Isolation and partial characterization of the ADP-ribosylated nuclear proteins from Ehrlich ascites tumor cells

Adamietz, Peter; Klapproth, Karin; Hilz, Helmuth

doi:10.1016/0006-291x(79)91199-9

Cited by 57 publications

(21 citation statements)

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“…The ADP-ribosylated histone H1 was separated from the unmodified protein by covalent chromatography on boronate cellulose (Merck, Darmstadt) (cf. [8,14,15]): 38 mg purified histone H1 including the (ADP-ribose), conjugates was applied to a column(0.6 x 9cm) of boronate cellulose equilibrated with 6 M guanidine hydrochloride, 50 mM morpholine/HCl pH 8.20. Unmodified molecules were removed by extensive washing with the same buffer.…”

Section: Isolation Of' (Adp-ribose) -Histone Hi Conjugatesmentioning

confidence: 99%

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ADP‐Ribosylated Histone H1

Braeuer

Adamietz

Nellessen

et al. 1981

European Journal of Biochemistry

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Conjugates between histone H1 and (ADP-ribose),, formed in isolated nuclei of Ehrlich ascites tumor cells, were purified free OF unmodified H1 using perchloric acid extraction, ion-exchange and boronate-cellulose chromatography. The isolated conjugates comprised 0.6 of the total protein-bound ADP-ribose residues, and about 1 % of the histone H1 population.Electrophoretic analysis in acid/urea gels revealed the presence of multiple components migrating slower than unmodified histone H1. They could be made visible by staining For protein or by fluorography of the t3H]ADPribose residues. The neighboring bands appeared to differ from each other by a single ADP-ribose residue. In most preparations a mean chain length of 2-3 was found. Removal of the ADP-ribosyl groups by treatment with alkali or phosphodiesterase shifted the bands to the position of unmodified histone H1. On higher-resolving gels the bands split into doublets representing different degrees of phosphorylation. The same microheterogeneity was also observed with the non-ADP-ribosylated control. This indicated that phosphorylation of histone H1 did not significantly influence the acceptor properties for ADP-ribosyl transfer.Studies on the lability of the (ADP-ribose), protein linkage showed that about 20% of the ADP-ribose was linked by an NHzOH/NaOH-sensitive bond, 70 % by an NHzOH-resistant/NaOH-sensitive bond, and the residual 10 % apparently by an additional, NaOH-resistant bond.Cleavage of the (ADP-ribose),-histone H1 conjugates by N-bromosuccinimide and gel electrophoretic analysis of the two Fragments revealed that by far the most ADP-ribose residues were linked (presumably at multiple sites) to the C-terminal fragment. Furthermore, a large fraction of the conjugates carried ADP-ribosyl groups exclusively either at the C-terminal fragment or at the N-terminal fragment.Covalent modification of proteins by ADP-ribosylation has been found in prokaryotic and eukaryotic systems. Besides toxin-mediated or phage-mediated transfer of single ADP-ribose residues to cytoplasmic or membraneous acceptor proteins, an enzymic system was described in nuclei and mitochondria of mammalian cells able to form protein conjugates with ADP-ribose or poly(ADP-ribose) (for a review see [l -31). Histones as well as non-histone proteins serve as acceptors in vitro (cf. [4-81). So far, however, only histone H1 has been identified unequivocally as being ADPribosylated in vivo [9 -111. Since only a small fraction of the total nuclear ADP-ribose residues appears to be associated with histone H1, [ll, 121, separation of ADP-ribosylated histone H1 from unmodified histones was essential for a characterization of these conjugates. This paper describes the first isolation of pure histone H1-(ADP-ribose), conjugates and reports on a partial characterization of these proteins from Ehrlich ascites tumor cell nuclei.Abbreviations. ADP-ribose, adenosine(5')diphospho(5')-/3-~-ribose; (ADP-ribose)., oligomers and polymers of ADP-ribose; EAT cells, Ehrlich ascites tumor cells; PhMeSOzF, phe...

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Section: Isolation Of' (Adp-ribose) -Histone Hi Conjugatesmentioning

confidence: 99%

“…Histone HI, including its (ADP-ribose), conjugates, was quantitatively [I 5,191, and to isolate the total acid-insoluble (ADP-ribose), conjugates [8].…”

Section: Isolation Of (Adp-ribose) -Histone Hi Conjugatesmentioning

confidence: 99%

ADP‐Ribosylated Histone H1

Braeuer

Adamietz

Nellessen

et al. 1981

European Journal of Biochemistry

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“…2). ADP-ribosylation and phosphorylation as reasons for this heterogeneity were ruled out by alkali treatment as in [50] and acidic phosphatase treatment as described in Methods. Gel electrophoresis of samples thus treated did not reveal any changes in the protein banding pattern.…”

Section: Structural Data Of Sarcoplasmic and Ribosomal 5s Rnp Complexesmentioning

confidence: 99%

“…To remove RNA from the 5s-RNA-protein complexes the RNA was alkali-hydrolyzed according to [50] with 0.25 M NaOH at 37°C. After 40 min the protein was precipitated with 10% trichloroacetic acid and the pellets were washed repeatedly with diethyl ether.…”

