Isolation and symmetrical splitting of desmosomal structures in 9 M urea

A new way of isolating desmosomal structures from various epithelia is described which takes advantage of the unusual resistance of the desmosomal plaque and parts of the desmosomal membrane domain to denaturing agents such as 9 M urea and 5 M guanidinium hydrochloride (Gdn-HCl). The fractions obtained have been examined by electron microscopy and by gel electrophoresis. When cytoskeletal fractions from epithelial cells, notably those from multistratified epithelia such as bovine epidermis or tongue mucosa, are treated with urea or Gdn-HCl most of the cytoskeletal protein, including cytokeratin material, is removed. The desmosomal structures, however, are retained with well preserved plaque organization and desmoglea components and can be harvested by centrifugation. This simple and rapid procedure for the enrichment of desmosomal structures and proteins also express internal desmosomal domains as the result of "splitting" of the desmosome along the midline structure. These split desmosomal halves reveal regular arrays of desmogleal particles of 8 to 15 nm diameter projecting from the membrane surface. Gel electrophoresis of the polypeptides present in these residual structures has shown prominent amounts of desmoplakins I and II as well as components 3 and 5 whereas glycoproteins 4a and 4b are significantly reduced in relation to untreated or citric acid-treated fractions. Using immunoelectron microscopy on desmosomes split in urea we have also demonstrated the specific localization of desmoplakin on the cytoplasmic side. The observations suggest that the architectural components of the desmosome are among the cell structures most resistant to protein-denaturing treatments. The value of this procedure for preparations of desmosomal proteins and for the production of antibodies specifically reacting with internal domains of junctions, i.e., tools that may interfere with cell-to-cell coupling, is discussed.     

Significance of two desmosome plaque-associated polypeptides of molecular weights 75 000 and 83 000.

Isolated desmosomes from bovine epidermis contain two major polypeptides of mol. wts. 75 000 (D6) and 83 000 (D5) which, like the desmoplakins of mol. wt. greater than 200 000, are associated with the insoluble desmosomal plaque structure. We have characterized these two polypeptides and examined their significance by peptide map comparisons and translation of bovine epidermal mRNA in vitro. Polypeptide D5 is different from polypeptide D6 by its apparent mol. wt., its isoelectric pH (approximately 6.35, whereas D6 is a basic polypeptide isoelectric at pH approximately 8.5) and its peptide map. By all these criteria desmosomal polypeptides D5 and D6 are also different from cytokeratins, desmoplakins and the glycosylated desmosomal proteins. Both polypeptides are synthesized from different mRNAs separable by gel electrophoresis on agarose: mRNA coding for polypeptide D5 is approximately 3500 nucleotides long, that for D6 is significantly shorter (estimated to 3050 nucleotides), and both contain relatively large proportions of non-coding sequences. The translational products of these mRNAs co-migrate, on two-dimensional gel electrophoresis, with the specific polypeptides from bovine epidermis, indicating that they are genuine polypeptides and are not the result of considerable post-translational processing or modification of precursor molecules. The cell and tissue distribution of these two cytoskeletal proteins and possible functions are discussed.     

Structure and biochemical composition of desmosomes and tonofilaments isolated from calf muzzle epidermis

Complexes of plasma membrane segments with desmosomes and attached tonofilaments were separated from the stratum spinosum cells of calf muzzle by means of moderately alkaline buffers of low ionic strength and mechanical homogenization. These structures were further fractionated by the use of various treatments including sonication, sucrose gradient centrifugation, and extraction with buffers containing high concentrations of salt, urea, citric acid, or detergents. Subfractions enriched in desmosome-tonofilament-complexes and tonofilament fragments were studied in detail. The desmosome structures such as the midline, the trilaminar membrane profile, and the desmosomal plaque appeared well preserved and were notably resistant to the various treatments employed. Fractions containing desmosome- tonofilament complexes were invariably dominated by the nonmembranous proteins of the tonofilaments which appeared as five major polypeptide bands (apparent molecular weights: 48,000; 51,000; 58,000; 60,000; 68,000) present in molar ratios of approx. 2:1:1:2:2. Four of these polypeptide bands showed electrophoretic mobilities similar to those of prekeratin polypeptides from bovine hoof. However, the largest polypeptide (68,000 mol wt) migrated significantly less in polyacrylamide gels than the largest component of the hoof prekeratin (approximately 63,000 mol wt). In addition, a series of minor bands, including carbohydrate-containing proteins, were identified and concluded to represent constituents of the desmosomal membrane. The analysis of protein-bound carbohydrates (total 270 microgram/mg phospholipid in desmosome-enriched subfractions) showed the presence of relatively high amounts of glucosamine, mannose, galactose, and sialic acids. These data as well as the lipid composition (e.g., high ratio of cholesterol to phospholipids, relatively high contents of sphingomyelin and gangliosides, and fatty acid pattern) indicate that the desmosomal membrane is complex in protein and lipid composition and has a typical plasma membrane character. The similarity of the desmosome-associated tonofilaments to prekeratin filaments and other forms of intermediate- sized filaments is discussed.     

