Protein phosphorylation in bovine oocytes following fertilisation and parthenogenetic activation in vitro

Zygote ◽  
1999 ◽  
Vol 7 (2) ◽  
pp. 135-142 ◽  
Author(s):  
R. C. Chian ◽  
S. L. Tan ◽  
M. A. Sirard
Keyword(s):  
Protein Phosphorylation ◽  
Gel Electrophoresis ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Parthenogenetic Activation ◽  
Two Samples ◽  
Sperm Penetration ◽  
Protein Dephosphorylation ◽  
Bovine Oocytes

This study examined the event of protein phosphorylation in bovine oocytes in response to sperm penetration and parthenogenetic activation. In vitro matured oocytes were labelled with [32P]orthophosphate at 3 h intervals from 3 h to 18 h or from 0 h to 12 h following in vitro fertilisation and parthenogenetic activation, respectively. The level of protein dephosphorylation, at approximately 43 kDa, was similar in fertilised and parthenogenetically activated bovine oocytes. However, the level of protein phosphorylation at 40 kDa, 23 kDa and 18 kDa was different between these two samples. There were no such changes of protein phosphorylation and dephosphorylation in the control oocytes. Further, by two-dimensional gel electrophoresis there is a difference in the level of protein phosphorylation at 18 kDa between the fertilised and activated oocytes. These results suggest that this protein phosphorylation may be related to the formation of the male pronucleus in bovine oocytes.

scholarly journals Reversible changes in protein phosphorylation during germinal vesicle breakdown and pronuclear formation in bovine oocytes in vitro

Zygote ◽  
2003 ◽  
Vol 11 (2) ◽  
pp. 119-129 ◽  
Author(s):  
R.C. Chian ◽  
J.T. Chung ◽  
K. Niwa ◽  
M.A. Sirard ◽  
B.R. Downey ◽  
...  
Keyword(s):  
Protein Phosphorylation ◽  
Gel Electrophoresis ◽  
Germinal Vesicle ◽  
Two Dimensional Gel Electrophoresis ◽  
Germinal Vesicle Breakdown ◽  
One Dimensional ◽  
Cell Cycle Transition ◽  
Pronuclear Formation ◽  
Bovine Oocytes

This study examined the event of protein phosphorylation in bovine oocytes during germinal vesicle breakdown (GVBD) and formation of pronuclei following fertilisation in vitro. Immature oocytes were obtained from abattoir materials and cultured in vitro. The oocytes were labelled with [32P]orthophosphate at 3 h intervals from 0 to 12 h following maturation in culture or from 3 to 18 h following insemination. One-dimensional gel electrophoresis indicated that levels of protein phosphorylation are low prior to GVBD. However, the levels of protein phosphorylation at approximately 40 kDa, 27 kDa, 23 kDa and 18 kDa increased substantially following GVBD and then decreased gradually as maturation in culture progressed. In contrast, the levels of protein phosphorylation increased gradually in the oocytes following pronucleus formation. Further, two-dimensional gel electrophoresis indicated that the protein at approximately 18 kDa reversibly changed in the oocytes during maturation and fertilisation. These results indicate that the reversible changes of this phosphoprotein may be related to either cell cycle transition or pronucleus formation during maturation and fertilisation in bovine oocytes.

scholarly journals Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of polytomella

1981 ◽  
Vol 91 (2) ◽  
pp. 352-360 ◽  
Author(s):  
TW McKeithan ◽  
JL Rosenbaum
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Cold Temperature ◽  
Two Dimensional ◽  
Multiple Forms ◽  
Cytoplasmic Fraction ◽  
Two Dimensional Gel Electrophoresis ◽  
Brain Tubulin ◽  
Β Tubulin

The alga polytomella contains several organelles composed of microtubules, including four flagella and hundreds of cytoskeletal microtubules. Brown and co-workers have shown (1976. J. Cell Biol. 69:6-125; 1978, Exp. Cell Res. 117: 313-324) that the flagella could be removed and the cytoskeletans dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell. In order to define the different species of tubulin in polytonella cytoplasm, a (35)S- labeled cytoplasmic fraction was subjected to two cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated polytomella flagella by two-dimensional gel electrophoresis showed that, whereas the β-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the two α-tubulins had distinctly different isoelectic points. As a second method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were found to be highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant α-tubulin of this in vitro- assembled cytoskeletal tubulin corresponded to the major cytoplasmic α-tubulin obtained by coassembly of labeled polytomella cytoplasmic extract with brain tubulin and was quite distinct from the α-tubulin of purified flagella. These results clearly show that two different microtubule-containing organelles from the same cell are composed of distinct tubulins.

