Two-dimensional polyacrylamide gel electrophoresis of chromatin proteins of normal rat liver and Novikoff hepatoma ascites cells

Chromatin was prepared from the citric acid nuclei of normal rat liver and Novikoff hepatoma ascites cells. After sulfuric acid extraction, the dehistonized chromatin was solubilized by digestion with deoxyribonuclease I. The proteins of normal liver and of Novikoff hepatoma chromatin fractions were analyzed by two-dimensional polyacrylamide gel electrophoresis. The liver pattern contained 69 components and the hepatoma pattern contained 84 components. Comparison of the two patterns revealed two dense protein spots migrating in the B region in the liver pattern that were absent from the tumor pattern and two dense protein spots migrating in the C region in the tumor pattern that were absent from the liver pattern.     

Localization of phosphoprotein C23 in nucleoli by immunological methods

Antiserum to a major phosphorylated nucleolar protein. C23 (MW 103000, pI 5.2) from Novikoff hepatoma was produced in rabbits. By immunodiffusion analysis, the antiserum produced precipitin bands and with various crude extracts of nucleoli, but not with extranucleolar or cytosol fractions. The specificity of the antibody was assessed using acid-urea polyacrylamide gel electropherograms of acid-soluble nucleolar proteins in which the separated proteins were transferred to nitrocellulose sheets. The purified antibody reacted predominantly with protein C23 as visualized by the immunoperoxidase procedure. By the indirect immunofluorescence technique, protein C23 was localized predominantly to nucleoli of Novikoff hepatoma or normal rat liver cells. In Novikoff hepatoma cells, traces of fluorescence were seen near the inner layer of the nuclear envelope. Additional narrow regions of fluorescence extended from the nucleoli into the extranucleolar areas of some Novikoff cells. The nucleolar areas of fluorescence were smaller but brighter in the normal liver than in Novikoff hepatoma, consistent with the small size of rat liver nucleoli. These data indicate that the major location of protein C23 is the nucleolus.     

Concanavalin A and wheat germ agglutinin receptor activity of sialoglycopeptides isolated from the surface of Novikoff hepatoma cells

Novikoff ascites hepatoma cells were highly agglutinable by the plant lectins concanavalin A and wheat germ agglutinin. Treatment of the intact cells with papain released from the cell surface a glycopeptide fraction which possessed concanavalin A and wheat germ agglutinin receptor activity, as judged by its ability to inhibit lectin-induced hemagglutination. A component of the cell-surface glycopeptide fraction, excluded from Sephadex G-50, possessed lectin receptor activities reflecting the cytoagglutination properties of the intact cells from which it was derived. Further resolution of this component by pronase digestion, gel filtration, and ion-exchange chromatography resulted in the isolation of sialoglycopeptides which exhibited potent and specific concanavalin A receptor activity.     

Preparation of eight ovalbumin subfractions by combined lectin affinity chromatography

Ovalbumin was fractionated by successive lectin affinity chromatography using concanavalin A/Sepharose and wheat germ agglutinin/Sepharose. Eight glycoprotein fractions, all behaving as ovalbumin on polyacrylamide gel electrophoresis, were obtained. To characterize the carbohydrate chains, the asparaginyl-carbohydrates were prepared from the Pronase digests of the ovalbumin fractions and their dansyl derivatives were analyzed by high-performance liquid chromatography. The elution profile of the dansylated asparaginyl-carbohydrates from each subfraction was compared with that from the unfractionated ovalbumin. The results indicated that the above eight subfractions could be separated from each other according to their carbohydrate chains and that three of the subfractions were homogeneous with respect to their carbohydrate chains.     

Two-dimensional polyacrylamide gel electrophoresis of acid extractable nuclear proteins of normal rat liver and Novikoff hepatoma ascites cells

The nuclear proteins of normal rat liver and Novikoff hepatoma nuclei were extracted with 0.4 N H₂SO₄ and subjected to two-dimensional polyacrylamide gel electrophoresis. A total of 98 protein components were found in the liver extract and 111 components in the tumor extract. A comparison of the patterns obtained revealed 11 qualitative differences and 5 quantitative differences.     

