Isolation and characterization of desmosome-associated tonofilaments from rat intestinal brush border

Epithelial cells of the small intestine, like those of other internal organs, contain intermediate-sized filaments immunologically related to epidermal prekeratin which are especially concentrated in the cell apex. Brush-order fractions were isolated from rat small intestine, and apical tonofilaments attached to desmosomal plaques and terminal web residues were prepared therefrom by extraction in high salt (1.5 M KCl) buffer and Triton X-100. The structure of these filaments was indistinguishable from that of epidermal tonofilaments and, as with epidermal prekeratin, filaments could be reconstituted from solubilized, denatured intestinal tonofilament protein. On SDS polyacrylamide gel electrophoresis of proteins of the extracted desmosome-tonofilament fractions, a number of typical brush-border proteins were absent or reduced, and enrichment of three major polypeptides of Mr 55,000, 48,000, and 40,000 was noted. On two- dimensional gel electrophoresis, the three enriched major polypeptides usually appeared as pairs of isoelectric variants, and the two smaller components (Mr 48,000, and 40,000) were relatively acidic (isoelectric pH values of 5.40 and below), compared to the Mr 55,000 protein which focused at pH values higher than 6.4. The tonofilament proteins were shown to be immunologically related to epidermal prekeratin by immunoreplica and blotting techniques using antibodies to bovine epidermal prekeratins. Similar major polypeptides were found in desmosome-attached tonofilaments from small intestine of mouse and cow. However, comparisons with epidermal tissues of cow and rat showed that all major polypeptides of intestinal tonofilaments were different from the major prekeratin polypeptides of epidermal tonofilaments. The results present the first analysis of a defined fraction of tonofilaments from a nonepidermal cell. The data indicate that structurally identical tonofilaments can be formed, in different types of cells, by different polypeptides of the cytokeratin family of proteins and that tonofilaments of various epithelia display tissue- specific patterns of their protein subunits.     

Biochemical and immunological identification of cytokeratin proteins present in hepatocytes of mammalian liver tissue

Hepatocytes of mammalian liver are known to contain intermediate-sized filaments of tonofilament morphology. Unlike many other epithelial cells, including cultured hepatocytes and hepatoma cells, hepatocytes present in normal liver tissue have been reported not to react, in significant intensity, with various preparations of antibodies to human and bovine epidermal prekeratin [2,6]. We have therefore examined, by biochemical and immunological methods, the cytoskeletal composition of hepatocytes grown in the body.Cytoskeletal preparations from hepatocytes of mouse and rat liver tissue resistant to high salt buffer and Triton X-100 are enriched in tangles of intermediate filaments and contain, besides some residual microfilamentous actin, a characteristic set of polypeptides. One- and two-dimensional gel electrophoresis reveals the presence of two major cytokeratin components, which appear as ‘pairs’ of isoelectric variants (component A, M_r 55 000, apparent pI values, 6.40 and 6.45; component D, M_r 49000, apparent pI values 5.43 and 5.38), and five minor components (M_r range from 41000 to 53 000), most of them also as ‘pairs’ of polypeptides slightly different in isoelectric pH value. These polypeptide patterns are very similar in mouse and rat liver although some minor but significant differences have been noted between the two species. The polypeptide patterns of liver cytoskeletons are also similar to—but clearly not identical with—the cytoskeletal protein patterns observed in other epithelial tissues and cells, including various lines of cultured rat hepatocytes and hepatoma cells.Guinea pig antibodies raised against individual cytokeratin proteins of mouse liver and against certain prekeratin polypeptides present in desmosome-attached tonofilaments of bovine muzzle are described which differ from previously described prekeratin antibodies. These prekeratin antibodies not only react with filament bundles of the prekeratin type present in many cultured epithelial cells (e.g. murine HEL, human HeLa, rat kangaroo PtK2) and various epithelial tissues, but also allow the detection of the cytokeratin components present in parenchymal cells of liver and pancreas of various species, man included. Immunofluorescence microscopy on frozen sections of liver using these antibodies reveals a novel structure, i.e. a three-dimensional filament meshwork extending throughout the whole cytoplasm of the hepatocyte, with higher intensity of staining in pericanalicular regions.The results show that parenchymal cells of normal liver and pancreas contain filaments of the cytokeratin type that are related to but not identical with epidermal prekeratin. The hepatocyte filaments appear to be different from prekeratin-type filaments present in epidermis and several other epithelial cells, both in some antigenic determinants exposed and in polypeptide composition. Our findings support the concept of the existence of a family of intermediate filament proteins, cytokeratins, containing many different polypeptides that are expressed in different epithelial cells in certain characteristic subsets in a cell type-specific mode.     

