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PubMed1993

Studies on oestrone sulphate sulphohydrolase from human placenta nuclear envelopes--solubilization and properties.

Kinetic properties of human placenta nuclear envelope oestrone sulphate sulphohydrolase were determined in envelope suspension, Triton X-100 solubilized enzyme preparations and in partially purified enzyme preparations. Purification of the enzyme(s) extracted with Triton X-100 was performed by three alternative procedures: Bio-gel A 15 m chromatography, DEAE-cellulose chromatography and Con-A-Sepharose chromatography. The nuclear envelope suspension revealed optimal hydrolysis of oestrone sulphate at pH 8.2; the Triton X-100 solubilized enzyme exhibited optimal activities at pH 6.6 and 8.6. Three distinct activity peaks of oestrone sulphate hydrolysis were found for the partially purified enzyme preparations: at pH 6.2 (5.8-6.6), at pH 7.0 and pH 8.4 (8.0-8.8). The influence of the oestrone sulphate concentration on the enzyme activity determined at pH 6.2 exhibited a sigmoid saturation kinetics with all enzyme preparations except that obtained after Con-A Sepharose chromatography. The value of Hill (h) coefficient was 2; S0.5 = 65 microM, the K'm = 4.9 x 10(-9) M. At pH 7.0 and 8.4, the influence of oestrone sulphate concentration on enzyme activity revealed a Michaelis-Menten saturation kinetics; the respective values of Km were 4.2 +/- 1.1 x 10(-6) M and 1.08 +/- 0.11 x 10(-4) M. The Km value of oestrone sulphate hydrolysis of the enzyme preparation after Con-A Sepharose chromatography determined at pH 6.2, 7.0 or 8.1 was 4.0 x 10(-4) M.

Arylsulfatases↗
PubMed1983

Importance of phospholipids to nitrosourea interactions with the nuclear envelope and associated nucleic acids.

Nuclease digestion of triton isolated HeLa cell nuclei has resulted in nuclear envelopes containing less than 10% of the nuclear macromolecule. Alkylation and carbamoylation of the nuclear envelope (NE) fraction by chloroethylnitrosoureas (HNU) was disproportionately high (approximately 35% of total nuclear alkylation and approximately 55% carbamoylation) suggesting that the nucleophilic species in the envelope were preferential targets for drug binding. Digestion of the envelope fraction with phospholipase C (PLC), followed by buoyant density gradient separation in Percoll (70%):0.15 M NaCl (30%), demonstrated that some envelope:nucleic acid attachments were destabilized as a result of phospholipid cleavage. In addition, the buoyant density of HNU-modified NE macromolecules was slightly altered by PLC digestion, suggesting that cleavage of polar phospholipids did not completely destabilize the chloroethylated products from the nuclear envelope.

Alkylation↗
PubMed1984

The role of protein phosphokinase and protein phosphatase during the nuclear envelope nucleoside triphosphatase reaction.

The activities of nuclear envelope-associated protein phosphokinase and protein phosphatase were determined in nuclear ghosts from liver and oviduct of quails. The protein kinase was found to be inhibited by poly(A) by 75%. During the kinase reaction proteins with molecular weights of 106 000 and 64 000 were phosphorylated. The phosphoprotein phosphatase from liver was stimulated to 190% by poly(A), whereas only a slight enhancing effect by this polymer was determined with the oviduct enzyme (to 125%). Comparative determinations of the nuclear ghost-associated enzyme activities revealed the following values (in nmol Pi/min per 10(8) ghosts); oviduct: phosphokinase, 0.015; phosphatase, 0.004 and nucleoside triphosphatase, 39.4; and liver: phosphokinase, 0.044; phosphatase, 0.012 and nucleoside triphosphatase, 11.7. These data indicate that phosphorylation/dephosphorylation proceeds independently of the nucleoside triphosphatase cycle. This assumption is supported by analytical results revealing that no marked dephosphorylation occurs after poly(A) binding to the nuclear envelope. Moreover, stoichiometrical data showed a nearly 1:1 molar ratio between ATP-binding and phosphorylation of nuclear envelope protein. From these findings a new model for the nucleoside triphosphatase-mediated poly(A)(+)mRNA efflux from nuclei is deducted, proposing phosphokinase and phosphatase only to modulate the affinity of the 'carrier structure' for poly(A) (+)mRNA, but not to constitute the nucleoside triphosphatase.

