while the concurrent incorporation rate of deoxycytidine was greatly stimulated. Incorporation of32 P-phosphate pulse label into nondialyzable DNA after irradiation mimicked that of thymidine; nucleotide analysis of enzymatically degraded DNA pulse-labeled with32 P-phosphate confirmed the results obtained with individual ribonucleosides and deoxyribonucleosides. The purine moieties from deoxyadenosine and deoxyguanosine were efficiently incorporated into RNA. Radiation had no detectable effect on either incorporation into RNA or on the relative distribution of radio...