Search PubMed⌕ Search

Biomedical subjects

P Traub

Publications and source records attributed to P Traub.

At least 127 records · Page 7Linked to original sources
PubMed1984

Large scale isolation, purification, and partial characterization of the intermediate filament-specific, Ca2+-activated proteinase from porcine kidney and Ehrlich ascites tumor cells: a comparative study.

The Ca2+-activated, neutral thiol proteinase specific for intermediate filament subunit proteins was isolated at large scale from the postribosomal supernatant of a low-ionic-strength extract of porcine kidney and Ehrlich ascites tumor (EAT) cells, respectively. The purification procedure encompassed DEAE-Sephacel ion exchange chromatography of the material precipitating between 23 and 55% (NH4)2SO4 saturation, followed by hydroxylapatite chromatography and activated thiol Sepharose 4B covalent chromatography. On the average, 25 mg of 62% pure enzyme was obtained from 500 g frozen kidney and 55 mg of 51% pure enzyme from 500 g EAT cells within a week. Both enzyme preparations were free of Ca2+-independent proteolytic activities and indistinguishable with respect to their physicochemical and functional properties; their catalytic properties were indistinguishable from those of enzyme purified to homogeneity on arginine methylester Sepharose 4B. Because of this identity, porcine kidney proves to be an inexpensive source for the Ca2+-activated proteinase which had previously been isolated and purified at small scale from EAT cells (W.J. Nelson and P. Traub, (1983) J. Biol. Chem. 257, 5544-5553). Despite a 38% protein contamination, the partially purified enzyme from porcine kidney is useful for the in vitro study of structure-function relationships of intermediate filaments and their subunit proteins. During affinity chromatography of the partially purified proteinase from EAT cells on arginine methylester Sepharose 4B, a 100-kDa protein was purified which has a high affinity for arginine residues. It also occurs in porcine kidney, although at a considerably lower concentration. Its cellular localization and function remain to be determined.

Animals↗
PubMed1984

Differential effect of arginine modification with 1,2-cyclohexanedione on the capacity of vimentin and desmin to assemble into intermediate filaments and to bind to nucleic acids.

When the intermediate filament proteins vimentin and desmin were reacted for a short period of time with the arginine-specific reagent 1,2-cyclohexanedione, the modification had a severe, inhibitory effect on the assembly of intermediate filaments and on the susceptibility of the basic, amino-terminal polypeptide of both proteins to degradation by the intermediate filament-specific, Ca2+-activated proteinase. However, it had only a slightly inhibitory effect on the binding of vimentin and desmin to ribosomal RNA from Ehrlich ascites tumour cells. Since the Ca2+-activated proteinase is very likely to be a trypsin-like enzyme, with a preference for arginyl and lysyl peptide bonds, the results indicate that the arginine residues of the amino-terminal polypeptide of vimentin and desmin are highly essential for filament assembly but largely dispensable for the binding of both proteins to nucleic acids. This was supported by the observation that two breakdown products of vimentin lacking a 5 X 10(3) Mr and an 8 X 10(3) Mr polypeptide from the amino terminus, respectively, did not assemble into intermediate filaments but were still capable of binding to rRNA. Both polypeptides also bound to single-stranded DNA-cellulose under non-denaturing conditions, but passed the affinity column in the presence of 6 M-urea. Thus, the binding of vimentin to nucleic acids appears to be based on two components: a non-specific electrostatic interaction mediated by the positively charged arginine residues of the amino-terminal polypeptide that is insensitive to denaturation by urea, and a specific interaction that is sensitive to denaturation by urea.

Arginine↗
PubMed1983

Isolation of glial fibrillary acidic protein from bovine brain white matter and its purification by affinity chromatography on single-stranded DNA-cellulose.

