In vitro [35S]methionine-labeled protein synthesis in microdissected discrete brain areas: Marked regional differences revealed by two-dimensional gel electrophoresis

1984 ◽  
Vol 5 (2) ◽  
pp. 116-121 ◽  
Author(s):  
Mark A. Gold ◽  
William E. Heydorn ◽  
G. Joseph Creed ◽  
Joan L. Weller ◽  
David C. Klein ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Regional Differences ◽  
Two Dimensional ◽  
Brain Areas ◽  
Two Dimensional Gel Electrophoresis

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.

Identification and quantitation of elongation factor EF-P in Escherichia coli cell-free extracts

1980 ◽  
Vol 58 (11) ◽  
pp. 1312-1314 ◽  
Author(s):  
Gynheung An ◽  
Bernard R. Glick ◽  
James D. Friesen ◽  
M. Clelia Ganoza
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Ribosomal Proteins ◽  
Elongation Factor ◽  
Peptide Bond ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis

EF-P, an elongation factor that stimulates peptide bond synthesis in vitro with some aminoacyl-tRNAs, has been identified by two-dimensional gel electrophoresis and the cellular content at three points in the growth curve has been measured. The molecular weight of EF-P is estimated to be 21 000. EF-P is a slightly acidic protein whose isoelectric point is close to RNA polymerase subunit α. The amount of EF-P present in Escherichia coli is about [Formula: see text]that of EF-G and the level is independent of the stage of cell growth; there is about one EF-P per 10 ribosomes. It is also shown that a highly purified preparation of EF-P is free of all known protein synthesis factors and ribosomal proteins.

scholarly journals Integrity of mitochondria in a mammalian cell mutant defective in mitochondrial protein synthesis.

1981 ◽  
Vol 90 (1) ◽  
pp. 108-115 ◽  
Author(s):  
K G Burnett ◽  
I E Scheffler
Keyword(s):  
Protein Synthesis ◽  
Mitochondrial Protein ◽  
Chinese Hamster ◽  
Two Dimensional ◽  
Mitochondrial Protein Synthesis ◽  
Two Dimensional Gel Electrophoresis ◽  
Translational Machinery ◽  
Sedimentation Behavior ◽  
Rrna Species

A defect in mitochondrial protein synthesis has previously been identified in the respiration-deficient Chinese hamster lung fibroblast mutant V79-G7. The present work extends the characterization of this mutant. A more sensitive analysis has shown that mutant mitochondria synthesize all mitochondrially encoded peptides, but in significantly reduced amounts. This difference is also seen when isolated mitochondria are tested for in vitro protein synthesis. To distinguish between a defect in the translational machinery and a defect in the transcription of mitochondrial DNA, we investigated the synthesis of the 16S and 12S mitochondrial rRNA species and found them to be made in normal amounts in G7 mitochondria. These rRNA species appear to be assembled into subunits whose sedimentation behavior is virtually indistinguishable from that of the wild-type subunits. We also examined the consequences of the defect in mitochondrial protein synthesis on mutant cells and their mitochondria-utilizing techniques of electron microscopy, two-dimensional gel electrophoresis and immunochemical analysis. G7 mitochondria have a characteristic ultrastructure distinguished by predominantly tubular cristae, but the overall biochemical composition of mitochondrial membrane and matrix fractions appears essentially unaltered except for the absence of a few characteristic peptides. Specifically, we identify the absence of two mitochondrially encoded subunits of cytochrome c oxidase on two-dimensional gels and demonstrate a drastic reduction of both cytoplasmically and mitochondrially synthesized subunits of enzyme in immunoprecipitates of G7 mitochondria.

Protein Synthesis During Oxygen Deprivation in Cotton

1996 ◽  
Vol 23 (3) ◽  
pp. 341 ◽  
Author(s):  
AA Millar ◽  
ES Dennis
Keyword(s):  
Molecular Weight ◽  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Major Part ◽  
Oxygen Deprivation ◽  
Two Dimensional ◽  
Root Tips ◽  
Two Dimensional Gel Electrophoresis ◽  
Molecular Masses ◽  
Adh Activity

The alteration of protein synthesis induced by oxygen deprivation has been examined in the root tips of cotton (Gossypium hirsutum cv. Siokra), a plant that is intolerant to anoxia. Using [35S]methionine labelling and two-dimensional gel electrophoresis it was demonstrated that 14 major polypeptides are being selectively synthesised during oxygen deprivation. These polypeptides have been designated the cotton anaerobic polypeptides (ANPs), and have estimated molecular masses that correspond to molecular masses of ANPs from other plant species. However, compared to maize, several of the major molecular weight classes are absent, suggesting that the response to oxygen deprivation in cotton is simpler than that of maize. Alcohol dehydrogenase (ADH) activity is induced by oxygen deprivation. Using western analysis we have determined that this increase in activity is correlated with the accumulation of the ADH polypeptide and that three of the major cotton ANPs are ADH, including the most intensely labelled ANP, demonstrating that the synthesis of ADH constitutes a major part of the response in cotton.

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.

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).

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