Intermediate filament proteins immunologically related to cytokeratins in the oocyte of the fish Cyprinus carpio

Zygote ◽  
1997 ◽  
Vol 5 (3) ◽  
pp. 207-212 ◽  
Author(s):  
Caterina Mencarelli ◽  
Franco Cotelli
Keyword(s):  
Cyprinus Carpio ◽  
Intermediate Filament ◽  
Mature Oocyte ◽  
Major Protein ◽  
Two Dimensional ◽  
Minor Components ◽  
Vertebrate Species ◽  
Intermediate Filament Proteins ◽  
Molecular Weights ◽  
Antigen Antibody

SummaryWe have used monoclonal antibodies specific for different sets of human cytokeratins and the anti-IFA (Intermediate Filament Antigen) antibody to investigate the expression of intermediate filament proteins in the mature oocyte of the teleostCyprinus carpio. Several polypeptides have been identified, showing molecular weights ranging from 43 to 65kDa. Two-dimensional analysis of the immunoreactive species revealed the presence of at least six major protein spots and a series of minor components, grouped in quite a narrow pI range from 5.52 to 6.28. The general complexity of the carp oocyte cytokeratin-related cytoskeleton appears to be higher than those described for oocytes of other vertebrate species.

Apolipoprotein C-II deficiency associated with nonfunctional mutant forms of apolipoprotein C-II

1984 ◽  
Vol 62 (9) ◽  
pp. 847-852 ◽  
Author(s):  
Graham F. Maguire ◽  
J. Alick Little ◽  
Gary Kakis ◽  
W. Carl Breckenridge
Keyword(s):  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Normal Subjects ◽  
Two Dimensional ◽  
Antibody Technique ◽  
Molecular Weights ◽  
Apolipoprotein C ◽  
Antigen Antibody ◽  
Mutant Forms

Two previously unidentified apolipoproteins (apo) designated apo C-II-X and C-II-Y have been found in plasma of homozygotes and obligate heterozygotes of a family with apo C-II deficiency. Because the plasmas of homozygotes do not activate lipoprotein lipase, apo C-II-X and C-II-Y are apparently nonfunctional. These apolipoproteins have isoelectric focusing points of 5.15 and 5.54, respectively, compared with 4.88 and 4.74 for the known isomorphs, C-II-1 and C-II-2, respectively. They have approximately similar molecular weights to apo C-II-1 and C-II-2 on two-dimensional sodium dodecyl sulphate – glycerol – polyacrylamide slab gel electrophoresis. They do not form insoluble antigen–antibody complexes with antibodies to apo C-II in single antibody immunodiffusion or electroimmunoassay systems. However, using a double-antibody technique in which immunoblotting is coupled with polyacrylamide isoelectric focusing slab gel electrophoresis, apo C-II-1, C-II-2, C-II-X, and C-II-Y have similar reactivity with antibodies to apo C-II. In this family the presence of apo C-II-X and C-II-Y discriminates obligate heterozygotes from normal subjects.

Characterisation of wool intermediate filament proteins separated by micropreparative two-dimensional electrophoresis

1997 ◽  
Vol 18 (3-4) ◽  
pp. 568-572 ◽  
Author(s):  
Ben R. Herbert ◽  
Mark P. Molloy ◽  
Jun X. Yan ◽  
Andrew A. Gooley ◽  
Warren G. Bryson ◽  
...  
Keyword(s):  
Intermediate Filament ◽  
Two Dimensional ◽  
Intermediate Filament Proteins ◽  
Two Dimensional Electrophoresis ◽  
Dimensional Electrophoresis

Molecular analysis of intermediate filament cytoskeleton--a putative load-bearing structure

1984 ◽  
Vol 246 (4) ◽  
pp. H566-H572 ◽  
Author(s):  
M. G. Price
Keyword(s):  
Skeletal Muscle ◽  
Molecular Analysis ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Direct Evidence ◽  
Two Dimensional ◽  
Myocardial Cells ◽  
Intermediate Filament Proteins ◽  
Bovine Myocardium ◽  
Bearing Structure

Myocardial cells contain a cytoskeleton of intermediate filaments connecting the myofibrils. The present molecular analysis of the myocardial cytoskeleton was designed to identify the intermediate filament proteins and examine their assembly properties. The intermediate filament proteins desmin and vimentin were isolated from adult bovine myocardium by sequential extraction, urea solubilization, and chromatography on hydroxylapatite and DEAE columns. Desmin was obtained virtually pure in one peak and in a mixture of desmin and vimentin in the trailing fractions. Intermediate filaments of different morphologies polymerized in the desmin and the desmin-vimentin fractions. Isolated myocardial desmin occurs as three isozymes and isolated myocardial vimentin as two isozymes, which co-migrate on two-dimensional gels with corresponding isozymes from bovine skeletal and smooth muscle. Polypeptides of 200,000 and 220,000 daltons that fractionate with myocardial desmin and vimentin are also present in cytoskeletons of smooth and skeletal muscle. The results provide direct evidence that myocardial desmin can assemble to form intermediate filaments, suggesting that desmin is the major component of the cytoskeletal filaments in cardiomyocytes.

