NUCLEAR CHROMATIN PROTEINS FROM RABBIT CEREBRUM, CEREBELLUM AND LIVER: SYNTHESIS AND PHOSPHORYLATION

Abstract— In order to investigate synthesis and phosphorylation of the various fractions of nuclear proteins. [³H]leucine and [³²P] phosphate incorporation were studied with tissue slices in vitro. Cerebral cortex and cerebellum were used to delineate the similarity and dissimilarity within CNS, and liver was taken to compare the extraneural organ. There were significant differences in [³H]leucine incorporation into nuclear proteins among those tissue sources examined, while [³²P]phosphate incorporation showed very similar results among them. Although the acidic chromatin protein demonstrated high activity in each tissue source for both synthesis and phosphorylation, 0.14M-NaCl soluble protein showed the activity as high as or even higher than the acidic chromatin protein. Both [³H]leucine incorporation and [³²P]phosphate incorporation were relatively low in histone. When the acidic chromatin protein was further fractionated with SDS-acrylamide gel electrophoresis, significant difference was found between CNS tissue and liver for synthesis and phosphorylation. However, considerable difference was also observed even between cerebral cortex and cerebellum. The present investigation demonstrated complicity and diversity of nuclear chromatin proteins in different organs, not only for their protein constituents but also for their synthesis and phosphorylation.     

NOREPINEPHRINE INCREASES THE [32P]LABELLING OF A SPECIFIC PHOSPHOLIPID FRACTION OF POST-SYNAPTIC PINEAL MEMBRANES

Abstract— Cultured pineal glands incorporated ³²P into membrane phospholipids. Treatment of cultured glands with norepinephrine, which is known to stimulate membrane- bound pineal adenyl cyclase and to increase the production and secretion of melatonin, stimulated the incorporation of ³²P into a phospholipid fraction of membranes and particulates containing phosphatidyl serine and phosphatidyl inositol. The labelling of other phospholipid fractions and the total ³²P in the gland were not changed by norepinephrine treatment. Experiments with chronically-denervated pineal glands indicated that the effect of norepinephrine on the [³²P]labelling of phospholipids occurred at a postsynaptic site. When norepinephrine-stimulated secretion of melatonin was partially inhibited by p -chlorophenylalanine (a compound which blocks the synthesis of melatonin precursors), the norepinephrine-stimulated labelling of phospholipids was still observed. Conversely, when melatonin secretion was stimulated in the absence of norepinephrine by treatment with the immediate precursor of melatonin, N -acetylserotonin, a stimulation of ³²P- labelling of phospholipids did not occur. These observations suggest that the increased [³²P]- labelling of a phospholipid fraction caused by the norepinephrine treatment is not related to the secretion of melatonin. This effect on phospholipids may be associated with the interaction of norepinephrine with a membrane-bound postsynaptic receptor. Stimulation by norepinephrine of [³²P]-incorporation into phospholipids has not been previously reported to occur in a tissue in which cholinergic fibres are absent.     

AN INVESTIGATION OF THE POSSIBLE ROLE OF PHOSPHOINOSITIDES AS REGULATORS OF ACTION POTENTIALS BY STUDYING THE EFFECT OF ELECTRICAL STIMULATION, TETRODOTOXIN AND CINCHOCAINE ON PHOSPHOINOSITIDE LABELLING BY 32P IN RABBIT VAGUS

Abstract— The effect of electrical stimulation, tetrodotoxin and cinchocaine (the latter two substances abolish action potentials) on the incorporation of [³²P]orthophosphate into the phosphoinositides of isolated rabbit vagus nerve has been studied. Electrical stimulation, or treatment with tetrodotoxin, had little significant effect on the incorporation of [³²P]orthophosphate into the phosphoinositides. Cinchocaine, however, caused a 3.5–4.4-fold increase ( P = < 0.001) in monophosphoinositide labelling. These findings are discussed in view of the possible function of the phosphoinositides in the nervous system.     

