Post-translational modification of the beta-subunit of the human fibronectin receptor

Monoclonal antibody DH12, directed against the beta-subunit of the fibronectin receptor recognizes a doublet of proteins (100 and 110 kDa) in Western blots of solubilized whole fibroblasts. Pulse-chase experiments with [35S]methionine in human skin fibroblasts suggested that the two proteins might be metabolically related as precursor (100 kDa) and product (110 kDa). Endo H digestion and [3H]fucose labeling suggested that maturation converted the high-mannose oligosaccharides (100 kDa) to the endoglycosidase H resistant complex type (110 kDa). This was supported by N-glycanase digestion and by chemical deglycosylation which showed a single polypeptide. Surface iodination of intact cells labeled only the presumed mature beta-subunit.     

Characterization, cloning and immunolocalization of a coronin homologue in Trichomonas vaginalis

On adhesion to host cells the flagellate Trichomonas vaginalis switches to an amoeboid form rich in actin microfilaments. We have undertaken the identification of actin-associated proteins that regulate actin dynamics. A monoclonal antibody 4C12 raised against a cytoskeletal fraction of T. vaginalis labeled a protein doublet at circa 50 kDa. These two bands were recognized by the antibody against Dictyostelium discoideum coronin. During cell extraction and actin polymerization, T. vaginalis coronin cosedimented with F-actin. By two-dimensional gel electrophoresis, the protein doublet was separated into two sets of isoforms covering two Ip zones around 6 and 7. By screening a T. vaginalis library with 4C12, two clones Cor 1 and Cor 2 were isolated. This gene duplicity is a particularity among unicellular organisms examined. The complete sequence of the gene Cor 1 encodes a 435-residue protein with a calculated molecular mass of 48 kDa and Ip of 5.58. The incomplete sequence Cor 2 was very similar but with a more basic calculated Ip than Cor 1 on the same region. T. vaginalis coronin had 50% similarity with the coronin family, possessing the five WD-repeats and a leucine zipper in its C-terminal part. Double immunofluorescence labeling showed that coronin mainly colocalized with actin at the periphery of the adherent amoeboid cells. However, coronin labeling displayed patches within a reticular array. Immunogold electron microscopy confirmed the coronin labeling in the actin-rich microfilamentous fringe beneath the plasma membrane, with accumulation in phagocytic zones and pseudopodial extensions. In T. vaginalis, one of the first emerging lineage of eukaryotes, coronin seems to play an important role in actin dynamics and may be a downstream target of a signaling mechanism for the cytoskeleton reorganization.     

Temporal and subcellular localization of PR-1 proteins in tomato stem tissues infected by virulent and avirulent isolates of Phytophthora capsici

Summary. Immunoblot analysis and immunogold labeling of PR-1 protein (pathogenesis-related protein 1) in tomato (Lycopersicon esculentum Mill.) were performed to examine the temporal and spatial expression patterns of PR-1 protein induced by Phytophthora capsici infection. Soluble proteins with molecular masses of 10, 17, 25, 27 and 75 kDa were induced and accumulated in P. capsici-infected stem tissues during the compatible and incompatible interactions. Western blot analysis revealed that expression of PR-1 protein (17 kDa), at 12 to 24 h after inoculation, occurred earlier in the incompatible than in the compatible interaction. Immunogold labeling of PR-1 proteins occurred over cell walls and cytoplasm of the host and the oomycete pathogen and at the interface between host and oomycete cell walls at 24 h after inoculation in the compatible interaction. In the incompatible interaction, numerous PR-1 proteins accumulated predominantly over oomycete cell walls and at the interface between host and oomycete cell walls. The quantity of PR-1 proteins deposited in both host and oomycete cells was much less in the compatible than the incompatible interaction. Healthy tomato stem tissue was nearly free of immunogold labeling of PR-1 proteins. Received October 9, 2001 Accepted January 18, 2002     

Characterization of caspase proteases in cytokine-dependent myeloid progenitor cells using enzyme affinity labeling