CHEMICAL CHARACTERIZATION OF ISOLATED EPIDERMAL DESMOSOMES

Desmosomes, isolated from cow nose epidermis by a method utilizing citrate buffer pH 2.6 and density gradient centrifugation, have been analyzed and found to contain approximately 76% protein, 17% carbohydrate, and 10% lipid. Nonpolar amino acids predominate in desmosomal protein, representing 456 residues per 1,000. The sialic acid content is 5 nM/mg of protein. The lipid fraction is composed of approximately 40% cholesterol and 60% phospholipids. Desmosomes are completely solubilized by incubation with 2% sodium dodecyl sulphate and 1% β-mercaptoethanol. Gel electrophoresis of the denatured desmosomal proteins reveals 24 bands, with mobilities corresponding to a molecular weight range of 15,000–230,000 daltons. Seven of these are considered to be major bands, together constituting 81% of the desmosomal protein. Bands 1 and 2, of molecular weights 230,000 and 210,000 daltons, together comprise 28% by weight of the desmosome. It is suggested that these protein chains are located in the desmosomal plaque. Bands 3 and 4 are PAS-positive, constitute 23% of the desmosomal protein, and have apparent molecular weights of 140,000 and 120,000 daltons, respectively. At least part of this material must originate from the carbohydrate-containing layer which is demonstrated, by histochemistry, to be present in the desmosomal interspace. The possible nature and origin of the remaining major bands, of molecular weights 90,000, 75,000, and 60,000 daltons, are discussed.     

The desmoplakin carboxyl terminus coaligns with and specifically disrupts intermediate filament networks when expressed in cultured cells

Specific interactions between desmoplakins I and II (DP I and II) and other desmosomal or cytoskeletal molecules have been difficult to determine in part because of the complexity and insolubility of the desmosome and its constituents. We have used a molecular genetic approach to investigate the role that DP I and II may play in the association of the desmosomal plaque with cytoplasmic intermediate filaments (IF). A series of mammalian expression vectors encoding specific predicted domains of DP I were transiently expressed in cultured cells that form (COS-7) and do not form (NIH-3T3) desmosomes. Sequence encoding a small antigenic peptide was added to the 3' end of each mutant DP cDNA to facilitate immunolocalization of mutant DP protein. Light and electron microscopical observations revealed that DP polypeptides including the 90-kD carboxy-terminal globular domain of DP I specifically colocalized with and ultimately resulted in the complete disruption of IF in both cell lines. This effect was specific for IF as microtubule and microfilament networks were unaltered. This effect was also specific for the carboxyl terminus of DP, as the expression of the 95-kD rod domain of DP I did not visibly alter IF networks. Immunogold localization of COS-7 cells transfected with constructs including the carboxyl terminus of DP demonstrated an accumulation of mutant protein in perinuclear aggregates within which IF subunits were sequestered. These results suggest a role for the DP carboxyl terminus in the attachment of IF to the desmosome in either a direct or indirect manner.     

Isolation of the intercellular glycoproteins of desmosomes

To characterize the desmosome components that mediate intercellular adhesion and cytoskeletal-plasma membrane attachment, we prepared whole desmosomes and isolated desmosomal intercellular regions (desmosomal "cores") from the living cell layers of bovine muzzle epidermis. The tissue was disrupted in a nonionic detergent at low pH, sonicated, and the insoluble residue fractionated by differential centrifugation and metrizamide gradient centrifugation. Transmission electron microscopic analyses reveal that a fraction obtained after differential centrifugation is greatly enriched in whole desmosomes that possess intracellular plaques. Metrizamide gradient centrifugation removes most of the plaque material, leaving the intercellular components and the adjoining plasma membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with methods that reveal carbohydrate-containing moieties on gels demonstrate that certain proteins present in whole desmosomes are glycosylated. These glycoproteins are specifically and greatly enriched in the desmosome cores of which they are the principal protein constituents, and thus may function as the intercellular adhesive of the desmosome.     

Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. I. Biochemical analysis

The functional interaction of cells in the formation of tissues requires the establishment and maintenance of cell-cell contact by the junctional complex. However, little is known biochemically about the mechanism(s) that regulates junctional complex assembly. To address this problem, we have initiated a study of the regulation of assembly of one component of the junctional complex, the desmosome, during induction of cell-cell contact in cultures of Madin-Darby canine kidney epithelial cells. Here we have analyzed two major protein components of the desmosomal plaque, desmoplakins I (Mr of 250,000) and II (Mr of 215,000). Analysis of protein levels of desmoplakins I and II by immunoprecipitation with an antiserum that reacts specifically with an epitope common to both proteins revealed that desmoplakins I and II are synthesized and accumulate at steady state in a ratio of 3-4:1 (in the absence or presence of cell-cell contact). The kinetics of desmoplakins I and II stabilization and assembly were analyzed after partitioning of newly synthesized proteins into a soluble and insoluble protein fraction by extraction of whole cells in a Triton X-100 high salt buffer. In the absence of cell-cell contact, both the soluble and insoluble pools of desmoplakins I and II are unstable and are degraded rapidly (t1/2 approximately 8 h). Upon induction of cell-cell contact, the capacity of the insoluble pool increases approximately three-fold as a proportion of the soluble pool of newly synthesized desmoplakins I and II is titrated into the insoluble pool. The insoluble pool becomes relatively stable (t1/2 greater than 72 h), whereas proteins remaining in the soluble pool (approximately 25-40% of the total) are degraded rapidly (t1/2 approximately 8 h). Furthermore, we show that desmoplakins I and II can be recruited from this unstable soluble pool of protein to the stable insoluble pool upon induction of cell-cell contact 4 h after synthesis; significantly, the stabilization of this population of newly synthesized desmoplakins I and II is blocked by the addition of cycloheximide at the time of cell-cell contact, indicating that the coordinate synthesis of another protein(s) is required for protein stabilization.     

Biochemical and immunological characterization of desmoplakins I and II, the major polypeptides of the desmosomal plaque

Epithelial cells contain complexes of cytokeratin filaments (tonofilaments) with specific domains of the plasma membrane that appear as symmetric junctions, i.e. desmosomes, or as asymmetric hemi-desmosomes. These regions of filament-membrane-attachment are characterized by 14 to 20 nm thick dense plaques (desmosomal plaque). In isolated desmosome-tonofilament complexes or other desmosomal fractions from various stratified squamous epithelia (e.g. bovine muzzle epidermis and tongue mucosa) desmosomal plaque structures are recognized and show a relatively high resistance to various extraction buffers and detergents. Such fractions enriched in desmosomal plaque material are also enriched in two prominent polypeptide bands of apparent molecular weights 250,000 (desmoplakin I) and 215,000 (desmoplakin II) which appear, on two-dimensional gel electrophoresis, as two distinct polypeptides isoelectric near neutral pH. These two polypeptides are present in almost equimolar amounts and each of them appears as a series of isoelectric variants, including some labeled by [32P]phosphate in tissue slices. The two desmoplakin polypeptides are closely related as shown by tryptic peptide map analysis and are different from keratin-like proteins and other major polypeptides of desmosome-rich fractions. Guinea pig antibodies raised against desmoplakins and specific for these proteins do not cross-react with other desmosomal antigen(s) or constituents of other types of junctions. Using desmoplakin antibodies we have identified desmoplakins as the major constituents of the desmosomal plaques present in epithelial and myocardiac cells of diverse species. The significance of this group of cell type-specific membrane-associated cytoskeletal proteins and their possible cytoskeletal functions are discussed.     