Effect of Protein Kinase C Activation on the Release of [3H]Acetylcholine in the Presence of Vesamicol

Abstract: The present work tested whether pharmacological activation of protein kinase C (PKC) influences the release of [³H]-acetylcholine ([³H]ACh) synthesized in the presence of vesamicol, an inhibitor of the vesicular acetylcholine transporter (VAChT). Newly synthesized [³H]ACh was released from hippocampal slices by field stimulation (15 Hz) in the absence of vesamicol, but as expected [³H]ACh synthesized during exposure to vesamicol was not released significantly by stimulation. Treatment of slices with the PKC activator phorbol myristate acetate (PMA) decreased the inhibitory effect of vesamicol on [³H]ACh release. The effect of PMA was dose-dependent, was sensitive to calphostin C, a PKC-selective inhibitor, and could not be mimicked by α-PMA, an inactive phorbol ester. PMA did not alter the release of [³H]ACh in the absence of vesamicol, suggesting that the site of PKC action could be related to the VAChT. In agreement with this observation, immunoprecipitation of VAChT from ³²P-labeled synaptosomes showed that phosphorylation occurs and that incorporation of ³²P in the VAChT protein increases in the presence of PMA. We suggest that PKC alters the output of [³H]ACh formed in the presence of vesamicol and also provide circumstantial evidence for a role of phosphorylation of VAChT in this process.     

Phosphorylation and Fucosylation of Myelin Protein In Vitro by Sciatic Nerve from Developing Rats

Abstract: Proteins of the paniculate fraction of sciatic nerve of rats ranging from 1 to 55 days of age were analyzed by polyacrylamide gel electrophoresis. The major myelin protein, P₀, could not be detected at 1 day of age, but by 10 days it comprised from 15 to 20% of the particulate protein, the same proportion as in adult rats. Growth of nerve continued throughout the period studied. Rat sciatic nerves were incubated with [³²P]orthophosphate or [³H]fucose. Particulate matter proteins from sciatic nerve (and in certain cases proteins of myelin purified from sciatic nerve) were separated by polyacrylamide disc gel electrophoresis and the distribution of protein and of radioactivity along the gels was determined. [³²P]Phosphate appeared to label all myelin proteins. Labeling with fucose was more specific; myelin basic proteins were not fucosylated. A developmental study showed that sciatic nerves from 2-day-old rats could incorporate radioactive fucose and [³²P]-phosphate into several proteins at the P₀ region of polyacrylamide gels. Specific radioactivity of [³H]fucose in P₀ protein was highest in preparations from 5-day-old rats and declined by 80% over the next 5 days as it was diluted by accumulating myelin. The specific radioactivity of incorporated [³²P] phosphate was high at the early age points and declined as a result of the accumulation of compact myelin. The results indicate an association of fucosylation and/or phosphorylation with some step in the formation of myelin.     

A 30-kDa Protein in the Surface Complex and Flagella of Euglena has Protein Kinase Activity

ABSTRACT A protein kinase (PK) was partially purified from NaCl extracts of the cell surface complex of Euglena using DEAE-cellulose chromatography. Tubulins extracted either from flagella or from the cell surface complexes of Euglena were readily phosphorylated when incubated with [γ-³²P]-ATP and the PK. Protein kinase activity was augmented with 5 mM Mn²⁺ or Mg² and was inhibited or had greatly reduced activity with 5 mM Ca²⁺, Co^2-, Cu²⁺ or Zn²⁺. Incorporation was much lower when [γ-³²P]-GTP was the phosphate donor. Serine and threonine were the major radiolabeled phosphoamino acids in tubulins; label was also found in phosphotyrosine. Alpha-tubulin solubilized from flagella was a relatively poor substrate for the PK, but a Euglena α-tubulin cDNA overexpressed as a Trx-fusion protein incorporated [γ-³²P]-ATP into serine and threonine when incubated with cell surface extracts. Alpha- and β-tubulins from cell surface complexes were equally good substrates for the PK. No incorporation was observed in intact microtubules either from the cell surface complex or from isolated flagella. In-gel assays identified a polypeptide of about 30 kDa that phosphorylated tubulins in extracts of both flagella and the cell surface complexes, and dephosphorylated casein was a competitive substrate for the partially purified kinase. In vivo incubation with [³²P]-orthophosphate produced numerous radiolabeled bands in acrylamide gels of NaCl extracts of the cell surface complex, but none of these bands could be positively related to tubulins extracted from surface complex microtubules.     