Animals↗
PubMed1979

Properties of mammalian nuclear-envelope nucleoside triphosphatase.

The nucleoside triphosphatase activities of the nuclear envelopes from rat liver, pig liver and simian-virus-40-transformed mouse-embryo 3T3 cells were shown to exhibit similar parperties. All three preparations hydrolyse ATP, 2'-dATP, 3'-dATP, GTP, CTP and UTP in the presence of Mg2+, Ca2+, Mn2+ and Co2+ with a pH optimum of 8.0, are sensitive to inhibition by mercurials, arsenicals, quercetin, proflavin and adenosine 5'-[gamma-thio]triphosphate and are partially inactivated by exposure to high ionic strength. The kinetic behaviour is similar for all substrates irrespective of the source of material. The typical Eadie-Hofstee plot, which is concave upwards at pH 8.0 when the ionic strength is 20mM, becomes linear when the pH is increased to 8.5 or the ionic strength to 160mM. The overall evidence, particularly the labelling of only one polypeptide by [gamma-32P]ATP, suggests that under the conditions of preparation and assay used only one class of nucleoside triphosphatase active sites is detectable in nuclear envelopes. The importance of these results for an understanding of the role of the enzyme in vivo is discussed.

Adenine↗
PubMed1999

Differential localization of 5- and 15-lipoxygenases to the nuclear envelope in RAW macrophages.

Leukotriene formation is initiated in myeloid cells by an increase in intracellular calcium and translocation of 5-lipoxygenase from the cytoplasm to the nuclear envelope where it can utilize arachidonic acid. Monocyte- macrophages and eosinophils also express 15-lipoxygenase, which converts arachidonic acid to 15(S)-hydroxyeicosatetraenoic acid. Enhanced green fluorescent 5-lipoxygenase (5-LO) and 15-lipoxygenase (15-LO) fusion proteins were expressed in the cytoplasm of RAW 264.7 macrophages. Only 5-lipoxygenase translocated to the nuclear envelope after cell stimulation, suggesting that differential subcellular compartmentalization can regulate the generation of leukotrienes versus 15(S)-hydroxyeicosatetraenoic acid in cells that possess both lipoxygenases. A series of truncation mutants of 5-LO were created to identify putative targeting domains; none of these mutants localized to the nuclear envelope. The lack of targeting of 15-LO was then exploited to search for specific targeting motifs in 5-LO, by creating 5-LO/15-LO chimeric molecules. The only chimera that could sustain nuclear envelope translocation was one which involved replacement of the N-terminal 237 amino acids with the corresponding segment of 15-LO. Significantly, no discrete targeting domain could be identified in 5-LO, suggesting that sequences throughout the molecule are required for nuclear envelope localization.

Amino Acid Sequence↗
PubMed2001

Binding of heterochromatin protein 1 to the nuclear envelope is regulated by a soluble form of tubulin.

We have previously shown that the mouse heterochromatin protein 1 homologue M31 interacts dynamically with the nuclear envelope. Using quantitative in vitro assays, we now demonstrate that this interaction is potently inhibited by soluble factors present in mitotic and interphase cytosol. As indicated by depletion and order-of-addition experiments, the inhibitory activity co-isolates with a 55-kDa protein, which binds avidly to the nuclear envelope and presumably blocks M31-binding sites. Purification of this protein and microsequencing of tryptic peptides identify it as alpha2/6:beta2-tubulin. Consistent with this observation, bona fide tubulin, isolated from rat brain and maintained in a nonpolymerized state, abolishes binding of M31 to the nuclear envelope and aborts M31-mediated nuclear envelope reassembly in an in vitro system. These observations provide a new example of "moonlighting," a process whereby multimeric proteins switch function when their aggregation state or localization is altered.

Amino Acid Sequence↗
PubMed1979

Dynamics of the nuclear envelope during cell cycle in plants.

Stereology of Allium cepa root meristem cells was done to evaluate changes in the nuclear envelope during cell cycle. A naturally synchronous population was labelled as binucleate by caffeine inhibition of cytokinesis. Growth of the nuclear envelope preferentially occurs from mid G2 to the next mid G1, most probably in relation to the reforming sister nuclei after mitosis. On the other hand, the number of nuclear pores doubles from mid G1 to mid G2, their growth rate being higher in the first half of interphase (from mid G1 to mid S). Hence, the new nuclear envelope probably lacks nuclear pores, which appear later.