Correlations among culture times,sugar composition and biological activities of Sporothrix schenckii antigens

Glycoproteins were isolated by ethanol precipitation, Con A-sepharose 4B and DEAE-sephadex A-50 chromatography from culture filtrates of Sporothrix schenckii ATCC 10268 at incubation periods of 2, 7, and 14 days, and their chemical and immunological properties investigated. Sugar composition of the isolated glycoproteins varied with time of culture, i.e. from mostly mannose on the 2nd day of culture to increasing amounts of rhamnose and small amounts of galactose in addition to mannose on the 7th and 14th day. The changes in sugar composition also were observed to be closely related to the growth morphology of the organisms. The isolated glycoproteins showed different serological reactivity in immunodiffusion tests against rabbit anti-S. schenckii antiserum. In addition, they showed varying degree of cross-reaction with rabbit anti Klebsiella pneumoniae K47, anti Cladosporium werneckii and anti Saccharomyces cerevisiae antisera. The immunodiffusion results correlate well with sugar composition and strongly suggest the possibility that rhamnose, galactose and mannose determinants participate in the serological reaction of S. schenckii. In delayed hypersensitivity skin tests in guinea pigs immunized with S. schenckii, only Con A-binding glycoproteins were reactive. These fractions also resembled each other in amino acid content. The results from the present work indicate that the immunochemical properties of S. schenckii glycoproteins vary with incubation period, and suggest the need for standardization of sporotrichin test antigens.     

Chemical and immunochemical characterization of caseins and the major whey proteins of rabbit milk.

Caseins were separated from whey proteins by acid precipitation of skimmed rabbit milk. Whole casein was resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis into three major bands with apparent relative molecular masses (Mr of 31 000, 29 000 and 25 000. On agarose/urea-gel electrophoresis whole casein gave three bands with electrophoretic mobilities alpha, beta and gamma. The three components were purified by DEAE-cellulose chromatography under denaturing and reducing conditions. Each was shown to have a different amino acid, hexose and phosphorus content, as well as non-identical peptide fragments after proteinase digestion. The 31 000 Da (dalton) protein, of alpha-electrophoretic mobility, had a high phosphorus content (4.38%, w/w); the 29 000 Da peptide, of gamma-mobility, had the highest hexose content (2.2%, w/w), contained 0.8 cysteine residue per 100 amino acid residues and was susceptible to chymosin digestion corresponding thus to kappa-casein; the 25 000 Da protein migrated to the beta-position. The rabbit casein complex is composed of at least three caseins, two of which (alpha- and kappa-caseins) are analogous to the caseins from ruminants. Although caseins are poor immunogens, specific antibodies were raised against total and purified polypeptides. The antiserum directed against whole casein recognized each polypeptide, each casein corresponding to a distinct precipitation line. The antisera directed against each casein polypeptide reacted exclusively with the corresponding casein and no antiserum cross-reaction occurred between the three polypeptides. From whey, several proteins were isolated, characterized and used as antigens to raise specific antibodies. An iron-binding protein with an apparent Mr of 80 000 was shown to be immunologically and structurally identical with serum transferrin.     

A constitutive transmembrane glycoprotein of Mr 165,000 (desmoglein) in epidermal and non-epidermal desmosomes. II. Immunolocalization and microinjection studies

Using two monoclonal antibodies described in the preceding paper we determined by immunofluorescence microscopy the distribution of an integral membrane protein of the desmosomal domain, the major glycopolypeptide of Mr 165,000 (bovine muzzle epidermal desmosome band 3; desmoglein) in various normal tissues, tumors and cultured cell lines from several mammalian species. This protein was detected in dotted or streak-like arrays along cell boundary structures which were known to contain non-membrane-integrated desmosomal plaque proteins such as desmoplakins. This is true for epithelial, i.e. cytokeratin-expressing cell types, for the desmin-producing myocardiac and Purkinje fiber cells of the heart, and for certain vimentin-containing cells such as arachnoidal and meningiomal cells and dendritic follicular cells of lymph nodes. However, on the basis of both immunoblot and immunocytochemical reactions, the protein is absent from non-desmosomal adhering junctions, including those devoid of desmoplakin but containing another plaque protein, plakoglobin ("band 5 protein"). We have used these antibodies to localize their epitopes with respect to the cell membrane. By immunoelectron microscopy we found that both epitopes are located in the desmosomal plaques, and this was confirmed by microinjection of purified antibodies into living cultured cells which resulted in labelling of the plaques. From these findings, taken together with previous analyses and localizations of the carbohydrate moieties of this glycoprotein, we conclude that desmoglein is a transmembrane glycoprotein which projects into--and contributes to--the desmosomal plaque structure. This glycoprotein represents a general component of true desmosomes and it is coexpressed with obligatory desmosome-specific plaque proteins such as desmoplakin I. The potential value of this glycoprotein as a desmosomal and cell type marker in histology and tumor diagnosis is discussed.     

