phosphorus-radioisotopes and Glioma

Topics: Animals; Brachytherapy; Chromium Compounds; Glioma; Half-Life; Heterografts; Humans; Mice; Microscopy, Electron, Scanning; Phosphates; Phosphorus Radioisotopes; Polylactic Acid-Polyglycolic Acid Copolymer; Positron Emission Tomography Computed Tomography; Radiopharmaceuticals; Tissue Distribution; Tomography, Emission-Computed, Single-Photon

A procedure for isolation of cyclic AMP (cAMP) by thin-layer chromatography on silica gel is described. One-dimensional ascending chromatograms were developed using [H(2)O/C(2)H(5)OH/NH(4)HCO(3) (30%:70%:0.2M)] as the mobile phase. This procedure separated [32P]cAMP from other radioactive metabolites of [32P]ATP in up to 19 samples on one sheet (20 x 10 cm) over 40-60 min at room temperature (21 degrees C). This simple and rapid isolation method provides a novel and convenient technique for the assay of adenylyl cyclase.

Topics: Adenylyl Cyclases; Animals; Autoradiography; Cholera Toxin; Chromatography, Thin Layer; Cyclic AMP; Glioma; Isoproterenol; Isotope Labeling; Pertussis Toxin; Phosphorus Radioisotopes; Rats; Silica Gel; Silicon Dioxide; Time Factors; Tumor Cells, Cultured

A ligand-independent activator of heterotrimeric brain G-protein was partially purified from detergent-solubilized extracts of the neuroblastoma-glioma cell hybrid NG108-15. The G-protein activator (NG108-15 G-protein activator (NG-GPA)) increased [(35)S]guanosine 5'-O-(thiotriphosphate) ([(35)S]GTPgammaS) to purified brain G-protein in a magnesium-dependent manner and promoted GDP dissociation from Galpha(o). The NG-GPA also increased GTPgammaS binding to purified, recombinant Galpha(i2), Galpha(i3), and Galpha(o), but minimally altered nucleotide binding to purified transducin. The NG-GPA increased GTPgammaS binding to membrane-bound G-proteins and inhibited basal, forskolin- and hormone-stimulated adenylyl cyclase activity in DDT(1)-MF-2 cell membranes. In contrast to G-protein coupled receptor-mediated activation of heterotrimeric G-proteins in DDT(1)-MF-2 cell membrane preparations, the action of the NG-GPA was not altered by treatment of the cells with pertussis toxin. ADP-ribosylation of purified brain G-protein also failed to alter the increase in GTPgammaS binding elicited by the NG-GPA. Thus, the NG-GPA acts in a manner distinct from that of a G-protein coupled receptor and other recently described receptor-independent activators of G-protein signaling. These data indicate the presence of unexpected regulatory domains on G(i)/G(o) proteins and suggest the existence of pertussis toxin-insensitive modes of signal input to G(i)/G(o) signaling systems.

Topics: Animals; Brain; Carrier Proteins; Cattle; Cell Line; Cell Membrane; Colforsin; Glioma; GTP-Binding Protein alpha Subunits, Gi-Go; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Isoproterenol; NAD; Neuroblastoma; Pertussis Toxin; Phosphorus Radioisotopes; Tumor Cells, Cultured