INCORPORATION OF 32P INTO THE PHOSPHOLIPIDS OF NEURONAL AND GLIAL CELL ENRICHED FRACTIONS ISOLATED FROM RABBIT CEREBRAL CORTEX: EFFECT OF NOREPINEPHRINE

Abstract— Glial cells isolated from rabbit cerebral cortex contained approximately one-third more phospholipids per unit protein than the neuronal cell bodies. The pattern of individual phospholipids was rather similar in both cell types. The incorporation of intracisternally administered ³²P into neuronal and glial phospholipid classes of rabbit brain was studied at intervals ranging from 5 to 60min. In general, for all investigated phospholipids the incorporation of the label was somewhat faster in neurons than in glial cells. Phosphatidylinositol showed the fastest and ethanolamine plasmalogen the slowest incorporation of ³²P in both neurons and glial cells. A lag phase of about 10 min could be observed before labelling of the glial phosphatidylcholine, phosphatidylethanolamine, ethanolamine plasmalogen, phosphatidylserine and sphingomyelin had occurred. Among the neuronal phospholipids a lag phase was found only for the labelling of the ethanolamine plasmalogen. Norepinephrine increased the incoropration of ³²P into phosphatidylinositol of both glia and neurons but had no effect on the specific radioactivity of ethanolamine plasmalogen and sphingomyelin. Labelling of phosphatidylcholine was slightly inhibited in both cell types by the administration of norepinephrine.     

Histones and the first cell cycle in maize germination

The timing of the onset of cell division during seed germination in maize and the role of histones for this process have been studied. Embryonic axes of maize seeds ( Zea mays L. hybrid H-30) were incubated in a sterile nutrient medium for different periods of time. For some experiments putrescine was also added. Mesocotyl, root tip and scutellar node were dissected at specific periods after incubation and the mitotic indices were determined in these tissues. Embryonic axes were incubated in the same medium either with [¹⁴C]-lysine or [³²P]-phosphate. The incorporation of either ¹⁴C or ³²P into histones was followed, both in postribosomal supernatant and in nuclei. It was found that during germination, there is specific timing for meristematic cells entering into cell division. Among the tissues tested, the mesocotyl meristem was the first to initiate this process. De novo synthesis of histones was detected as early as after 6 h of imbibition and the rate increased up to 12 h. Putrescine stimulated cell division and phosphorylation of the histones. The implications of these findings are discussed.     

EFFECTS OF ACETYLCHOLINE ON THE INCORPORATION OF [32P]ORTHOPHOSPHATE IN VITRO INTO THE PHOSPHOLIPIDS OF NERVE-ENDING PARTICLES FROM GUINEA PIG BRAIN

Abstract— Guinea pig brain nerve-ending particles (synaptosomes) were incubated with [³²P]orthophosphate in a medium with or without 10⁻⁴M-acetylcholine and 10⁻⁴ M-eserine. Phospholipids were then extracted and separated by chromatography. About 60 per cent of the ³²P was found in phosphatidic acid and about 20 per cent in triphosphoinositide. Acetylcholine significantly increased the specific radioactivity of phosphatidic acid but had no effect on that of phosphatidylinositol or the nucleotide fraction. Labelling of the other phospholipids, including diphosphoinositide and triphosphoinositide, was not altered significantly by acetylcholine. Labelling of the nucleotide fraction and the polyphosphoinositides reached a peak at 40 min, that of phosphatidic acid at 80 min, while that of phosphatidylinositol was still rising at 160 min.     