Immunochemical comparison of prekeratin and alcoholic hyalin intermediate filaments

Alcoholic hyalin is an hepatocellular aggregate composed of filaments apparently related to the prekeratin intermediate filament subclass. The relationship between these two filament preparations was determined immunochemically using guinea pig antisera derived against alcoholic hyalin, prekeratin, and major prekeratin polypeptides. Immunocrossreactivities were determined using sensitive solid-phase enzyme-immunoassays. These assays indicated that antisera derived against a given filament preparation reacted 10–1000 times better with that preparation than with the other system. The nature of crossreactive meterial was determined using antisera derived against the larger prekeratin polypeptides (M_r 61,000 and 51,000). When tested against these two antisera, alcoholic hyalin appeared to react better with the serum derived against the larger prekeratin component. Moreover, anti-alcoholic hyalin antiserum bound four to five times better to the 61,000 dalton component than to the 51,000 dalton polypeptide in the enzyme-immunoassay. Our results indicate that antigenic determinants related to prekeratin can be detected in alcoholic hyalin, but that these determinants are present in relatively low concentrations in purified alcoholic hyalin. In addition, it appears that the relative concentrations of prekeratin components in alcoholic hyalin do not reflect those in purified prekeratin.     

Incorporation of [3H]glucosamine into keratin-related polypeptides in pig epidermis

Metabolic labelling studies have provided evidence for glycosylated keratins in cultured pig epidermis. [3H]Glucosamine was incorporated into five major particulate polypeptides of Mr 68 000, 61 000, 57 000, 53,000 and 48,000. Radioactivity was present in protein-bound carbohydrate. Non-enzymic glycation was excluded. Labelling was largely unaffected by tunicamycin indicating that radioactivity was incorporated mainly into O-linked oligosaccharides. These [3H]glucosamine-labelled components were closely related to keratins since they had a similar electrophoretic mobility to polypeptides of purified pig prekeratin, they were immunoprecipitated by anti-prekeratin serum and they were incorporated into reconstituted, intermediate-sized, keratin filaments.     

Ejectisins: tough and tiny polypeptides are a major component of cryptophycean ejectisomes

Fragments of discharged ejectisomes were isolated from two Cryptomonas and a Chroomonas species by detergent treatment followed by Percoll density gradient centrifugation. The fragments withstand high concentrated detergent solutions, reducing agents and freeze-thawing. Disintegration was achieved in 6 M guanidine hydrochloride. Reassembly into long, filamentous, ejectisome-like structures occurred after dialysis. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the polypeptide patterns of isolated ejectisome fragments and of reconstituted ejectisome-like structures were dominated by polypeptides with relative molecular weights of approximately 6 kDa. The polypeptides were not glycosylated and did not cross-react with antisera directed against recombinant Reb polypeptides which constitute the R-bodies of Caedibacter taeniospiralis. A polyclonal antiserum directed against reconstituted, ejectisome-like filaments cross-reacted with the 6-kDa polypeptides and immunolabeled extruded ejectisome filaments. Twenty amino acid residues, obtained by N-terminal amino acid sequence analysis, matched to polypeptide sequences deduced from cDNA sequences of the cryptophyte Guillardia theta. The term “ejectisins” is introduced for the 6-kDa polypeptides which represent a major component of cryptophycean ejectisomes.     

Epidermal proteins of cultured human and bovine keratinocytes.

