scholarly journals Degradation of glucagon in isolated liver endosomes. ATP-dependence and partial characterization of degradation products

1991 ◽  
Vol 280 (1) ◽  
pp. 211-218 ◽  
Author(s):  
F Authier ◽  
B Desbuquois
Keyword(s):  
Degradation Products ◽  
Glucagon Receptor ◽  
Free System ◽  
Chloroquine Treatment ◽  
Endosomal Acidification ◽  
A Cell ◽  
Poly Ethylene ◽  
Gradient Fractionation

Endosomes have recently been identified as one major site of glucagon degradation in intact rat liver. In this study, a cell-free system has been used to assess the role of ATP-dependent acidification in endosomal glucagon degradation and identify the glucagon products generated. Percoll gradient fractionation of Golgi-endosomal fractions prepared 10-30 min after injection of [125I]iodoglucagon showed a time-dependent shift of the radioactivity towards high densities. Regardless of time, the radioactivity was less precipitable by trichloroacetic acid (Cl3Ac) at high densities than at low densities. Chloroquine treatment slightly increased the density shift of the radioactivity and decreased its Cl3Ac-precipitability throughout the gradient. Incubation of endosomal fractions containing [125I]iodoglucagon in 0.15 M-KCl at 30 degrees C resulted in a time- and pH-dependent generation of Cl3Ac-soluble radioactivity, with a maximum at pH 4 (t1/2, 7 min). At pH 5, 1,10-phenanthroline, bacitracin and p-chloromercuribenzoic acid partially inhibited [125I]iodoglucagon degradation. At pH 6-7, ATP stimulated [125I]iodoglucagon degradation by 5-10-fold and caused endosomal acidification as judged from Acridine Orange uptake. The effects of ATP were inhibited by chloroquine, monensin, N-ethylmaleimide and dansylcadaverine. Poly(ethylene glycol) (PEG) precipitation of the radioactivity associated with endosomes showed that lowering the pH below 5.5 caused dissociation of the glucagon-receptor complex, and that, regardless of incubation conditions, all degraded [125I]iodoglucagon diffused extraluminally. On h.p.l.c., at least three products less hydrophobic than [125I]iodoglucagon were identified in incubation mixtures along with monoiodotyrosine. Radiosequence analysis of the products revealed one major cleavage located C-terminally to Tyr-13 and two minor cleavages affecting Thr-5-Phe-6 and Phe-6-Thr-7 bonds. It is concluded that glucagon degradation in liver endosomes is functionally linked to ATP-dependent endosomal acidification and involves several cleavages in the glucagon sequence.

A somatic cell-derived system for studying both early and late mitotic events in vitro

1989 ◽  
Vol 94 (3) ◽  
pp. 449-462
Author(s):  
J. Nakagawa ◽  
G.T. Kitten ◽  
E.A. Nigg
Keyword(s):  
Chromatin Condensation ◽  
Nuclear Lamina ◽  
Free System ◽  
Cell Free System ◽  
Protein Substrates ◽  
Nuclear Envelope Breakdown ◽  
Biochemical Fractionation ◽  
A Cell

We describe a cell-free system for studying mitotic reorganization of nuclear structure. The system utilizes soluble extracts prepared from metaphase-arrested somatic chicken cells and supports both the disassembly and subsequent partial reassembly of exogenous nuclei. By fluorescence microscopy, biochemical fractionation, protein phosphorylation assays and electron microscopy, we show that chicken embryonic nuclei incubated in extracts prepared from metaphase-arrested chicken hepatoma cells undergo nuclear envelope breakdown, lamina depolymerization and chromatin condensation. These prophase-like events are strictly dependent on ATP and do not occur when nuclei are incubated in interphase extracts. Compared to interphase extracts, metaphase extracts show increased kinase activities toward a number of nuclear protein substrates, including lamins and histone H1; moreover, they specifically contain four soluble phosphoproteins of Mr 38,000, 75,000, 95,000 and 165,000. Following disassembly of exogenous nuclei in metaphase extracts, telophase-like reassembly of a nuclear lamina and re-formation of nuclear membranes around condensed chromatin can be induced by depletion of ATP from the extract. We anticipate that this reversible cell-free system will contribute to the identification and characterization of factors involved in regulatory and mechanistic aspects of mitosis.

Isolation and partial characterization of amphibian tyrosine oxidase polysomes

Development ◽  
1970 ◽  
Vol 24 (1) ◽  
pp. 109-118
Author(s):  
E. L. Triplett ◽  
R. Herzog ◽  
L. P. Russell
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Enzymic Activity ◽  
Antigenic Determinants ◽  
Free System ◽  
A Cell ◽  
Generating System ◽  
Soluble Components

A population of polysomes isolated from frogskinis capable of supporting protein synthesis in a cell-free system containing an energy generating system, ‘soluble components’, and amino acids. These polysomes catalyse the oxidation of DOPA after gentle trypsinization, and they also have antigenic determinants attributable to tyrosine oxidase. Skin polysomes sedimented in 10–30 % sucrose gradients contain tyrosine oxidase peaks of enzymic activity at the bottom and top of the tube and in the 250 S regions. A peak of tyrosine oxidase antigenic acitvity is found in the 250–350S region of the gradient. Polysomes resolved on the gradient retain the ability to support protein synthesis in a cellfree system. All 250–350S particles capable of supporting the incorporation of [14C]amino acid into tyrosine oxidase are precipitable with tyrosine oxidase antibodies. It is probable that 250–350S tyrosine oxidase antibody precipitates contain only polysomes for this protein.

