scholarly journals Specific changes in the protein composition of rat liver in response to the peroxisome proliferators ciprofibrate, Wy-14,643 and di-(2-ethylhexyl)phthalate

1985 ◽  
Vol 227 (3) ◽  
pp. 767-775 ◽  
Author(s):  
T Watanabe ◽  
N D Lalwani ◽  
J K Reddy
Keyword(s):  
Gel Electrophoresis ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferation ◽  
Peroxisome Proliferators ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Basic Proteins ◽  
Acidic Proteins ◽  
Liver Homogenates ◽  
Ethylhexyl Phthalate

The hypolipidaemic agents ciprofibrate and Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid) and the phthalate-ester plasticizer di-(2-ethylhexyl)-phthalate (DEHP), like other peroxisome proliferators, produce a significant hepatomegaly and induce the peroxisomal fatty acid beta-oxidation enzyme system together with profound proliferation of peroxisomes in hepatic parenchymal cells. Changes in the profile of liver proteins in rats following induction of peroxisome proliferation by ciprofibrate, Wy-14,643 and DEHP have been analysed by high-resolution two-dimensional gel electrophoresis. The proteins of whole liver homogenates from normal and peroxisome-proliferator-treated rats were separated by two-dimensional gel electrophoresis using isoelectric focusing for acidic proteins and nonequilibrium pH gradient electrophoresis for basic proteins. In the whole liver homogenates, the quantities of six proteins in acidic gels and six proteins in the basic gels increased following induction of peroxisome proliferation. Peroxisome proliferator administration caused a repression of three acidic proteins in the liver homogenates. By the immunoblot method using polyspecific antiserum against soluble peroxisomal proteins and monospecific antiserum against peroxisome proliferation associated Mr 80000 polypeptide (polypeptide PPA-80), the majority of basic proteins induced by these peroxisome proliferators appeared to be peroxisomal proteins. Polypeptide PPA-80 becomes the most abundant protein in the total liver homogenates of peroxisome-proliferator-treated rats. These results indicate that ciprofibrate, DEHP and Wy-14,643 induce marked changes in the profile of specific hepatic proteins and that some of these changes should serve as a baseline to identify a set of gene products that may assist in defining the specific ‘peroxisome proliferator domain’.

Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue

PROTEOMICS ◽  
2004 ◽  
Vol 4 (1) ◽  
pp. 27-30 ◽  
Author(s):  
Kyla Pennington ◽  
Emma McGregor ◽  
Clare L. Beasley ◽  
Ian Everall ◽  
David Cotter ◽  
...  
Keyword(s):  
Human Brain ◽  
Brain Tissue ◽  
Gel Electrophoresis ◽  
Two Dimensional ◽  
Human Brain Tissue ◽  
Two Dimensional Gel Electrophoresis ◽  
Basic Proteins

scholarly journals The characterization of an acidic calmodulin-binding protein in brain cytoskeleton and membrane fractions

1986 ◽  
Vol 240 (2) ◽  
pp. 593-596
Author(s):  
P Strocchi ◽  
J M Gilbert
Keyword(s):  
Intermediate Filament ◽  
Gel Electrophoresis ◽  
Binding Protein ◽  
Two Dimensional ◽  
Proteolytic Digestion ◽  
Two Dimensional Gel Electrophoresis ◽  
Intermediate Filament Proteins ◽  
Calmodulin Binding ◽  
Acidic Proteins

One of the most abundant acidic proteins in rat brain has an Mr of 68,000 and a pI of 5.6 (68K 5.6 protein) when analysed by two-dimensional gel electrophoresis. The 68K 5.6 protein was found in large relative amounts in brain cytoskeleton preparations and in membrane and supernatant fractions. High-salt washing and proteolytic digestion did not remove this protein from the membrane elements. The 68K 5.6 protein was also found in the microtubule-associated protein fraction of purified microtubules and was present in large relative amounts in preparations of intermediate-filament proteins. The 68K 5.6 protein binds to calmodulin in the presence of Ca2+ ions, and we found it to be an abundant acidic calmodulin-binding protein in brain tissue.

scholarly journals A search for differential polypeptide synthesis throughout the cell cycle of HeLa cells.