Bovine and human epidermal cells were cultured on mitomycin C treated fibroblasts. The cells were carried through four passages and found to synthesize fibrous proteins and insoluble cell envelopes. Acid buffer soluble fibrous protein, prekeratin, and urea soluble fibrous protein were both identified and the latter was the major component in older cultures. Some of the prekeratin polypeptides of intact tissue were not found in cultured cells, but the ones that were present corresponded to those of whole tissue. X-ray diffraction, amino acid analysis and immunological techniques were used to establish that the polypeptides were keratins. The insoluble cell envelopes had a higher proline and 1/2 cystine content than the fibrous protein, similar to what is found in whole epidermis. Histidase, a characteristic enzyme marker of whole epidermis, was not observed in cultured cells. These studies indicate that differentiation occurs in cultured cells but it may not be as complete as in intact tissue.     

Role of microtubules and intermediate filaments in maintaining the shape of epithelial cells

The role of microtubules and intermediate filaments in control of cell shape of cultured cells of hepatomas McA-RH-7777 and 27 was investigated. Indirect immunofluorescence with specific polyclonal antibodies against tubulin and monoclonal antibodies against prekeratin with molecular weight 49 kD and vimentin was used. Incubation of cells in colcemid, resulting in specific distribution of microtubules did not change either prekeratin or vimentin distribution in cells of both the hepatomas, but reversed polarization of elongated McA-RH-7777 cells. These data suggest that the effect of disruption of microtubular system on the cell shape is not mediated by alterations of intermediate filaments.     

Modification of human prekeratin during epidermal differentiation.

The polypeptide-chain components of human epidermal prekeratin and keratin were analysed by high-resolution SDS (sodium dodecyl sulphate)/polyacrylamide-gradient-gel electrophoresis. Size heterogeneity existed amongst prekeratin components and at least ten polypeptides, in the molecular-weight range 46,000-70,000, were observed in 0.1 M-citric acid/sodium citrate buffer (pH 2.65) extracts of scale epidermis. Prekeratin from scalp pilosebaceous ducts was identical with that from the contiguous epidermis, and no prekeratin was found in extracts of scale dermis. Prekeratin from plantar epidermis contained additional polypeptide chains, but only slight anatomical variation existed between the non-callus sites examined. Keratin differed from prekeratin in at least two major respects: (a) many major components did not co-electrophorese on high-resolution SDS/polyacrylamide slab gels, and (b) keratin, but not prekeratin, required denaturing and reducing conditions for extraction. Keratin extracted from scale epidermis after complete removal of prekeratin was identical with forearm stratum-corneum keratin. Palmar and plantar keratin contained additional polypeptide chains and had a different size distribution compared with forearm and scalp keratin components. Modification of prekeratin components to produce the keratin polypeptide profile occurred during epidermal differentiation, and these changes appeared to take place in the granular-layer region of the epidermis.     

The intermediate-sized filaments in rat kangaroo PtK2 cells. II. Structure and composition of isolated filaments.

When cultured cells of the rat kangaroo cell line PtK2 grown on plastic or glass surfaces are lysed and extracted with combinations of low and high salt buffers and the non-ionic detergent Triton X-100 cytoskeletal preparations are obtained that show an enrichment of 6 to 11 nm thick filaments. The arrays of these filaments have been examined by various light and electron microscopic techniques, including ultrathin sectioning, whole mount transmission electron microscopy, negative staining, and indirect immunofluorescence microscopy. In addition, 6 to 11 nm filaments isolated from these cells with similar extraction procedures and with centrifugation techniques have been examined by electron microscopy. The arrays of these isolated intermediate-sized filaments, their ultrastructure and their specific decoration by certain antibodies present in normal rabbit sera as well as by guinea pig antibodies against purified bovine prekeratin is demonstrated. When preparations enriched in these intermediate-sized filaments are examined by SDS-polyacrylamide gel electrophoresis a corresponding enrichment of three polypeptide bands with apparent molecular weights of about 45 000, 52 000 and 58 000 (the latter component sometimes appears split into two bands) is observed, besides some residual actin and a few high molecular weight bands. The morphology of the isolated filaments, their immunological reaction with antibodies decorating prekeratin-containing structures, and the sizes of their constitutive polypeptides suggest that these filaments are closely related to prekeratin-containing filaments observed in a variety of epithelial cells.     