SOLUBLE AND MEMBRANE LECTIN-BINDING GLYCOPROTEINS OF THE CHROMAFFIN GRANULE

Abstract— Fluorescein isothiocyanate-labelled lectins were used to identify lectin-binding glycoproteins of the chromaffin granule after electrophoresis of the membrane and soluble granule proteins on sodium dodecyl sulphate polyacrylamide slab gels. The glycoprotein nature of all lectin-binding bands was confirmed by staining the gels for carbohydrates, and the specificity of the lectin-binding was demonstrated by hapten sugar inhibition of binding. In samples of granule membrane proteins reduced with dithiothreitol 10 concanavalin A (Con A), 5 wheat germ agglutinin, 8 Ricinus communis agglutinin-60, and 7 Ricinus communis agglutinin-120 (RCA-120) binding glycoproteins were identified. Molecular weights of these glycoproteins varied from 20,000 to 200,000 daltons. All but two of the Con A-binding bands and one of the RCA-120 binding bands appeared to react with more than one lectin, suggesting possible carbohydrate heterogeneity in these membrane glycoproteins. The band identified as dopamine β-hydroxylase reacted most intensely with all four lectin tested, and in the soluble core material this enzyme was the sole significant lectin binding glycoprotein.     

Evidence for coupled synthesis of mRNA for ribosomal proteins and rRNA.

Two-dimensional gel electrophoresis revealed quantitative differences in the 35S-methionine labeled proteins synthesized in vitro in the wheat germ system containing poly A(+) mRNA of normal liver, regenerating liver or Novikoff hepatoma cells. A group of low molecular weight proteins which comigrated with proteins of 40S ribosomal subunits were synthesized in a much greater concentration with the poly A(+) mRNA from the latter two sources. This correlates with increased synthesis of ribosomal proteins in regenerating liver or tumors which is temporally coupled with the increased nucleolar synthesis of rRNA precursors.     

Trypanosoma brucei brucei and Trypanosoma brucei gambiense: stage specific differences in wheat germ agglutinin binding and in endoglycosidase H sensitivity of glycoprotein oligosaccharides

Gel electrophoresis, lectin affinity blotting, and endoglycosidase H digestion have been used to analyze the glycoprotein profiles of bloodstream and procyclic forms of Trypanosoma brucei brucei and T. b. gambiense. Proteins resolved by polyacrylamide gel electrophoresis were stained with silver nitrate or electrophoretically transferred to nitrocellulose and probed with a horseradish peroxidase conjugate of either concanavalin A or wheat germ agglutinin. Silver staining showed, as expected, that the expression of the variant specific glycoprotein was restricted to the bloodstream forms. Twenty-three concanavalin A binding proteins were resolved in blots of bloodstream forms. Concanavalin A binding molecules corresponding in electrophoretic mobility to 21 of these 23 bloodstream form glycoproteins were detected in blots of procyclic forms. The two concanavalin A binding glycoproteins present only in bloodstream form extracts were variant specific glycoprotein and an 81-kDa protein designated glycoprotein 81b. One concanavalin A binding molecule of 84 kDa, glycoprotein 84p, was detected only in procyclic forms. The 19 major wheat germ agglutinin binding glycoproteins expressed by bloodstream forms were not detected in procyclic forms; only small proteins or protein fragments in procyclic form extracts bound wheat germ agglutinin. Incubating transferred proteins in endoglycosidase H eliminated subsequent binding of concanavalin A to most of the 22 common glycoproteins of bloodstream forms. Three major concanavalin A binding glycoproteins of bloodstream forms, variant specific glycoprotein, glycoprotein 81b, and a 110-kDa molecule (glycoprotein 110b), and other minor glycoproteins carried sugar chains that resisted endoglycosidase H digestion. In contrast, concanavalin A did not bind to any procyclic form glycoproteins, including a 110-kDa concanavalin A binding molecule (glycoprotein 110p) after endoglycosidase H treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lectin-Reactive Components in White Matter Membranes from Normal and Multiple Sclerosis Brains

Abstract: Polypeptides derived from human white matter membranes reacted with the radioiodinated lectins concanavalin A, Lens culinaris phytohemagglutinin, Ricinus communis agglutinin and wheat germ agglutinin after electrophoresis in polyacrylamide pore gradient gels. The molecular weights of these lectin-reactive bands were estimated by comparison with radioiodinated protein standards by using the linear relationship between log of the molecular weight and log of the gel concentration reached by the protein after electrophoresis in a polyacrylamide gradient gel. The molecular weight estimates for components reactive with concanavalin A were 176,800, 141,200, 72,800, 52,800, 44,700, 40,000, 24,800 and 23,900. The molecular weights of the bands reactive with both wheat germ agglutinin and Lens culinaris phytohemagglutinin were 138,000, 113,500, 92,100, 52,800, 44,700, 24,800 and 23,900. Wheat germ agglutinin was bound also to a band with a molecular weight of 72,800. Ricinus communis agglutinin bound to bands with estimated molecular weights of 138,000, 72,800, 52,800, 44,700, 24,800 and 23,900. The electrophoretic pattern of lectin-reactive polypeptides derived from normal-appearing white matter of multiple sclerosis brains was not qualitatively different from the lectin-binding pattern of control brain membrane polypeptides.     