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 Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells

1982 ◽  
Vol 2 (9) ◽  
pp. 1104-1114
Author(s):  
D L Gard ◽  
E Lazarides
Keyword(s):  
Cyclic Amp ◽  
Intermediate Filament ◽  
Specific Activity ◽  
Fold Increase ◽  
Major Protein ◽  
Myogenic Cells ◽  
Intermediate Filament Proteins ◽  
Dependent Protein

The intermediate filament proteins desmin and vimentin and the muscle tropomyosins were the major protein phosphate acceptors in 8-day-old myotubes incubated for 4 h in medium containing radiolabeled phosphate. The addition of isoproterenol or 8-bromo-cyclic AMP (BrcAMP) resulted in a two- to threefold increase in incorporation of 32PO4 into both desmin and vimentin, whereas no changes in the incorporation of 32PO4 into tropomyosin or other cellular proteins were observed. The BrcAMP- or hormonally induced increase in 32PO4 incorporation into desmin and vimentin was independent of protein synthesis and was not caused by stimulation of protein phosphate turnover. In addition, BrcAMP did not induce significant changes in the specific activity of the cellular ATP pool. These data suggest that the observed increase in 32PO4 incorporation represented an actual increase in phosphorylation of the intermediate filament proteins desmin and vimentin. Two-dimensional tryptic analysis of desmin from 8-day-old myotubes revealed five phosphopeptides of which two showed a 7- to 10-fold increase in 32PO4 incorporation in BrcAMP-treated myotubes. Four of the phosphopeptides identified in desmin labeled in vivo were also observed in desmin phosphorylated in vitro by bovine heart cAMP-dependent protein kinase. Although phosphorylation of desmin and vimentin was apparent in myogenic cells at all stages of differentiation, BrcAMP- and isoproterenol-induced increases in phosphorylation of these proteins were restricted to mature myotubes. These data strongly suggest that in vivo phosphorylation of the intermediate filament proteins desmin and vimentin is catalyzed by the cAMP-dependent protein kinases and that such phosphorylation may be regulated during muscle differentiation.

scholarly journals Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins.

1981 ◽  
Vol 91 (2) ◽  
pp. 410-413 ◽  
Author(s):  
H R Kaslow ◽  
V E Groppi ◽  
M E Abood ◽  
H R Bourne
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Intermediate Filament ◽  
Gel Electrophoresis ◽  
Cytoskeletal Proteins ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Intermediate Filament Proteins ◽  
Adp Ribosylation ◽  
Human Foreskin Fibroblasts

Cholera toxin catalyzes transfer of radiolabel from [32P]NAD+ to several peptides in particulate preparations of human foreskin fibroblasts. Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of Mr = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase. The radiolabeling of another group of peptides (Mr = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins. This suggestion was confirmed by showing that incubation with cholera toxin and [32P]NAD+ caused radiolabeling of purified microtubule and intermediate filament proteins.

scholarly journals Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells.

1982 ◽  
Vol 2 (9) ◽  
pp. 1104-1114 ◽  
Author(s):  
D L Gard ◽  
E Lazarides
Keyword(s):  
Cyclic Amp ◽  
Intermediate Filament ◽  
Specific Activity ◽  
Fold Increase ◽  
Major Protein ◽  
Myogenic Cells ◽  
Intermediate Filament Proteins ◽  
Dependent Protein

