Protein composition of nuclear matrix preparations from HeLa cells: an immunochemical approach

1986 ◽  
Vol 80 (1) ◽  
pp. 103-122
Author(s):  
R. Verheijen ◽  
H. Kuijpers ◽  
P. Vooijs ◽  
W. van Venrooij ◽  
F. Ramaekers
Keyword(s):  
Nuclear Matrix ◽  
Connective Tissue Diseases ◽  
Protein Composition ◽  
Cytoskeletal Proteins ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Core Proteins ◽  
Nuclear Rna ◽  
Hela S3 ◽  
Protein Components

Procedures for the isolation of HeLa S3 nuclear matrices were re-examined with special emphasis on the use of various nucleases and detergents as well as on the ionic strength of the final salt extraction. The protein composition of the resulting nuclear matrix preparations was analysed by one- and two-dimensional gel electrophoresis and found to be extremely reproducible. By means of co-electrophoresis several typical cytoskeletal proteins (actin, vimentin and cytokeratins) and heterogeneous nuclear RNA (hnRNA)-associated core proteins (hnRNP) were shown to be present in such nuclear matrix preparations. The nature of some other protein components was elucidated using two-dimensional immunoblotting and immunofluorescence. For this purpose mouse monoclonal antibodies to cytoskeletal components (vimentin, cytokeratins), small nuclear RNP (70 X 10(3) Mr protein of U1-RNP), hnRNP (C1/C2) and the pore-complex lamina (lamins A, B and C) were used next to human autoimmune sera obtained from patients with connective tissue diseases and directed against the residual nucleoli and the internal fibrillar mass. These antibodies enabled us to identify a number of proteins present specifically in the nuclear matrix and to show that part of the cytoskeletal proteins are still present in the isolated structures.

Cytoskeletal proteins from human skin fibroblasts, peripheral blood leukocytes, and a lymphoblastoid cell line compared by two-dimensional gel electrophoresis.

1982 ◽  
Vol 28 (4) ◽  
pp. 955-961 ◽  
Author(s):  
C S Giometti ◽  
K E Willard ◽  
N L Anderson
Keyword(s):  
Gel Electrophoresis ◽  
Lymphoblastoid Cell Line ◽  
Protein Pattern ◽  
Cytoskeletal Proteins ◽  
Cytoskeletal Protein ◽  
Two Dimensional ◽  
Human Skin Fibroblasts ◽  
Peripheral Blood Leukocytes ◽  
Blood Leukocytes ◽  
Two Dimensional Gel Electrophoresis

Abstract Differences in proteins between cells grown as suspension cultures and those grown as attached cultures were studied by comparing the proteins of detergent-resistant cytoskeletons prepared from peripheral blood leukocytes and a lymphoblastoid cell line (GM607) (both grown as suspension cultures) and those of human skin fibroblasts (grown as attached cultures) by two-dimensional gel electrophoresis. The major cytoskeletal proteins of the leukocytes were also present in the protein pattern of GM607 cytoskeletons. In contrast, the fibroblast cytoskeletal protein pattern contained four groups of proteins that differed from the patterns of the leukocytes and GM607. Three groups (Cytoskf:8--10, :14--16, and :17--18) showed qualitative differences, and the fourth group (Cytoskf:11 and :13) showed quantitative differences. In addition, surface labeling of GM607 and human fibroblasts with 125I demonstrated that substantial amounts of vimentin and actin are exposed at the surface of the attached fibroblasts, but there is little evidence of similar exposure at the surface of the suspension-grown GM607. Cytoskf:11 and :13 in fibroblast preparations were also labeled with the 125I. These results demonstrate some differences in cytoskeletal protein composition between different types of cells could be related to their ability or lack of ability to grow as attached cells in tissue culture.

scholarly journals Identification of phagocytosis-associated surface proteins of macrophages by two-dimensional gel electrophoresis.

1982 ◽  
Vol 92 (2) ◽  
pp. 283-288 ◽  
Author(s):  
F D Howard ◽  
H R Petty ◽  
H M McConnell
Keyword(s):  
Cell Surface ◽  
Gel Electrophoresis ◽  
Protein Composition ◽  
Surface Protein ◽  
Surface Proteins ◽  
Two Dimensional ◽  
Cell Surface Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Reducing Conditions ◽  
Membrane Components

Two-dimensional PAGE (P. Z. O'Farrell, H. M. Goodman, and P. H. O'Farrell. 1977. Cell. 12:1133-1142) has been employed to assess the effects of antibody-dependent phagocytosis on the cell surface protein composition of RAW264 macrophages. Unilamellar phospholipid vesicles containing 1% dinitrophenyl-aminocaproyl-phosphatidylethanolamine (DNP-cap-PE) were used as the target particle. Macrophages were exposed to anti-DNP antibody alone, vesicles alone, or vesicles in the presence of antibody for 1 h at 37 degrees C. Cell surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination at 4 degrees C. After detergent solubilization, membrane proteins were analyzed by two-dimensional gel electrophoresis. The resulting pattern of spots was compared to that of standard proteins. We have identified several surface proteins, not apparently associated with the phagocytic process, which are present either in a multichain structure or in several discretely charged forms. After phagocytosis, we have observed the appearance of two proteins of 45 and 50 kdaltons in nonreducing gels. In addition, we have noted the disappearance of a 140-kdalton protein in gels run under reducing conditions. These alterations would not be detected in the conventional one-dimensional gel electrophoresis. This evidence shows that phagocytosis leads to a modification of cell surface protein composition. Our results support the concept of specific enrichment and depletion of membrane components during antibody-dependent phagocytosis.

scholarly journals Variations in the Methionine-rich Protein Composition of the Genus Arachis1