PHOSPHORYLATION OF NUCLEAR PROTEINS FROM RABBIT CEREBRUM, CEREBELLUM AND LIVER IN VITRO

Abstract— Phosphorylation of nuclear protein was investigated with isolated nuclei from rabbit cerebral cortex, cerebellum and liver by using [γ-³²P]ATP. The results were compared with the previously reported findings on phosphorylation with tissue slices and [³²P]phosphate. Cerebral cortex showed a very high level of phosphorylation, while liver showed the lowest, the difference being several fold in magnitude. With each tissue source, the extent of phosphorylation was maximum at incubation period for 2–3 min with steady decline afterwards. When nuclear proteins were further fractionated into 0.14 m -NaCl-soluble, 0.25 n -HCl-soluble (mainly histone) and acidic phenol-soluble proteins, NaCl-soluble protein showed the highest phosphorylation while HCl-soluble the lowest. The ratio among these tissue sources studied and the ratio among various protein fractions in each tissue source were strikingly similar to what had been shown with tissue slices. Further separation of acidic phenol-soluble protein with polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed retention of the characteristic difference of the pattern of phosphorylation between liver and the CNS tissue as having been observed with tissue slices, although phosphorylation of proteins with molecular weights of less than 40,000 was much reduced with the isolated nuclei. Although other methods with extracted protein kinase or chromatic protein fractions might be more desirable under ordinary situations, the system for nuclear protein phosphorylation with isolated nuclei and [γ-³²P]ATP may be useful under certain experimental conditions provided the incubation condition is carefully selected.     

Chronic Administration of Lithium Chloride Increases Immunodetectable Glial Fibrillary Acidic Protein in the Rat Hippocampus

Abstract: We studied the effect of treating rats with lithium salts on the content and in vitro phosphorylation rate of the astrocyte cell marker, glial fibrillary acidic protein (GFAP), in brain slices. Rats were fed a diet incorporating lithium chloride until the concentration of Li⁺ in serum reached 0.6–1.2 m M , a range similar to that achieved in clinical practice. Hippocampal tissue was analyzed for immunoreactive GFAP by a dot assay, and slices of hippocampus and caudate nucleus were labeled with [³²P]-phosphate to determine the in vitro rate of phosphorylation of GFAP. Compared with controls, the level of immunoreactive GFAP in the hippocampus from lithium-treated rats was increased 34%, and GFAP in hippocampal slices incorporated 39% more ³²P. This effect of lithium was apparently not confined to the hippocampus because the in vitro rate of phosphorylation of GFAP in caudate slices was also increased in the treated rats.     

Receptor-mediated increases in phosphatidylinositol turnover in neuron-like cell lines

Abstract: Muscarinic receptors found in the N IE-115 mouse neuroblastoma cell line were tested for their ability to mediate stimulation of phosphatidylinositol (PI) turnover. This study was facilitated by the development of a new solvent system (acetone: butanol: acetic acid: water, 5: 5: 1: 1) for the rapid and consistent separation of PI by one-dimensional thin-layer chromatography. Cholinergic stimulation caused as much as a 680% increase in the incorporation of ³²P into PI. Enhanced incorporation of ³²P into PI could be measured as early as 4 min after stimulation began. By 20 min, the rate of incorporation by stimulated cells had decreased to that of unstimulated cells, indicating desensitization. The magnitude of the response was dependent on the extent of receptor occupancy and the response elicited by a saturating dose of carbamylcholine was blocked completely by 10⁻⁷ M at-ropine, a specific muscarinic antagonist. Chronic stimulation, known to cause a loss of receptor binding sites, led to a 90% decrease in the maximum response even after a 40-min withdrawal period. Replacement of Na⁺ ions in the medium with choline or K⁺ severely impaired the ability of the cells to incorporate added ³²P into PI (90 and 50%, respectively). Removal of the putative second messenger Ca²⁺ for short periods of time by the addition of excess EGTA did not alter either basal or muscarinic-stimulated PI turnover.     