Cell Cycle↗
PubMed1976

Selective effects of nonionic detergent and salt solutions in dissolving nuclear envelope protein.?20U.

Protein has been selectively extracted from isolated chicken erythrocyte nuclear envelope by (1) dilute MgCl2/Triton X-100 followed by (2) concentrated MgCl2/Triton X-100 solutions. Certain proteins appear to be selectively dissolved in the first solvent and may occur in the nuclear envelope primarily as lipoproteins. Among the proteins insoluble in the low MgCl2/Triton X-100 wash, as well as in 500 mM MgCl2 without Triton previously used in the preparation of the envelope fraction, the quantitatively major polypeptides dissolve in a combination of high MgCl2 and Triton X-100. Further, much of this dissolved protein precipitates when the MgCl2 concentration is lowered by dialysis. The insolubility of these proteins appears to result from a combination of ionic and hydrophobic interactions and may explain the resistance of nuclei to various manipulative procedures including nonionic detergent washes. The procedures described provide a route for gently and selectively dissolving representative proteins from the nuclear envelope lipoprotein matrix and from the envelope "residual" protein.

Animals↗
PubMed1996

Characterization of p18, a component of the lamin B receptor complex and a new integral membrane protein of the avian erythrocyte nuclear envelope.

Employing avian erythrocytes, we have previously isolated a multimeric complex consisting of the lamin B receptor (LBR, or p58), the nuclear lamins, an LBR-specific kinase, a 34-kDa protein, and an 18-kDa polypeptide termed p18. As the LBR kinase and the 34-kDa component have been recently characterized, we now proceed in the characterization of p18. We show here that p18 is an integral membrane protein specific to the erythrocyte nuclear envelope which binds to LBR and B-type lamins. NH2-terminal sequencing indicates that p18 is distinct from other nuclear envelope components, but has similarity to the mitochondrial isoquinoline-binding protein. In situ analysis by immunoelectron microscopy and examination of digitonin-permeabilized cells by indirect immunofluorescence show that p18, unlike LBR and other lamin-binding proteins, is equally distributed between the inner and outer nuclear membrane. Furthermore, cycloheximide inhibition experiments reveal that the fraction of p18 that resides in the outer nuclear membrane does not represent nascent chains en route to the inner nuclear membrane, but rather material in equilibrium with the p18 that partitions with the inner nuclear membrane. The paradigm of p18 suggests that transmembrane complexes formed by the nuclear lamins and LBR provide potential docking sites for integral membrane proteins of the nuclear envelope that equilibrate between the rough endoplasmic reticulum and the inner nuclear membrane.

Amino Acid Sequence↗
PubMed2006

Guilt by association: the nuclear envelope proteome and disease.

The discovery that many inherited diseases are linked to interacting nuclear envelope proteins has raised the possibility that human genetic studies could be assisted by a fusion with proteomics. Two principles could be applied. In the first, the proteome of an organelle associated with a genetically variable disease is determined. The chromosomal locations of the genes encoding the organellar proteins are then determined. If a related disease is linked to a large chromosomal region that includes a gene identified in the organelle, then that gene has an increased likelihood of causing the disease. Directly sequencing this allele from patient samples might speed identification compared with further genetic linkage studies as has been demonstrated for multiple diseases associated with the nuclear envelope. The second principle is that if an organelle has been implicated in the pathology of a particular disorder, then comparison of the organelle proteome from control and patient cells might highlight differences that could indicate the causative protein. The distinct, tissue-specific pathologies associated with nuclear envelope diseases suggest that many tissues will have a set of disorders linked to this organelle, and there are numerous as yet unmapped or partially mapped syndromes that could benefit from such an approach.

Disease↗
PubMed2003

The first 238 amino acids of the human lamin B receptor are targeted to the nuclear envelope in plants.