The extraction and characterization of bovine epidermal alpha-keratin.

1. The alpha-fibrous protein (alpha-keratin) component of bovine epidermis has been extracted and characterized. 2. Prekeratin, a multichain unit of the epidermal tonofilaments, was shown to consist of six different polypeptide chains on polyacrylamide-gel systems containing sodium dodecyl sulphate or sodium decyl sulphate with discontinuous gel buffers, but only three chains were seen when a gel system containing sodium dodecyl sulphate with a continuous gel buffer was used. 3. Extraction of the 'keratinized' stratum corneum and the living part of the epidermis with urea buffers at pH 7.6 or 9.0 released 60% of the total dry weight of the tissues in the form of alpha-helical polypeptides. 4. The numbers, relative amounts and properties of the extracted polypeptides were the same as the subunits of prekeratin and thus are derived from the tonofilaments in situ. 5. The subunits of prekeratin and the polypeptides extracted from the living cell layers contained an average of six cysteine residues, but those from the stratum corneum contained an average of three intrachain disulphide bonds. 6. The polypeptide chains aggregated through non-covalent interactions in vitro into filaments that were similar to the tonofilaments. 7. Since the polypeptides could be released from the stratum corneum without breaking covalent bonds, it is concluded that such bonds do not cross-link the tonofilaments and non-fibrous keratohyalin. It is suggested that the tonofilaments and keratohyalin of bovine epidermis are associated by secondary bonding forces.     

Zein of maize grain: I — isolation by gel filtration and characterization of monomeric and dimeric species

Unreduced zein chromatographed on Sephadex G 200 in 8 M urea, on G 100 in 1.5 or 2.5% sodium dodecyl sulfate (SDS) and on hydroxypropylated G 100 in 70% ethanol was resolved into two minor fractions A and B and two major ones D and M irrespective of the medium. The quantitative importance of the fraction M was dependent on the isolation conditions of zein. It decreased from 53% of the proteins contained in ethanolic extract and chromatographed as they were extracted, to 40% of the purified zein. The molecular weight values obtained from SDS-polyacrylamide gel electrophoresis and amino acid compositional data indicated that fractions D and M, as isolated from purified zein in the presence of ethanol, represented respectively dimeric and monomeric forms of a mixture of M_r 22 000 and 24 000 polypeptides with threonine or phenylalanine as NH₂-terminal residue.Electrophoretic analysis of selectively carbamylated fraction M on starch gel at pH 3.5 revealed that zein subunits comprised several polypeptides differing in the number and the nature of basic amino acids. At least one of these polypeptides contained one lysyl residue.     

Prekeratin biosynthesis in human scalp epidermis.

Analysis of human scalp epidermal prekeratin polypeptides by two-dimensional gel electrophoresis revealed that each of the bands observed in one-dimensional electrophoresis consisted of three to five polypeptides of the same molecular weight but differing in isoelectric points. It was possible to divide the polypeptides into two families, with isoelectric points in the ranges pH 6.0-8.0 and pH 5.0-5.5 respectively. Incorporation of radiolabelled amino acids into freshly excised pieces of scalp epidermis showed that some of the polypeptides had relatively greater contents of glycine and serine than others. Radiolabelled methionine and leucine were, in contrast, incorporated more or less uniformly into all the polypeptides. After incubation with 32P-labelled orthophosphate, relatively more intense labelling by 32P was observed in the higher molecular weight bands of each family. The most basic of the isoelectric variants in each case did not take up phosphate, implying that at least some of the variation in charge was due to different degrees of phosphorylation. Polyadenylated RNA isolated from scalp epidermis was translated in an RNA-dependent reticulocyte haemolysate system followed by immunoprecipitation and electrophoresis. The polypeptides isolated by using anti-(human scalp prekeratin) immunoglobulin G had similar electrophoretic mobilities in sodium dodecyl sulphate/polyacrylamide gels to authentic prekeratin polypeptides, but had different isoelectric properties. This suggested that the products of keratin gene expression undergo post-translational modification.     

