scholarly journals Immunological localization of a major karyoskeletal protein in nucleoli of oocytes and somatic cells of Xenopus laevis.

1982 ◽  
Vol 94 (3) ◽  
pp. 749-754 ◽  
Author(s):  
G Krohne ◽  
R Stick ◽  
J A Kleinschmidt ◽  
R Moll ◽  
W W Franke ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Xenopus Oocytes ◽  
Specific Protein ◽  
Nuclear Bodies ◽  
Core Complex ◽  
Antibody Staining ◽  
First Case ◽  
Strong Staining ◽  
Triton X ◽  
Peptide Maps

Oocyte nuclei of Xenopus laevis contain two major karyoskeletal proteins characterized by their resistance to extractions in high salt buffers and the detergent Triton X-100, i.e. a polypeptide of 68,000 mol wt which is located in the core complex-lamina structure and a polypeptide of 145,000 mol wt enriched in nucleolar fractions. Both proteins are also different by tryptic peptide maps and immunological determinants. Mouse antibodies were raised against insoluble karyoskeletal proteins from Xenopus oocytes and analyzed by immunoblotting procedures. Affinity purified antibodies were prepared using antigens bound to nitrocellulose paper. In immunofluorescence microscopy of Xenopus oocytes purified antibodies against the polypeptide of 145,000 mol wt showed strong staining of nucleoli, with higher concentration in the nucleolar cortex, and of smaller nucleoplasmic bodies. In various other cells including hepatocytes, Sertoli cells, spermatogonia, and cultured kidney epithelial cells antibody staining was localized in small subnucleolar granules. The results support the conclusion that this "insoluble" protein is a major nucleus-specific protein which is specifically associated with--and characteristic of--nucleoli and certain nucleolus-related nuclear bodies. It represents the first case of a positive localization of a karyoskeletal protein in the nuclear interior, i.e. away from the pore complex-lamina structure of the nuclear cortex.

scholarly journals Ca2+-dependent interaction of the growth-associated protein GAP-43 with the synaptic core complex

1997 ◽  
Vol 325 (2) ◽  
pp. 455-463 ◽  
Author(s):  
Tatsumi HARUTA ◽  
Noboru TAKAMI ◽  
Manami OHMURA ◽  
Yoshio MISUMI ◽  
Yukio IKEHARA
Keyword(s):  
Pc12 Cells ◽  
Synaptic Vesicles ◽  
Specific Protein ◽  
Cross Linking ◽  
Core Complex ◽  
Synaptic Vesicle Exocytosis ◽  
Kinase C ◽  
Vesicle Exocytosis ◽  
Chemical Cross Linking ◽  
Dependent Interaction

The synaptic vesicle exocytosis occurs by a highly regulated mechanism: syntaxin and 25 kDa synaptosome-associated protein (SNAP-25) are assembled with vesicle-associated membrane protein (VAMP) to form a synaptic core complex and then synaptotagmin participates as a Ca2+ sensor in the final step of membrane fusion. The 43 kDa growth-associated protein GAP-43 is a nerve-specific protein that is predominantly localized in the axonal growth cones and presynaptic terminal membrane. In the present study we have examined a possible interaction of GAP-43 with components involved in the exocytosis. GAP-43 was found to interact with syntaxin, SNAP-25 and VAMP in rat brain tissues and nerve growth factor-dependently differentiated PC12 cells, but not in undifferentiated PC12 cells. GAP-43 also interacted with synaptotagmin and calmodulin. These interactions of GAP-43 could be detected only when chemical cross-linking of proteins was performed before they were solubilized from the membranes with detergents, in contrast with the interaction of the synaptic core complex, which was detected without cross-linking. Experiments invitro showed that the interaction of GAP-43 with these proteins occurred Ca2+-dependently; its maximum binding with the core complex was observed at 100 μM Ca2+, whereas that of syntaxin with synaptotagmin was at 200 μM Ca2+. These values of Ca2+ concentration are close to that required for the Ca2+-dependent release of neurotransmitters. Furthermore we observed that the interaction invitro of GAP-43 with the synaptic core complex was coupled with protein kinase C-mediated phosphorylation of GAP-43. Taken together, our results suggest a novel function of GAP-43 that is involved in the Ca2+-dependent fusion of synaptic vesicles.

