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.

A stress-inducible 40 kDa protein (hsp40): purification by modified two-dimensional gel electrophoresis and co-localization with hsc70(p73) in heat-shocked HeLa cells

1993 ◽  
Vol 104 (3) ◽  
pp. 629-638 ◽  
Author(s):  
H. Hattori ◽  
T. Kaneda ◽  
B. Lokeshwar ◽  
A. Laszlo ◽  
K. Ohtsuka
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Hela Cells ◽  
Gel Electrophoresis ◽  
Recovery Period ◽  
Chinese Hamster ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Terminal Amino ◽  
Kinetics Of

We have previously reported that a novel 40 kDa protein is induced by heat shock and several environmental stresses in mammalian and avian cells and that the N-terminal amino acid sequence of this 40 kDa protein has homology with the bacterial DnaJ heat-shock protein. We have purified this protein (40 kDa heat-shock protein, hsp40) from HeLa cells by modified two-dimensional gel electrophoresis and generated a polyclonal antibody against hsp40. This antibody was highly specific for human hsp40 and cross-reacted weakly with rat and Chinese hamster hsp40. Indirect immunofluorescence revealed that the hsp40 in HeLa cells accumulates in the nucleus, especially in the nucleolus, during heat shock and returns to the cytoplasm during the recovery period. The kinetics of the accumulation in the nucleoli and subsequent return to the cytoplasm of hsp40 was similar to that of hsp70. In addition, hsp40 was co-localized with hsc70(p73) in heat-shocked HeLa cells as demonstrated by double immunofluorescence staining. These results suggest that hsp40 (a DnaJ homologue) and hsp70 (a DnaK homologue) may act in concert to repair (refold) denatured proteins and protein aggregates in the nuclei and nucleoli of heat-shocked HeLa cells.

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 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’.

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.

Age- and ploidy-related changes in rat liver nuclear proteins as assessed by one- and two-dimensional gel electrophoresis

1982 ◽  
Vol 60 (3) ◽  
pp. 204-214 ◽  
Author(s):  
Klaus Brasch
Keyword(s):  
Gel Electrophoresis ◽  
Minor Component ◽  
Subsequent Stage ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Nonhistone Proteins ◽  
Stage Of Development ◽  
Age Related ◽  
Associated Proteins ◽  
A Minor

Hepatocyte nuclei from young (3–5 week), mature (8–12 week), and aged (over 32 weeks) rats were isolated and characterized by flow cytometry. Nuclei were bulk separated into diploid (2C), tetraploid (4C), and octoploid (8C) enriched fractions on sucrose gradients. Total, 0.35 M NaCl soluble, and residual proteins were prepared from all nuclear stages and examined by one- and two-dimensional gel electrophoresis. Within limits of sensitivity of these techniques, the following general features emerged. (a) A majority of proteins visualized were common to and present in similar relative quantities in nuclei from all age and ploidy groups. (b) A relatively higher proportion of nonhistone proteins (NHP) were saline-soluble in 2C nuclei from young rats than at any subsequent stage of development. (c) Several age-related and to a lesser extent ploidy-related fluctuations in pattern among the NHP were evident. These reflected primarily differences in solubility rather than major quantitative changes among individual proteins. (d) Exceptions to the foregoing included a group of high molecular weight components (> 100 000), a major and a minor component between 45 000 and 50 000, and a heterogeneous group of proteins in 2C nuclei from very young animals. There were no obvious differences among the histones, although these proteins were not examined in detail. The complex pattern of changes observed are discussed in terms of known aspects of hepatocyte differentiation and are related to possible changes in nucleoplasmic, nuclear matrix and Hn-RNP associated proteins.

Up-dated catalogue of HeLa cell proteins: percentages and characteristics of the major cell polypeptides labeled with a mixture of 16 14C-labeled amino acids.

1982 ◽  
Vol 28 (4) ◽  
pp. 766-781 ◽  
Author(s):  
R Bravo ◽  
J E Celis
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
High Resolution ◽  
Gel Electrophoresis ◽  
Neoplastic Transformation ◽  
Cellular Distribution ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Gradient Electrophoresis ◽  
Protein Map

Abstract A total of 1357 polypeptides [946 acidic (isoelectric focusing) and 411 basic (nonequilibrium pH-gradient electrophoresis)] from human HeLa cells have been separated and catalogued with use of high-resolution two-dimensional gel electrophoresis. Of these polypeptides, 1266 were detected by labeling cells with [35S]methionine, while the rest were revealed by silver staining or by labeling with a mixture of 16 14C-labeled amino acids. For convenience, all these polypeptides have been numbered and are indicated in a large fold-out protein map. The percentages of some of the major 14C-labeled proteins have been determined, and for some we list a few characteristics such as: variation during the cell cycle; cellular distribution in cytoplasts and karyoplasts; presence in Triton- and salt-extracted cytoskeletons; and phosphorylation and sensitivity to neoplastic transformation.

scholarly journals The synthesis of ninety proteins including actin throughout the HeLa cell cycle.

1978 ◽  
Vol 79 (3) ◽  
pp. 833-838 ◽  
Author(s):  
C Milcarek ◽  
K Zahn
Keyword(s):  
Cell Cycle ◽  
Total Synthesis ◽  
Hela Cell ◽  
Gel Electrophoresis ◽  
Similar Proportion ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Time Points ◽  
Cytoplasmic Proteins ◽  
Actin Protein

Abundant cytoplasmic proteins pulse-labeled with [35S]methionine at specific times throughout the HeLa cell cycle were analyzed with two-dimensional gel electrophoresis. More than 300 proteins could be resolved in this way. The frequency of appearance of label in the most abundant 90 proteins, ranging from 4% to less than 0.1% of the total methionine incorporated, was determined at six time points in the cell cycle. 84 of these proteins were made as a similar proportion of the total at all times during the cell cycle. A nonmuscle actin protein (spot 1) identified by molecular weight and isoelectric point represented 2-4% of the total methionine incorporated at all the time points. Only six proteins were found which varied by greater than fourfold during cell division, four appearing to represent a greater proportion of the total synthesis during the period at or immediately surrounding M (spots 31b, 44, 53, and 70d). Two appear to represent a smaller percentage of total synthesis during the early (spot 78) or the total (spot 74) G2 period.

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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