1984 ◽  
Vol 11 (1) ◽  
pp. 1-3 ◽  
Author(s):  
Sheikh M. Basha ◽  
Sunil K. Pancholy
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Protein Composition ◽  
Electrophoretic Analysis ◽  
Two Dimensional ◽  
Weight Group ◽  
Two Dimensional Gel Electrophoresis ◽  
One Dimensional ◽  
Molecular Weights ◽  
Isoelectric Points

Abstract Methionine-rich proteins (MRP) from seeds of different species of the Genus Arachis were isolated and analyzed by gel electrophoresis to detect possible compositional differences. One-dimensional gel electrophoretic analysis showed presence of quantitative and qualitative variations among the MRP-fractions. Following two-dimensional gel electrophoresis, the MRP-fractions were found to contain three groups of polypeptides with apparent molecular weights of approximately 21,000; 19,000 and 16,000, and isoelectric points between 5.1 and 5.8. Within each molecular weight group the number of polypeptides varied between 1 and 3.

scholarly journals Developmental association of the beta-galactoside-binding protein galectin-1 with the nuclear matrix of rat calvarial osteoblasts

1998 ◽  
Vol 111 (20) ◽  
pp. 3035-3043 ◽  
Author(s):  
J.Y. Choi ◽  
A.J. van Wijnen ◽  
F. Aslam ◽  
J.D. Leszyk ◽  
J.L. Stein ◽  
...  
Keyword(s):  
Nuclear Matrix ◽  
Osteoblast Differentiation ◽  
Matrix Protein ◽  
Binding Protein ◽  
Protein Composition ◽  
Matrix Proteins ◽  
Specific Gene ◽  
Two Dimensional Gel Electrophoresis ◽  
Nuclear Matrix Proteins ◽  
Differential Retention

The protein composition of the nuclear matrix changes significantly as the osteoblast matures from a proliferating pre-osteoblast to an osteocyte embedded in a mineralized matrix. These matrix protein are the result of developmental stage-specific gene expression during osteoblast differentiation. To isolate nuclear matrix proteins unique to the bone phenotype we analyzed nuclear matrix preparations from cultures of rat calvarial osteoblasts by high resolution two-dimensional gel electrophoresis at two different stages: proliferation (day 3) and differentiation (day 18, mineralized). We characterized one protein (14 kDa; pI 5.0), that was detectable only in the nuclear matrix of differentiated osteoblasts. By mass spectrometry and microsequencing, this protein was identified as the beta -galactoside-binding protein galectin-1. Both immunofluorescence staining of nuclear matrix preparations with the galectin-1 antibody and western blot analysis of subcellular fractions confirmed that galectin-1 is only associated with the nuclear matrix in differentiated osteoblasts as the result of differential retention. Galectin-1 protein and mRNA are present throughout osteoblast differentiation. Galectin-1 is present in the cytoplasmic and nuclear fractions in both proliferating and differentiated osteoblasts. However, its only stable binding is to the nuclear matrix of the differentiated osteoblast; but, in proliferating osteoblasts, galectin-1 is not retained in the nuclear matrix. Taken together, our results suggest that developmental association of galectin-1 with the nuclear matrix reflects differential subnuclear binding of galectin-1 during osteoblast differentiation.

Characterization of Myocardial Protein Composition in Dilated Cardiomyopathy by Two-Dimensional Gel Electrophoresis

1994 ◽  
Vol 15 (suppl D) ◽  
pp. 37-44 ◽  
Author(s):  
M. Knecht ◽  
V. Regitz-Zagrosek ◽  
K.-P. Pleissner ◽  
P. Jungblut ◽  
C. Steffen ◽  
...  
Keyword(s):  
Dilated Cardiomyopathy ◽  
Gel Electrophoresis ◽  
Protein Composition ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis

Nuclear RNA-associated proteins and their relationship to the nuclear matrix and related structures in HeLa cells

1985 ◽  
Vol 63 (6) ◽  
pp. 631-643 ◽  
Author(s):  
Dominique Bouvier ◽  
Jean Hubert ◽  
Annie-Pierre Sève ◽  
Michel Bouteille
Keyword(s):  
Nuclear Matrix ◽  
Dnase I ◽  
High Salt ◽  
Relative Mass ◽  
Two Dimensional Gel Electrophoresis ◽  
Basic Proteins ◽  
Salt Extraction ◽  
Nuclear Rna ◽  
Associated Proteins ◽  
Rnase Digestion

The ultrastructure and the polypeptide composition of residual nuclear substructures including nuclear matrices, nuclear ghosts, and residual envelopes were investigated by means of electron microscopy and two-dimensional gel electrophoresis. Nuclear matrices were prepared by digesting isolated nuclei with DNase I alone, followed by high-salt extraction in 2 M NaCl. Nuclear ghosts were obtained by high-salt extraction of nuclei previously digested with DNase and RNase in MgCl2-containing buffers. To prepare residual envelopes, nuclei were first digested with RNase in the presence of EDTA, then digested with Mg2+-activated DNase I, and extracted in 2 M NaCl. The results of this comparative study support the conclusion that the intranuclear matrix is made of two distinct RNA-containing elements. One of these elements appears on ultrathin sections as a thin fibrillar network. It disappears from RNase-digested nuclei, together with numerous basic proteins, whatever the conditions of digestion. Although this element is present in extranucleolar territories, it is a major component of residual nucleoli. The second element appears as coarse-beaded fibers absent from the nucleolar areas. Its preservation in residual nuclear substructures depends on the presence of Mg2+ ions during RNase digestion of nuclei. It is enriched in two minor basic proteins of relative mass 49 000 and 70 000. The involvement of this fibrogranular element in heterogeneous nuclear RNA attachment to the nuclear matrix is discussed.

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