Cyclic cytoplasmic activity of non-nucleate egg fragments of Xenopus controls the morphology of injected sperms

1983 ◽  
Vol 63 (1) ◽  
pp. 69-76
Author(s):  
M. Sakai ◽  
A. Shinagawa
Keyword(s):  
Xenopus Laevis ◽  
Sperm Nucleus ◽  
Cyclic Activity ◽  
Triton X

Triton X-100-treated sperms were injected into non-nucleate egg fragments of Xenopus laevis to determine whether the structure of the injected sperm nucleus is affected by the cyclic activity of the cytoplasm. Swollen vesicular nuclei were very frequently observed when the sperms were injected and incubated during the ‘rounding-up’ phase of the recipient fragment, whereas no such structures were found when they were incubated during the ‘relaxing’ phase.

Uptake of biotin by native Xenopus laevis oocytes

1990 ◽  
Vol 259 (3) ◽  
pp. C397-C401 ◽  
Author(s):  
H. M. Said ◽  
L. Polenzani ◽  
S. Khorchid ◽  
D. Hollander ◽  
R. Miledi
Keyword(s):  
Xenopus Laevis ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Sulfhydryl Group ◽  
Maximum Velocity ◽  
Significant Inhibition ◽  
Metabolic Inhibitors ◽  
Xenopus Laevis Oocytes ◽  
Uptake System

The present study examined biotin uptake by Xenopus laevis oocytes in vitro. Uptake of low (0.03 microM) and high (10 microM) concentrations of biotin was linear with time for up to 4 h of incubation and occurred with little initial binding to oocytes. Uptake of biotin was dependent on extracellular Na+ concentration [Na+]o and was severely inhibited when Na+ was replaced by other monovalent cations [choline, tetraethylammonia, Li+, and tris(hydroxymethyl)aminomethane]. The initial rate of biotin uptake was saturable as a function of concentration with an apparent Michaelis constant of 3.9 +/- 0.5 microM and maximum velocity of 1,559 +/- 70 fmol.oocyte-1.h-1. Addition to the incubation medium of biotin structural analogues desthiobiotin and thioctic acid caused significant and concentration-dependent inhibition in the uptake of [3H]biotin. This inhibition was found to be competitive in nature with inhibition constant values of 9 and 17.5 microM. In contrast, neither the structural analogue biocytin nor biotin methyl ester (compounds in which the carboxyl group of the valeric acid moiety is blocked) showed any effect on the uptake of [3H]biotin. Biotin uptake was significantly blocked by the metabolic inhibitors dinitrophenol, cyanide, and azide and by incubation at 4 degrees C. Also, the sulfhydryl group blocker p-(chloromercuri)phenylsulfonate caused significant inhibition in biotin uptake. These results demonstrate that Xenopus oocytes possess an uptake system for biotin in its cell membrane that is Na+, energy, and temperature dependent. These characteristics of biotin uptake are similar to those reported in mammalian cells. It is suggested that Xenopus oocytes might be a useful in vitro model system to study the details of the mechanisms and regulation of biotin movement across biological membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Two structurally and functionally different forms of the transforming protein of PRC II avian sarcoma virus.

1982 ◽  
Vol 2 (8) ◽  
pp. 890-896 ◽  
Author(s):  
B Adkins ◽  
T Hunter
Keyword(s):  
Virus Genome ◽  
Specific Protein ◽  
Protein Kinase Activity ◽  
Avian Sarcoma Virus ◽  
Nonionic Detergent ◽  
Sarcoma Virus ◽  
Infected Cells ◽  
Avian Sarcoma ◽  
Peptide Maps ◽  
Specific Protein Kinase

The primary translation product of the PRC II avian sarcoma virus genome is a protein of 105,000 daltons (P105), and we have previously shown that approximately 50% of the P105 molecules are converted to molecules of 110,000 daltons (P110) by posttranslational modification. Fractionation of PRC II-infected cells showed that P105 was contained primarily in a nonionic detergent-soluble compartment, whereas P110 partitioned almost exclusively with a nonionic detergent-insoluble or crude cytoskeletal fraction. The tyrosine-specific protein kinase activity previously observed in immunoprecipitates which presumably contained both P110 and P105 was found predominantly in the P110-containing immunoprecipitates made from the cytoskeletal fraction and was essentially absent from the P105-containing immunoprecipitates prepared from the soluble fraction. Individual analysis of 32P-labeled P110 and P105 prepared by this fractionation technique revealed that P110 contained more phosphotyrosine per mole of protein than did P105. Examination of the tryptic peptide maps of 32P-labeled P110 and P105 suggested that the additional phosphotyrosine in P110 resulted from phosphorylation at discrete sites within the protein. From these experiments, we conclude that PRC II-infected cells contain two discrete forms, P105 and P110, of the transforming protein and that each of these proteins exhibits distinct structural and functional characteristics.