Bone marrow-derived myeloid progenitor cells are dependent on the presence of cytokines such as interleukin-3 (IL-3) for their survival. The withdrawal of IL-3 from IL-3-dependent myeloid progenitors results in death via an apoptotic program. Previous studies have shown that IL-3 withdrawal induces the activities of caspase proteases. However, the molecular identities of myeloid progenitor caspases have not been determined. In this study, we used an affinity labeling reagent (biotin-YVAD-acyloxymethyl ketone) that binds to processed active caspase subunits, to study caspase activation in 32D and FDCP-1 myeloid progenitor cells. After IL-3 withdrawal, we detected affinity labeling of caspase subunits of 20, 17, and 16 kDa in both cell lines. Surprisingly, affinity labeling of the 20- and 17-kDa proteins, but not the 16-kDa protein, was also detected in healthy cells maintained in the presence of IL-3. By contrast, in cytokine-independent cell lines, affinity labeling of caspase subunits was detected only after treatment with an apoptotic stimulus. Immunoblotting experiments showed that caspase-3 constitutes at least a portion of the 20- and 17-kDa affinity-labeled proteins detected in the myeloid progenitor cell lines. Taken together, these data provide direct evidence of caspase activation in cytokine-dependent myeloid progenitors, and suggest that unique apoptotic pathways may exist in these cells.     

Isoprenylated proteins in cultured cells: subcellular distribution and changes related to altered morphology and growth arrest induced by mevalonate deprivation

In the presence of lovastatin (mevinolin), an inhibitor of endogenous mevalonate synthesis, C1300 murine neuroblastoma cells incorporated (2-14C)mevalonate into several discrete polypeptides that were separable by SDS-PAGE. The electrophoretic pattern of the labeled proteins did not vary substantially when cells were homogenized with Ca++, Mg++, high concentrations of NaCl or phosphatase inhibitor, or when cells were lysed immediately in trichloroacetic acid. When cells that had been prelabeled with (14C)mevalonate were incubated with lovastatin and simultaneously deprived of exogenous mevalonate, there was a 50-60% decline in the concentration of protein-bound isoprenoid label within 17 h. In contrast, there was little change in the radioactivity in the sterol, dolichol, or ubiquinone fractions. The time course of the decline in mevalonate-derived label in cellular polypeptides paralleled the onset of neurite outgrowth and preceded the decline of DNA synthesis, suggesting that a decreased intracellular concentration of protein-bound isoprenoid groups may contribute to the well-documented effects of mevalonate deprivation on cell morphology and cell cycling. Fractionation of neuroblastoma cells by differential centrifugation and sucrose density-gradient centrifugation revealed that mevalonate-labeled proteins of 53 kDA, 22-26 kDa, and 17 kDa were concentrated in the cytosol. Proteins migrating at 45 kDa were found in both the soluble and particulate fractions, including those enriched in mitochondria and plasma membrane. The isoprenylated proteins migrating at approximately 66 kDa were localized exclusively in the nuclear fraction. When chromatin was removed from the nuclei by extraction with 2 M NaCl, the 66 kDa isoprenylated proteins remained associated with the residual components of the nuclear matrix and lamina. Isoprenylated proteins with electrophoretic mobilities similar to those observed in neuroblastoma cells were detected in a variety of established cell lines. However, there was considerable variation among cell lines in the overall efficiency of protein labeling with (14C) mevalonate and in the prominence and mobilities of specific labeled proteins in the 45-70 kDa range. Comparisons of paired transformed vs. nontransformed fibroblast cell lines suggested that the profile of mevalonate-labeled proteins in a given cell line is not altered by malignant transformation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization and purification of the hyaluronan-receptor on liver endothelial cells.