Immunochemical characterization of related families of glycoproteins in desmosomes

Using several biochemical approaches, we have characterized the relatedness of the various glycoprotein components of the bovine epidermal desomosome. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of purified epidermal desmosomes reveals 12 proteins, of which 8 are glycosylated. Analysis with monoclonal antibodies indicates that the 8 glycoproteins comprise 3 antigenically distinct protein families. Members of the highest molecular weight glycoprotein family (a triplet of Mr = 150,000) were not distinguishable by partial proteolytic peptide mapping. At least 6 different monoclonal antibodies have been identified that recognize unique antigenic determinants shared by these proteins. Members of a 97,000-118,000-dalton glycoprotein family (about 4 bands) generate very similar but not identical partial proteolytic peptide maps. At least 3 different monoclonal antibodies have been identified that recognize unique antigenic determinants shared by these proteins. A Mr = 22,000 glycoprotein is immunologically unrelated to either of the high molecular weight glycoprotein families. Lectin-binding profiles indicate that within each immunologically related family the glycoproteins are similar in their oligosaccharide composition. Some lectins distinguish among the families. These glycoproteins probably mediate the specific intercellular recognition and adhesive functions of the desmosome.     

Dietary level of protein regulates glyceraldehyde-3-phosphate dehydrogenase content and synthesis rate in mouse liver cytosol

The content of liver cytosolic proteins was studied in mice subjected to protein depletion followed by refeeding with a normal diet. Depletion elicited either the accumulation or the decrease of several polypeptides, being the early increase of a Mr 36 000 polypeptide the most pronounced change observed. The refeeding with a normal diet for 2 days caused a return of the cytosol protein composition to that of normally fed animals. The Mr 36 000 polypeptide was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Its molecular weight, the sequence of its first twenty amino acid residues, its amino acid composition and its antigenic properties were found to be similar with those of GAPDH from different mammalian cells. During the first 2 days of protein depletion, both the GAPDH polypeptide content and activity increased. Thereafter, the enzymatic activity of GAPDH decreased, whereas GAPDH protein mass decreased in a lesser extent. The accumulation of GAPDH and other particular polypeptides in the cytosols of protein depleted mice was associated with an increased synthesis. The refeeding with a normal diet caused an immediate return to the synthesis pattern of normal livers.     

Haemocyanins in spiders, III. Chemical and physical properties of the proteins in Dugesiella and Cupiennius blood.

The haemolymph of the tarantulas, Dugesiella (Eurypelma) californica and Dugesiella (Eurypelma) helluo contains high molecular weight haemocyanin (80-82% of total blood proteins) and a second protein not related to haemocyanin (18-20%). In the Lycosid spider, Cupiennius salei, haemocyanin (75% of total blood protein) occurs in two states of association. The haemocyanins were isolated by ultracentrifugation, gel filtration, isoelectric focusing, or preparative gel electrophoresis. Their sedimentation constants are 36.7 S (both tarantulas), 23.4 S and 15.9 S (Cupiennius). After alkaline dissociation, polypeptides sedimenting at 5.8 S (D. californica) and 4.7 S (Cupiennius) were obtained. The molecular weight of the intact functional subunit is (by sedimentation equilibrium) 70 300 (D. californica) and 69 900 (Cupiennius). Copper analysis results in closely similar values. By sodium dodecylsulphate gel electrophoresis, molecular weights of 71 000 (D. californica), 72 000 (Cupiennius) and 74 000 (D. helluo) were obtained. Denaturation with various agents did not lead to smaller polypeptides. The amino acid composition of the haemocyanins was determined (Table 1). The amino end group is blocked. The haemocyanins contain 1.2-1.5% of neutral carbohydrates and 0.3-0.5% of glucosamine (possibly acetylated). The neutral carbohydrates were identified with glucose, mannose, fucose, and arabinose, glucose being the dominant species. Neuraminic acid was not detected. The haemocyanins of the three species cannot be distinguished by their carbohydrate moieties, while there is a significant difference in amino acid composition between tarantula and Cupiennius haemocyanins. The second, non-respiratory protein isolated from spider blood sediments with 16.1 S (Dugesiella) or 15.9 S (Cupiennius). Its isoelectric point is at pH 5.5 It is stable in weakly alkaline solutions but can be denatured to yield polypeptide chains with molecular weights of 95 000 and 110 000. The amino acid composition is reported. As in the haemocyanins, the N-terminus is blocked. The carbohydrate content is 0.9%, glucose being the only sugar identified.     

