Biomedical subjects

J T Sawyer

Publications and source records attributed to J T Sawyer.

6 recordsLinked to original sources

PubMed1994

Dithiothreitol treatment induces heterotypic aggregation of newly synthesized secretory proteins in HepG2 cells.

To analyze the importance of the endoplasmic reticulum oxidizing state for the folding and aggregation of newly made polypeptides, we have incubated intact HepG2 hepatoma cells in the presence of dithiothreitol. When dithiothreitol-treated cells were extracted under nondenaturing conditions immunoprecipitates of newly synthesized albumin showed a complex polypeptide profile. Using direct and sequential immunoprecipitation protocols we identified eight other polypeptide chains, transferrin, plasminogen, ceruloplasmin, alpha 2-macroglobulin, the three fibrinogen chains, and haptoglobin, that were among the proteins co-immunoprecipitated with albumin. The heterotypic aggregates are larger than several hundred kilodaltons and are stabilized by noncovalent interactions. Of the 10 polypeptide chains we examined, only one, alpha 1-antitrypsin, failed to aggregate. This protein is distinguished from the others by the absence of disulfide bonds. We propose a model in which the function of the oxidizing conditions of the endoplasmic reticulum is to promote the rapid formation of disulfide bonds that stabilize adhesive domains in a buried state, thus preventing "global" heterotypic aggregation of newly synthesized chains.

Dithiothreitol↗

PubMed1992

Early disulfide bond formation prevents heterotypic aggregation of membrane proteins in a cell-free translation system.

We previously demonstrated that a heterotypic complex of the two rat asialoglycoprotein receptor subunits was assembled during cell-free translation (Sawyer, J. T., and D. Doyle. 1990. Proc. Natl. Acad. Sci. USA. 87:4854-4858). We have characterized this system further by analyzing polypeptide interactions under both reducing and oxidizing translation conditions. This report shows that the complex represents a heterogeneous interaction between reduced membrane proteins rather than a specific oligomeric structure. In the reduced state membrane proteins interact in this system to form aggregates of diverse size and composition. The aggregated nascent polypeptides interact with the immunoglobulin heavy chain binding protein but this protein is not an integral component of the aggregate. Aggregation occurs via the exoplasmic domain, rather than the transmembrane domain, and the folding of this domain by the formation of intramolecular disulfides, prevents the interaction from occurring. Additionally, the folded molecules containing intramolecular disulfides lack high affinity binding activity and thus appear to resemble the earliest folding intermediates seen in vivo (Olson, J. T., and M. D. Lane. 198. FASEB (Fed. Am. Soc. Exp. Biol.) J. 3:1618-1624). These results lead us to suggest that the formation of intramolecular disulfides during early biogenesis serves to prevent nonspecific associations between nascent polypeptides.

Animals↗

PubMed1990

Assembly of a heterooligomeric asialoglycoprotein receptor complex during cell-free translation.

We have translated RNAs for the two rat asialoglycoprotein receptor polypeptides together in a cell-free system containing dog pancreatic microsomes and immunoprecipitated the products with antibodies that distinguish the two proteins. In this system the proteins oligomerize, as judged by their coprecipitation with either of the subunit-specific antisera. Oligomerization does not occur between subunits synthesized without microsomes or between subunits synthesized on separate microsomes mixed during detergent solubilization. Thus, oligomerization occurs within the microsomal membrane. We calculate that oligomerization proceeds with an efficiency of approximately 85%. The receptor complex appears to represent a specific oligomer because it excludes a third membrane glycoprotein synthesized in the same reaction. Oligomerization of the asialoglycoprotein receptor in vitro should provide a useful system to study the assembly of a membrane-protein complex.

Animals↗

PubMed1988

Identification of a complex of the three forms of the rat liver asialoglycoprotein receptor.

