Isolation of cDNA coding for the placental protein 15 (PP15)

Grundmann, U.; Nerlich, Claudia; Rein, Theo; Lottspeich, Friedrich; Küpper, Hans

doi:10.1093/nar/16.10.4721

Cited by 18 publications

(15 citation statements)

References 2 publications

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“…7). This peptide sequence was highly homologous to a human protein of unknown function called placental protein 15 (pplS), which is a homodimer of 14,478-Da subunits (10)(11)(12) permeabilized cells. In the presence of fraction A, p1O and Ran were the only two proteins from the original cytosolic B fraction required to support import at levels comparable to unfractionated cytosol (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…7) with ppl5, a human placental protein of unknown function. As pplS is a homodimer of 14,478-Da subunits (10)(11)(12), and as p1O fractionates as a 30-kDa species upon gel filtration, the actual mass of p1O (as opposed to its SDS/PAGE-estimated size) may be -15 kDa rather than 10 kDa. The 50-to 60-kDa peak of B activity observed on gel filtration (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Purification of a Ran-interacting protein that is required for protein import into the nucleus.

Moore

Blobel

1994

Proc. Natl. Acad. Sci. U.S.A.

301

207

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Previously we reported the isolation of two cytosolic fractions (A and B) from Xenopus ovary that are required sequentially to support protein import into the nuclei of digitonin-permeabilized cells. Fraction A is required for recognition of the nuclear localization sequence and targeting to the nuclear envelope, whereas fraction B is required for the subsequent translocation of the bound substrate into the nucleus. The first protein required for fraction B activity to be purified was the small GTPase Ran (ms-related nuclear protein). Here we report the purification of the second (and final) protein required for fraction B activity. By The first component required for fraction B activity to be purified was Ran, a member of the Ras superfamily of small GTP-binding proteins (5). Experiments utilizing guanine nucleotide analogs indicated that Ran had to be in the GTPbound form in order to catalyze nuclear import and that GTP hydrolysis was also required for import (though not necessarily by Ran). Melchior et al. (6) also reported a requirement for both GTP hydrolysis and Ran in nuclear import in vitro. Our results further indicated that purified Ran was by itself, however, insufficient to reconstitute full fraction B activity, indicating that a second necessary factor had been lost during Ran purification. This second factor has been designated (on the basis ofactivity) as the B-2 component, Ran being B-1 (3).Here we report the purification and characterization of the protein that supplies the B-2 activity. MATERIALS AND METHODSNuclear Import Assay. The nuclear import assay was performed in digitonin-permeabilized BRL (buffalo rat liver) cells as originally described (7), with our modifications (4, 5). The import substrate was rhodanine-labeled human serum albumin coupled to peptide containing the NLS ofthe simian virus 40 large tumor (T) antigen (4 activity contained recombinant Ran (100 pg/ml) and fraction A (2 mg/ml). Units of B-2 activity (see Table 1) were as described for B activity except that the import obtained just with fraction A and Ran was subtracted as background (4).Puriication of p1O. All procedures were performed at 40C unless otherwise stated. The starting material for this purification was the ovarian tissue (-300 ml of settled ovary) obtained from 18 adultXenopus laevis females (no. LM535M; Nasco, Fort Atkinson, WI). Homogenization and isolation of the 146,000 X g supernatant in buffer A (20 mM Hepes-KOH, pH 7.3/2 mM dithiothreitol) containing 2 mM Mg(OAc)2 were performed as described (4). The high-speed supernatant (280 ml) was brought to 150 mM KOAc by dropwise addition (with stirring) ofbuffer A/1 M KOAc/2 mM Mg(OAc)2 and loaded onto a 100-ml DE-52 column (Whatman; 4.5 cm x 6.3 cm) equilibrated in buffer A/150 mM KOAc/2 mM Mg(OAc)2 at a flow rate of 300 ml/hr. The column was washed with 500 ml of the same buffer. The first 60 ml of the flowthrough was discarded and the next 570 ml of the flowthrough and wash fractions were pooled. These pooled fractions were brought to 10 mM EDTA by...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Purification of a Ran-interacting protein that is required for protein import into the nucleus.

Moore

Blobel

1994

Proc. Natl. Acad. Sci. U.S.A.

301

207

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“…Although the molecular mass predicted from its sequence is 14 kDa, it has been reported that in solution, native NTF2 has an apparent molecular mass of 28 kDa (Grundmann et al, 1988;Moore & Blobel, 1994;, consistent with its existing as a dimer. We used gel-filtration chromatography to confirm that the bacterially expressed rat NTF2 had also dimerized.…”

Section: Expression and Characterization Of Ntf2mentioning

confidence: 90%

The 1.6 Å Resolution Crystal Structure of Nuclear Transport Factor 2 (NTF2)

Bullock

Clarkson

Kent

et al. 1996

Journal of Molecular Biology

143

127

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“…The appearance of a homolog of testis‐specific gene A2 might be associated with male meiotic metaphase chromosome [40]. Surprisingly, a homolog of placental protein 15 was present in the male seahorse cDNA library, whose roles either as nuclear transport factor 2 [41] or others are unclear.…”

Section: Resultsmentioning

confidence: 99%

Molecular profile of the unique species of traditional Chinese medicine, Chinese seahorse (Hippocampus kuda Bleeker)

Zhang

Mou

et al. 2003

FEBS Letters

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A cDNA library of male Chinese seahorse (Hippocampus kuda Bleeker) was constructed to investigate the molecular pro¢le of seahorse as one of the most famous traditional Chinese medicine materials, and to reveal immunological and physiological mechanisms of seahorse as one of the most primitive vertebrates at molecular level. A total of 3372 expressed sequence tags (ESTs) consisting of 1911 unique genes (345 clusters and 1566 singletons) were examined in the present study. Identi¢cation of the genes related to immune system, paternal brooding and physiological regulation provides not only valuable insights into the molecular mechanism of immune system in teleost ¢sh but also plausible explanations for pharmacological activities of Chinese seahorse. Furthermore, the occurrence of high prevalent C-type lectins suggested that a lectin-complement pathway might exert a more dominant function in the innate immune system of teleost than mammal. Carbohydrate recognition domain (CRD) without a collagen-like region in the lectins of seahorse was likely an ancient characteristic of lectins similar to invertebrates. ß

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Isolation of cDNA coding for the placental protein 15 (PP15)

Cited by 18 publications

References 2 publications

Purification of a Ran-interacting protein that is required for protein import into the nucleus.

Purification of a Ran-interacting protein that is required for protein import into the nucleus.

The 1.6 Å Resolution Crystal Structure of Nuclear Transport Factor 2 (NTF2)

Molecular profile of the unique species of traditional Chinese medicine, Chinese seahorse (Hippocampus kuda Bleeker)

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