Fibrin-I Degradation Products with a Molecular Weight Higher than That of Fibrinogen

1973 ◽  
Vol 29 (01) ◽  
pp. 122-129 ◽  
Author(s):  
René von Hugo ◽  
Henner Graeff
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Degradation Products ◽  
Gel Filtration ◽  
Agarose Gel ◽  
Two Dimensional ◽  
Plasma Clot ◽  
Two Dimensional Gel Electrophoresis ◽  
Parent Molecule

SummaryThe observation of intravascular lysis of fibrin deposits and of fibrinogen derivatives with a molecular weight higher than the parent molecule in human cases of disseminated intravascular coagulation (DIC) initiated the following in vitro study. Following streptokinase induced plasma clot solubilization fibrinogen derivatives were investigated after ß-alanine precipitation of the plasma samples by polyacrylamide (PAA) gel electrophoresis, intra gel immunoprecipitation, two dimensional gel electrophoresis and by agarose gel filtration. Three fibrin-i degradation products were observed and characterized according to their relative electrophoretic mobility in 5% PAA gel: 0.23, 0.35, 0.46 (fibrinogen: 0.43) x 10-5 cm2/V x sec. They could also be demonstrated after electrophoresis in the presence of 5 M urea. Agarose gel filtration yielded one peak at 180 ml of effluent volume. The 0.23 derivative was eluted in the peak fractions, whilst the 0.35 and 0.46 derivatives were eluted together at approximately 201 ml of the effluent volume (fibrinogen: 225 ml). This indicates, that the three fibrin-i degradation products described are molecular entities with molecular weights higher than fibrinogen and, that the 0.46 derivative has an increased charge/molecular size ratio in comparison with fibrinogen. Corresponding data were obtained by two dimensional gel electrophoresis in gels of different pore size.

Changes in patterns of protein synthesis in axolotl oocytes during progesterone-induced maturation

Development ◽  
1986 ◽  
Vol 92 (1) ◽  
pp. 103-113
Author(s):  
Jean Gautier ◽  
Renée Tencer
Keyword(s):  
Protein Synthesis ◽  
Protein Phosphorylation ◽  
Cholera Toxin ◽  
Oocyte Maturation ◽  
Gel Electrophoresis ◽  
De Novo ◽  
Protein Pattern ◽  
Ambystoma Mexicanum ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis

Patterns of protein phosphorylation and synthesis during axolotl (Ambystoma mexicanum) oocyte maturation were studied by incorporation of [32P]orthophosphate and [35S]methionine into polypeptides, followed by two-dimensional gel electrophoresis. Various alterations were observed after progesterone treatment: de novo appearance of [35S]methionine-labelled polypeptides, a quantitative increase in previously synthesized proteins and a quantitative decrease in or disappearance of other previously synthesized proteins. Changes in 32P- and 35S-labelling were observed very early during maturation. Neither prior oocyte enucleation nor α-amanitin treatment had a significant effect on these changes. Stimulation with MPF provided the same final protein pattern as PG treatment. However, cholera toxin inhibited all the changes seen during maturation. Comparisons between the patterns of [35S]methionine- and [32P]phosphatelabelling provide further information on the biochemical events that take place during oocyte maturation.

Protein phosphorylation in bovine oocytes following fertilization and parthenogenetic activation in vitro

1997 ◽  
Vol 47 (1) ◽  
pp. 206
Author(s):  
H.M. Chung ◽  
R.C. Chian ◽  
J.J. Ko ◽  
T.K. Yoon ◽  
K.Y. Cha
Keyword(s):  
Protein Phosphorylation ◽  
Parthenogenetic Activation ◽  
Bovine Oocytes

scholarly journals Dog pancreatic microsomal-membrane polypeptides analysed by two-dimensional gel electrophoresis

1984 ◽  
Vol 217 (1) ◽  
pp. 145-157 ◽  
Author(s):  
M A Kaderbhai ◽  
B M Austen
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Microsomal Membrane ◽  
Translation System ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Microsomal Preparation ◽  
Microsomal Membranes

The two-dimensional polyacrylamide-gel electrophoresis technique of O'Farrell [(1975) J. Biol. Chem 250, 4007-4021] was applied to resolve and analyse the polypeptide composition of dog pancreatic rough microsomal membranes, which were shown to be active in co-translational processing of preprolactin synthesized from pituitary mRNA in a translation system in vitro. About 100 polypeptides are resolved. Treatment of rough microsomal membranes with EDTA and high KCl concentration yielded membranes stripped of their ribosomes with retention of activity for translocation and processing. Stripped microsomal membranes showed a selective concentration of approximately 25 polypeptides in the membranes when analysed by two-dimensional polyacrylamide-gel electrophoresis. The two-dimensional electrophoretic profile was catalogued into polypeptides that are glycoproteins, those that contain free thiol groups disposed at the cytosolic surface of microsomal vesicles and those that are of secretory origin but have been entrapped in the microsomal preparation. Several secretory components, including amylase, procarboxypeptidases, lipase and anionic trypsinogen, were tentatively identified among the microsomal polypeptides. The rough and stripped microsomal membranes from dog pancreas show a characteristic set of seven major acidic polypeptides, which are also identifiable in microsomal-membrane preparations isolated from dog liver and rat liver. One of these polypeptides was identified as protein disulphide-isomerase (EC 5.3.4.1).