We have generated antibodies against synthetic peptides which represent the carboxyl terminus of either the major, or the two minor, forms of the rat hepatic lectin which recognizes galactose-terminated glycoproteins (asialoglycoproteins). The antibodies were shown to be specific for the form of the lectin containing the immunizing peptide sequence by the following: reaction with purified lectin after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoprecipitation of sodium dodecyl sulfate-denatured lectin, immunoprecipitation of lectin synthesized in vitro. These antibodies, however, precipitated all three rat hepatic lectin forms from nonionic detergent extracts of hepatocytes labeled with 125I via the lactoperoxidase catalyzed technique. A similar result was obtained if antibody was bound to intact cells prior to solubilization with detergent and collection of the immune complexes. We conclude that at least the plasma membrane-associated fraction of the rat hepatic lectin forms exists as a heterotypic complex.

Animals↗

PubMed1986

Differential redistribution of lectin receptor classes on clonal rat myotubes and myoblasts.

To evaluate the relative mobilities of cell surface glycoconjugates during myogenesis we have studied the redistribution of fluorescein-conjugated plant lectins on L6 rat myogenic cells. Previous experiments had demonstrated that the receptors for the lectins soybean agglutinin (SBA), wheat germ agglutinin, concanavalin A and Lens culinaris agglutinin all were relatively uniformly distributed on both myoblasts and myotubes, and that SBA receptors were capable of rapid redistribution on myotubes but not myoblasts at 4 degrees C (Sawyer & Akeson, 1983). Here we show that when SBA-labelled myoblasts are incubated at 37 degrees C, or for extended times at 4 degrees C, the lectin aggregates as on myotubes. So it appears that SBA-binding components show a quantitative rather than qualitative change in their mobility during L6 differentiation. In addition, the redistribution of the three other lectins on myoblasts and myotubes was either less prominent (i.e. showing fewer apparent surface clusters) or occurred less rapidly than with SBA. None of these three lectins showed striking differences in mobility between myoblasts and myotubes. Thus, it appears that SBA binds to a subset of surface glycoconjugates that is relatively highly mobile, and that this mobility is specifically enhanced with differentiation.

Animals↗

PubMed1983

Clonal myoblasts and myotubes show differences in lectin-binding patterns.

To determine changes in distribution or mobility of cell-surface glycoconjugates during myogenesis the binding of fluorescein-conjugated plant lectins to myoblasts and myotubes of the L6 rat skeletal muscle cell line has been studied. Binding has been carried out at 4 degrees C on either live or glutaraldehyde-fixed cells. Fluorescein conjugates of soybean agglutinin (Fl-SBA), wheat germ agglutinin (Fl-WGA), concanavalin A (Fl-conA) and Lens culinaris agglutinin (Fl-LCA) produced predominantly uniform fluorescence on both live and fixed myoblasts. On fixed myotubes, Fl-LCA, Fl-conA and Fl-SBA again produced predominantly uniform fluorescence, whereas Fl-WGA showed a pattern of diffuse, irregular spots in addition to uniform fluorescence. Fl-conA, Fl-LCA and Fl-WGA binding to live myotubes resulted in patterns quite similar to those on fixed myotubes; the only differences being the presence of weak patterns of diffuse spots with Fl-LCA and Fl-conA and an enhanced pattern of diffuse spots with Fl-WGA. Fl-SBA, however, showed a unique pattern on live myotubes which consisted of discrete, round spots and minimal uniform fluorescence. With shorter labeling times, Fl-SBA produced relatively more prominent uniform fluorescence on live myotubes. It appears, therefore, that the native distribution of SBA, conA and LCA-binding sites is similar and predominantly random on L6 myoblasts and myotubes, whereas some WGA-binding sites may be aggregated on myotubes. The results also suggest that SBA-binding sites readily cluster at 4 degrees C on myotubes but not myoblasts, whereas the other lectin sites undergo little or no redistribution on either cell type. Thus the mobility of SBA-binding sites may increase with differentiation.

Animals↗