Myelinated axons, which had been prepared from bovine brain white matter employing the flotation method, were extracted with Triton X-100 in a low ionic strength buffer containing 4 mM Mg2+. After delipidation of the detergent-resistant, residual material with chloroform-methanol, glial fibrillary acidic protein (GFAP) was solubilized with 6 M urea. It was enriched by ion exchange chromatography in the presence of 6 M urea on CM-Sepharose CL-6B at pH 5 and on DE52-cellulose at pH 7.6, respectively. The final purification was achieved by affinity chromatography on single-stranded DNA-cellulose in 6 M urea. Employing SDS-polyacrylamide gel electrophoresis, the molecular weight of the purified GFAP was determined to be 51,000. 2D-polyacrylamide gel electrophoresis revealed a major protein constituent of pI 4.7 to 4.8, accompanied by 3 acidic isoelectric variants. Upon incubation at 37 degrees C in the presence of 150 mM KCl, GFAP assembled into 10 nm-filaments.

Animals↗
PubMed1983

The turnover of vimentin in Ehrlich ascites tumour cells.

The turnover of vimentin and vimentin-derived peptides has been examined in logarithmically growing Ehrlich ascites tumour cells. Cells were pulse-labelled with [35S]methionine for 30 min and then chased for up to 60 h. It was found that the specific radioactivity of the main isoelectric variant of vimentin decreased to half the original value in 15.3 h which was close to the division time of the cells (16 h). The protein moiety of the phosphorylated variant of vimentin also turned over very slowly, in contrast to the turnover rate of the phosphate group itself which has a half-life of 1.4 h. The role of the intermediate filament-specific, Ca2+-activated proteinase has been considered in relationship to the slow turnover of vimentin.

Animals↗
PubMed1983

The interaction in vitro of the intermediate filament protein vimentin with naturally occurring RNAs and DNAs.

The binding of the intermediate filament protein vimentin to a variety of naturally occurring RNAs and DNAs was studied. The relative capacities of the various nucleic acids to associate with pure [3H]vimentin were determined in competition experiments with 28 S rRNA from Ehrlich ascites tumor cells. The reaction products were analyzed by sucrose gradient centrifugation at low ionic strength and in the presence of EDTA. Under these ionic conditions, vimentin reacted preferentially with single-stranded nucleic acids, particularly with those of high (G + C) content. The vimentin binding potentials of single-stranded RNAs and DNAs were largely comparable. However, when the concentrations of mono- and divalent cations were raised to physiological and higher values, only single-stranded DNA retained its vimentin binding capacity. With increasing KCl concentrations at 0 to 1 mM Mg2+, increasing amounts of vimentin were detected in complexes which sedimented considerably faster than the bulk of the DNA, suggesting cooperative binding of vimentin. The salt optimum of this cooperativity was at 200 mM KCl. Thus, the capability of vimentin to discriminate between single-stranded RNA and DNA under physiological ionic conditions points to specificity of the interaction of vimentin with nucleic acids.

Animals↗
PubMed1983

Synthesis of vimentin in a reticulocyte cell-free system programmed by poly(A)-rich RNA from several cell lines and rat liver.

Poly(A)-rich RNA has been isolated from Ehrlich ascites tumor (EAT) cells and translated in a rabbit reticulocyte cell-free system. The intermediate filament protein, vimentin, was found to be a major translation product. Fractionation of the poly(A)-rich RNA by sucrose gradient centrifugation showed that the vimentin mRNA had a sedimentation coefficient of about 18 S corresponding to a molecular size of about 2000 nucleotides. This means that it must possess significant non-coding regions. Vimentin synthesized in vitro was identical to native vimentin with regard to its precipitability with ammonium sulphate, extent of phosphorylation and susceptibility to digestion by the vimentin-specific, Ca2+-activated proteinase. Poly(A)-rich RNA was also isolated from a number of tissue-culture cells and rat liver, which contain varying amounts of vimentin in situ. It was found that the amount of vimentin synthesized by these RNA preparations in a rabbit reticulocyte cell-free system is proportional to the amount of vimentin detectable in situ, suggesting that the amount of cellular vimentin may be controlled at the level of transcription.

Animals↗
PubMed1983

Isolation, purification and characterization of the intermediate filament protein desmin from porcine smooth muscle.