The intermediate filament proteins desmin and vimentin and the muscle tropomyosins were the major protein phosphate acceptors in 8-day-old myotubes incubated for 4 h in medium containing radiolabeled phosphate. The addition of isoproterenol or 8-bromo-cyclic AMP (BrcAMP) resulted in a two- to threefold increase in incorporation of 32PO4 into both desmin and vimentin, whereas no changes in the incorporation of 32PO4 into tropomyosin or other cellular proteins were observed. The BrcAMP- or hormonally induced increase in 32PO4 incorporation into desmin and vimentin was independent of protein synthesis and was not caused by stimulation of protein phosphate turnover. In addition, BrcAMP did not induce significant changes in the specific activity of the cellular ATP pool. These data suggest that the observed increase in 32PO4 incorporation represented an actual increase in phosphorylation of the intermediate filament proteins desmin and vimentin. Two-dimensional tryptic analysis of desmin from 8-day-old myotubes revealed five phosphopeptides of which two showed a 7- to 10-fold increase in 32PO4 incorporation in BrcAMP-treated myotubes. Four of the phosphopeptides identified in desmin labeled in vivo were also observed in desmin phosphorylated in vitro by bovine heart cAMP-dependent protein kinase. Although phosphorylation of desmin and vimentin was apparent in myogenic cells at all stages of differentiation, BrcAMP- and isoproterenol-induced increases in phosphorylation of these proteins were restricted to mature myotubes. These data strongly suggest that in vivo phosphorylation of the intermediate filament proteins desmin and vimentin is catalyzed by the cAMP-dependent protein kinases and that such phosphorylation may be regulated during muscle differentiation.

scholarly journals Dual regulation of intermediate filament phosphorylation.

1984 ◽  
Vol 98 (3) ◽  
pp. 1144-1149 ◽  
Author(s):  
M E Gilmartin ◽  
J Mitchell ◽  
A Vidrich ◽  
I M Freedberg
Keyword(s):  
Growth Factor ◽  
Cyclic Amp ◽  
Intermediate Filament ◽  
Cross Reactivity ◽  
Dibutyryl Cyclic Amp ◽  
Intermediate Filament Proteins ◽  
Molecular Weights ◽  
Cytoskeletal Network ◽  
Epidermal Growth ◽  
Keratin Proteins

Intermediate filament proteins have been isolated from ME-180, cells of a human cervical carcinoma. Eight of these proteins have been identified as keratins by immunologic cross-reactivity to antibodies raised against authentic human epidermal keratins. The ME-180 keratin proteins consist of two major subunits designated MEK-1 and MEK-2 with approximate molecular weights of 58,000 and 53,000, respectively, and six minor subunits of 59, 57, 52.5, 50.5, 45, and 40 kilodaltons. When ME-180 cells were incubated for 2-24 h in the presence of [32P]orthophosphate, MEK-1 and MEK-2 as well as the 52.5- and 40-kilodalton keratins were phosphorylated at their serine residues. V8 protease digests revealed that phosphorylation of MEK-2 is restricted to one peptide representing approximately half the molecule. Regulation of MEK-1 and MEK-2 phosphorylation has been studied by prelabeling the cells for 2 h in 32P-labeled medium. This was followed by up to 2 h of continued incubation in the same medium after the addition of a variety of perturbing agents. The phosphorylation of MEK-2 increased in the presence of 10(-4) M dibutyryl cyclic AMP (twofold), 1 mM methylisobutylxanthine (2.5-fold), 10(-5) M isoproterenol (fivefold), and 10(-9) M cholera toxin (sevenfold). In contrast, MEK-1 phosphorylation was unaffected by these agents. Neither cyclic GMP, Ca++, hydrocortisone, nor epidermal growth factor had any effect on the phosphorylation of MEK-1 or MEK-2. The results indicate that the phosphorylation of these two keratins is independently controlled by cyclic AMP-dependent kinase for MEK-2 and by cyclic nucleotide-independent kinase for MEK-1. The observed differences in control suggest distinct functions for MEK-1 and MEK-2 within the cytoskeletal network.

scholarly journals Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization.

1982 ◽  
Vol 92 (3) ◽  
pp. 665-673 ◽  
Author(s):  
E B Lane
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Cell Line ◽  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Antigenic Determinant ◽  
Protein Antigens ◽  
Molecular Weight Range ◽  
Intermediate Filament Proteins ◽  
Molecular Weights

The tonofilament-associated protein antigens recognized in epithelial cells by a group of six monoclonal antibodies have been studied by immunofluorescence and gel immunoautoradiography. The monoclonal antibodies were generated against detergent insoluble cytoskeleton extracts from a cultured simple epithelium derived cell line, Ptk1 cells. They show various tissue specificities, and while they all recognize components at the low end of the molecular weight range for intermediate filament proteins, they confirm that single antibody species can react with multiple polypeptides of different molecular weights in the tonofilament complex. The monoclonal antibodies described here demonstrate the presence of a simple epithelium antigenic determinant associated with intermediate filaments that is not detectable in the specialized cells of squamous and keratinizing epithelia but can reappear in such cells after transformation.

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