The retinoblastoma (RB) tumor suppressor is a nuclear phosphoprotein important for cell growth control and able to bind specifically to viral oncoproteins such as the SV40 large tumor antigen (T-ag). Human RB possesses a bipartite nuclear localization sequence (NLS) consisting of two clusters of basic amino acids within amino acids 860 -877, also present in mouse and Xenopus homologs, which resembles that of nucleoplasmin. The T-ag NLS represents a different type of NLS, consisting of only one stretch of basic amino acids. To compare the nuclear import kinetics conferred by the bipartite NLS of RB to those conferred by the T-ag NLS, we used ␤-galactosidase fusion proteins containing the NLSs of either RB or T-ag. The RB NLS was able to target ␤-galactosidase to the nucleus both in vivo (in microinjected cells of the HTC rat hepatoma line) and in vitro (in mechanically perforated HTC cells). Mutational substitution of the proximal basic residues of the NLS abolished nuclear targeting activity, confirming its bipartite character. Nuclear accumulation of the RB fusion protein was half-maximal within about 8 min in vivo, maximal levels being between 3-4-fold those in the cytoplasm, which was less than 50% of the maximal levels attained by the T-ag fusion protein, while the initial rate of nuclear import of the RB protein was also less than half that of T-ag. Nuclear import conferred by both NLSs in vitro was dependent on cytosol and ATP and inhibited by the nonhydrolyzable GTP analog GTP␥S. Using an ELISA-based binding assay, we determined that the RB bipartite NLS had severely reduced affinity, compared with the T-ag NLS, for the high affinity heterodimeric NLS-binding protein complex importin 58/97, this difference presumably representing the basis of the reduced maximal nuclear accumulation and import rate in vivo. The results support the hypothesis that the affinity of NLS recognition by NLS-binding proteins is critical in determining the kinetics of nuclear protein import.All passive and active transport into and out of the nucleus occurs through the nuclear pore complex (NPC) 1 (1-3). Proteins larger than 45 kDa require a nuclear localization sequence (NLS) (4) to be targeted to the nucleus. NLSs are defined as the sequences sufficient and necessary for nuclear import of their respective proteins (5-7) and are generally functional in targeting heterologous, normally cytoplasmic proteins to the nucleus. NLS-dependent protein transport can be divided into two steps. The first is energy-independent and involves recognition and targeting of the NLS-containing protein to the NPC by a heterodimeric protein complex consisting of importin 58/97 (8, 9) or ␣/␤ (10, 11). The second, energy-dependent step is the translocation of the NLS-bearing protein through the NPC into the nucleus (12, 13) and requires GTP hydrolysis mediated by the GTP-binding protein Ran/TC4 (14 -16) and the interacting factor p10/NTF2 (17-19). We were interested in the nuclear import kinetics of tumor suppressor proteins, one of which is the retino...

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A polysaccharide was extracted in high yield from tamarind (Tamarindus indica L.) seed (TSP) by acidic hot water extraction and ethanol precipitation. It was composed of 86.2% neutral polysaccharide, 5.4% uronic acid and 1.3% protein. The molecular weight of TSP was estimated to be about 1735 kDa, with glucose, xylose, and galactose in a molar ratio of 2.9:1.8:1.0 as the major monosaccharides. The steady shear and viscoelastic properties of TSP aqueous solutions were investigated by dynamic rheometry. Results revealed that TSP aqueous solution at a concentration above 0.5% (w/v) exhibited non-Newtonian shear-thinning behavior. Dynamic oscillatory analysis revealed that 10% (w/v) TSP showed as a “weak gel” structure. Apparent viscosities and viscoelastic parameters of TSP solutions decreased drastically in an alkaline solution of pH > 10, but slightly influenced by acidic solution, high temperature and the presence of salt ions and sucrose. These results indicated that TSP possessed excellent pH-resistance and thermo-stability, which might be suitable for applications in acidic beverages and high-temperature processed foodstuffs.

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