Interaction of GTP with proteins during the cell cycle of Streptomyces coelicolor

Abstract Six putative GTP binding proteins were detected by ultraviolet light in the presence of [α-³²P]GTP during the developmental cycle of Streptomyces coelicolor . Four out of six were true GTP binding proteins. Immunological reactions carried out with antiserum which recognizes the α-common subunit of G regulatory proteins identified two bands of 67 kDa and 30 kDa. Studies with [γ-³²P]GTP showed significant changes in protein phosphorylation during the cell cycle. The results show that at least three different systems of GTP protein interaction are present in S. coelicolor .     

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.     

Phosphorylation of phospholipase B in the plasma membrane of Saccharomyces cerevisiae

Abstract Plasma membrane vesicles from Saccharomyces cerevisiae were incubated with [γ-³²P]ATP. Several phosphorylated protein bands were separated by LiDS polyacrylamide gel electrophoresis. One of these bands with an apparent M _r of 145 000 was identified by immunoprecipitation as a membrane-bound phospholipase.     

Sensitization of G Protein-Coupled Benzodiazepine Receptors in the Striatum of 6-Hydroxydopamine-Lesioned Rats

Abstract: The nonselective benzodiazepine (BZ) agonist diazepam is a potent inhibitor of adenylyl cyclase (AC) activity in the rat striatum. To examine this inhibitory action of diazepam further, its effects were examined in 6-hydroxydopamine-lesioned animals, which reportedly exhibit sensitization of the striatal AC pathway. As previously observed, inhibition of AC activity by diazepam was biphasic, with the first phase being receptor-mediated, whereas the second phase involves a direct action on the enzyme itself. In the presence of NaCl (120 m M ), a marked sensitization to the receptor-mediated inhibitory effect of diazepam on AC activity was observed in striatal membranes of lesioned animals. EC₅₀ values were 10.4 ± 1.1 and 4.8 ± 0.9 n M ( p < 0.05) for intact and lesioned striata, respectively. An examination of [³H]diazepam binding revealed a significant increase in the density of binding sites in denervated striata, with no change in affinity. A time-dependent increase in [α-³²P]GTP labeling of two distinct striatal proteins with apparent molecular masses of 40 and 45 kDa, suggestive of the α subunits of G_i and G_s, respectively, was observed. There was a significant increase in basal [α-³²P]GTP binding to both proteins in lesioned striata. In addition, diazepam stimulated [α-³²P]GTP binding to the 40-kDa protein, especially in lesioned striata. These data indicate that the sensitization of the receptor-mediated inhibitory effect of diazepam on AC activity in denervated striata may involve up-regulation of BZ receptors as well as enhanced functional coupling of these receptors to inhibitory G proteins.     

Intraplastidial lipid trafficking: Regulation of galactolipid release from isolated chloroplast envelope

A method is described for cell-free studies of lipid release from isolated chloroplast envelope. The isolated membrane fraction incorporated radiolabeled galactose into galactolipids, predominantly monogalactosyldiacylglycerol, prior to immobilization of the membrane vesicles onto strips of nitrocellulose. The strips with immobilized membrane were individually incubated with various co-factors and the incubations were terminated by removing the strips. Radioactivity was determined for the strips with immobilized membrane as well as for the material released during the assay. The release of galactolipids from immobilized chloroplast envelope was time- and temperature dependent, required stroma protein(s) and was further stimulated by hydrolysable ATP, GTP and ≤50 μ M acyl-CoAs, of which 16:1-CoA was the most stimulative. To investigate whether guanine nucleotide-binding proteins could be involved, stroma and envelope were independently or together incubated with [ α -³²P]GTP or [ Γ -³²P]GTP. Stroma and envelope proteins were phosphorylated and the envelope fraction contained GMP/GDP binding proteins as well. When the fractions were co-incubated, the patterns of protein phosphorylation and guanine nucleotide binding was different compared to the additive effects of the separate fractions, suggesting that guanine nucleotides may have roles in galactolipid release in addition to providing energy. The results point to several similarities between the regulation of galactolipid release from isolated chloroplast envelope and the regulation of vesicular trafficking among animal and yeast cytosolic membranes, although other mechanisms for lipid release cannot, at this stage, be ruled out.     