Lectin affinity chromatography of cell surface proteins of novikoff tumor cells

Novikoff hepatocellular carcinoma cells were radioiodinated by a cell surface-specific method using lactoperoxid ase/¹²⁵I. The iodinated proteins were solubilized in 0.5% Nonidet P-40 and subjected to affinity chromatography on Sepharose-conjugated lectins (Ricinus communis agglutinins I or II, soybean agglutinin, concanavalin A, or wheat germ agglutinin) and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Almost all the iodinated proteins bound to one or more of the Sepharose-conjugated lectins, presumptive evidence that these peptides are glycosylated. Lectin affinity chromatography resolved defined subsets of iodinated glycoproteins and suggested that certain glycoproteins could be fractionated on the basis of heterogeneity of their heterosaccharide moieties. Incubation of the iodinated cells with neuraminidase resulted in increased binding of iodinated proteins to Sepharose-conjugated Ricinus communis agglutinins I and II and soybean agglutinin and decreased binding to Sepharose-conjugated wheat germ agglutinin. Binding of iodinated proteins to concanavalin A was unaffected by neuraminidase treatment of the cells. These studies demonstrate the utility of lectins for the multicomponent analysis of plasma membrane proteins.     

Differential effect of ATP on RNA and DNA release from nuclei of normal and neoplastic liver.

Nuclei isolated from differentiated normal liver, moderately differentiated Hepatoma 5123 and poorly differentiated Novikoff hepatoma have been compared with respect to ATP-dependence of messenger-like RNA release and resistance to lysis (DNA release) in a cell-free system containing homologous cytosol. The release of RNA from the nuclei of liver, Hepatoma 5123D and the Novikoff hepatoma was totally ATP-dependent, partially ATP-dependent and ATP-independent, repectively. The sensitivity of the nuclear RNA transport in the presence of ATP to beryllium nitrate, an inhibitor of a nuclear pore phosphatase, paralleled their ATP-dependence. Although RNA release from the nuclei of both liver and Novikoff hepatoma has an absolute requirement for cytosol proteins, the structural integrity of liver, but not Novikoff hepatoma nuclei in the presence of ATP, is dependent on macromolecules in the cytosol.     

The effect of oral contraceptives on rat platelet membrane glycoproteins

Female rats were administered oral contraceptives and the levels of sialic acid on platelet membrane and granule glycoproteins were compared to controls using a sialic acid assay and a fluorescein-conjugated wheat germ agglutinin binding assay and also by measuring the binding of ¹²⁵I-labelled wheat germ agglutinin to glycoprotein bands from platelets separated by polyacrylamide electrophoresis. The contraceptive-treated rats showed increased levels of glycoprotein sialylation which may partly explain the altered physiological function of the platelets.     

gp110—A major sialoglycoprotein of human cells: Isolation and partial characterization from a malignant melanoma cell line

A major sialoglycoprotein (gp110) was isolated from NP-40 extracts of the human melanoma cell line SK-MEL-37 by concanavalin A-Sepharose and wheat germ agglutinin-Sepharose affinity chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A rabbit antiserum was prepared to this concanavalin A- and wheat germ agglutinin-binding glycoprotein and used to study the biochemical properties and distribution of gp110 in human cells. gp110 is highly acidic (pI ~ 3.8–4.0) and located on the cell surface in melanoma cells. It contains sialylated, N-linked complex chains as well as sialylated, O-linked carbohydrate chains. gp110 was detected as a major glycoprotein on all human cell lines tested (except erythrocytes), although its apparent molecular weight varied from cell line to cell line. The pI of gp110 from normal and malignant human kidney epithelial cells was identical, indicating that gp110's from two cell types do not substantially differ in their sialylated carbohydrate moieties.     

