scholarly journals Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution.

1978 ◽  
Vol 79 (2) ◽  
pp. 546-566 ◽  
Author(s):  
L Gerace ◽  
A Blum ◽  
G Blobel
Keyword(s):  
Rat Liver ◽  
Nuclear Periphery ◽  
Sodium Dodecyl ◽  
Double Diffusion ◽  
Immunoperoxidase Staining ◽  
Nuclear Pore ◽  
Intense Staining ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Pore Complexes

This laboratory has previously isolated a fraction from rat liver nuclei consisting of nuclear pore complexes associated with the proteinaceous lamina which underlies the inner nuclear membrane. Using protein eluted from sodium dodecyl sulfate (SDS) gels, we have prepared antibodies in chickens to each of the three predominant pore complex-lamina bands. Ouchterlony double diffusion analysis shows that each of these individual bands cross-reacts strongly with all three antisera. In immunofluorescence localization performed on tissue culture cells with these antibodies, we obtain a pattern of intense staining at the periphery of the interphase nucleus, with little or no cytoplasmic reaction. Electron microscope immunoperoxidase staining of rat liver nuclei with these antibodies labels exclusively the nuclear periphery. Furthermore, reaction occurs in areas which contain the lamina, but not at the pore complexes. While our isolation procedure extracts the internal contents of nuclei completely, semiquantitative Ouchterlony analysis shows that it releases negligible amounts of these lamina antigens. Considered together, our results indicate that these three bands represent major components of a peripheral nuclear lamina, and are not structural elements of an internal "nuclear protein matrix." Fluorescence microscopy shows that the perinuclear interphase localization of these lamina proteins undergoes dramatic changes during mitosis. Concomitant with nuclear envelope disassembly in prophase, these antigens assume a diffuse localization throughout the cell. This distribution persists until telophase, when the antigens become progressively and completely localized at the surface of the daughter chromosome masses. We propose that the lamina is a biological polymer which can undergo reversible disassembly during mitosis.

scholarly journals A modified procedure for the isolation of a pore complex-lamina fraction from rat liver nuclei.

1976 ◽  
Vol 70 (3) ◽  
pp. 581-591 ◽  
Author(s):  
N Dwyer ◽  
G Blobel
Keyword(s):  
Nuclear Envelope ◽  
Rat Liver ◽  
Nuclear Pore Complex ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Envelope Membrane ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Pore Complexes

A modified procedure for the isolation of a nuclear pore complex-lamina fraction from rat liver nuclei is described. Evidence is provided that the isolated lamina, a 150-A thick, proteinaceous structure, apposes the inner nuclear envelope membrane, connecting nuclear pore complexes and surrounding the entire nucleus.

scholarly journals Cytochemical studies on the relation of nucleoside triphosphatase activity to ribonucleoproteins in isolated rat liver nuclei.

1980 ◽  
Vol 28 (1) ◽  
pp. 27-35 ◽  
Author(s):  
A Vorbrodt ◽  
G G Maul
Keyword(s):  
Nuclear Envelope ◽  
Rat Liver ◽  
Enzyme Reaction ◽  
Point Of View ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Isolated Rat Liver ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Isolated Rat

Cytochemical tests for nucleosidetriphosphatase (NTPase) and Bernhard's preferential staining for ribonucleoproteins (RNP) were applied to isolated rat liver nuclei. The strongest and most easily reproducible positive reaction for NTPase was detected at pH 7.7 with ATP and GTP. This reaction was activated by Mg2+ and Ca2+ and inhibited by Be2+, Zn2+, quercetin, and ribonuclease. The major sites of enzyme reaction were intranuclear RNA-containing structures. Incubation of nuclei in ATP-stimulated RNA-release medium eliminated a considerable part of the material showing both NTPase reaction and staining for RNP; the perichromatin granules disappeared, while interchromatin granules remained. NTPase activity in the nuclear envelope seems to be associated with the annular part of nuclear pore complexes (permanent component) and with RNP particles translocated through nuclear pores or attached to the surface of nuclei (transitional component). From a morphological point of view, these observations support previous biochemical data suggesting the existence of a connection between NTPase activity and the translocation of RNP particles through the nuclear envelope.

scholarly journals Rat liver nuclear skeleton and ribonucleoprotein complexes containing HnRNA.