Protein Methylation in Cerebellar Synaptosomes

Abstract: Synaptosomes from five regions of adult rat brain were isolated, analyzed for methyl acceptor proteins, and probed for methyltransferases by photoaffinity labeling. Methylated proteins of 17 and 35 kDa were observed in all regions, but cerebellar synaptosomes were enriched in a 21–26-kDa family of methyl acceptor proteins and contained a unique major methylated protein of 52 kDa and a protein of 50 kDa, which was methylated only in the presence of EGTA. When cerebellar and liver subcellular fractions were compared, the cytosolic fractions of each tissue contained methylated proteins of 17 and 35 kDa; liver membrane fractions contained few methylated proteins, whereas cerebellar microsomes had robust methylation of the 21–26-kDa group. Differential centrifugation of lysed cerebellar synaptosomes localized the 17- and 35-kDa methyl acceptor proteins to the synaptoplasm, the 21–26-kDa family to the synaptic membranes, and the 52-kDa to synaptic vesicles. The 21–26-kDa family was identified as GTP-binding proteins by [α-³²P]GTP overlay assay; these proteins contained a putative methylated carboxyl cysteine, based on the presence of volatile methyl esters and the inhibition of methylation by acetylfarnesylcysteine. The 52-kDa methylated protein also contained volatile methyl esters, but did not bind [α-³²P]GTP. When synaptosomes were screened for putative methyltransferases by S -adenosyl-L-[ methyl -³H]methionine photoaffinity labeling, a protein of 24 kDa was detected only in cerebellum, and this labeled protein was localized to synaptic membranes.     

Detection and characterization of GTP-binding proteins in the chloroplast envelope of spinach (Spinacia oleracea)

Proteins binding guanosine triphosphate (GTP) have emerged as important regulators in several cellular processes in plants. To investigate any role of such proteins in chloroplast functions, we subjected envelope, stroma and thylakoid fractions isolated from spinach chloroplasts to two different GTP-binding assays. With both methods, we detected GTP-specific binding only in the envelope fraction. Two chloroplast envelope proteins with the apparent molecular weights of 30.5 and 33.5 kDa, respectively, bound [α-³²P]GTP after SDS-PAGE followed by electroblotting onto a PVDF-membrane and renaturation. Both proteins were intrinsic proteins located in the outer chloroplast envelope. Also, when the fractions were incubated with [α-³²P]GTP, followed by periodate oxidation and borohydride reduction to cross-link GTP to proteins, two proteins in the envelope fraction, of apparent molecular weights of 28 and 39 kDa, appeared to specifically bind GTP. When agents that stimulate heterotrimeric G-proteins, cholera toxin or the mastoparan analogue mas7, were added to isolated chloroplast envelope, the binding of radiolabelled GTP to the 39 kDa protein, a protein of the inner chloroplast envelope, was stimulated, whereas GTP-binding of the 28 kDa protein, a protein of the outer envelope, was unchanged. Mas7 also stimulated synthesis of monogalactosyl diacylglycerol in isolated chloroplast envelope. The occurrence and regulation of GTP-binding proteins in the chloroplast envelope suggests that GTP-binding proteins could be involved in communication with the extraplastidic compartment during chloroplast biogenesis and development.     

Characterization of the protein kinase activity in beet root plasma membranes

Two protein kinase activities were found in plasma membrane-enriched preparations from red beet ( Beta vulgarix L.). The kinases in these preparations produced the phosphorylation of several membrane polypeptides. These kinases also phosphorylated histone III-S and casein. The activities of two different kinases could be distinguished: one was half-maximally stimulated by 1 μ M free Ca²⁺ phosphorylated histone III-S better than casein, showed half-maximal activity at an ATP concentration of 0.071 m M . had an optimum pH of 7, and was poorly inhibited by GTP, CTP or UTP. Another, much lower, kinase activity that phosphorylated casein was also observed; it was Ca²⁺ independent, showed half-maximal activity at ATP concentrations of 0.017 and 0.287 m M , exhibited a broad pH optimum about pH 7 and was inhibited by GTP, CTP, UTP or GDP to a greater extent than the calcium-stimulated activity. When plasma membrane proteins were solubilized with lysophosphatidyicholine and treated with [γ-³²P]ATP at several dilutions, a 125-kDa polypeptide was autophosphorylated in the absence of Ca²⁺, while 77-, 71- and 65-kDa polypeptides were autophosphorylated in its presence. Autophosphorylation in gels after electrophoresis showed a Ca²⁺-stimulated phosphoprotein band at 64 kDa.     