In order to characterize the proteins on liver endothelial cells that bind hyaluronan (HYA), liver endothelial cells were surface-iodinated with 125I, solubilized by Triton X-100 and passed through a column containing HYA coupled to agarose. The column was washed and eluted with HYA-oligosaccharides. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted material, followed by autoradiography, showed a major band with a molecular mass of 100 kDa, that upon reduction gave major bands of 20 and 35 kDa, and minor doublet bands at 60 and 80 kDa. Two-dimensional electrophoresis of liver endothelial cell membrane proteins revealed that the 100 kDa protein has a pI of 6.6-6.8. The protein was purified by preparative SDS-PAGE of liver endothelial cell membrane proteins. The 100 kDa protein was excised from the gel and used for immunization of rabbits. Antiserum from immunized rabbits specifically recognized only the 100 kDa protein on immunoblots of liver endothelial cell membrane proteins separated by SDS-PAGE. The binding of 3H-HYA to liver endothelial cells and liver endothelial cell membranes could be specifically inhibited by Fab-fragments of the antibodies. When we tried to isolate the receptor in large scale by affinity chromatography of proteins from purified liver endothelial cell membranes, the 100 kDa protein could often not be detected on immunoblots or by silver staining following SDS-PAGE of the eluted material. Instead, proteins with molecular masses of 55 and 15 kDa were detected, but the antibodies reacted specifically with these proteins. Thus the 100 kDa protein is apparently susceptible to cleavage into distinct subcomponents.     

Density Dependent Change of Myristoylated Proteins in C3H10T1/2 Fibroblasts and Their Transformants

We have examined the pattern of protein myristoylation in C3H10T1/2 fibroblasts during cell growth. During the growing phase of 10T1/2 cells, several proteins were radiolabelled with [³H]myristate, and among them proteins with molecular masses of 22, 35, a doublet of 42–45 and 67 kDa were labelled predominantly. The extent of myristoylation in each of these proteins changed with cell density. The amount of radioactivity incorporated into the 22 kDa protein in 10T1/2 cells decreased with increasing cell density and remained at a low level during the stationary phase. In contrast, the incorporation into the 67 kDa protein increased parallel to cell density. The density-dependent change of myristoylation was not observed in any of the transformants of 10T1/2 cells thus far examined. The 67 kDa protein was identified as MARCKS (myristoylated alanine-rich C kinase substrate) by immunoprecipitation with an anti-MARCKS antibody. By Western blot analysis, we found that the amount of MARCKS in 10T1/2 cells increased significantly analogous with cell density. Therefore, it is possible that MARCKS and the 22 kDa protein play a role in contact-mediated cell signalling in 10T1/2 cells, but the mechanism is lost in transformed cells. © 1997 John Wiley & Sons, Ltd.     

Identification of cyclosporin binding sites in rat liver plasma membranes, isolated hepatocytes, and hepatoma cells by photoaffinity labeling using [3H]cyclosporin-diaziridine

[3H]Cyclosporin diaziridine, a new photoaffinity label, enters rat liver cells in the dark. Photoaffinity labeling of isolated rat liver-cell plasma membranes with this probe modifies several polypeptides with molecular mass of 200, 85, 54, 50, 34 kDa. The major labeled protein of 85 kDa represents 2% of the total plasma membrane protein. A 50 kDa protein is heavily labeled in freshly isolated rat hepatocytes at low temperature and after short incubation in the dark. The 85 kDa protein becomes substituted after longer preincubation periods at temperatures above 10 degrees C. This suggests a localisation at the cytoplasmic side of the membrane. Several controls point to a specific interaction with the above mentioned proteins. Comparison of [3H]cyclosporin-diaziridine- and isothiocyanatobenzamido[3H] cholic acid-labeled membrane proteins reveals identity of binding proteins with the exception of the 85 kDa protein. However, the interaction of bile acids with the 85 kDa protein became apparent at higher concentrations as demonstrated by the differential photoaffinity labeling experiments. In the cytosol of rat liver cells, further [3H]cyclosporin-diaziridine binding proteins could be identified. In particular, a 17 kDa polypeptide was found which appears similar to cyclophilin, a protein known to be present in T-lymphocytes (R. Handschumacher et al. (1984) Science 226, 544-547: Cyclophilin. A specific cytosolic binding protein for cyclosporin A). Proteins with molecular mass of 90, 56, 30, 24, 20 kDa are labeled in AS-30D ascites hepatoma cells and those with molecular mass of 200, 150, 80, 70, 42, 25 kDa in Ehrlich ascites tumor cells.     