The presence of a nucleotide pyrophosphatase (EC 3.6.1.9) on the plasma membrane of rat C6 glioma has been demonstrated by analysis of the hydrolysis of ATP labeled in the base and in the alpha- and gamma-phosphates. The enzyme degraded ATP into AMP and PPi and, depending on the ATP concentration, accounted for approximately 50-75% of the extracellular degradation of ATP. The association of the enzyme with the plasma membrane was confirmed by ATP hydrolysis in the presence of a varying concentration of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a membrane-impermeable inhibitor of the enzyme. PPADS concentration above 20 microM abolished the degradation of ATP into AMP and PPi. The nucleotide pyrophosphatase has an alkaline pH optimum and a Km for ATP of 17 +/- 5 microM. The enzyme has a broad substrate specificity and hydrolyzes nucleoside triphosphates, nucleoside diphosphates, dinucleoside polyphosphates, and nucleoside monophosphate esters but is inhibited by nucleoside monophosphates, adenosine 3',5'-bisphosphate, and PPADS. The substrate specificity characterizes the enzyme as a nucleotide pyrophosphatase/phosphodiesterase I (PD-I). Immunoblotting and autoadenylylation identified the enzyme as a plasma cell differentiation antigen-related protein. Hydrolysis of ATP terminates the autophosphorylation of a nucleoside diphosphate kinase (NDPK/nm23) detected in the conditioned medium of C6 cultures. A function of the pyrophosphatase/PD-I and NDPK in the purinergic and pyrimidinergic signal transduction in C6 is discussed.

Topics: Adenosine Triphosphate; Animals; Astrocytes; Enzyme Inhibitors; Extracellular Space; Glioma; Guanosine Triphosphate; Hydrolysis; Nucleoside-Diphosphate Kinase; Phosphorus Radioisotopes; Phosphorylation; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Pyrophosphatases; Rats; Receptors, Purinergic; Stem Cells; Tumor Cells, Cultured

Topics: Affinity Labels; Animals; Autoradiography; Azides; Electrophoresis, Polyacrylamide Gel; Glioma; GTP-Binding Proteins; Guanosine Triphosphate; Indicators and Reagents; Macromolecular Substances; Molecular Structure; Phosphorus Radioisotopes; Recombinant Proteins; Synaptic Membranes; Tubulin; Tumor Cells, Cultured

We have found that the small stress protein, hsp27, exists in extracts of U251 MG human glioma cells in two forms: a large or aggregated form (L-hsp27, 300-400 kDa) and a small or dissociated form (S-hsp27, < 70 kDa), as indicated by centrifugation on sucrose density gradients. Dissociation of L-hsp27 to S-hsp27 was enhanced by incubation of cells with phorbol 12-myristate-13 acetate, interleukin-1 alpha, tumor necrosis factor alpha, or okadaic acid, all of which are known to enhance or mimic the effects of phosphorylation of hsp27 without stimulation of its synthesis. Exposure of cells to chemical stressors, namely, NaAsO2 and CdCl2, also enhanced the dissociation of L-hsp27. hsp27 that had been labeled with [32P]H3PO4 in U251 MG cells was detected mostly in fractions that contained S-hsp27, and the incorporation of radioactivity to S-hsp27 was enhanced under conditions that stimulated the dissociation of L-hsp27. L-hsp27 present in the (NH4)2SO4 fraction (0-50% saturation) of cell extracts were dissociated to 32P-labeled hsp27 when incubated in the presence of [gamma-32P]ATP and Mg2+. These results indicate that the molecular configuration of hsp27 in cells is determined in part by phosphorylation and dephosphorylation of this protein by protein kinase(s) and phosphatase(s) and, moreover, that the rapid dissociation of the aggregated form of hsp27 by phosphorylation might be involved in a cellular defense mechanism for protection against stress.

Topics: Arsenates; Cadmium; Cadmium Chloride; Cell Line; Centrifugation, Density Gradient; Chlorides; Cholera Toxin; Electrophoresis, Polyacrylamide Gel; Glioma; Heat-Shock Proteins; Hot Temperature; Humans; Interleukin-1; Phosphates; Phosphorus Radioisotopes; Phosphorylation; Protein Kinases; Protein Tyrosine Phosphatases; Recombinant Proteins; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

A 116 kDa protein in NG108-15 homogenates is labelled in the presence of [32P]NAD+. This protein was found to be poly(ADP-ribosyl)ated and appears to be a poly(ADP-ribosyl)transferase which has poly(ADP-ribosyl)ated itself. Sodium nitroprusside, an NO generating agent, also stimulates the labelling of this protein by [32P]NAD+, but this can only be seen in the presence of thymidine, which inhibits poly(ADP-ribosyl)transferase activity. Sodium nitroprusside also stimulates the labelling of this protein by [3H-nicotinamide]NAD+, indicating that NO facilitates the formation of an adduct between this protein and NAD+. The insensitivity of the linkage between the protein and NAD+ to mercuric ions indicates that the adduct does not involve thiol groups.