Further Studies of Pollen Wall Glycoproteins in Cucurbita pepo L.

Samples of pollen wall protein of Cucurbita pepo were prepared as reported in previous paper. Gas chromoatographic analyses snowed that the carbohydrate fraction of the pollen wall glycoprotcin contained 20.4% rhamnose, 15.3% fucose, 11% mannose, 11% galactose, 31% glucose, 4% arabinose and traces of xylose. The glycoproteins were further purified by Con. A affinity chromatography, Isoelectric focussing electrophoresis of the purified sample showed 3 PAS-positive bands, with respective PI 5.2, 6.0 and 6.3. The glycoprotein samples were subjected to hydrolysis with 6N HC1. After hydrolysis, the mixture was analyzed for amino acid composition with Backman 121-MB automatic amino acid analyzer, Results show the amino acid composition of the 3 glycoprotein was very similar, They all have glycine, glutamic acid and serine as their major component (these three amino acids constitute 50–60% of the total amino acids); and they all contain very small amount of methionine, phenylalanine, isoleucine and tyrosine. The lysine content of each glycoprotein is consistent with its respective PI, the glycoprotein which contains more lysine has higher PI.     

Carbohydrate Composition of Variant-Specific Surface Antigen Glycoproteins from Trypanosoma brucei

SYNOPSIS. The carbohydrate of variant-specific surface antigen glycoproteins from bloodstream forms of 13 cloned variants of Trypanosoma brucei was analyzed by gas-liquid chromatography. The glycoproteins contained from 6 to 17% carbohydrate by weight, and all contained the same 4 sugars: mannose, galactose, glucose, and glucosamine (probably as N-acetyl-glucosamine). The glycoprotein from variant 048, strain 427 contained (±20%) 11 mannose, 4 galactose, 4 glucose, and 5 glucosamine residues/mole of glycoprotein (molecular weight 65,000). Glucose was an integral component of the glycoproteins, not dissociable by sodium dodecyl sulphate, 8 M urea, or 1 M acetic acid. Some of the glucose was dissociated by trichloroacetic acid. Most of the glycoproteins formed precipitin bands with concanavalin A in Ouchterlony double diffusion, but none formed such bands with wheat germ agglutinin or Ricinus communis lectin (molecular weight 120,000).     

Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity

Human factor VIII was isolated from commercial factor VIII concentrates and found to consist of multiple polypeptides with molecular weights ranging from 80 000 to 210 000. Immunological and amino acid sequence data identified these polypeptides as subunits of factor VIII. N-Terminal amino acid sequence analysis determined that the Mr 210 000 and 80 000 proteins are derived from the N- and C-terminal portions of factor VIII, respectively; Mr 90 000-180 000 polypeptides are derived from the Mr 210 000 polypeptide by C-terminal cleavages. Treatment of purified factor VIII with thrombin resulted in proteolysis of Mr 80 000-210 000 proteins and the generation of polypeptides of Mr 73 000, 50 000, and 43 000. Maximum coagulant activity of thrombin-activated factor VIII was correlated with the generation of these polypeptides. The proteolysis as well as activation of factor VIII by thrombin was found to be markedly dependent on CaCl2 concentration. Proteolysis of factor VIII with activated protein C (APC) resulted in degradation of the Mr 90 000-210 000 proteins with the generation of an Mr 45 000 fragment. This cleavage correlated with inactivation of factor VIII by APC. The Mr 80 000 protein was not degraded by APC. Factor Xa cleaved the Mr 80 000-210 000 factor VIII proteins, resulting in the generation of fragments of Mr 73 000, 67 000, 50 000, 45 000, and 43 000. Factor Xa was found to initially activate and subsequently inactivate factor VIII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Organization of desmosomal plaque proteins in cells growing at low calcium concentrations