Use of antibodies in the analysis of connexin 43 turnover and phosphorylation

A series of antipeptide antibodies designed to recognize specific sequences of the gap junction protein connexin 43 (Cx43) were developed and characterized immunochemically and immunohistologically. These antibodies bound to gap junctions and, on Western blots, to 43-kDa (often resolved as a doublet) and 41-kDa proteins in samples from heart, leptomeningeal cells, and brain. Relatively little of the 41-kDa protein was detectable in heart homogenates. Cultured rat leptomeningeal cells expressed high levels of the gap junction protein Cx43 and were used to analyze its turnover and phosphorylation. Pulse-chase experiments in leptomeningeal cells with [(35)S]methionine indicated that the 41-kDa form of connexin 43 was the first immunoprecipitable translation product. Radiolabel subsequently appeared in the lower band of the doublet at 43 kDa, followed by a shift into the higher band and turnover of the protein with a t(1/2) of 2.7 h. Pulse-chase labeling with [(32)P]P(i) indicated that phosphorylation of connexin 43 was limited to the 43-kDa protein, with a t(1/2) of 1.7 h. Treatment with alkaline phosphatase shifted the apparent molecular mass of the 43-kDa protein doublet such that it comigrated with the 41-kDa form. Hence, the 43-kDa protein observed on Western blots of both leptomeningeal cells and heart arises by phosphorylation of the 41 kDa precursor. Phosphorylation of serine residues accounts for most, if not all, of Cx43 phosphorylation in this system.     

Studies of the cyclic adenosine monophosphate chemoreceptor ofParamecium

Summary A doublet of proteins (48,000M _r) from theParamecium cell body membrane fits several criteria for the external cAMP chemoreceptor. These criteria include: (i) selective elution from a cAMP affinity column, matching a specificity that could be predicted from the behavioral response and whole-cell binding; (ii) binding to wheat germ agglutinin indicating the presence of carbohydrate moieties indicating surface exposure; and (iii) selective inhibition of the intact cells' chemoresponse to cAMP by antibodies against the doublet. Additional evidence for the existence of a receptor, in general, comes from selective elimination of the cAMP chemoresponse by photoaffinity labeling of whole cells with 8-N₃-cAMP. The doublet proteins are not identical to the regulatory subunit of a cAMP-dependent protein kinase fromParamecium, theDictyostelium cAMP chemoreceptor, or the 42–45 kDa range proteins related to the large surface glycoprotein inParamecium. The doublet proteins are not readily separable and, as inDictyostelium, may represent two different covalent modification states of the same protein. Amino acid analysis indicates that the proteins are similar, but does not distinguish between the possibilities of proteolysis and covalent modification. Once cloned, this doublet may prove to be only the fifth external, eukaryotic chemoreceptor to be identified.     

Identification of several cyclosporine binding proteins in lymphoid and non-lymphoid cells in vivo.

The immunosuppressant cyclosporine A (CSA) has been shown to bind to the ubiquitous cellular protein, cyclophilin, and to inhibit its rotamase activity. In the present study, 3H-cyclosporine diazirine analogue was used to photolabel viable human cells of lymphoid and fibroblast origin in order to identify the intracellular targets for the drug. While cyclophilin was strongly labeled in situ, additional minor cyclosporine-protein complexes of 25, 40, 46 and 60 kDa were identified in the T cell leukemia cell line Jurkat. These proteins bound specifically, since only active CSA but not inactive CSH or FK506 competed for binding. Photolabeling of MRC5 cells, a CSA resistant human fibroblast cell line, revealed a 25 kDa complex as the major product, while the 46 and 60 kDa bands were not detectable and cyclophilin labeling was only faint, even though both MRC5 and Jurkat cells contain similar cyclophilin concentrations. Thus, our data suggest that the intracellular targets of CSA and/or the accessibility to cyclophilin varies considerably in drug sensitive and resistant cell types, which may contribute to explaining the lymphocyte selectivity of the drug.     