Topics: Animals; Electrophoresis, Polyacrylamide Gel; Glioma; Hybrid Cells; Kinetics; Mice; Molecular Weight; NAD; Neuroblastoma; Nitroprusside; Phosphorus Radioisotopes; Poly(ADP-ribose) Polymerases; Rats; Tumor Cells, Cultured

DNA from human brain tumor samples was analysed by the 32P-postlabeling technique for the presence of aromatic DNA adducts. Thirteen out of 16 samples showed low levels of adducts at 0.14-3.53 adducts per 10(9) nucleotides. Inter-individual variations in the patterns of these aromatic adducts were observed. On the other hand, none of 5 brain samples from epilepsy patients revealed any evidence of such adducts. The data demonstrated the presence of low level, large molecule aromatic DNA adducts in malignant brain tissues and these adducts may either result from environmental exposure to an undetermined genotoxic agent or from the aging process.

Topics: Adenoma; Adult; Aged; Brain Chemistry; Brain Neoplasms; Carcinogens; Carcinoma, Squamous Cell; Chromatography, Thin Layer; Cytochrome P-450 Enzyme System; DNA Damage; DNA, Neoplasm; Epilepsy; Female; Glioma; Humans; Male; Meningioma; Middle Aged; Neurilemmoma; Phosphorus Radioisotopes; Pituitary Neoplasms; Polycyclic Compounds

To suppress cyst formation in 42 brain tumors, 32P has been stereotactically instilled in doses calculated to deliver 20,000-40,000 rad to the cyst wall, assuming uniform dispersal of the radioisotope. However, samples of cyst fluid obtained at varying intervals after injection showed lower than expected activity levels, suggesting early 'plating' of 32P. To accommodate this phenomenon, a surface-area-dependent dosimetric calculation is compared with a volume-dependent calculation which assumes uniform dispersal. These two approaches represent lower and upper extremes. It appears that in small cysts there is less difference in the required administered dose, but in larger cysts potentially very large differences exist and caution should be exercised if uniform suspension is assumed.

Topics: Brain Neoplasms; Chromium; Chromium Compounds; Colloids; Cysts; Glioma; Humans; Phosphates; Phosphorus Radioisotopes; Radiosurgery; Stereotaxic Techniques

Five separate guanine nucleotide-binding proteins (G proteins) were immunologically identified in membranes from neuroblastoma x glioma NG108-15 hybrid cells. These alpha subunit proteins were Gi2 alpha, two isoforms of Gi3 alpha, and two isoforms of Go alpha. The G proteins that interacted with delta-opioid receptors in these membranes were identified using cholera toxin (CTX)-induced ADP-ribosylation and antisera selective for various G protein alpha subunits. In the presence of delta-opioid agonists, CTX induced the incorporation of [32P]ADP-ribose into three pertussis toxin substrates. Using antisera generated against peptide sequences from G alpha subunits, these three pertussis toxin substrates were identified as Gi2 alpha, Go2 alpha, and one isoform of Gi3 alpha, which has yet to be identified. This CTX-induced labeling was demonstrated to be mediated via the delta-opioid receptor in these hybrid cells by the observation that delta agonists D-Ala2-D-Leu5-enkephalin (DA-DLE) and D-Pen2-D-Pen5-enkephalin, as well as the nonselective agonists etorphine and bremazocine, were active, but the mu agonist PL017 and the kappa agonist U-50-488H did not show this activity. This incorporation into all three substrates induced by DADLE was dose dependent, with EC50 (95% confidence interval) values ranging from 12 (3-52) to 183 (65-520) nM, which compared with the Kd value of 10 +/- 1.5 nM for this agonist, a dose that produces maximal inhibition of adenylate cyclase activity. Furthermore, pretreatment of the cells with pertussis toxin or treatment of the membranes with the antagonist naloxone blocked the incorporation induced by DADLE. Incorporation of [32P]ADP-ribose into all three substrates decreased 35-83% in membranes in which the receptors had been down-regulated by chronic treatment of the cells with DADLE. Thus, a single opioid receptor type can interact with three separate G proteins.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Analgesics; Animals; Autoradiography; Cell Line; Cell Membrane; Cholera Toxin; Electrophoresis, Polyacrylamide Gel; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Enkephalins; Glioma; GTP-Binding Proteins; Hybrid Cells; Kinetics; Mice; Molecular Weight; Neuroblastoma; Pertussis Toxin; Phosphorus Radioisotopes; Rats; Receptors, Opioid; Receptors, Opioid, delta; Virulence Factors, Bordetella