Desmin was isolated from porcine stomach smooth muscle, which had been treated with 0.6 M KCl in the presence of Triton X-100, by extraction with 6 M urea and chromatographed on CM-Sepharose CL-6B at pH 5. After delipidation with chloroform-methanol, the protein was purified by affinity chromatography on arginine methylester Sepharose 4B and single-stranded DNA-cellulose, respectively. Chromatography on single-stranded DNA-cellulose removed a considerable amount of vimentin which had been extracted and enriched together with desmin. The molecular weight of the purified desmin was 55,000 as determined by polyacrylamide gradient slab gel electrophoresis in the presence of Na-dodecylsulfate. Two-dimensional polyacrylamide gel electrophoresis revealed one major polypeptide of pI 5.3 to 5.4 accompanied by two to three acidic, isoelectric variants. During incubation in the presence of 150 mM KCl, desmin assembled into 10 nm filaments. This method allows the isolation of large amounts of pure desmin in a relatively short time with only minimal denaturation of the protein.

Animals↗
PubMed1983

Proteolysis of vimentin and desmin by the Ca2+-activated proteinase specific for these intermediate filament proteins.

The degradation of vimentin and desmin by the Ca2+-activated proteinase specific for these intermediate filament proteins proceeds in two stages in the form of a limited proteolysis. At first, the reaction is very rapid, with the stepwise and complete removal of a peptide (ca. 9,000 daltons) from the N-terminal of vimentin and desmin. This results in the production of a characteristic "staircase" of degradation products, as seen in two-dimensional polyacrylamide gel electrophoresis. The second stage of proteolysis is characterized by the accumulation of peptides which are resistant to further proteolysis; this is due not to product inhibition but to the fact that these peptides are not substrates for the proteinase and therefore do not protect the latter from inactivation (autodigestion). In vitro phosphorylation of the substrates does not affect proteinase activity, probably because the phosphorylation site is located towards the C-terminal of the molecules. The specific and limited proteolysis of vimentin and desmin results in the deletion of the nucleic acid binding and filament assembly site of these proteins, indicating that the Ca2+-activated proteinase plays a role in regulating the function(s) of these intermediate filament proteins, rather than their simple turnover during the cell cycle.

Amino Acid Sequence↗
PubMed1983

Polyacrylamide gel electrophoretic screening of mammalian cells cultured in vitro for the presence of the intermediate filament protein vimentin.

A total of 53 different cell lines originating from a variety of mammalian species were cultured in vitro and analysed for the presence of vimentin, employing polyacrylamide gradient slab gel electrophoresis in urea/acetic acid as buffer system. Irrespective of the cell culture conditions, and the growth potential and morphology of the cells, vimentin was expressed in all cell lines examined, with two exceptions: MPC-11 mouse myeloma and MOPC-31C mouse plasmacytoma cells. Immunoblotting with the monoclonal antibody alpha-IFA, which is directed against an antigenic determinant shared by all classes of intermediate filaments, did not detect any other of the known intermediate filament proteins in MPC-11 and MOPC-31C cells. Vimentin synthesized by various cell lines was characterized by four different criteria: (1) its extractability with Triton X-100 under various ionic conditions; (2) its behaviour in (NH4)2SO4 fractionation of cellular extracts; (3) its electrophoretic mobility in polyacrylamide gel electrophoresis in urea/acetic acid; and (4) the co-isolation of polypeptides of higher electrophoretic mobility, which, by comparison with degradation products of vimentin obtained with the Ca2+-activated proteinase specific for intermediate filament proteins in vitro, were identified as products of Ca2+-dependent proteolysis of vimentin. Although the degradation products occurred in different ratios in extracts of different cell lines, they constituted the same characteristic set of proteins whenever degradation of vimentin was observed. The formation of proteolytic breakdown products could be partially to totally suppressed when the cells were harvested, washed and processed in the presence of EGTA and proteinase inhibitors. The experimental data show that: (1) vimentin, as well as the Ca2+-activated proteinase specific for intermediate filament proteins, is highly conserved during the evolution of mammalian species; (2) the proteolytic breakdown products of vimentin, which give rise to a characteristic 'staircase' in two-dimensional gel electrophoresis, are probably artefacts of isolation; (3) the expression of vimentin is neither a prerequisite for nor necessarily indicative of rapid cell proliferation in vitro; and (4) the techniques described can be used for the routine identification of vimentin in cells and tissues in case vimentin-specific antibodies are not available.