Section: Protein Sequencingmentioning

confidence: 99%

5S-rRNA-containing ribonucleoproteins from rabbit muscle and liver. Complex and partial primary structures

et al. 1987

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A 5s-rRNA-containing ribonucleoprotein was purified to homogeneity from a rabbit muscle extract through its affinity to phosphofructokinase-1 and then stucturally characterized. This RNP was compared to the 5S-rRNA-containing ribonucleoprotein extracted from rabbit liver ribosomal 60s subunits with EDTA. Analytical gel filtration revealed a molecular mass of 70 -80 kDa for both complexes. Gel electrophoresis of the ribosomal complex revealed three protein components, one migrating as a band of 35 kDa and two other small polypeptides of apparently 16.5 kDa and 17.5 kDa. In the sarcoplasmic RNP these small polypeptides were absent. However, besides a major component of 35 kDa, up to five slightly larger and smaller species of 31.5-36.5 kDa were detected. Despite this heterogeneity, only one N-terminal amino acid sequence was obtained for the isolated sarcoplasmic protein, suggesting a C-terminal heterogeneity of one single polypeptide. Within the first 46 amino acid residues no difference between the sequences of the isolated 35-kDa components of sarcoplasmic and ribosomal complexes was found. Homology criteria indicated that this component belongs to the ribosomal protein L5 family. The RNA was identified by complete enzymatic sequencing as 5s rRNA; it was also identical in both complexes and is strongly homologous to 5s rRNA of man. Both L5-5s-RNA complexes could be resolved by hydroxyapatite chromatography into three species still consisting of both protein and RNA. 5'-Terminal dephosphorylation experiments showed that this heterogeneity is exclusively due to the differing number (1 -3 ) of 5'-terminal phosphates. The two additional low-molecular-mass proteins were stably associated to the ribosomal RNP at high salt concentrations in a stoichiometry of about 2: 1. They were identified as the acidic phosphoproteins P2/P3 by N-terminal sequencing. High phosphate concentrations facilitated their dissociation from the L5 -5s-RNA complex. For the sarcoplasmic L5 -5s-RNA complex a hitherto unknown interaction with phosphofructokinase-1 , affecting the enzymatic properties, was demonstrated.Eubacterial, archaebacterial, and eukaryotic 5s ribosomal RNAs can be isolated as defined complexes with proteins and these complexes are essential for the ribosomal function and in eukaryotes of importance in rRNA transcription.In the 50s subunit of Escherichiu coli ribosomes three proteins, E-L5, E-L18, and E-L25, with molecular masses of 20, 13 and 11 kDa, respectively, form a stable complex with 5s RNA [l -51. In Bacillus stearothermophilus the two pro- Ahhreviutions. PFK, phosphofructokinase-1 ; CaM, calmodulin; 5s RNP,, sarcoplasmic 5s RNP; 5s RNP,, ribosomal 5s RNP; TP60, total protein of the 60s subunit.Enzymes. Phosphofructokinase-1 (EC 2.7.1 .I 1); alkaline phosphatase (EC 3.1.3.1); acidic phosphatase (EC 3.1.3.2); proteinase K (EC 3.4.21.14).In Xenopus laevis oocytes, at early stages of oogenesis, most of the 5s RNA is complexed with three abundant nonribosomal proteins to form two types of cytoplasmic particles. One par...

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“…In nuclei and nucleosomes this enzyme can undergo extensive poly(ADP-ribosylation) [4,5] and modifies preferentially histone HI [6-101 and in some cases core histone H2B [11,12]. It has also been shown that at high N A D concentrations the enzyme was able to catalyse the formation of an histone HI 'dimer' or complex [7,13,14].…”

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Adenosine Diphosphate Ribosylation of Chicken‐Erythrocyte Histones H1, H5 and High‐Mobility‐Group Proteins by Purified Calf‐Thymus Poly(adenosinediphosphate‐ribose) Polymerase

Poirier

Niedergang

Champagne

et al. 1982

European Journal of Biochemistry

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Poly(ADP-ribosylation) of histones H I , H5 and non-histone chromosomal high-mobility-group proteins H M G 1, 2, 14 and 17 from chicken erythrocytes by purified calf thymus poly(ADP-ribose) polymerase was studied using acid/urea/Triton gel electrophoresis and autoradiography.With histone H I , besides ADP-ribosylated HI supporting short chains of polymer, the appearance of H1 'dimer' was observed and this reaction was dependent on NAD concentration and incubation time. In addition, highly modified and/or aggregated species of histone H I were observed.Histone HS was slightly ADP-ribosylated at low NAD concentrations. At higher NAD concentrations or after longer incubations the formation of H5 'dimer' and of more modified forms of H5 could be observed.HMG 1 and H M G 2 were found to be ADP-ribosylated, the reaction being dependent on NAD concentration and time. Here again some discrete intermediates appeared. HMG 14 and HMG 17 were only slightly ADP-ribosylated under our experimental conditions.These results indicate that the purified DNA-independent poly(ADP-ribose) polymerase can catalyse the formation of H I 'dimer' as in nuclei and nucleosomes and that H5 and HMG proteins can also be ADPribosylated and produce well-defined higher complexes. These modifications of nuclear proteins may provide a means of localized conformational changes of the chromatin structure in vivo.

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Isolation and partial characterization of the ADP-ribosylated nuclear proteins from Ehrlich ascites tumor cells

Cited by 57 publications

References 13 publications

ADP‐Ribosylated Histone H1

ADP‐Ribosylated Histone H1

5S-rRNA-containing ribonucleoproteins from rabbit muscle and liver. Complex and partial primary structures

Adenosine Diphosphate Ribosylation of Chicken‐Erythrocyte Histones H1, H5 and High‐Mobility‐Group Proteins by Purified Calf‐Thymus Poly(adenosinediphosphate‐ribose) Polymerase

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