This paper describes a simple and direct procedure for assaying Ca(2+)-dependent protein kinase C (PKC) activity in membrane fractions isolated from purified murine B lymphocytes (B cells) treated with phorbol 12-myristate 13-acetate (PMA). The results indicate that membrane-bound PKC in B cells, treated with PMA, can be measured directly in the presence of 0.5% Brij 58 by assaying the transfer of 32P from [gamma-32P]ATP to histone type III-S. This method obviates the need for partial purification of the protein kinase by ion-exchange chromatography prior to assaying PKC activity. The properties of membrane-associated PKC activity in B cells have been characterized, and the kinetics of PMA-induced translocation of PKC in cultured murine B cells, the rat glial tumor clone C6, and primary neonatal osteoblastic cells have been defined by this direct assay. The results obtained with B cells and the other cell lines indicate that this direct assay procedure could be useful for studies on the factors controlling PKC translocation in a variety of cultured mammalian cells.

Topics: Adenosine Triphosphate; Animals; B-Lymphocytes; Cell Fractionation; Cell Membrane; Cells, Cultured; Cetomacrogol; Cytosol; Detergents; Female; Glioma; Kinetics; Mice; Mice, Inbred DBA; Osteoblasts; Phosphorus Radioisotopes; Protein Kinase C; Radioisotope Dilution Technique; Rats; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Pertussis toxin (PTX) catalyzes the ADP-ribosylation of the alpha-subunit of GTP-binding proteins (G-proteins) in the presence of NAD+. Pertussis toxin also decreases the electrophoretic mobility of the alpha-subunit on urea SDS PAGE. This effect of PTX has been suggested to be a property of the toxin different from its ability to catalyze ADP-ribosylation. However, the present report provides evidence to the contrary; ie, this mobility shift required the ADP-ribosylation of alpha-subunits. This conclusion was based on: (1) in the presence of increasing concentrations of NAD+ (0.026-1.3 microM), there was a linear increase in the formation of the slower migrating alpha-subunit as measured by immunoblotting with selective antisera, (2) addition of NADase to the incubation mixture completely eliminated the formation of this protein, and (3) increasing concentrations of nicotinamide (50-250 mM), which inhibits ADP-ribosylation, decreased the amount of the slower migrating alpha-subunit. Thus, in addition to PTX, NAD+ was required for the mobility shift and the slower migrating alpha-subunit is likely the ADP-ribosylated form.