Vinblastine photoaffinity labeling of a high molecular weight surface membrane glycoprotein specific for multidrug-resistant cells

Photoactive radioactive analogues of vinblastine were used to photoaffinity label membranes of Chinese hamster lung drug-sensitive (DC-3F), multidrug-resistant sublines selected for resistance to vincristine (DC-3F/VCRd-5L) or actinomycin D (DC-3F/ADX), and revertant (DC-3F/ADX-U) cells. A radiolabeled doublet (150-180 kDa) consisting of a major and minor band which was barely detectable in parental drug-sensitive cells was increased up to 150-fold in the drug-resistant variants but only 15-fold in the revertant cells. Photoaffinity labeling in the presence of 200-fold excess vinblastine reduced radiolabeling of the 150-180-kDa species up to 96%, confirming its Vinca alkaloid binding specificity. The radiolabeled doublet comigrated with a Coomassie Blue stained polypeptide doublet in the drug-resistant cells and was immunoprecipitated with polyclonal antibody which is specific for the 150-180-kDa surface membrane glycoprotein in multidrug-resistant cell lines. The identification of this Vinca alkaloid acceptor in multidrug-resistant plasma cell membranes suggests the possibility of a direct functional role for the 150-180-kDa surface membrane protein in the development of multidrug resistance.     

The multidrug resistance protein is photoaffinity labeled by a quinoline-based drug at multiple sites

Tumor cells overcome cytotoxic drug pressure by the overexpression of either or both transmembrane proteins, the P-glycoprotein (P-gp) and the multidrug resistance protein (MRP). The MRP has been shown to mediate the transport of cytotoxic natural products, in addition to glutathione-, glucuronidate-, and sulfate-conjugated cell metabolites. However, the mechanism of MRP drug binding and transport is at present not clear. In this study, we have used a photoreactive quinoline-based drug, N-(hydrocinchonidin-8'-yl)-4-azido-2-hydroxybenzamide (IACI), to show the photoaffinity labeling of the 190 kDa protein in membranes from the drug resistant SCLC H69/AR cells. The photoaffinity labeling of the 190 kDa protein by IACI was saturable and specific. The identity of the IACI-photolabeled protein as the MRP was confirmed by immunoprecipitation with the monoclonal antibody QCRL-1. Furthermore, a molar excess of leukotriene C(4), doxorubicin, colchicine, and other quinoline-based drugs, including MK571, inhibited the photoaffinity labeling of the MRP. Drug transport studies showed lower IACI accumulation in MRP-expressing cells which was reversed by depleting ATP levels in H69/AR cells. Mild digestion of the purified IACI-photolabeled MRP with trypsin showed two large polypeptides ( approximately 111 and approximately 85 kDa). The 85 kDa polypeptide which contains the QCRL-1 and MRPm6 monoclonal antibody epitopes corresponds to the C-terminal half of the MRP (amino acids approximately 900-1531) containing the third multiple spanning domain (MSD3) and the second nucleotide binding site. The 111 kDa polypeptide which contains the epitope sequence of the MRPr1 monoclonal antibody encodes the remainder of the MRP sequence (amino acids 1-900) containing the MSD1 and MSD2 plus the first nucleotide binding domain. Cleveland maps of purified IACI-labeled 85 and 111 kDa polypeptides revealed 6 kDa and approximately 6 plus 4 kDa photolabeled peptides, respectively. In addition, resolution of the exhaustively digested IACI-photolabeled MRP by HPLC showed two major and one minor radiolabeled peaks that eluted late in the gradient (60 to 72% acetonitrile). Taken together, the results of this study show direct binding of IACI to the MRP at physiologically relevant sites. Moreover, IACI photolabels three small peptides which localize to the N- and C-halves of the MRP. Finally, IACI provides a sensitive and specific probe for studying MRP-drug interactions.     

Characterisation of membrane oligonucleotide-binding proteins and oligonucleotide uptake in keratinocytes.

Inadequate cellular compartmentalisation of plasmid DNA and antisense oligodeoxynucleotides (ODNs) is generally considered as a major limitation in their use. In this study, an approach combining in situ visual-isation of rhodamine-labelled ODNs and affinity modification of proteins by radiolabelled-alkylating ODN derivatives has been used to investigate the uptake of ODNs into keratinocytes. We confirm here that unmodified ODNs are efficiently taken up and accumulate in cell nuclei in primary keratinocytes as well as in HaCaT and A431 keratinocyte cell lines. Uptake is fast, irreversible, saturable and not significantly altered by incubation at low temperature. Affinity modification studies in keratinocyte cell lines has revealed two high-affinity, cell-specific interactions between ODNs and proteins of 61-63 kDa and 35 kDa. Trypsin pre-treatment of A431 cells and pre-incubation with polyanions, or with unlabelled nucleic acid competitors, inhibited the accumulation of rhodamine-labelled ODNs in nuclei as well as the affinity labelling of the 61-63 kDa doublet and 35 kDa ODN-binding proteins by reactive ODN derivatives. Finally, cell fractionation studies indicated that these ODN-binding proteins were essentially localised in the plasma membrane. Our results suggest that these ODN-binding proteins might be involved in the recognition and transport of ODNs into keratinocytes.     