Differential inhibition of AE1 and AE2 anion exchangers by oxonol dyes and by novel polyaminosterol analogs of the shark antibiotic squalamine

1998 ◽  
Vol 76 (5) ◽  
pp. 799-806 ◽  
Author(s):  
Seth L Alper ◽  
Marina N Chernova ◽  
Jon Williams ◽  
Michael Zasloff ◽  
Foon-Yee Law ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Anion Exchange ◽  
Xenopus Oocytes ◽  
Red Cells ◽  
Xenopus Laevis Oocytes ◽  
Red Cell ◽  
Cell Type ◽  
Anion Exchangers ◽  
Oxonol Dyes ◽  
Assay Conditions

Oxonol and polyaminosterol drugs were examined as inhibitors of recombinant mouse AE1 and AE2 anion exchangers expressed in Xenopus laevis oocytes and were compared as inhibitors of AE1-mediated anion flux in red cells and in HL-60 cells that express AE2. The oxonols WW-781, diBA(5)C4, and diBA(3)C4 inhibited HL-60 cell Cl-/Cl- exchange with IC50 values from 1 to 7 µM, 100-1000 times less potent than their IC50 values for red cell Cl-/anion exchange. In Xenopus oocytes, diBA(5)C4 inhibited AE1-mediated Cl- efflux several hundred times more potently than that mediated by AE2. Several novel squalamine-related polyaminosterols were also evaluated as anion exchange inhibitors. In contrast to diBA(5)C4, polyaminosterol 1361 inhibited oocyte-expressed AE2 8-fold more potently than AE1 (IC50 0.6 versus 5.2 µM). The 3-fold less potent desulfo-analog, 1360, showed similar preference for AE2. It was found that 1361 also partially inhibited Cl- efflux from red cells, whereas neither polyaminosterol inhibited Cl efflux from HL60 cells. Thus, the oxonol diBA(5)C4 is >100-fold more potent as an inhibitor of AE1 than of AE2, whereas the polyaminosterols 1360 and 1361 are 8-fold more potent as inhibitors of AE2 than of AE1. Assay conditions and cell type influenced IC50 values for both classes of compounds.Key words: band 3, oxonols, squalamine, Xenopus laevis oocytes, HL-60 cells.

A calcium-independent chloride current activated by hyperpolarization in Xenopus oocytes

1988 ◽  
Vol 233 (1271) ◽  
pp. 191-199 ◽  
Keyword(s):  
Xenopus Laevis ◽  
Xenopus Oocytes ◽  
Chloride Ions ◽  
Chloride Current ◽  
Calcium Dependent ◽  
External Calcium

Hyperpolarization of oocytes of Xenopus laevis usually elicits mainly passive currents. However, when polarized to potentials more negative than about – 100 mV, oocytes obtained from some donors show a relatively well maintained current that is carried mainly by chloride ions. This response does not depend upon external calcium, and is thus clearly different from the calcium-dependent transient chloride current previously described.

scholarly journals Identification of a novel protein (G210) specific to the Golgi apparatus.

1989 ◽  
Vol 37 (8) ◽  
pp. 1177-1182 ◽  
Author(s):  
R Hogue-Angeletti ◽  
R Y Xu ◽  
J O Gonatas ◽  
A Stieber ◽  
N K Gonatas
Keyword(s):  
Golgi Apparatus ◽  
Specific Protein ◽  
Light Microscopic Level ◽  
Gut Epithelium ◽  
Carbonate Solutions ◽  
Microsomal Membranes ◽  
Relative Molecular Weight ◽  
Immunohistochemical Studies ◽  
Triton X ◽  
Novel Protein

A monoclonal antibody, 3C9, has enabled the detection of a novel Golgi-specific protein in bovine tissues. Immunohistochemical studies at the light microscopic level have detected the 3C9 antigen only in certain cells: exocrine pancreas, gut epithelium, and thymus epithelium. Examination of gut and pancreas by immunoelectron microscopy showed a localization exclusive to the Golgi apparatus. The relative molecular weight of the antigen detected by immunoblotting is 210,000 daltons. The antigen is not extracted from microsomal membranes of bovine gut epithelium by sodium carbonate solutions. Furthermore, the 3C9 antigen enters into the detergent phase when Triton X-114 partitioning methods are used. These data strongly suggest that this novel antigen is an intrinsic membrane protein, resident in the Golgi apparatus of certain cells. Moreover, they enhance the hypothesis that the distribution of enzymes and polypeptides in the Golgi apparatus is cell specific.

scholarly journals Association of methionyl-tRNA synthetase with detergent-insoluble components of the rough endoplasmic reticulum.