Membrane glycoproteins of the nerve growth cone: diversity and growth regulation of oligosaccharides

A subcellular fraction prepared from fetal rat brain and enriched in growth cone membranes is analyzed for its lectin-binding proteins. Growth-associated glycoproteins are identified by comparing the growth cone glycoproteins with those of synaptosomes. Protein was resolved in one- or two-dimensional gels, electroblotted, and blots probed with radioiodinated concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinins I and II. In one-dimensional gels, each lectin recognizes approximately 20 polypeptides (with substantial overlap) most of which migrate diffusely and have relatively high molecular masses (range 30-200 kD). The seven major Coomassie-staining proteins of the membrane fraction (34-52 kD) are not the major lectin-binding proteins. In two-dimensional gels, the lectin-binding proteins are either streaked across the pH gradient or exist as multiple spots, indicating broad charge heterogeneity. Seven wheat germ agglutinin- and Ricinus communis agglutinin II-binding glycoproteins are present in greater abundance in growth cone fractions compared with synaptosomes. Most notably, an acidic, sialic acid-rich protein (27-30 kD, pI 4.0; termed gp27-30) is most abundant at postnatal day 4, but absent from adult brain. The protein's very acidic isoelectric point is due, at least in part, to its high sialic acid content. Growth regulation of specific protein-linked oligosaccharides suggests that they play a special role in growth cone function. In addition, the great diversity of growth cone glycoproteins from whole brain suggests glycoprotein heterogeneity among growth cones from different neuron types.     

Antibodies to dehistonized chromatin of normal and fetal rat liver

Antisera to dehistonized adult or fetal rat liver chromatin were treated with electrophoretically separated chromosomal proteins of adult and fetal liver, Novikoff hepatoma and adult rat kidney. Both types of antisera reacted with numerous antigens in both tissue chromatins. However, immunoabsorption experiments have shown that while adult rat liver shared most of its nuclear antigens with other tissues, antisera to dehistonized fetal liver chromatins were more specific. In terms of antigenic specificity, the fetal rat liver and Novikoff hepatoma chromatins were similar; however, the former contained several antigens which could not be absorbed with Novikoff hepatoma chromatin.     

Isolation and characterization of gastric microsomal glycoproteins. Evidence for a glycosylated beta-subunit of the H+/K(+)-ATPase.

Detergent-solubilization of hog gastric microsomal membrane proteins followed by affinity chromatography using wheat germ agglutinin or Ricinus communis I agglutinin resulted in the isolation of five glycoproteins with the apparent molecular masses on sodium dodecyl sulfate polyacrylamide gels of (in kDa): 60-80 (two glycoproteins sharing this molecular mass); 125-150; and 190-210. In the nonionic detergent Nonidet P-40 (NP-40), the 94 kDa H+/K(+)-ATPase was recovered exclusively in the lectin-binding fraction; however, in the cationic detergent dodecyltrimethylammonium bromide, most of the ATPase was recovered in the nonbinding fraction. Detection of glycoproteins either by periodic acid-dansyl hydrazine staining of carbohydrate in polyacrylamide gels or by Western blots probed with lectins indicated that the majority of the ATPase molecules are not glycosylated. In addition, in the absence of microsomal glycoproteins, the NP-40-solubilized ATPase does not bind to a lectin column. Taken together, these results suggest that the recovery of NP-40-solubilized ATPase in the lectin-binding fraction is due to its noncovalent interaction with a gastric microsomal glycoprotein. Immunoprecipitation of the ATPase from NP-40-solubilized microsomal membrane proteins resulted in the co-precipitation of a single 60-80 kDa glycoprotein. Characterization of the 60-80 kDa glycoprotein associated with the ATPase revealed that: it is a transmembrane protein; it has an apparent core molecular mass of 32 kDa; and, it has five asparagine-linked oligosaccharide chains. Given its similarity to the glycosylated beta-subunit of the Na+/K(+)-ATPase, this 60-80 kDa gastric microsomal glycoprotein is suggested to be a beta-subunit of the H+/K(+)-ATPase.     