Animals↗
PubMed1983

Involvement of the N-terminal polypeptide of vimentin in the formation of intermediate filaments.

The potential to form intermediate filaments of a 54 X 10(3) molecular weight (Mr) polypeptide derived from vimentin by cleavage by the intermediate filament-specific, Ca2+-activated proteinase was investigated. Under physiological conditions of assembly, the breakdown product did not form intermediate filaments. Electron microscopy revealed short, rod-like structures similar to those described by Geisler et al. for a 38 X 10(3) Mr alpha-helical core particle derived from desmin. Since the specific, Ca2+-activated proteinase degrades vimentin preferentially from its N terminus, this result suggests the involvement of the basic, N-terminal polypeptide of vimentin in the assembly of intermediate filaments. This was supported by the observation that arginine inhibits the formation of intermediate filaments from intact vimentin. Whereas lysine had very little effect on the assembly process, guanidinium hydrochloride was effective at the same concentration as arginine. On the basis of these findings, an affinity chromatography method for the identification and isolation of intermediate filament subunit proteins was developed. Beside vimentin, desmin, the 68 X 10(3) Mr neurofilament triplet protein, the glial fibrillary acidic protein and cytokeratins also bound to arginine methylester Sepharose 4B in a salt-stable manner and could be eluted with arginine. The 145 X 10(3) Mr neurofilament triplet protein exhibited reduced binding activity, whereas the 210 X 10(3) Mr subunit did not bind to the affinity matrix. Among the degradation products of vimentin produced by the specific, Ca2+-activated proteinase, only those with molecular weights higher than 40 X 10(3) bound to arginine methylester Sepharose 4B. The same applied to the high molecular weight degradation products of desmin with a protein-resistant 37 X 10(3) Mr polypeptide as the major component. The results suggest that arginine residues of the non-alpha-helical, N-terminal polypeptides of intermediate filament subunit proteins play an important role in filament assembly.

Animals↗
PubMed1983

The interaction in vitro of the intermediate filament protein vimentin with synthetic polyribo- and polydeoxyribonucleotides.

In a continuation of our studies on the association of the intermediate filament protein vimentin with unfolded ribosomal subunits, rRNA and various naturally occurring RNAs and DNAs, we have investigated the binding of vimentin to a variety of synthetic polyribo- and polydeoxyribonucleotides. The vimentin-binding potentials of the different nucleic acids were determined in competition with 28S rRNA from Ehrlich ascites tumor cells. The reaction products were analysed by sucrose gradient centrifugation at low ionic strength and in the presence of EDTA. Among the homopolynucleotides tested, poly(rG), poly(rX), poly(rI), poly(rs4U) and poly(dG) were very good competitors, whereas poly(dC) was moderately so. Poly(rA), poly(rU), poly(rC), poly(dA), poly(dU), poly(dT) and poly(dI) were very weak competitors. Annealing of the active homopolymers to their complementary homopolymer strands resulted in a reduction of the vimentin-binding potential; however, the resulting duplexes still had substantial vimentin-binding capacity. A similar reduction was observed when the mononucleotides of the active homopolymers were randomly copolymerized with their complementary nucleotides or incorporated into strictly alternating, double-stranded DNAs. DNA-type copolymers, duplexes and alternating copolymers containing the nuclear bases adenine, thymine and uracil were moderate competitors, but, in general, they were significantly more active than the corresponding homopolymers. This result, together with those of the analysis of a series of random heteropolyribonucleotides, indicates that there is some influence of the base sequence and/or secondary structure of the nucleic acids on their vimentin-binding capacities. Whereas the minimum length of oligo(dG) for efficient vimentin binding was between 11 and 18 nucleotides, (dT)18 was still incapable of interacting with vimentin. In order to obtain information on the salt stability of vimentin-nucleic acid adducts, we have also performed affinity chromatographies on single-stranded calf thymus DNA-cellulose and on rRNA and homopolyribonucleotides covalently coupled to agarose. Although because of unspecific secondary interaction of vimentin with the matrix, these nucleic acid-substituted absorbents could not be used for this purpose under non-denaturing conditions, in the presence of 6M urea a relationship between the nucleic acid substitution and the salt stability of vimentin binding was observed. Finally, the nucleic acid-binding properties of vimentin are compared with those of cytosolic steroid hormone receptors, particularly with those of the androgen receptor.