Agonist-Induced Phosphorylation of the κ-Opioid Receptor

Abstract: Antipeptide antibodies against the κ-opioid receptor were used to test whether acute or chronic exposure to κ agonists altered the phosphorylation state of the κ-opioid receptor. Immunoprecipitation of the κ receptor from guinea pig hippocampal slices preincubated in [³²P]orthophosphoric acid revealed a basal phosphorylation of the κ-opioid receptor. The amount of ³²P incorporation into the receptor was increased following a 75-min treatment with the κ agonist U50,488H. This effect was blocked by the selective κ receptor antagonist norbinaltorphimine. The time course of this change in the phosphorylation state of the receptor correlated with a desensitization of the electrophysiological response to κ agonists measured in the dentate gyrus of hippocampal slices. The phosphorylation state of the κ-opioid receptor was also elevated in brain slices from guinea pigs made tolerant to U50,488H by 5 days of continuous exposure and then maintained in κ agonist to avoid acute opiate withdrawal. The results of this study show that the κ-opioid receptor was phosphorylated in an agonist-dependent manner in brain slices taken from untreated and U50,488H-tolerant animals.     

EFFECTS OF DENERVATION ON THE INCORPORATION OF 32P INTO PHOSPHATIDYL INOSITOL AND OTHER PHOSPHOLIPIDS OF RAT DIAPHRAGM

Abstract— At 24 h after denervation of the rat hemidiaphragm, incorporation of ³²P into phosphatidyl inositol was depressed relative to incorporation of ³²P into phosphatidyl choline (measured 75 min after injection of the isotope intraperitoneally). The ratio of the specific radioactivity of phosphatidyl choline to the specific radioactivity of P_i was unaffected by denervation which implies that denervation had depressed incorporation of isotope into phospatidyl inositol. Denervation did not cause a measurable change in the pool size of phosphatidyl inositol relative to that of phosphatidyl choline. The effect of denervation on incorporation of ³²P into phosphatidyl inositol was not entirely a direct consequence of the cessation of ACh release at the motor end-plate since the effect was clearly manifest in strips of muscle not containing motor end-plates, but the magnitude of the denervation effect was slightly greater in the strips of denervated hemidiaphragm which contained motor end-plates.     

DEMONSTRATION OF ACETYLCHOLINE RELEASE BY MEASURING EFFLUX OF LABELLED CHOLINE FROM CEREBRAL CORTICAL SLICES

Abstract— To demonstrate release of ACh in the absence of inhibition of cholinesterase, slices of cerebral cortex were incubated with [³H]choline, after which they were placed in a tissue bath for superfusion. Hemicholinium (HC-3) increased the spontaneous efflux of [³H]choline. Electrical stimulation at 4/s increased the efflux of [³H]choline to the same extent whether the slices were stimulated early or late during superfusion. The effect of stimulation on efflux of [³H]choline was abolished by tetrodotoxin and by the absence of calcium. The extent of choline efflux resulting from stimulation, as calculated from the specific radioactivity of the incubation medium, was the same when the slices were incubated with 0.1 or 1.0mM choline, but was less with lower concentrations of choline. We conclude that the increased efflux of [³H]choline evoked by stimulation probably originates from stores of [³H]ACh synthetized during incubation.     

The Phosphorylation of Proteins in Hippocampal Slices: Effects of Noradrenaline and of Pretreatment with Kainic Acid

Abstract: Hippocampal slices were incubated in the presence of [³²P]P_i, and protein phosphorylation was examined by means of sodium dodecyl sulfate-gel electrophoresis. Incubation for at least 30 min with 300 μCi of [³²P)P_i/brain slice gave rise to the phosphorylation of 8–10 protein bands. Most of these bands showed enhanced phosphorylation in response to noradrenaline. The basal phosphorylation of kainic acid-pretreated hippocampal slices was enhanced two- to threefold compared with controls. There was also an additional increase in kainic acid-pretreated slices in the response to noradrenaline. 8-Br-Cyclic AMP and phosphodiesterase inhibitors, such as papaverine or isobutylmethyl-xanthine, had no effect on the phosphorylation patterns.     