Topics: Adenosine Diphosphate Ribose; Animals; Autoradiography; Cell Line; Electrophoresis, Polyacrylamide Gel; Glioma; GTP-Binding Proteins; Hybrid Cells; Immunoblotting; Kinetics; Macromolecular Substances; NAD; Neuroblastoma; Niacinamide; Pertussis Toxin; Phosphorus Radioisotopes; Virulence Factors, Bordetella

The major route of phosphatidylcholine (PtdCho) biosynthesis in mammalian cells is the sequence: choline (Cho)----phosphocholine (PCho)----cytidinediphosphate choline (CDP-Cho)----PtdCho. Recently, we have found that intermediates of this pathway are not freely diffusible in cultured rat glioma (C6) cells but are channeled towards PtdCho biosynthesis (George et al. (1989). Biochim. Biophys. Acta. 1004, 283-291). Channeling of intermediates in other mammalian systems is thought to be mediated through adsorption of enzymes to membranes and cytoskeletal elements to form multienzyme complexes. In this study, agents which perturb the structure and function of cytoskeletal elements were tested for effects on phospholipid metabolism in glioma cells. The filament-disrupting agent cytochalasin B (CB), but not other cytochalasins or the microtubule depolymerizer colchicine inhibited PtdCho and phosphatidylethanolamine (PtdEtn) biosynthesis as judged by dose-dependent reduction of labeling from [3H]Cho and [14C]ethanolamine (Etn). 32Pi pulse-labeling indicated that CB selectively decreased PtdCho and PtdEtn biosynthesis without affecting synthesis of other phospholipids. Synthesis of water-soluble intermediates of PtdCho metabolism was unaffected but the conversion of phosphoethanolamine to CDP-ethanolamine was reduced by CB. Effects of CB on phospholipid biosynthesis were not due to inhibition of glucose uptake as shown by experiments with 2-deoxyglucose, glucose-starved cells and other cytochalasins. Experiments with Ca(2+)-EGTA buffers and digitonin-permeabilized cells, and the Ca(2+)-channel blocker verapamil suggest that effects of CB on PtdCho and PtdEtn biosynthesis are due to alteration of intracellular Ca2+. Taken together, these results suggest that CB acts at sites distinct from glucose transport and cellular microfilaments to specifically inhibit PtdCho and PtdEtn biosynthesis by mechanisms dependent on intracellular Ca2+.

Topics: Animals; Biological Transport, Active; Calcium; Carbon Radioisotopes; Cell Line; Choline; Cytochalasin B; Deoxyglucose; Ethanolamine; Ethanolamines; Glioma; Glucose; Kinetics; Phosphates; Phosphatidylcholines; Phosphatidylethanolamines; Phosphorus Radioisotopes; Rats; Tritium; Verapamil

Topics: Animals; Autoradiography; Cell Line; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Neuroblastoma; Phosphatidylinositols; Phosphorus Radioisotopes; Radioligand Assay; Tritium; Type C Phospholipases

We have obtained a cDNA fragment to human glial fibrillary acidic protein (GFAP) by immunoscreening a lambda gt11 human brain cDNA library with antibody to bovine GFAP. The highly homologous nucleotide sequence of this clone with that of the mouse GFAP enabled the identification of this cDNA as one encoding GFAP. As this cDNA hybridized with a single major RNA species in Northern blots of RNA from human and mouse brain tissues and gave one or two bands in Southern blots of human genomic DNA, it was considered to be specific for GFAP. Using this cDNA as a probe we investigated the levels of GFAP expression in ten human glioma cell lines. A 3.5-kb GFAP mRNA was detected in five of the ten glioma cell lines, one of which was U-251 MG cell line and the other four were clones derived from the same tumor (CL1, 2, 3, and 4). There was a difference in the amount of GFAP mRNA among U-251 MG and the four clonal cell lines. Quantitative evaluation of this difference by RNA dot blot analysis revealed that the amount of GFAP mRNA expressed in CL3 was about 1/5 and in CL4 about 1/10 the amount expressed in U-251 MG, CL1, and CL2. Semiquantitative Western blot analysis showed that GFAP levels corresponded to the GFAP mRNA levels in these cell lines. By Southern blot analysis of genomic DNA the GFAP gene was similarly detected in all of these cell lines regardless of the level of GFAP expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Amino Acid Sequence; Blotting, Northern; Blotting, Southern; Blotting, Western; Cell Line; DNA; DNA Restriction Enzymes; Glial Fibrillary Acidic Protein; Glioma; Humans; Immunohistochemistry; Molecular Sequence Data; Neoplasm Proteins; Phosphorus Radioisotopes; RNA; Sequence Homology, Nucleic Acid; Tumor Cells, Cultured

