Phosphorylation and DNA binding of nuclear rat liver proteins soluble at low ionic strength

Crane, Paula M.; Busch, Harris

doi:10.1021/bi00647a027

Biochemistry

1976

DOI: 10.1021/bi00647a027

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Phosphorylation and DNA binding of nuclear rat liver proteins soluble at low ionic strength

Paula M. Crane¹,

Harris Busch²

Abstract: Proteins were extracted from isolated rat liver nuclei with 0.15 M NaCl and 0.35 M NaCl at pH 8.0. The number of phosphoproteins in these extracts was determined by labeling with 32P and autoradiography after two-dimensional gel electrophoresis. Two proteins, B22p and B24p, contained small amounts of 32P and sedimented with the 30S nuclear informofer particle. With the exception of two phosphoproteins, CB and CN', all of the phosphoproteins found in the 0.35 M NaCl extract. Approximately 20% of the 0.15 M NaCl… Show more

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Cited by 22 publications

(7 citation statements)

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“…Using high resolution Laemmli-type (29) slab gel electrophoresis, Comings and Harris (10) showed that the proteins removed by a 0.35 M wash of nuclei previously washed with 0.15 M NaC1 were virtually identical to the nonhistone proteins remaining on the chromatin. Similar conclusions on the relationship of "loosely bound" 0.35 M NaC1 wash proteins to nonhistone chromosomal proteins have been reached by others (23,24,28,37).…”

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confidence: 83%

Nuclear proteins. II. Similarity of nonhistone proteins in nuclear sap and chromatin, and essential absence of contractile proteins from mouse liver nuclei.

Comings¹,

Harris²

1976

The Journal of Cell Biology

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High resolution SDS slab gel electrophoresis has been used to examine the distribution of nonhistone proteins (NHP) in the saline-EDTA, Tris, and 0.35 M NaCI washes of isolated mouse liver nuclei. These studies led to the following conclusions: (a) all the prominent NHP which remain bound to DNA are also present in somewhat similar proportions in the saline-EDTA, Tris, and 0.35 M NaCI washes of nuclei; (b) a protein comigrating with actin is prominent in the first saline-EDTA wash of nuclei, but present as only a minor band in the subsequent washes and on washed chromatin; (e) the presence of nuclear matrix proteins in all the nuclear washes and cytosol indicates that these proteins are distributed throughout the cell; (d) a histone-binding protein (J2) analogous to the HMG1 protein of K. V. Shooter, G. H. Goodwin, and E. W. Johns (Eur J. Biochem. 4"/:263-270) is a prominent nucleoplasmic protein; (e) quantitation of the major NHP indicates that they are present in a range of 2.2 • 105-5.2 • l06 copies per diploid nucleus. Most of the electrophoretically visible NHP are probably structural rather than regulatory proteins; 09 actin, myosin, tubulin, and tropomyosin, if present at all, constitute a very minor fraction of the nuclear NHP. Contractile proteins constitute a major portion of the NHP only when the chromatin is prepared from crude cell lysates instead of from purified nuclei. These studies support the conclusion that there are no clear differences between many nucleoplasmic and chromatin-bound nonhistone proteins. Except for the histones, many of the intranuclear proteins appear to be in equilibrium between DNA, HnRNA, and the nucleoplasm.Most studies of nonhistone proteins begin with chromatin purified by repeated washing in dilute buffers. The washes are usually discarded. Since the development of high-resolution SDS gel electrophoresis there have been few studies to examine whether the proteins removed by the nuclear washes represent a unique set of proteins or whether they are essentially identical to the nonhistone proteins that remain bound to DNA. Using a relatively low resolution SDS gel electropho-440

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supporting

confidence: 83%

Nuclear proteins. II. Similarity of nonhistone proteins in nuclear sap and chromatin, and essential absence of contractile proteins from mouse liver nuclei.

Comings¹,

Harris²

1976

The Journal of Cell Biology

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“…Two‐dimensional gel electrophoresis (2‐DE) has been widely used to systematically analyze total cellular proteins [3–5]. In combination with metabolic labeling of phosphoproteins using radioactive inorganic phosphate, 2‐DE has successfully identified phosphoproteins [6]. However, nonphosphorylated proteins comigrating with the radioactive spots have hindered MS identification of phosphorylated proteins.…”

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confidence: 99%

Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis

et al. 2007

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Prefractionation procedures facilitate the identification of lower‐abundance proteins in proteome analysis. Here we have optimized the conditions for immobilized metal affinity chromatography (IMAC) to enrich for phosphoproteins. The metal ions, Ga(III), Fe(III), Zn(II), and Al(III), were compared for their abilities to trap phosphoproteins; Ga(III) was the best. Detailed analyses of the pH and ionic strength for IMAC enabled us to determine the optimal conditions (pH 5.5 and 0.5 m NaCl). When whole cell lysates were fractionated in this way, about one‐tenth of the total protein was recovered in the eluate, and the recovery of phosphorylated extracellular signal‐regulated kinase (ERK) was more than 90%. Phosphorylated forms of ribosomal S6 kinase (RSK) and Akt were also enriched efficiently under the same conditions. Our Ga(III) IMAC and a commercially available purification kit for phosphoproteins performed similarly, with a slight difference in the spectrum of phosphoproteins. When phosphoproteins enriched from NIH3T3 cells in which ERK was either activated or suppressed were analyzed by two‐dimensional fluorescence difference gel electrophoresis, phosphorylated ERK was detected as discrete spots unique to ERK‐activated cells, which overlapped with surrounding spots in the absence of prefractionation. We applied the same technique to search for Akt substrates and identified Abelson interactor 1 as a novel potential target. These results demonstrate the efficacy of phosphoprotein enrichment by IMAC and suggest that this procedure will be of general use in phosphoproteome research.