Characterization of cells of amniotic fluids by immunological identification of intermediate-sized filaments: Presence of cells of different tissue origin

Summary Antibodies against intermediate-sized filaments, of the prekeratin or vimentin type, were used to investigate the presence of these filaments by indirect immunofluorescence microscopy in cultured and non-cultured amniotic fluid cells, in frozen sections of the placenta and in isolated cells of the amniotic epithelium. Two major classes of cells can be cultured from amniotic fluids, namely cells of epithelial origin containing filaments of the prekeratin type and cells of different origin which contain filaments of the vimentin type but are negative when tested with antibodies to epidermal prekeratin. The presence of prekeratin type filaments correlates with the morphology of colonies of amniotic fluid cell cultures in vitro as classified by Hoehn et al. (1974). Cells of E-type colonies are shown to be of epithelial origin. In contrast our data indicate a different origin of almost all cells of F-type colonies and of the large majority of cells of AF-type colonies. Cells of epithelial origin and positively stained with antibodies to epidermal prekeratin are occasionally scattered in F-type colonies and in variable percentages (up to 30%) in AF-type colonies. Surprisingly, cryostat sections of the amniotic epithelium and isolated groups of amniotic cells showed positive reactions with both antibodies to vimentin and prekeratin. The possibility that amniotic cells may be different from other epithelial cells in that they contain both types of filaments simultaneously already in situ is presently under investigation.Part of this work is included in the doctoral thesis of Irmgard Treiss to be submitted to the Faculty of Medicine of the University of Heidelberg     

Isolation and in vitro assembly of the components of the outer S layer of Lampropedia hyalina.

The outermost component of the S layer of Lampropedia hyalina, the punctate layer, is assembled onto an inner perforate layer. The punctate layer is composed of long, tapered cylindrical units centered on p6 symmetry axes and connected by six fine linking arms, joining at the axis of threefold symmetry to create a hexagonal layer with a lattice constant of 25.6 +/- 0.5 nm (J. A. Chapman, R. G. E. Murray, and M. R. J. Salton, Proc. R. Soc. London Ser. B 158:498-513, 1963; R. G. E. Murray, Can. J. Microbiol. 9:593-600, 1963). Extraction of cell envelopes with 100 mM Tris buffer (pH 8) containing 2% deoxycholate resulted in the release of several proteins, but left the S layers intact. The punctate layer was then extracted with 3 M guanidine hydrochloride or 6 M urea, leaving the perforate layer intact. This treatment led to the release of three polypeptides with molecular weights of 60,000, 66,000, and 240,000 (60K, 66K, and 240K polypeptides). These three polypeptides reassembled on the perforate layer as a template to form the S-layer complex or self-assembled to form the punctate layer alone after dialysis of the extract against 50 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (pH 7.5) containing 10 mM CaCl2. The self-assemblies were composed of a 240K polypeptide and a 60K polypeptide. The 240K and 60K polypeptides were separated by column chromatography and examined by electron microscopy. The 240K polypeptide appeared in negative stain as a long, flexible structure and assembled into loose arrays with sixfold symmetry with obvious Y-shaped linking elements, while fractions containing both the 60K and 240K polypeptides showed assemblies closely resembling the punctuate layer. Immunoelectron microscopy was used to confirm the presence of both the 60K and 240K polypeptides as components of the punctuate layer.     

Further investigations on the polypeptides and reconstitution of prasinophycean ejectisomes

Ejectisome fragments were isolated from the prasinophyte Pyramimonas grossii and subjected to different treatments, i.e. Percoll density gradient centrifugation, incubation at pH 2.5 or at pH 10.8, or incubation in 6 M guanidine hydrochloride. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that Percoll density gradient centrifugation did not improve the purity of the ejectisome fragment-enriched fractions. The ejectisome fragments withstood pH 2.5 and pH 10.8 treatment, and no loosely bound polypeptides became detached. The disintegration of ejectisome fragments was achieved in 6 M guanidine hydrochloride, and reassembly into filamentous, ejectisome-like structures occurred after dialysis against distilled water. Fractions enriched either in ejectisome fragments or in reconstituted ejectisome-like structures were dominated by three polypeptides with relative molecular weights of approximately 12.5–19 kDa and two additional polypeptides of 23 and 26 kDa. A polyclonal antiserum directed against an ejectisome fragment-enriched fraction weakly cross-reacted with these polypeptides, and no significant immuno-labelling of ejectisome fragments was registered. A positive immuno-label was achieved using immunoglobulin (IgG) fractions which were gained by selectively incubating nitrocellulose stripes of these polypeptides with the antiserum.     