1978 ◽  
Vol 76 (3) ◽  
pp. 675-691 ◽  
Author(s):  
T E Miller ◽  
C Y Huang ◽  
A O Pogo
Keyword(s):  
Rat Liver ◽  
Sulfonyl Chloride ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Morphological Aspect ◽  
Ribonucleoprotein Complexes ◽  
Liver Nuclei ◽  
Rnase Treatment ◽  
Labeled Rna ◽  
Pore Complexes

Rat liver nuclei deprived of chromatin and nucleoplasm show a spongelike network which preserves its connection with nucleoli, the inner membrane of the nuclear envelope, and nuclear pore complexes. It contains all of the HnRNA, provided the endogenous proteolytic activity is inhibited by a proteolytic inhibitor such as phenylmethyl sulfonyl chloride (PMSC) or the fluoride form (PMSF). In the absence of these proteolytic inhibitors, HnRNA is dissociated from the spongelike network and sediments in a sucrose gradient as polydispersed ribonucleoprotein complexes. Furthermore, purified HnRNA as well as rRNA do not bind to the spongelike network when added to these nuclei. These observations demonstrate that the association of HnRNA to the nuclear skeleton is not an artifact. RNase treatment of the spongelike network digests the majority of the rapidly labeled RNA but does not alter the morphological aspect nor the architecture of this network. EDTA and heparin treatments affect neither the attachment of HnRNA nor the structural organization of this network. Electron microscope studies of the network reveal a characteristic flexuous configuration. Its relationship with diffused and condensed chromatin is discussed.

scholarly journals Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores.

1987 ◽  
Vol 104 (2) ◽  
pp. 189-200 ◽  
Author(s):  
D R Finlay ◽  
D D Newmeyer ◽  
T M Price ◽  
D J Forbes
Keyword(s):  
Rat Liver ◽  
Protein Transport ◽  
Nuclear Protein ◽  
Nuclear Transport ◽  
Nuclear Pore ◽  
Major Mechanism ◽  
Rat Liver Nuclei ◽  
Egg Extract ◽  
Liver Nuclei

Selective transport of proteins is a major mechanism by which biochemical differences are maintained between the cytoplasm and nucleus. To begin to investigate the molecular mechanism of nuclear transport, we used an in vitro transport system composed of a Xenopus egg extract, rat liver nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. With this system, we screened for inhibitors of transport. We found that the lectin, wheat germ agglutinin (WGA), completely inhibits the nuclear transport of fluorescently labeled nucleoplasmin. No other lectin tested affected nuclear transport. The inhibition by WGA was not seen when N-acetylglucosamine was present and was reversible by subsequent addition of sugar. When rat liver nuclei that had been incubated with ferritin-labeled WGA were examined by electron microscopy, multiple molecules of WGA were found bound to the cytoplasmic face of each nuclear pore. Gel electrophoresis and nitrocellulose transfer identified one major and several minor nuclear protein bands as binding 125I-labeled WGA. The most abundant protein of these, a 63-65-kD glycoprotein, is a candidate for the inhibitory site of action of WGA on nuclear protein transport. WGA is the first identified inhibitor of nuclear protein transport and interacts directly with the nuclear pore.

Labelling of Proteins by Isolated Rat Liver Nuclei

1972 ◽  
Vol 50 (2) ◽  
pp. 190-199 ◽  
Author(s):  
K. M. Anderson ◽  
M. Slavik ◽  
O. P. Elebute
Keyword(s):  
Protein Synthesis ◽  
Rat Liver ◽  
Nuclear Protein ◽  
Sucrose Solution ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rat Liver Nuclei ◽  
Teleological Argument ◽  
Liver Nuclei ◽  
Triton X

Rat liver nuclei, isolated in hypertonic sucrose solution and washed with Triton X-100, incorporate radioactive amino acids into hot trichloroacetic acid insoluble materials.Optical and biochemical evidence of nuclear purity is presented. The temperature-dependent incorporation continued for 20–30 min, and was proportional to the concentrations of both nuclear protein between 0.5–1.5 mg/ml, and radioactive amino acid. The radioactive product was degraded by pronase, and a number of inhibitors reduced incorporation, but only if present at [Formula: see text]. Proteins extracted from labelled nuclei and microsomes and examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis at pH 7.2 exhibited different patterns of radioactivity. This provides further support for the concept of protein synthesis intrinsic to rat liver nuclei.A teleological argument for the function of nuclear protein synthesis is discussed.

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