Multiple GTP-binding proteins in sea urchin sperm: Evidence for Gs and small G-proteins

Sea urchin sperm plasma membranes isolated from heads and flagella were used to examine the presence of Gs (stimulatory guanine nucleotide-binding regulatory protein) and small G-proteins. Flagellar plasma membranes incubated with [³²P]NAD and cholera toxin (CTX) displayed radiolabeling in a protein of 48 kDa, which was reactive by immunoblotting with a specific antibody against mammalian Gs. CTX-catalyzed [³²P]ADP-ribosylation in conjunction with immunoprecipitation with anti-Gs, followed by electrophoresis and autoradiography, revealed one band of 48 kDa. Head plasma membranes, in contrast, did not show substrates for ADP-ribosylation by CTX. In flagellar and head plasma membranes pertussis toxin (PTX) ADP-ribosylated the same protein described previously in membranes from whole sperm; the extent of ADP-ribosylation by PTX was higher in flagellar than in head membranes. Small G-proteins were investigated by [³²P]GTP-blotting. Both head and flagellar plasma membranes showed three radiolabeled bands of 28, 25 and 24 kDa. Unlabeled GTP and GDP, but not other nucleotides, interfered with the [α-³²P]GTP-binding in a concentration-dependent manner. A monoclonal antibody against human Ras p21 recognized a single protein of 21 kDa only in flagellar membranes. Thus, sea urchin sperm contain a membrane protein that shares characteristics with mammalian Gs and four small G-proteins, including Ras . Gs, Gi and Ras are enriched in flagellar membranes while the other small G-proteins do not display a preferential distribution along the sea urchin sperm plasma membrane. The role of these G-proteins in sea urchin sperm is presently under investigation.     

ATP-induced Secretion in PC12 Cells and Photoaffinity Labeling of Receptors

Abstract— Secretion of catecholamines by rat PC12 cells is strongly stimulated by extracellular ATP via a P₂-type pur-inergic receptor. ATP-induced norepinephrine release was inhibited 80% when extracellular Ca²⁺ was absent. Only four nucleotides, ATP, ATPγS, benzoylbenzoyl ATP (BzATP), and 2-methylthio-ATP, gave substantial stimulation of norepinephrine release from PC12 cells. ATP-induced secretion was inhibited by Mg²⁺, and this inhibition was overcome by the addition of excess ATP suggesting that ATP^4-was the active ligand. ATP-induced secretion of catecholamine release was enhanced by treatment of cells with pertussis toxin or 12- O -tetradecanoylphorbol 13-acetate. The stimulatory effects of 12- O -tetradecanoyl-phorbol 13-acetate and pertussis toxin on norepinephrine release were additive. After brief exposure of intact cells to the photoaffinity analog, [α-³²P]BzATP, two major proteins of 44 and 50 kDa and a minor protein of 97 kDa were labeled. An excess of ATP-γS and BzATP but not GTP blocked labeling of the proteins by [³²P]BzATP. Labeling of the 50-kDa protein was more sensitive to competition by 2-methylthio-ATP than the other labeled proteins, suggesting that the 50-kDa protein represents the P₂ receptor responsible for ATP-stimulated secretion in these cells.     

Identification of an Ectokinase Activity in Cerebellar Granule Primary Neuronal Cultures

Abstract: Primary neuronal cultures from 8-day-old rat cerebellum were incubated in the presence of exogenously added 16 n M [γ-³²P]ATP. Phosphorylation of a 45-kDa endogenous protein was detected within 1 min and increased linearly for ∼20 min. Unlike what was seen with [γ-³²P]ATP, in the presence of [³²P] orthophosphate no visible phosphorylation of protein was detected after 10 min, but a different pattern of phosphorylation was obtained in 30 min. The phosphorylation of the 45-kDa protein was reduced by 80–90% in the presence of 1 µ M unlabeled ATP, 5 U/ml of apyrase, or 0.01% trypsin but not 1 m M PO₄³⁻. Phosphorylation was inversely proportional to cell density and was unaffected by addition to the cells of 56 m M KCl or 100 µ M glutamate for 3 min. The presence of exogenously added cellular protein extracts or pretreatment of the cells for up to 20 min in phosphorylation buffer also did not affect the observed phosphorylation of the 45-kDa protein. The phosphorylation was found to be insensitive to MgCl₂ but inhibited in the presence of MnCl₂ or NaF and in the absence of CaCl₂. Analogues of ATP suppressed phosphorylation of the 45-kDa protein by 80–90%. A similar inhibition was obtained in the presence of ADP or AMP. In this study, we establish via several different means that the phosphorylation of the 45-kDa protein in primary neuronal granule cultures occurs extracellularly through an ectokinase activity, which is furthermore distinguishable from a series of other presently characterized ecto-protein enzymes and intracellular kinases.     