A protease-like permeability factor in the guinea pig skin. 2. In vitro activation of the latent form permeability factor by weakly acidic phosphate buffer.

Conditions for the in vitro activation of the latent form of a protease-like permeability factor in the pseudoglobulin fraction from guinea pig skin were examined. (1) The factor was activated by dialysis against 67 mM phosphate buffer at pH 5.8--6.4, not at pH 7.0--8.0. (2) High salt concentration (200 mM or greater phosphate buffer or 67 mM phosphate buffer containing 200 mM or greater KCl or NaCl) prevented the activation at pH 6.2. (3) High osmotic pressure (sucrose at 1 M) did not affect activation at pH 6.2. (4) Reconversion of the activated permeability factor into an inactive form was not observed under high salt conditions, under which the latent permeability factor was stable in its own form. (5) The molecular size of the latent permeability factor was estimated as approx. 80 000 by Sephadex G-100 gel filtration at high salt concentration.     

Protein filaments may initiate the assembly of the Bacillus subtilis spore coat.

The Bacillus subtilis spore coat consists of three morphological layers: a diffuse undercoat, a striated inner coat and a densely staining outer coat. These layers are comprised of at least 15 polypeptides and the absence of one in particular, CotE, had extensive pleiotropic effects. Only a partial inner coat was present on the spores which were lysozyme-sensitive. The initial rate of germination of these spores was the same as for the wild type but the overall optical density decrease was greater apparently due to the loss of the incomplete spore coat from germinated spores. Suppressors of the lysozyme-sensitive phenotype had some outer coat proteins restored as well as some novel minor polypeptides. These spores still lacked an undercoat and germinated as did those produced by the cotE deletion strain. The CotE protein was synthesized starting at stage II-III of sporulation, long before the appearance of the coat on spores at stage IV-V. Despite its apparent hydrophilic properties, this protein was present in the crude insoluble fraction from sporulating cells. CotE was not solubilized by high or low ionic strength buffers not by detergents used for the solubilization of membrane proteins. Either 8 M urea or 6 M guanidine HC1 was required and dialysis against a low ionic strength buffer resulted in aggregation into long, sticky filaments. Both the CotE and CotT spore coat proteins appeared to be necessary for the formation of these filaments. Each of these proteins contains sequences related to a bovine intermediate filament protein so their interaction could result in an analogous structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Minor lamin polypeptides from rat liver nuclei can be cross-linked into heteropolymers by disulfide bridges

The peripheral lamina of rat liver nuclei is characterized by the presence of three major polypeptides called lamins A, B, and C. Recent studies have identified in rat liver lamina two quantitatively minor polypeptides that have some of the biochemical and immunological properties of the lamins and were tentatively called minor lamin species. We have further characterized these minor lamin polypeptides. Both minor lamin species copurified quantitatively with the major lamins in dissociation-reassociation experiments and shared epitopes with all three major lamins as well as with intermediate filament proteins, including an epitope involved in coiled-coil interactions in lamina and filaments. Minor lamins generated partial peptide maps very similar to each other but completely different from those of lamins A, B, and C. The two minor lamin species could be cross-linked into heteropolymers containing a constant ratio of both polypeptides by exposure to O-phenanthroline - cupric ion complexes, although they did not appear to be cross-linked by disulfide bonds in the native envelope. Preliminary results suggest that the cross-linked minor lamins could be preferentially associated with lamin B. It therefore appears that in addition to the network of lamins A, B, and C, the peripheral lamina is characterized by the presence of two closely juxtaposed minor lamin polypeptides. The molecular interactions between these various polypeptides and their respective roles remain to be identified.     