The major G-protein of rat glioma C6BU1 cells corresponds immunologically to Gi2. In the absence of guanine nucleotides, this protein is shown to be a substrate for ADP-ribosylation catalysed by both cholera and pertussis toxins. Under these conditions, a receptor for a growth factor, which has previously been shown to be activated by foetal calf serum, modulated the effects of both cholera and pertussis toxins on the G-protein. These ligand-mediated alterations of cholera and pertussis toxin-catalysed ADP ribosylation demonstrate that, in this system, the growth factor receptor interacts functionally with Gi2.

Topics: Adenosine Diphosphate Ribose; ADP-Ribosylation Factors; Animals; Cholera Toxin; Gene Expression Regulation, Neoplastic; Glioma; Growth Substances; GTP Phosphohydrolases; GTP-Binding Proteins; Molecular Weight; NAD; Pertussis Toxin; Phosphorus Radioisotopes; Rats; Tumor Cells, Cultured; Virulence Factors, Bordetella

Hydroxylamine stability has been used to classify (ADP-ribose)protein bonds into sensitive and resistant linkages, with the former representing (ADP-ribose)glutamate, and the latter, (ADP-ribose)arginine. Recently, it was shown that cysteine also serves as an ADP-ribose acceptor. The hydroxylamine stability of [cysteine([32P]ADP-ribose)]protein and [arginine([32P] ADP-ribose)]protein bonds was compared. In transducin, pertussis toxin catalyzes the ADP-ribosylation of a cysteine residue, whereas choleragen (cholera toxin) modifies an arginine moiety. The (ADP-ribose)cysteine bond formed by pertussis toxin was more stable to hydroxylamine than was the (ADP-ribose)arginine bond formed by choleragen. The (ADP-ribose)cysteine bond apparently represents a third class of ADP-ribose bonds. Pertussis toxin ADP-ribosylates the inhibitory guanyl nucleotide-binding regulatory protein (Gi) of adenylate cyclase, whereas choleragen modifies the stimulatory guanyl nucleotide-binding regulatory protein (Gs). These (ADP-ribose)protein linkages are identical in stability to those formed in transducin by the two toxins, consistent with the probability that cysteine and arginine are modified in Gi and Gs, respectively. Bonds exhibiting differences in hydroxylamine-stability were found in membranes from various non-intoxicated mammalian cells following incubation with [32P]NAD, which may reflect the presence of endogenous NAD:protein-ADP-ribosyl-transferases.

Topics: Adenosine Diphosphate Ribose; Animals; Cattle; Cell Line; Cell Membrane; Glioma; GTP-Binding Proteins; Hybrid Cells; Hydroxylamine; Hydroxylamines; Kinetics; Mice; Neuroblastoma; Nucleoside Diphosphate Sugars; Phosphorus Radioisotopes; Rats; Retina; Structure-Activity Relationship

A method for the intraoperative detection of brain tumor propagation is described. Based on the well-known radiophosphorus test, a very sensitive semiconductor probe was tested in 16 brain tumor operations. With this miniaturized sensor, the beta-emission of 32P could be measured with a high topographical resolution. Especially in high-grade gliomas, in meningiomas and in metastases a good discrimination of normal and tumor-infiltrated tissue was possible. The perspectives of a technical improvement of this method and the application of more specific tumor markers are discussed.