Brain β-Spectrin Phosphorylation: Phosphate Analysis and Identification of Threonine-347 as a Heparin-Sensitive Protein Kinase Phosphorylation Site

Abstract: Phosphorylation of brain spectrin was studied by a combination of in vivo and in vitro approaches. Chemical analysis of phosphate groups on electrophoretically purified mouse brain β-spectrin yielded a stoichiometry of 3.2 ± 0.18 mol of PO₄/mol of β-spectrin. The spectrin isolated by chromatographic methods from mouse brain, pig brain, and human erythrocytes yielded 4.1, 5.6, and 3.2 mol of PO₄/mol of spectrin heterodimer, respectively. The ³²P labeling of spectrin in retinal ganglion cell neurons or NB 2a/d1 neuroblastoma cells with [³²P]orthophosphate showed phosphorylation of only β-spectrin in vivo. Two-dimensional phosphopeptide map analyses showed that most of the in vivo sites on β-spectrin were phosphorylated by either a heparin-sensitive endogenous cytoskeleton-associated protein kinase or protein kinase A. Phosphoamino acid analysis of in vivo and in vitro phosphorylated β-spectrin showed that [³²P]phosphate groups were incorporated into both serine (>90%) and threonine residues. In vitro, phosphate groups were incorporated into threonine residues by the heparin-sensitive endogenous protein kinase. The amino acid sequence VQQQLQAFNTY of an α-chymotryptic ³²P-labeled peptide phosphorylated by the heparin-sensitive cytoskeleton-associated endogenous protein kinase corresponded to amino acid residues 338–348 on the β1 repeat of β-spectrin_G (βSPIIa) gene. These data suggest that phosphorylation of Thr³⁴⁷, which is localized on the presumptive synapsin I binding domain of β-spectrin_G, may play a role in synaptic function by regulating the binding of spectrin to synaptic vesicles.     

Effects of dichloroisopropylarterenol on respiration, nucleotide ∼ P and phospholipids of slices of cerebral cortex

In a Recent report Hokin (1969) showed that norepinephrine stimulated the incorporation of ³²P into phosphatidic acid (PA) of slices of cerebral and cerebellar cortex and into phosphaditylinositol (PI) of slices of cerebral cortex. In the course of our experiments on agents affecting the metabolism of phospholipids, the effects of epinephrine and its antagonist, dichloroisopropylarterenol (DCI), on the labelling of energy-rich nucleotides and phospholipids of cerebral tissue have been measured. Epinephrine had no significant effect but DCI stimulated the incorporation of ³²P into PA by 165 per cent and into PI by 90 per cent.     

Indole-3-acetic acid production by bacteroids from soybean root nodules

Purine nucleotide and RNA synthesis have been investigated at the different growth stages of carrot ( Daucus carota L.) cells grown in suspension cultures. At the early growth stages an increase in the content of RNA was observed, although at later stages RNA was degraded. The highest rates of incorporation of [¹⁴C]-labelled adenosine into ATP and GTP were observed at the late growth sttages. This indicated that purine slavage was more importnt at the late growth stages, while de novo synthesis was dominant during the initial growth stages. This pattern was also reflected by increased levels, in the cell dividison phase, of theenzymes glycinamide ribonucleotide synthetase (EC 6.3.1.3.) and phosphoribosylpyrophosphate amido-transferase (EC 2.4.2.14) involved in de novo purine synthesis. The activities of the purine salvage enzymes varied little during growth. Cells in the stationary phase, that were starved for sucrose and phosphate, showed a dramatic increase in cellular metabolism, as judged from a rapid uptake and incorporation of [³²P]-labelled phosphate into nucleotides and RNA, when incubated in fresh medium.     

THE EFFECT OF DENERVATION ON INCORPORATION OF 32P AND [3H]GLYCEROL BY THE MUSCLE MEMBRANE

Abstract— The incorporation in vivo of ³²P₁ was significantly increased in all glycerophosphatide of preparations of denervated muscle membrane in frogs. There was no increase in incorporation of ³²P₁ into sphingomyelin. Disuse induced by tenotomy did not significantly increase incorporation of ³²P₁ into phospholipids of the muscle membrane. The phospholipid content of muscle membranes remained unchanged as a result of denervation or tenotomy. Denervation produced an increase in the incorporation of [2-³H]glycerol into all glycerophosphatides in parallel with the increase in ³²P₁ incorporation. Although the stimulated incorporation of ³²P₁ was increased in the regions of the muscle membrane rich in endplates, the most marked effect was in the endplate-poor region where activity in phosphatidylserine was most markedly increased.