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.     

Chemosensory Adaptation to Lysozyme and GTP Involves Independently Regulated Receptors in Tetrahymena thermophila

ABSTRACT. Chemosensory adaptation is seen in Tetrahymena thermophila following prolonged exposure (ten minutes) to micromolar concentrations of the chemorepellents lysozyme or guanosine triphosphate (GTP). Since these cells initially show repeated backward swimming episodes (avoidance reactions) in these repellents, behavioral adaptation is seen as a decrease in this repellent-induced behavior. The time course of this behavioral adaptation is paralleled by decreases in the extents of surface binding of either [³²P]GTP or [³H]lysozyme in vivo. Scatchard plot analyses of repellent binding in adapted cells suggests the behavioral adaptation is due to a dramatic decrease in the number of surface binding sites, as represented by decreased Bmax values. The estimated K_D values for nonadapted cells are 6.6 μM and 8.4 μM for lysozyme and GTP binding, respectively. Behavioral adaptation and decreased surface receptor binding are specific for each repellent. The GTP adapted cells (20 μM for ten minutes) still respond behaviorally to 50 μM lysozyme and bind [³H]lysozyme normally. Lysozyme adapted cells (50 μM for ten minutes) still bind [³²P]GTP and respond behaviorally to GTP. All the behavioral and binding changes seen are also reversible (deadaptation). Neomycin was shown to be a competitive inhibitor of [³H]lysozyme binding and lysozyme-induced avoidance reactions, but it had no effect on either [³²P]GTP binding or GTP-induced or avoidance reactions. These results are consistent with the hypothesis that there are two separate repellent receptors, one for GTP and the other for lysozyme, that are independently downregulated during adaptation to cause specific receptor desensitization and consequent behavioral adaptation.     

Myelin P0 Glycoprotein: Identification of the Site Phosphorylated In Vitro and In Vivo by Endogenous Protein Kinases

Abstract: Myelin membrane prepared from mouse sciatic nerve possesses both kinase and substrates to incorporate [³²P]PO₄³⁻ from [γ-³²P]ATP into protein constituents. Among these, P₀ glycoprotein is the major phosphorylated species. To identify the phosphorylated sites, P₀ protein was in vitro phosphorylated, purified, and cleaved by CNBr. Two ³²P-phosphopeptides were isolated by HPLC. The exact localization of the sequences around the phosphorylated sites was determined. The comparison with rat P₀ sequence revealed, besides a Lys¹⁷² to Arg substitution, that in the first peptide, two serine residues (Ser¹⁷⁶ and Ser¹⁸¹) were phosphorylated, Ser¹⁷⁶ appearing to be modified subsequently to Ser¹⁸¹. In the second peptide, Ser¹⁹⁷, Ser¹⁹⁹, and Ser²⁰⁴ were phosphorylated. All these serines are clustered in the C-terminal region of P₀ protein. This in vitro study served as the basis for the identification of the in vivo phosphorylation sites of the C terminal region of P₀. We found that, in vivo, Ser¹⁸¹ and Ser¹⁷⁶ are not phosphorylated, whereas Ser¹⁹⁷, Ser¹⁹⁹, Ser²⁰⁴, Ser²⁰⁸, and Ser²¹⁴ are modified to various extents. Our results strongly suggest that the phosphorylation of these serine residues alters the secondary structure of this domain. Such a structural perturbation could play an important role in myelin compaction at the dense line level.     

Phosphorylation of Endogenous Proteins by Adenosine 3':5'-Monophosphate-Dependent Protein Kinase in Mouse Neuroblastoma Cells