Characterization of two molecules at 30,33 kDa as major target antigens of natural thymocytotoxic autoantibodies

We have attempted to characterize, by immunoblotting, the cell surface determinants which are recognized by natural thymocytotoxic autoantibodies (NTA). NTA-positive sera from spontaneously autoimmune mice and from mice rendered autoreactive by neonatal induction of tolerance were used as probes on blots of thymocytes. Fifty percent of the sera screened under these conditions reacted with a doublet of 30,33 kDa molecular weight present on membranes, but not detectable on blots of total cell extracts. Additional bands of various molecular weights were detected by NTA-positive sera at a much lower frequency than the 30,33 kDa determinants. Direct evidence of identity between the antibodies inducing thymocytotoxicity and those detecting the doublet in immunoblotting was provided by immunoadsorption tests. Among a panel of immunoadsorbents prepared with thymocyte membrane proteins of various molecular weights, only the one containing the 30,33 kDa molecules efficiently adsorbed cytotoxic antibodies whereas the other preparations containing some of the bands occasionally detected in immunoblotting had practically no effect. In addition we showed that the 30,33 kDa doublet is also detected on membrane preparations of T and B lymphocytes but not on preparations of a nonlymphoid cell line, and is composed of two independent molecules not covalently linked, in their native configuration, by disulfide bonds. Altogether these results strongly suggest that the 30,33 kDa molecules represent major cell surface determinants for thymocytotoxic autoantibodies.     

Multiple forms of SppI (secreted phosphoprotein, osteopontin) synthesized by normal and transformed rat bone cell populations: regulation by TGF-beta

Metabolic labeling has revealed that rat bone cell populations in culture synthesize several forms of the secreted phosphoprotein, SppI. Most cell populations produced two major [32PO4]-labeled forms that behaved anomolously on SDS-PAGE migrating at 60 kDa and 56 kDa on 10% gels and 55 kDa and 44 kDa on 15% gels. Minor forms of intermediate sizes were also resolved. In normal bone cells the 60 kDa form was predominant and was the only form produced by the clonal bone cell line, RCA 11, whereas the 56 kDa a form predominated in the transformed bone cell line, ROS 17/2.8. In all populations [35S]-methionine-labeling revealed SppIs at approximately 60 kDa but no 56 kDa form. Each form of SppI was specifically cleaved by thrombin which generated fragments of approximately 28 kDa. Transforming growth factor beta 1 increased SppI mRNA levels 3 to 6-fold within 24 h in the normal bone cells, but no increase occurred in the ROS 17/2.8 cells. The elevated expression of SppI was reflected in a selective increase in the synthesis of the [32PO4]-and [35S]-methionine-labeled 60 kDa SppIs.     

Outer membrane proteins as major antigens of Fusobacterium nucleatum

The immunochemical reactions of rabbit polyclonal antibodies directed to different preparations of Fusobacterium nucleatum i.e, whole cells, peptidoglycan associated proteins, a peptidoglycan-protein complex and a purified 40 kiloDalton (kDa) protein, were investigated on outer membrane preparations of Fusobacterium species and a restricted number of Leptotrichia buccalis after their separation on sodium dodecyl sulphate polyacrylamide gels and electrotransfer to nitrocellulose. All F. nucleatum strains had identical reaction patterns with the immune sera tested. Surface exposed parts of a restricted number of proteins with apparent molecular weights at 70 kDa (a doublet band), 60 kDa, 55 kDa and 40 kDa seemed to be major immunogens. Antigenic related proteins either of identical or slightly deviating electrophoretic mobilities to the 40-kDa protein were observed with the other members of Bacteroidaceae tested. The characteristic 70-kDa protein doublet seemed to be restricted to F. nucleatum although single protein bands of near identical molecular weights belonging to the other species tested also reacted. The data also indicate that the 60-kDa and 55-kDa polypeptides might be present in other species of Fusobacterium.     