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“…For many purposes this definition has been satisfactory. However, many studies on the solubility of nuclear acidic proteins in low ionic-strength buffers have demonstrated that nuclear proteins soluble in, for example, 0.35 M NaCl closely resemble the residual chromosomal nonhistones (30, 31, 35, 44, THE JOURNAL OF CELL BIOLOGY " VOLUME 86 JULY 1950 135-155 ©The Rockefeller University Press " 0021-9525/80/07/0135/21 $1 .00 45,62,87) . This suggested that what many researchers have normally regarded as cytoplasmic or nucleoplasmic "contamination" of the chromosomal nonhistone fraction, are, in fact, loosely bound nonhistones.…”

mentioning

confidence: 99%

Two-dimensinal gel electrophoresis of rat liver nuclear washes, nuclear matrix, and hnRNA proteins.

Peters¹,

Commings²

1980

The Journal of Cell Biology

140

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The proteins of rat liver cytoplasm, nuclear washes, matrix, membrane, heterogeneous nuclear (hn)RNA proteins and chromatin were examined by two-dimensional gel electrophoresis . The inclusion in the gels of six common protein standards of carefully selected molecular weight and isoelectric point allowed us to clearly follow the distribution of specific proteins during nuclear extraction . In the nuclear washes and chromatin, we observed five classes of proteins : (a) Exclusively cytoplasmic proteins, present in the first saline-EDTA wash but rapidly disappearing from subsequent washes ; (b) ubiquitous proteins of 75,000, 68,000, 57,000, and 43,000 mol wt, the latter being actin, found in the cytoplasm, all nuclear washes and the final chromatin pellet ; (c) proteins of 94,000, 25,000, 22,500, and 20,500 mol wt specific to the nuclear washes ; (d) proteins present in the nuclear washes and final chromatin, represented by species at 62,000, 55,000, 54,000, and 48,000 mol wt, primarily derived from the nuclear matrix ; and (e) two proteins of 68,000 mol wt present only in the final chromatin . The major 65,000-75,000-mol wt proteins seen by one-dimensional gel electrophoresis of nuclear matrix were very heterogeneous and contained a major acidic, an intermediate, and a basic group . A single 68,000-mol wt polypeptide constituted the majority of the membrane-lamina fraction, consistent with immunological studies indicating that a distinct subset of matrix proteins occurs, associated with heterochromatin, at the periphery of the nucleus . Actin was the second major nuclear membrane-lamina protein . Two polypeptides at 36,000 and 34,000 mol wt constituted 60% of the hnRNP. Approximately 80% of the mass of the nonhistone chromosomal proteins (NHP) from unwashed nuclei is contributed by nuclear matrix and hnRNPs, and essentially the same patterns were seen with chromatin NHP . The concept of NHP being a distinct set of DNA-bound proteins is unnecessarily limiting . Many are derived from the nuclear matrix or hnRNP particles and vary in the degree to which they share different intracellular compartments .The study of the nonhistone chromosomal proteins (NHP) has been hindered by the tremendous diversity of these proteins with respect to size, charge, solubility, and function (for reviews, see references 23 and 40) . The term "nonhistone" itself emphasizes that these proteins have historically been viewed more in terms of what they are not than what they are. They have been operationally defined as a heterogeneous group of proteins which along with histones, DNA, and various species of RNA, make up the interphase chromosome, or chromatin (18,23) . For many purposes this definition has been satisfactory. However, many studies on the solubility of nuclear acidic proteins in low ionic-strength buffers have demonstrated that nuclear proteins soluble in, for example, 0.35 M NaCl closely resemble the residual chromosomal nonhistones (30, 31, 35, 44, THE JOURNAL OF CELL BIOLOGY " VOLUME 86 JULY 1950 135-155 ©The Roc...

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Phosphorylation and DNA binding of nuclear rat liver proteins soluble at low ionic strength

Cited by 22 publications

References 48 publications

Nuclear proteins. II. Similarity of nonhistone proteins in nuclear sap and chromatin, and essential absence of contractile proteins from mouse liver nuclei.

Nuclear proteins. II. Similarity of nonhistone proteins in nuclear sap and chromatin, and essential absence of contractile proteins from mouse liver nuclei.

Purification of phosphoproteins by immobilized metal affinity chromatography and its application to phosphoproteome analysis

Two-dimensinal gel electrophoresis of rat liver nuclear washes, nuclear matrix, and hnRNA proteins.

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