A set of non‐histone proteins isolated from the nuclei of various rat tissues

Abstract

A set of non-histone proteins has been identified in the nuclei from liver, brain, spleen and testis tissues of the rat. Following moderate digestion of thoroughly washed nuclei with DNase 1 or micrococcal nuclease, EDTA was added to 5 m M to the reaction mixture and the preparation centrifuged. We found that the supernatant contained a limited amount of non-histone proteins (fraction SI). Sodium dodecyl sulfate (SDS) gel electrophoresis revealed S1 to be composed of a remarkably simple set of proteins resolved into four groups (A -D) each possessing closely spaced doublets or a triplet. Their molecular weights were A, 76100-80000; B, 48200-49500; C, 44500-45200 and D, 39500-41 500. The yield suggested that these proteins were structural constituents; however, they did not coincide with the known structural proteins of the cell nucleus. Two-dimensional gel electrophoresis further resolved each of the SDS bands into as many as nine spots, according to various charges. Some were labelled with [32P]orthophosphate in vivo, or with [y-32P]ATP and purified nuclear protein kinase NII in vitro. The released proteins B -D had fairly constant relative molar ratios at various times of digestion, thereby indicating possible localizations a t similar sites in the nucleus. The kinetic data together with the aggregation property at neutral pH values and the solubility in 5 mM EDTA suggest that proteins B -D constitute a group of proteins that have several common characteristics.The abundance of protein species in the cell nucleus varies with the protein; some are structural or regulatory in function while others are catalytic in various biochemical processes. Isolation and characterization of these nuclear proteins should greatly aid in understanding the structure and function of the nucleus as well as the molecular properties of the protein in question. Isolation of a group of proteins which share molecular properties may be advantageous, as a common biological function may be involved. The precedent of this can be illustrated with histones and high mobility group (HMG) proteins. Histones, the building molecules of the nucleosomes [I], can be readily extracted in a set from the chromatin with dilute mineral acids [2]; H M G proteins, which confer a structural characteristic of the active Chromatin [3], are extracted as a group from chromatin with 0.35 M NaCl and remain soluble in 2There have been numerous studies on various nuclear architectures such as chromatin, nuclear matrix and nuclear envelopes. One approach is to use DNA-hydrolyzing enzymes ; for example, DNase I has been used to distinguish the transcriptionally active from the bulk chromatin in differential digestion kinetics [5], or to isolate the nuclear pore complexlamina fraction [6] and nuclear matrix [7].In our studies on nuclear proteins we were interested in protein components liberated from the nuclei by digestion with DNA-hydrolyzing enzymes. We report here that a novel and remarkably simple set of non-histone proteins was isolated from the nuclei by nucle...

S1 proteins C2 and D2 are novel hnRNPs similar to the transcriptional repressor, CArG box motif‐binding factor A

“…We found S1 proteins in 1983. They constitute another such group of nuclear proteins [4]. They are present at sites sensitive to RNase as well as DNase, and extracted at pH 4.9 from the reaction mixture of nuclei treated mildly with either enzyme.…”

“…The S1 proteins are separated into four doublets, A, B, C, and D in SDS/PAGE: A1 (migrating with an apparent molecular mass of 74.5 kDa), A2 (69.5); B1 (47.4), B2 (46.5); C1 (43.9), C2 (42.8), D1 (40.8), and D2 (39.4). Proteins B1, B2, C1, C2, D1, and D2 are liberated from nuclei with very similar kinetics in DNase I digestion, suggesting that they are present at similar sites in the nucleus [4]. S1 proteins are widely found, not only in all rat tissues examined, but also in many species [4,7] (T. Watanabe & A. Inoue, unpublished results).…”

Molecular cloning of a RNA binding protein, S1-1

“…They are liberated from nuclei with closely similar kinetics on DNase I digestion, suggesting that the S1 proteins are present in the same or very similar sites in the nucleus. They have been found in all rat tissues examined so far (5) and in mammals, a bird (chicken), a fish (carp), an amphibian (frog; unpublished data) and an echinoderm (starfish; 8). Polyclonal antibodies raised in two rabbits with protein B as immunogen both reacted with proteins B2, C1 and D1 (9,10).…”

Association of hnRNP S1 proteins with vimentin intermediate filaments in migrating cells

“…Reference S1 proteins were prepared from rat liver nuclei as described (Inoue et al, 1983;Tsugawa et al, 1997). ARL cells were solubilized in a standard SDS sample buffer (SDS and 2-mercaptoethanol were at 2% and 1% respectively) and the lysates sonicated and heated at 95°C for 5 minutes.…”

“…In fact, VFs become prerequisite not only in contraction and reorganization but also in cell motility of connective tissues, and all of these are mandatory events in wound healing (Eckes et al, 1998) (reviewed by Gailit and Clark, 1994). S1 proteins A-D are ubiquitously found in animal cells (Inoue et al, 1983;Emura et al, 1992; and unpublished results), and extracted as a group of proteins at pH 4.9 from the nuclei treated with either RNase A or DNase I (Inoue et al, 1983;Higashi et al, 1984;Inoue et al, 1986). They are resolved by SDS-PAGE, each as doublets: A1 (an apparent molecular mass of 74.5 kDa), A2 (69.5 kDa); B1 (47.4 kDa), B2 (46.5 kDa); C1 (43.9 kDa), C2 (42.8 kDa); D1 (40.8 kDa) and D2 (39.4 kDa).…”