Topics: Brain Neoplasms; Female; Glioma; Humans; Male; Meningeal Neoplasms; Meningioma; Middle Aged; Phosphorus Radioisotopes; Radionuclide Imaging; Tomography, X-Ray Computed

Evidence is presented that isoproterenol treatment of rat C6 glioma cells, under conditions that increase glioma cell cAMP levels, causes the phosphorylative modification of several RNA polymerase II subunits. RNA polymerase II in control and isoproterenol-stimulated 32Pi-labeled confluent glioma cells was immunoprecipitated from ribonuclease-treated nuclear extracts with hen anti-calf RNA polymerase II antiserum conjugated to Sepharose. The immunoprecipitated RNA polymerase II was analyzed for 32P-labeled subunits by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Using this technique, we have shown that isoproterenol causes a time-dependent increase of phosphate incorporation into RNA polymerase II subunits of 214,000, 180,000, 140,000, 35,000, 28,000, and 16,500 daltons. Phosphate incorporation occurred exclusively on serine in all of the six subunits. About 0.5-2 mol of phosphate/mol of RNA polymerase II subunit were incorporated. Dibutyryl cAMP (10(-3)M) mimics the stimulatory action of isoproterenol and mediates increased phosphate incorporation into the six subunits. (RS)-propranolol (10(-4)M) prevents the isoproterenol-mediated phosphorylative changes. These data indicate that isoproterenol, via cAMP, mediates a transient structural modification of RNA polymerase II subunits in rat C6 glioma cells which may possibly lead to a modulation of RNA polymerase II function(s).

Topics: Amino Acids; Animals; Bucladesine; Cell Line; Electrophoresis, Polyacrylamide Gel; Glioma; Isoproterenol; Kinetics; Macromolecular Substances; Phosphorus Radioisotopes; Phosphorylation; Rats; RNA Polymerase II

Coelectrophoresis in two-dimensional gels of rat glial fibrillary acidic protein (GFA) and 32P-labeled whole cell extracts of rat C-6 glioma cells showed that the GFA migrated in close proximity to a previously noted phosphoprotein, 50K-6.1, of these cells. GFA electrophoresed as a 50K polypeptide with at least four charge variants, the most acidic of which coelectrophoresed with 50K-6.1. Exposure of the C-6 cultures to dibutyryl cyclic AMP (dbcAMP) for 48 h increased the relative abundance of the endogenous polypeptide associated with 50K-6.1 by threefold, consistent with the hypothesis that 50K-6.1 was GFA. Norepinephrine stimulated 50K-6.1 phosphorylation 3.2-fold in dbcAMP-induced cultures. Peptide mapping with V8 protease and subtilisin was used to test the hypothesis that GFA and 50K-6.1 were identical polypeptides. With V8 protease, the peptides generated from the [35S]methionine labeled putative GFA spot of the C-6 cells were indistinguishable from the stained bands derived from authentic GFA in mixed samples of the two proteins. Likewise, the 35S-labeled acidic satellite to the putative GFA spot also yielded a peptide map that matched that of the authentic GFA. 32P-labeled peptides derived from the 50K-6.1 protein were a subset of those from authentic GFA. With three subtilisin concentrations, 32P-labeled 50K-6.1 was degraded to peptides which were again a subset of the stained GFA peptides. A cytoskeletal fraction from 32P-labeled C-6 cells contained a 50K phosphoprotein.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cell Line; Electrophoresis, Polyacrylamide Gel; Glial Fibrillary Acidic Protein; Glioma; Intermediate Filament Proteins; Kinetics; Norepinephrine; Peptide Fragments; Phosphorus Radioisotopes; Phosphorylation; Rats; Spinal Cord; Sulfur Radioisotopes

Topics: Brain Neoplasms; Craniopharyngioma; Glioma; Humans; Meningioma; Phosphorus Radioisotopes; Radionuclide Imaging; Sella Turcica; Semiconductors

Topics: Adenine Nucleotides; Carbon Radioisotopes; Cell Adhesion; Cell Fractionation; Cell Line; Cell Membrane; Chromatography, Ion Exchange; Cytosine Nucleotides; Glioma; Humans; Kinetics; L-Lactate Dehydrogenase; Neuroglia; Nucleotides; Phosphorus Radioisotopes; Phosphotransferases; Spectrophotometry, Ultraviolet; Time Factors; Uracil Nucleotides