1980 ◽  
Vol 84 (3) ◽  
pp. 795-802 ◽  
Author(s):  
R Bravo ◽  
J E Celis
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Gel Electrophoresis ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Nonhistone Proteins ◽  
Polypeptide Synthesis ◽  
Acidic Proteins ◽  
Filament Protein

The polypeptides synthesized during the cell cycle of HeLa cells were analyzed by means of two-dimensional gel electrophoresis followed by fluorography under conditions in which the position of 700 polypeptides (acidic and basic) could be reproducibly assessed. Mitotic cells obtained by mechanical detachment and synchronized cells in other stages of the cell cycle were labeled with [35S]methionine for 30-min pulses or for long terms starting at the beginning of each phase. Visual comparison of the polypeptide maps obtained in the different stages of the cell cycle showed that these were strikingly similar, and there was no indication that the synthesis of any of the detected polypeptides was confined to only one of the cell cycle phases. Quantitation of 99 abundant polypeptides (acidic and basic) in pulse-labeled and long-term labeled cells revealed that the relative amount (i.e., the rate of synthesis) of most polypeptides, including total actin, alpha-actinin, 6 abundant basic nonhistone proteins, and 13 major acidic proteins present in Triton cytoskeletons, remains constant throughout the cell cycle. Among the few variable polypeptides (markers), we have identified alpha- and beta-tubulin (increase in M), the subunit of the 100-A filament protein "fibroblast type" (decreases in M), and a 36,000 mol wt acidic cytoarchitectural protein that increases in S. A few other unidentified polypeptides have also been found to vary in M and in M and G2, but no marker was found in G1.

scholarly journals Evidence for a phosphorylated form of calmodulin in chicken brain and muscle

1983 ◽  
Vol 3 (8) ◽  
pp. 1412-1420
Author(s):  
Y D Plancke ◽  
E Lazarides
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Brain Slices ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Conventional Procedure ◽  
Phosphorylated Form ◽  
Acidic Proteins

Phosphocalmodulin (PCaM) was identified after analysis of calmodulin (CaM) preparations by two-dimensional gel electrophoresis by using a modified ampholyte system to resolve very acidic proteins. The analysis of CaM prepared by the conventional procedure based upon its heat resistance and acidity as well as the analysis of whole urea extracts from brain showed that PCaM was a major component in this tissue. PCaM was 1 pH unit more acidic than CaM, and its electrophoretic mobility, unlike CaM, was not changed by either calcium or ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. In urea extracts of brain prepared in buffers containing phosphate and sodium fluoride, PCaM was as prominent as CaM; it was partially converted into CaM after elution from the gel and reelectrophoresis. Amino acid analysis of PCaM and CaM purified by two-dimensional gel electrophoresis showed the same composition for the two proteins, including their trimethyllysine content. Incorporation of 32P occurred exclusively into the acidic variant when brain slices were incubated with H332PO4; amino acid analysis showed that the phosphate was bound to serine residues. CaM was found also to be phosphorylated in vitro by a phosphorylase kinase preparation from skeletal muscle.

scholarly journals Evidence for a phosphorylated form of calmodulin in chicken brain and muscle.

1983 ◽  
Vol 3 (8) ◽  
pp. 1412-1420 ◽  
Author(s):  
Y D Plancke ◽  
E Lazarides
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Brain Slices ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Conventional Procedure ◽  
Phosphorylated Form ◽  
Acidic Proteins

Phosphocalmodulin (PCaM) was identified after analysis of calmodulin (CaM) preparations by two-dimensional gel electrophoresis by using a modified ampholyte system to resolve very acidic proteins. The analysis of CaM prepared by the conventional procedure based upon its heat resistance and acidity as well as the analysis of whole urea extracts from brain showed that PCaM was a major component in this tissue. PCaM was 1 pH unit more acidic than CaM, and its electrophoretic mobility, unlike CaM, was not changed by either calcium or ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid. In urea extracts of brain prepared in buffers containing phosphate and sodium fluoride, PCaM was as prominent as CaM; it was partially converted into CaM after elution from the gel and reelectrophoresis. Amino acid analysis of PCaM and CaM purified by two-dimensional gel electrophoresis showed the same composition for the two proteins, including their trimethyllysine content. Incorporation of 32P occurred exclusively into the acidic variant when brain slices were incubated with H332PO4; amino acid analysis showed that the phosphate was bound to serine residues. CaM was found also to be phosphorylated in vitro by a phosphorylase kinase preparation from skeletal muscle.

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