Halophilic Nuclease of a Moderately Halophilic Bacillus sp.: Production, Purification, and Characterization

A moderately halophilic bacterium, Bacillus sp., isolated from rotting wood on the seashore in Nauru, produced an extracellular nuclease when cultivated aerobically in media containing 1 to 2 M NaCl. The enzyme was purified from the culture filtrate to an electrophoretically homogeneous state by ethanol precipitation, DEAE-Sephadex A-50 column chromatography, and Sephadex G-200 gel filtration. The enzyme consisted of two charge isomers and showed both RNase and DNase activities. Molecular weight was estimated to be 138,000 by Sephadex G-200 gel filtration. The enzyme had marked halophilic properties, showing maximal activities in the presence of 1.4 to 3.2 M NaCl or 2.3 to 3.2 M KCl. The enzyme hydrolyzed thymidine-5′-monophosphate-p-nitrophenyl ester at a rate that increased with NaCl concentration up to 4.8 M. In the presence of both Mg²⁺ and Ca²⁺, activity was greatly enhanced. The activity was lost by dialysis against water and low-salt buffer, but it was protected when 10 mM Ca²⁺ was added to the dialysis buffer. When the inactivated enzyme was dialyzed against 3.5 M NaCl buffer as much as 68% of the initial activity could be restored. The enzyme exhibited maximal activity at pH 8.5 and at 50°C on DNA and at 60°C on RNA and attacked RNA and DNA exonucleolytically and successively, producing 5′-mononucleotides.     

Heterotypic tetramer (A2D2) complexes of non-epidermal keratins isolated from cytoskeletons of rat hepatocytes and hepatoma cells

Cytoskeletal residues obtained after extraction of rat liver and cultured rat hepatoma cells (line MH1C1) were used to isolate cytokeratin subunit complexes by solubilization in low salt buffer containing 4 M-urea. Alternatively, the complexes were prepared by solubilization of total cytoskeletal proteins in 9.5 M-urea or 6 M-guanidinium hydrochloride (Gu . HCl), followed by separation using reversed phase high pressure liquid chromatography and dialysis first against either 9.5 M-urea or 6 M-Gu . HCl and then against buffers containing either 4 M-urea or 2 M-Gu . HCl, respectively. The complexes contained only two cytokeratin polypeptides in a 1 : 1 ratio as demonstrated by electrophoresis and isoelectric focusing, i.e. components A (Mr 55,000; isoelectric point in 9.5 M-urea, pH 6.4) and D (Mr 49,000; isoelectric point, pH 5.38) which were separated from each other at urea concentrations higher than 7 M. The complex had a sedimentation coefficient S25,w of 4.96 S in 2 M-Gu . HCl. Sedimentation equilibrium analysis gave an average Mr value of 207,000 which was interpreted as a tetramer containing two chains each of A and D. This complex was also directly demonstrated by gel electrophoresis under non-dissociating conditions. Using dimethyl suberimidate to cross-link the complex in solution of 4 M-urea or 2 M-Gu . HCl, we identified covalently linked heterodimers of A and D, and a tetrameric unit containing equal amounts of A and D which was the largest cross-link product obtained. This complex was similar to the tetrameric complex of rat and human vimentin formed under the same conditions. The constituents of the cross-linked products were identified by two-dimensional ("diagonal") gel electrophoresis, involving the cleavage of the bis(amidine) cross-links after the initial separation in the first dimension. Identical cross-link products were recognized when cytokeratin filaments were used. By electron microscopy the complexes appeared as threads of 2 to 3 nm diameter with a mean length of approximately 48 nm. On dialysis to low salt buffer, the complexes formed 2 to 3 nm protofilaments, intertwisted 3 to 4 nm protofilaments and typical 7 to 11 nm intermediate-sized filaments. Complexes formed from equivalent cytokeratins of other species such as man and cow, as well as heterologous recombinations such as human component A mixed with bovine component D and vice versa, showed the same characteristics.(ABSTRACT TRUNCATED AT 400 WORDS)