In plants, the nuclear envelope (NE) is one of the least characterized cellular structures. In particular, little is known about its dynamics during the cell cycle. This is due to the absence of specific markers for in vivo studies. To generate such an in vivo marker, the suitability of the human lamin B receptor (LBR) was tested. When the first 238 amino acids of the LBR, fused to the green fluorescent protein (GFP), were expressed in tobacco plants, fluorescence accumulated only at the NE of leaf epidermal cells. This was confirmed by electron microscopy. The protein was shown to be membrane-integral by phase separation. Distribution of fluorescence was compared with two ER markers, GFP-calnexin and GFP-HDEL. While co-localization of all three markers was noted at the NE, only LBR-GFP was specific to the NE, while the other two also showed fluorescence of the cortical ER. These results suggest that common targeting mechanisms to those in animals and fungi exist in plants to direct and locate proteins to the NE. This chimaeric construct is the first available fluorescent integral membrane protein marker to be targeted exclusively to the plant NE and it provides a novel opportunity to investigate the dynamics of this membrane system in vivo. With it, the cell cycle was followed in tobacco BY-2 cells stably expressing the fusion protein. The interphase labelling of the NE altered in metaphase into an ER-like meshwork, suggesting the dispersal of the NE to ER as in animal cells. Finally, the meshwork of fluorescent membranes was lost and new fluorescent NE formed around the daughter nuclei.

Cell Line↗
PubMed1996

Export of ribosomal subunits from resealed rat liver nuclear envelopes.

We have previously described the rat liver resealed nuclear envelope model system for the study of the selective import of nuclear proteins, and the export of poly(A)-containing mRNA [Riedel, N., Bachmann, M., Richter, H. & Fasold, H. (1987) Proc. Natl Acad. Sci. USA 83, 3540-3544]. The vesicles still respond to the importin-ATP signal for the uptake of nuclear-location-sequence (NLS)-carrying proteins. During the preparation of the vesicles and extraction of the chromatin from nuclei in cold hypotonic heparin solution, ribosomal subunits may be introduced into these envelopes, and after resealing remain stably included. Efflux from the resealed nuclear envelopes is effected by a cytoplasmatic protein fraction, and strongly enhanced in the presence of ATP. The heterogeneous nuclear RNP (hnRNP) A1, the components of importin, or GTP showed no influence on this export. The ATP-dependent efflux of mRNA is not affected by these cytoplasmic proteins in this model system.

Adenosine Triphosphate↗
PubMed1995

Purification and molecular shape of a 144 kDa protein bearing N-acetylglucosamine residues from rat liver nuclear envelopes.

A 144 kDa protein was purified from the WGA-Sepharose bound fraction of a rat liver nuclear envelope salt-extract by hydroxyapatite HPLC (HAP HPLC). Two other, 120 and 86 kDa, proteins were also partially purified from the fraction by a combination of DEAE- and HAP-HPLCs. It was suggested that the 144, 120, and 86 kDa proteins bear GlcNAc residues, and are nucleoporins, because they were purified from nuclear envelopes, reacted with WGA-HRP, and cross-reacted with an antibody against p62 nucleoporin complexes. The sedimentation coefficients and Stokes' radii of these GlcNAc-bearing proteins were determined by glycerol density gradient centrifugation and gel filtration in the presence of 500 mM NaCl. The molecular masses calculated from these values suggested that these three proteins each exist as a monomer under the conditions employed. The axial ratios of the purified 144, 120, and 86 kDa GlcNAc-proteins were estimated to be 35, 31, and 31, respectively. These values suggested that they are rod-shaped molecules. The axial ratio of a purified nucleoporin-complex consisting of 62, 60, and 54 kDa components bearing GlcNAc was shown to be 20. This nucleoporin complex seems to be a rod-shaped complex. From these results, a rod shape is proposed to be a common characteristic of GlcNAc-proteins in nuclear envelopes.

Acetylglucosamine↗
PubMedDate not supplied

The use of field emission in-lens scanning electron microscopy to study the steps of assembly of the nuclear envelope in vitro.

At mitosis the nuclear envelope (NE) is disassembled to allow chromosome separation. In telophase it is reassembled as the chromosomes decondense. Cell-free extracts of Xenopus eggs have been used extensively to study assembly of the NE and the nuclear pore complexes (NPCs), providing several models for the steps involved. The NE is a surface structure which in cell-free extracts is easily exposed. It is appropriate, therefore, to use a surface imaging technique to study NE dynamics. Field emission in-lens scanning electron microscopy (FEISEM) provides the opportunity to image surfaces, directly, and to visualise details of structures such as the NPC. Here we show the feasibility and value of FEISEM to study the steps of NE formation. Nuclei have been assembled in vitro and fixed at different time points during assembly, followed by conductive staining, platinum coating, and visualisation by FEISEM. Changes on the nuclear surface with time are shown. Details of the surface of chromatin and the cytoplasmic face of NPC structure are demonstrated without the need to isolate the structures from the nucleus.