scholarly journals Peptide analysis of collagen produced from cDNA by transcription and translation in vitro

1987 ◽  
Vol 245 (2) ◽  
pp. 393-398 ◽  
Author(s):  
J F Bateman ◽  
S Lamande ◽  
D Chan ◽  
W G Cole
Keyword(s):  
Gel Electrophoresis ◽  
Protein Translation ◽  
Normal Fibroblast ◽  
Cdna Clones ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Rabbit Reticulocyte Lysate System ◽  
Cnbr Cleavage ◽  
Cdna Construct

When collagen CNBr-cleavage peptides are analysed by two-dimensional gel electrophoresis each peptide is resolved into a reproducible set of charged forms. To test whether this peptide heterogeneity resulted from polymorphic mRNA, collagen was produced by transcription and translation in vitro of a collagen cDNA clone, and the peptides were mapped by two-dimensional gel electrophoresis. A cDNA construct was produced by ligation of the 5′ end of the rat phenylalanine hydroxylase cDNA [Dahl & Mercer (1986) J. Biol. Chem. 261, 4148-4153], containing the translation-initiation codon, to a human alpha 1(I) cDNA [Chu, Myers, Bernard, Ding & Ramirez (1982) Nucleic Acids Res. 10, 5925-5934] coding for a large portion of helical region including the complete CB7 and CB3 CNBr-cleavage peptides. This cDNA construct was ligated into the transcription vector pSP65, and cell-free translation of the mRNA transcribed from the pSP65 plasmid was performed with a rabbit reticulocyte lysate system. After CNBr cleavage of the hybrid protein translation products, the collagen CB7 and CB3 peptides were resolved by two-dimensional electrophoresis into the same multiple charged forms whether the mRNA was produced from the cDNA construct or was extracted from normal fibroblast cultures. This result demonstrated that the multiple peptide spots were not due to polymorphic mRNA species. The heterogeneity must result from some uncharacterized specific post-translational modification or chemical alterations during sample preparation. This method of expression and analysis of proteins from cDNA clones should be of considerable use in the identification and characterization of clones that code for mutant proteins.

Identification of proteins and mRNAs in isolated yellow crescents of ascidian eggs

Development ◽  
1985 ◽  
Vol 89 (1) ◽  
pp. 275-287
Author(s):  
William R. Jeffery
Keyword(s):  
Gel Electrophoresis ◽  
In Vitro Translation ◽  
Supernatant Fraction ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Mass Fractionation ◽  
Specific Subset ◽  
Mesenchyme Cells ◽  
Tail Muscle

The yellow crescent or myoplasm is a localized cytoplasmic region in eggs of the ascidian Styela that is partitioned to the larval tail muscle and mesenchyme cells during embryonic development. To determine whether the myoplasm contains a specific subset of maternal macromolecules, its abundant proteins and mRNAs were identified and compared to those present in the remainder of the egg. This was accomplished by exploiting a newly developed method for the mass fractionation of yellow crescents which is based on the presence of a unique cytoskeletal domain in the yellow crescent region. The fractionation yields a yellow crescent fraction (YCF) representing the myoplasm and a supernatant fraction representing the nonmyoplasmic regions. The YCF comprises structures which contain the characteristic myoplasmic organelles and about 10% of the protein, 8% of the RNA, and 1–3% of the poly (A) of whole eggs. Two-dimensional gel electrophoresis indicated that the YCF contains 15 polypeptides that are undetectable in the supernatant fraction and 43 polypeptides that are significantly depleted in the latter fraction. The proteins restricted to the YCF are both of cytoskeletal and noncytoskeletal origin. In vitro translation of RNA in a message-dependent lysate and analysis of [35S]methionine-labelled polypeptide products by two-dimensional gel electrophoresis did not reveal qualitative differences between the YCF and the supernatant fraction. Furthermore, the mRNAs coding for two polypeptides that were localized in the myoplasm were not restricted to the YCF. The results suggest that qualitative differences in proteins but not in prevalent mRNAs exist between the yellow crescent and the other cytoplasmic regions of Styela eggs.

Sign in / Sign up

Export Citation Format

Share Document