scholarly journals The Epigenetic Repertoire of Daphnia magna Includes Modified Histones

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Nicole F. Robichaud ◽  
Jeanette Sassine ◽  
Margaret J. Beaton ◽  
Vett K. Lloyd
Keyword(s):  
Life Cycle ◽  
Histone H3 ◽  
Dependent Manner ◽  
Nurse Cells ◽  
Important Species ◽  
Clonal Population ◽  
A Cell ◽  
Cycle Dependent

Daphnids are fresh water microcrustaceans, many of which follow a cyclically parthenogenetic life cycle. Daphnia species have been well studied in the context of ecology, toxicology, and evolution, but their epigenetics remain largely unexamined even though sex determination, the production of sexual females and males, and distinct adult morphological phenotypes, are determined epigenetically. Here, we report on the characterization of histone modifications in Daphnia. We show that a number of histone H3 and H4 modifications are present in Daphnia embryos and histone H3 dimethylated at lysine 4 (H3K4me2) is present nonuniformly in the nucleus in a cell cycle-dependent manner. In addition, this histone modification, while present in blastula and gastrula cells as well as the somatic cells of adults, is absent or reduced in oocytes and nurse cells. Thus, the epigenetic repertoire of Daphnia includes modified histones and as these epigenetic forces act on a genetically homogeneous clonal population Daphnia offers an exceptional tool to investigate the mechanism and role of epigenetics in the life cycle and development of an ecologically important species.

Characterization of phospholipase D in a cell-free system of cultured cells derived from rat frontal cortex

1992 ◽  
Vol 595 (1) ◽  
pp. 12-16 ◽  
Author(s):  
Akira Nishida ◽  
Masami Shimizu ◽  
Yasunori Kanaho ◽  
Yoshinori Nozawa ◽  
Shigeto Yamawaki
Keyword(s):  
Frontal Cortex ◽  
Phospholipase D ◽  
Cultured Cells ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Rat Frontal Cortex

Familial Overexpression of β Antithrombin Caused by an Asn135Thr Substitution

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4242-4247
Author(s):  
T.A. Bayston ◽  
A. Tripodi ◽  
P.M. Mannucci ◽  
E. Thompson ◽  
H. Ireland ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Consensus Sequence ◽  
Wild Type ◽  
Antithrombin Deficiency ◽  
Free System ◽  
Heparin Interaction ◽  
A Cell ◽  
Variant Protein ◽  
Very High

We have investigated the basis of antithrombin deficiency in an asymptomatic individual (and family) with borderline levels (≈70% antigen and activity) of antithrombin. Direct sequencing of amplified DNA showed a mutation in codon 135, AAC to ACC, predicting a heterozygous Asn135Thr substitution. This substitution alters the predicted consensus sequence for glycosylation, Asn-X-Ser, adjacent to the heparin interaction site of antithrombin. The antithrombin isolated from plasma of the proband by heparin-Sepharose chromatography contained amounts of β antithrombin (the very high affinity fraction) greatly increased (≈20% to 30% of total) above the trace levels found in normals. Expression of the residue 135 variant in both a cell-free system and COS-7 cells confirmed altered glycosylation arising as a consequence of the mutation. Wild-type and variant protein were translated and exported from COS-7 cells with apparently equal efficiency, in contrast to the reduced level of variant observed in plasma of the affected individual. This case represents a novel cause of antithrombin deficiency, removal of glycosylation concensus sequence, and highlights the potentially important role of β antithrombin in regulating coagulation.

scholarly journals Aster migration determines the length scale of nuclear separation in the Drosophila syncytial embryo

2012 ◽  
Vol 197 (7) ◽  
pp. 887-895 ◽  
Author(s):  
Ivo A. Telley ◽  
Imre Gáspár ◽  
Anne Ephrussi ◽  
Thomas Surrey
Keyword(s):  
Early Embryo ◽  
Length Scale ◽  
Free System ◽  
Nuclear Separation ◽  
A Cell ◽  
Distinct Distance ◽  
Spindle Elongation ◽  
Insect Embryos ◽  
Microtubule Aster

In the early embryo of many species, comparatively small spindles are positioned near the cell center for subsequent cytokinesis. In most insects, however, rapid nuclear divisions occur in the absence of cytokinesis, and nuclei distribute rapidly throughout the large syncytial embryo. Even distribution and anchoring of nuclei at the embryo cortex are crucial for cellularization of the blastoderm embryo. The principles underlying nuclear dispersal in a syncytium are unclear. We established a cell-free system from individual Drosophila melanogaster embryos that supports successive nuclear division cycles with native characteristics. This allowed us to investigate nuclear separation in predefined volumes. Encapsulating nuclei in microchambers revealed that the early cytoplasm is programmed to separate nuclei a distinct distance. Laser microsurgery revealed an important role of microtubule aster migration through cytoplasmic space, which depended on F-actin and cooperated with anaphase spindle elongation. These activities define a characteristic separation length scale that appears to be a conserved property of developing insect embryos.

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