Animals↗
PubMed2005

The nuclear envelope: form and reformation.

The membrane system that encloses genomic DNA is referred to as the nuclear envelope. However, with emerging roles in signaling and gene expression, these membranes clearly serve as more than just a physical barrier separating the nucleus and cytoplasm. Recent progress in our understanding of nuclear envelope architecture and composition has also revealed an intriguing connection between constituents of the nuclear envelope and human disease, providing further impetus to decipher this cellular structure and the dramatic remodeling process it undergoes with each cell division.

Animals↗
PubMed1989

Statin, a nonproliferation-specific protein, is associated with the nuclear envelope and is heterogeneously distributed in cells leaving quiescent state.

Statin, a protein of 57,000 daltons, is present primarily in the nuclei of nonproliferating cells of terminally differentiated tissues or of in vitro aged fibroblast cultures. In young growing cells, the protein can be induced to appear in the nuclei once the cell-cycle traverse is blocked by various tissue culture manipulations, such as serum starvation; this expression, however, can be rapidly removed by addition of serum. The disappearance of statin in cells leaving the quiescent state is not uniform along the periphery of the nucleus; it can be distributed in various patterns, such as caps, nodules, patches, or irregular granules. This unusual distribution seems to suggest that preferential sites exist at the region of the nuclear envelope where statin presence may residually remain. The concentration of statin at the nuclear envelope region in cells at G0-quiescent phase is confirmed by the intense staining of fluorescent antibody at the periphery of isolated rat liver nuclei. Further examination of the isolated nuclei reveals that the protein is associated with the lamina compartment of the nuclear envelope; this is evidenced by the results of immunoblotting experiments showing statin presence in the fraction enriched for lamins A-C. Immunogold labelling studies show that the protein is located in the general area of the nuclear envelope. These results suggest that statin in G0-quiescent cells is located predominantly at the nuclear envelope region and that in this vicinity there may exist geometrically sites of statin concentration as evidenced by the heterogeneous distribution in those cells experiencing the departure from the quiescent state.

Animals↗
PubMed1984

Nuclear envelope localization of an adenovirus tumor antigen maintains the integrity of cellular DNA.

The adenovirus early-region 1B 19,000-molecular-weight tumor antigen is required for oncogenic transformation of cells by adenovirus. We have demonstrated that this tumor antigen is located in the nuclear envelope of infected and transformed cells and that a fraction of the protein within the nuclear envelope is associated with the nuclear lamina. During cell division in the transformed cells, the nuclear envelope containing the tumor antigen dissociates at metaphase and then reforms around the separated daughter chromosomes at telophase. Adenovirus mutants carrying lesions in the gene encoding this tumor antigen cause degradation of host cell chromosomal DNA, and in these mutants, the intracellular localization of the 19,000-dalton protein is altered. These results demonstrate that components of the nuclear envelope function in the organization of chromatin in infected and transformed cells and that a virus-encoded protein plays a critical role in this process.

Adenoviridae↗
PubMedDate not supplied

[Effect of glucocorticoids and adrenaline on insulin receptors in plasma membranes and nuclear envelopes of the rat liver].

To elucidate the course of regulation of insulin receptors in nuclear envelope and its relationship with insulin receptors in the plasma membrane a comparative study of these receptors in both subcellular fractions was conducted under the influence of the involved cell surface receptor factor. It is found that under adrenalectomy the number of nuclear envelope receptors and degree of their affinity did not increase as this occurs in plasma membrane receptors. Hydrocortisone replacement therapy in these animals lowers the receptor number in the both fractions. Hydrocortisone-induced hypercorticism does not change 125I-insulin binding by nuclear envelope but decreases its binding (accounted for the number of receptors and affinity) by the plasma membrane. Under hyperadrenalinemia the number of receptors decreases in the both subcellular fractions. The results suggest no independent regulation of insulin receptors on the surfaces of nuclei and cells.

Adrenalectomy↗