1983 ◽  
Vol 96 (4) ◽  
pp. 1138-1147 ◽  
Author(s):  
C V Dang ◽  
D C Yang ◽  
T D Pollard
Keyword(s):  
Endoplasmic Reticulum ◽  
Fluorescent Antibody ◽  
Insoluble Fraction ◽  
Trna Synthetase ◽  
Insoluble Residue ◽  
Synthetase Activity ◽  
Fluorochrome Staining ◽  
Antibody Staining ◽  
Triton X ◽  
Methionyl Trna Synthetase

Using fluorescent antibody staining, we have established the association of methionyl-tRNA synthetase with the endoplasmic reticulum in PtK2 cells. After Triton X-100 extraction, 70% of the recovered aminoacyl-tRNA synthetase activity was found in the detergent-insoluble fraction. This fraction of the enzyme remained localized with insoluble endoplasmic reticulum antigens and with ribosomes, which were stained with acridine orange. By both fluorescence microscopy and electron microscopy the organization of the detergent-insoluble residue was found to depend on the composition of the extracting solution. After extraction with a microtubule-stabilizing buffer containing EGTA, Triton X-100, and polyethylene glycol (Osburn, M., and K. Weber, 1977, Cell, 12:561-571) the ribosomes were aggregated in large clusters with remnants of membranes. After extraction with a buffer containing Triton X-100, sucrose, and CaCl2 (Fulton, A. B., K. M. Wang, and S. Penman, 1980, Cell, 20:849-857), the ribosomes were in small clusters and there were few morphologically recognizable membranes. In both cases the methionyl-tRNA synthetase and some endoplasmic reticulum antigens retained approximately their normal distribution in the cell. Double fluorochrome staining showed no morphological association of methionyl-tRNA synthetase with the microtubule, actin, or cytokeratin fiber systems of PtK2 cells. These observations demonstrate that detergent-insoluble cellular components, sometimes referred to as "cytoskeletal" preparations, contain significant amounts of nonfilamentous material including ribosomes, and membrane residue. Caution is required in speculating about intermolecular associations in such a complex cell fraction.

scholarly journals Distinct distribution of vimentin and cytokeratin in Xenopus oocytes and early embryos

1992 ◽  
Vol 101 (1) ◽  
pp. 151-160 ◽  
Author(s):  
N.P. Torpey ◽  
J. Heasman ◽  
C.C. Wylie
Keyword(s):  
Spinal Cord ◽  
Amino Acid ◽  
Xenopus Oocytes ◽  
Germ Plasm ◽  
Predicted Amino Acid Sequence ◽  
Larval Stages ◽  
Functional Differences ◽  
Early Embryos ◽  
Strong Staining ◽  
Distinct Distribution

We report the identity of a major component of Triton-insoluble extracts from Xenopus oocytes and early embryos. In a previous paper we showed that an antibody, Z9, cross-reacts with two polypeptides from such extracts (Mr 56,000 and 57,000) as well as Xenopus vimentin. Direct microsequencing of the Mr 57,000 protein shows near identity of three tryptic fragments with regions of the predicted amino acid sequence of XCK1(8), a basic cytokeratin whose mRNA is known to be expressed in Xenopus oocytes. We have raised an antibody, CK7, against a fusion protein generated from this cDNA. The specificity of this antibody has been tested using 1- and 2-dimensional immunoblotting, which show that it is specific for the Mr 56,000 and 57,000 proteins, suggesting that these two proteins may be the products of two non-allelic XCK1(8) genes. The antibody does not cross-react with vimentin. We have used CK7 to follow the distribution of XCK1(8) throughout development by immunoblotting and immunocytochemistry. In larval stages, strong staining is seen in the notocord, the apical epithelia of the gut, the mesentery, and a few cells in the spinal cord. In oocytes and early embryos, two distinct intermediate filament (IF) networks can be distinguished: a cortical cytokeratin network, and a deeper vimentin one. In addition, the oocyte germ plasm stains with Z9 but not CK7. We propose that such distinct distributions of each IF protein reflect functional differences during early development.

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