Desmosomes are not formed in epithelial cell cultures growing in media with low (less than or equal to 0.1 mM) concentrations of Ca2+ (LCM) but appear rapidly upon shift to media of normal calcium concentrations (NCM). Previous authors using immunolocalization of desmoplakin, a marker protein for the desmosomal plaque, in LCM-grown cells have interpreted positively stained, dense, cytoplasmic aggregates on intermediate filaments (IF) bundles as preformed plaque units which upon NCM shift would move to the plasma membrane and contribute to desmosome formation. Studying various cell cultures, including primary mouse keratinocytes and human A-431 cells, we show that most, probably all, desmoplakin-positive aggregates in LCM-grown cells are associated with membranous structures, mostly vesicles, and also contain other desmosomal markers, including desmoglein, a transmembrane glycoprotein. We interpret such vesicles as residual desmosome-derived domains endocytosed upon cell dissociation. Only keratinocytes grown for long times (2-4 wk) in LCM are practically free from such vesicles. In addition, we demonstrate that certain cells such as A-431 cells, when passaged in LCM and in the absence of stable junctions, are able to continually assemble "half-desmosomes" on the plasma membrane which in turn can be endocytosed as plaque-bearing vesicles. We also show that in LCM the synthesis of several desmosomal proteins (desmoplakins I and II, plakoglobin, desmoglein, "band 6 protein") continues and that most of the plaque protein, desmoplakin, is diffusely spread over the cytoplasm, apparently in a soluble monodisperse form of approximately 9S. From our results we propose that the plaque proteins occur in small, discrete, diffusible entities in the cytoplasm, in concentrations that are relatively high in LCM and low in NCM, from which they assemble directly, i.e., without intermediate precursor aggregates on IFs in the cytoplasm, on certain plasma membrane domains in a Ca2+ dependent process.     

The phenoloxidases of the ascomycete Podospora anserina. Structural differences between laccases of high and low molecular weight.

In order to investigate the extent of the relationship between the three copper-containing glycoproteins, laccases I, II and III (Mr70000, 80000 and 390000 respectively) of Podospora anserina, the following experiments were carried out on laccases II and III: (a) determination of amino acid composition; (b) determination of N-terminal and C-terminal amino acid; (c) determination of sugar composition; (d) dissociation studies on native and denatured laccases and also after removal of copper from the enzymes; (e) digestion of the carbohydrate moieties with the aid of glycosylhydrolases. A comparison between the results of these experiments and data previously obtained with laccase I allows the following conclusions to be drawn. 1. Laccases II and III are not identical. 2. Neither of these low molecular weight laccases are as complete molecules subunits of the oligomeric laccase I. 3. The possibility of partial identity of amino acid sequences of laccases I and III can not be excluded. 4. Laccase II possibly consists of subunits of Mr37000 whereas laccase III does not. 5. Digestion of 50% of the carbohydrate content leads to complete loss of serological specificity (serological reaction and cross reaction). This finding is discussed with regard to the possible role of the carbohydrate moiety as antigenic determinants and thus as the reason for the immunological relationship. As a consequence, at least three independent structural genes for laccases must be assumed.     

Stratifin (14-3-3 σ) Limits Plakophilin-3 Exchange with the Desmosomal Plaque

Desmosomes are prominent cell-cell adhesive junctions in stratified squamous epithelia and disruption of desmosomal adhesion has been shown to have dramatic effects on the function and integrity of these tissues. During normal physiologic processes, such as tissue development and wound healing, intercellular adhesion must be modified locally to allow coordinated cell movements. The mechanisms that control junction integrity and adhesive strength under these conditions are poorly understood. We utilized a proteomics approach to identify plakophilin-3 associated proteins and identified the 14-3-3 family member stratifin. Stratifin interacts specifically with plakophilin-3 and not with other plakophilin isoforms and mutation analysis demonstrated the binding site includes serine 285 in the amino terminal head domain of plakophilin-3. Stratifin interacts with a cytoplasmic pool of plakophilin-3 and is not associated with the desmosome in cultured cells. FRAP analysis revealed that decreased stratifin expression leads to an increase in the exchange rate of cytoplasmic plakophilin-3/GFP with the pool of plakophilin-3/GFP in the desmosome resulting in decreased desmosomal adhesion and increased cell migration. We propose a model by which stratifin plays a role in regulating plakophilin-3 incorporation into the desmosomal plaque by forming a plakophilin-3 stratifin complex in the cytosol and thereby affecting desmosome dynamics in squamous epithelial cells.