Enzymatic modification of Novikoff hepatoma lamins A and C

A significant increase in the molecular weights of lamin A and more so of lamin C was observed when isolated Novikoff hepatoma chromatin was incubated in the presence of Ca2+. This increase did not occur to any significant degree in similar preparations of normal rat liver nuclei. Although detectable in Coomassie Brilliant Blue stained gels, this increase to a higher molecular weight (by approximately 2000 Mr) was much more visible when the electrophoretically separated lamins were transferred to nitrocellulose sheets and stained (using peroxidase-antiperoxidase) with polyclonal antiserum to the three major lamin proteins. This modification could also be induced when whole Novikoff hepatoma cell lysates were incubated in the presence of calcium. Again, this change did not occur in normal rat liver cells treated in the same manner. Further analysis has provided evidence that this modification is most likely mediated by the transaminating activity of an intrinsic nuclear transglutaminase forming a cross-link between the affected lamins and an unknown low molecular weight (approximately equal to 2000 Mr) moiety.     

In vitro translation of rat liver and Novikoff hepatoma cytokeratin mRNAs

Summary Cytoplasmic poly(A)⁺ RNA was isolated from normal rat liver and Novikoff ascites hepatoma cells, translated in vitro using rabbit reticulocyte lysate system and the translational products were assayed by immunoprecipitation with antibodies specific for Novikoff hepatoma principal cytokeratins p39, p49 (a group of hepatic cytokeratins C, D, and E) and p56. The identity of the precipitated antigens was further confirmed by two-dimensional polyacrylamide gel electrophoresis. Only the Novikoff hepatoma poly(A)⁺ RNA contained translatable mRNA coding for the p39 cytokeratin while the p49 and p56 cytokeratins were translated from both the normal rat liver and Novikoff hepatoma poly(A)⁺ RNAs. Immunoprecipitations employing monoclonal antibody specific for p39 also recovered significant quantities of p56 and 49K cytokeratins, presumably due to oligomeric associations of these proteins with p39 immediately after in vitro synthesis. Similar results were observed after experiments with anti-p56 monoclonal antibody in which p39, not reactive with this antibody, was recovered in immunoprecipitates. Overall, the two-dimensional gel fluorograms of cytokeratins synthesized in vitro from NAH or liver poly(A)⁺ RNA are quite similar to isolated antigenic and cytokeratin profiles reported previously. These results suggest that overt posttranslational processing is not likely responsible for the diversity of cytokeratins observed in the liver.Abbreviations NAH Novikoff ascites hepatoma - HEPES N-2hydroxyethylpiperazine-N-2-ethane sulfonic acid - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid     

Poly (adnenosine diphosphoribose) synthase activity of isolated nuclei of normal and leukemic leukocytes.

Poly(adenosine diphosphoribose) (ADPR) synthase activities of nuclei isolated from normal human and leukemic leukocytes were assayed by incubation with radioactive NAD+. The synthase activity of leukemic leukocyte nuclei was significantly higher than that of normal leukocyte nuclei. The average length of polymers formed by isolated leukemic nuclei under the prescribed experimental condition ranged from 3.1 to 5.3 ADPR residues per chain, while those produced by normal leukocytes nuclei was 1.7 and 2.6 residues per chain. Isolated leukemic and normal leukocyte nuclei were incubated with and without NAD+ and the ability to carry out DNA synthesis was measured. The endogenous DNA synthesis of NAD-treated and untreated nuclei was the same. This finding parallels the result obtained with Novikoff hepatoma cell nuclei and differs from the observation with rat liver or testis nuclei.     

Glycoproteins in the whorls of membrane produced by oligodendroglia in culture

Two glycoproteins of 99 kDa and 77 kDa which exhibit intense binding to wheat germ agglutinin have been purified from the whorls of membrane produced by oligodendroglia in culture. The whorls of membrane were isolated by gradient centrifugation from purified bovine oligodendroglia maintained in culture. The two glycoproteins were solubilized from the membranes using a non-ionic detergent and purified by Sephadex LH-60 chromatography, wheat germ agglutinin affinity chromatography, and SDS-polyacrylamide pore gradient gel electrophoresis. HPLC peptide mapping of the 99-kDa and 77-kDa glycoproteins revealed structural differences between the two proteins. Peptide mapping suggested that the 99-kDa glycoprotein from the whorls of membrane may be homologous to that from the plasma membranes. The 77-kDa glycoproteins from both sets of membrane may also be structurally related. Lectin binding studies showed that both glycoproteins from the whorls of membrane bound to wheat germ agglutinin, succinylated wheat germ agglutinin, concanavalin A, and lentil lectin, indicating the presence of high mannose and hybrid type oligosaccharide side-chains.