Abstract: Increased intracellular adenosine 3':5'-monophosphate (cAMP) levels and activation of cAMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) in vivo were correlated in mouse neuroblastoma cells grown in the presence of 1 mM-⁶ N.O ²-dibutyryl 3':5'-monophosphate (Bt₂cAMP). The time course for activation showed that cAMP-dependent protein kinases were activated by 30 min. A heat-stable inhibitor protein inhibited a majority of activated cAMP-dependent protein kinase. Activation of cAMP—dependent protein kinase caused additional phosphorylation of proteins when compared with untreated control cells, as demonstrated by endogenous phosphorylation of proteins in vitro using [γ-³²P]ATP and analysis by two—dimensional polyacrylamide gel electrophoresis. The phosphorylation data show selective phosphorylation of specific proteins by cAMP-independent and cAMP-dependent protein kinase. Among the proteins in the postmitochondrial supernatant fraction phosphorylated by cAMP-dependent protein kinases, two proteins with a molecular weight of 43,000 were heavily phosphorylated. It is suggested that phosphorylation of cellular proteins by cAMP-dependent protein kinases might be involved in the cAMP-modulated biochemical changes in neuroblastoma cells.     

A Comparison of In Vitro- and In Vivo Phosphorylated Neurofilament Polypeptides

Abstract: Neurofilament polypeptides phosphorylated in vitro by incubation of neurofilament-enriched preparations from rat CNS with [γ-³²P]ATP were compared with the corresponding polypeptides labeled in vivo by injection of ³²P_i into the lateral ventricles of rats. Autoradiography of sodium dodecyl sulfate (SDS)-polyacrylamide gels revealed that the major phosphorylated species in both preparations were the three neurofilament subunits, which have molecular weights of 200K, 145K, and 68K. However, the relative levels of ³²P detected in the three in vitro -labeled subunits differed from the relative in vivo levels. The two larger neurofilament polypeptides displayed similar ³²P isoprotein distribution patterns on two-dimensional gels, whereas additional isoproteins were seen in the in vitro -labeled 68K species. Limited proteolysis in SDS-polyacrylamide gels revealed the presence of common phosphopeptides in the corresponding pairs of in vitro- and in vivo-labeled subunits, but the in vivo -labeled 145K and in vitro -labeled 200K polypeptides contained additional digestion products. Two-dimensional peptide mapping of the 68K polypeptide digested with a mixture of trypsin and chymotrypsin indicated that this component was phosphorylated at a single, identical site, both in vivo and in vitro. These results indicate that the protein kinase that copurifies with neurofilament preparations may be involved in their in vivo phosphorylation.     

Experimental Galactose Toxicity: Effects on Synaptosomal Phosphatidylinositol Metabolism

Abstract Experimental galactose toxicity was induced by weaning rats onto an isocaloric 40% galactose diet. Synaptosomes were prepared from cerebra of rats at 2-9 weeks post-weaning and incubated with [³³P]P_i and myo -[2-³H]inositol in the presence or absence of 0.2 mM-acetylcholine. The acetylcholine-stimulated [³³P]P_i labeling of phosphatidylinositol and the changes in amounts of phosphatidylinositol were similar in the normal and galactose-toxic rats; however, acetylcholine-stimulated myo -[2-³H]inositol labeling of phosphatidylinositol was markedly decreased in the galactose-toxic rats. The impairment of acetylcholine-stimulated myo -[2-³H]inositol incorporation into phosphatidylinositol observed after 2 weeks on the diet did not vary after more prolonged exposure to galactose.     

ATP-Binding Proteins on the External Surface of Synaptic Plasma Membranes: Identification by Photoaffinity Labeling

Abstract: 8-Azidoadenosine triphosphate labeled in the α or γ position with ³²P was used as a photoaffinity reagent for identifying ATP binding sites on the external surface of intact rat brain synaptosomes. As revealed by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns, UV irradiation of intact synaptosomes in the presence of the above radioactive compounds at 5–10 µ M resulted in the formation of several major radioactive conjugates with approximate molecular masses of 29, 45/46, 58, and 93 kDa. Minor bands of 20, 39, 52/54, 82/84, 120, and 140 kDa were also consistently labeled in these experiments. The possibility that labeling of these proteins was due to the presence of contaminating subcellular particles or intrasynaptosomal proteins was excluded. The major 8-azidoadenosine [α-³²P]triphosphate-labeled protein complex of ∼45/46 kDa was resolved into several subbands that are labeled differently depending on the type of divalent cations added to the photoaffinity reaction. In the presence of magnesium only, the major labeled band appeared at 45 kDa. With calcium, two additional subbands (43 and 46 kDa) could be distinguished. In the presence of 1 m M EDTA, a band at 44 kDa was labeled within this ATP-binding complex. The labeling pattern of the subbands of this 45/46-kDa complex is consistent with these bands being extracellular ATP-binding proteins on the surface of the synaptosome.     

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.