Animals↗
PubMed1982

Interaction of the intermediate filament protein vimentin with ribosomal subunits and ribosomal RNA in vitro.

If in a low ionic strength extract of Triton X-100-resistant residual cell structures derived from Ehrlich ascites tumour (EAT) cells Mg2+ was chelated by EDTA, vimentin became associated with unfolded ribosomal subunits. The first molecular characterization of this association has shown that (1) vimentin binds to the RNA moiety of the ribosomes, (2) vimentin has a higher affinity for unfolded small ribosomal subunits or 18S rRNA than for unfolded large ribosomal subunits or 28S rRNA, (3) the limited degradation of vimentin by the vimentin-specific, Ca2+-activated proteinase, with the formation of a 48 Kd breakdown product, abolishes its affinity for rRNA, (4) the association products are rather sensitive to moderate concentrations of KCl and Mg2+, and (5) reductive alkylation of vimentin with pyridoxal-5'-phosphate and NaBH4 has no effect on the affinity of vimentin for rRNA. Actin and tubulin do not interact with EAT cell rRNA under the above ionic conditions.

Animals↗
PubMed1982

Purification and further characterization of the Ca2+-activated proteinase specific for the intermediate filament proteins vimentin and desmin.

A calcium (Ca2+)-activated, neutral proteinase has been purified from Ehrlich ascites tumor cells. The protocol used has resulted in a 3,600-fold purification of the enzyme in a yield of 21% from the Ehrlich ascites tumor cell postnuclear supernatant. The purified proteinase has a high substrate specificity for the intermediate filament subunit proteins, vimentin and desmin, and showed no activity towards other intermediate filament proteins except a 60,000-dalton protein of the cytokeratins. Also, there was no degradation of actin, tubulin, the major constituent proteins of myofibrils and several standard proteins. Characterization of the purified proteinase has shown that it is activated by Ca2+ (10 to 100 microM), is probably calmodulin-independent and irreversibly loses activity when incubated in the presence of Ca2+ without substrate. The enzyme has a Km of 1.7 x 10(-8) M for vimentin and 5.2 x 10(-7) M for desmin. The proteinase has a major subunit of 72,000 daltons which has the catalytic center and a minor component of 29,000 daltons; by gel permeation chromatography it has an apparent molecular weight of 100,000. It requires a reduced sulfhydryl group for activity and can be inhibited by sulfhydryl-blocking reagents. The high substrate specificity of the proteinase indicates that it is involved in the regulation of the distribution and turnover of vimentin- and desmin-containing intermediate filaments.

Animals↗
PubMed1982

Effect of the ionic environment on the incorporation of the intermediate-sized filament protein vimentin into residual cell structures upon treatment of Ehrlich ascites tumour cells with triton X-100. II. Ultrastructural analysis.

Ehrlich ascites tumour cells were extracted in buffers containing Triton X-100 and mono-di- and polyvalent cations and then analysed by phase-contrast and electron microscopy. The results of this ultrastructural analysis confirm those of the biochemical analysis in the accompanying paper that the stability of intermediate-sized filaments is dependent on the ionic environment. Furthermore, the organization of filaments in long parallel arrays is dependent on the presence of divalent cations and can be inhibited, to some extent, by the presence of monovalent cations. The stability of other detergent-resistant structures, the boundary lamina, microfilaments, microtubules, centrioles, polyribosomes and the nuclear cortex, is also affected by the ionic environment but to a lesser extent.

Animals↗