The synthesis and secretion of collagen by cultured sea urchin micromeres

Circumstantial evidence in several previous studies has suggested that sea urchin embryo micromeres, the source of primary mesenchyme cells which produce the embryonic skeleton, contribute to the extracellular matrix of the embryo by synthesizing collagen. A direct test of this possibility was carried out by culturing isolated micromeres of the sea urchin Stronglyocentrotus purpuratus in artificial sea water containing 4% (v/v) horse serum. Under these conditions the micromeres divide and differentiate to produce spicules with the same timing as intact embryos. Collagen synthesis was determined by labeling cultures with [3H]proline or [35S]methionine and the medium and cell layer were assayed for collagen. The results indicate that by the second day in culture micromeres synthesize and secrete a collagenase-sensitive protein doublet with a molecular weight of about 210 kDa. Densitometry indicates a 2:1 ratio of the respective bands in the doublet which is characteristic of Type I collagen. The doublet is insensitive to digestion with pepsin. This differential sensitivity is characteristic of collagen. Over 90% of the collagen synthesized by micromeres is soluble in the seawater culture medium. On days 2-4 in culture, collagen accounts for 5% of the total protein synthesized and secreted. Additional collagenase-sensitive bands are noted at 145 and 51 kDa. The relationship of the described collagen metabolism to previously characterized collagen gene expression in sea urchin embryos is discussed.     

Immunopurification and structural analysis of a putative epithelial Cl- channel protein isolated from bovine trachea.

We have purified to homogeneity a 38-kDa protein (called p38) from bovine tracheal epithelium. This protein, when reconstituted into liposomes, mediates stilbene disulfonate-sensitive 125I- conductive uptake. On nonreduced or partially reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this protein associates into a doublet of 62-64 kDa. In some experiments a multimer of 141 kDa was also observed. Rabbit polyclonal anti-P38 antibodies have been produced and used to immunopurify the native transporter. Upon reconstitution of the immunoaffinity-purified protein into liposomes, a 260-fold enhancement of 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate and valinomycin-sensitive 125I- uptake was observed as compared to proteoliposomes containing unseparated material. On Western blots of total solubilized tracheal membrane proteins or semipurified fractions, the antibody recognized the 62-64-kDa doublet much better than the original 38-kDa antigen. Similar protein bands were detected in T84 and CFPAC cells as well. However, if apical membrane proteins were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, the antibody recognized major bands at 140 and approximately 240 kDa. Upon partial reduction, immunolabeling of these proteins diminished with the concomitant appearance of the 62-64-kDa doublet. Upon complete reduction, the appearance of 32- and 38-kDa proteins was evident with the disappearance of the 62-64-kDa doublet. We hypothesize that the native Cl-channel is a heteromer containing at least four subunits connected by S-S bridges.     

Outer membrane proteins as major antigens of Fusobacterium nucleatum

Abstract The immunochemical reactions of rabbit polyclonal antibodies directed to different preparations of Fusobacterium nucleatum i.e, whole cells, peptidoglycan associated proteins, a peptidoglycan-protein complex and a purified 40 kiloDalton (kDa) protein, were investigated on outer membrane preparations of Fusobacterium species and a restricted number of Leptotrichia buccalis after their separation on sodium dodecyl sulphate polyacrylamide gels and electrotransfer to nitrocellulose. All F. nucleatum strains had identical reaction patterns with the immune sera tested. Surface exposed parts of a restricted number of proteins with apparent molecular weights at 70 kDa (a doublet band), 60 kDa, 55 kDa and 40 kDa seemed to be major immunogens. Antigenic related proteins either of identical or slightly deviating electrophoretic mobilities to the 40-kDa protein were observed with the other members of Bacteroidaceae tested. The characteristic 70-kDa protein doublet seemed to be restricted to F. nucleatum although single protein bands of near identical molecular weights belonging to the other species tested also reacted. The data also indicate that the 60-kDa and 55-kDa polypeptides might be present in other species of Fusobacterium .