Iodination of hepatitis A virus reveals a fourth structural polypeptide.
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Hepatitis A virus present in the feces of two patients with naturally acquired hepatitis A was purified, radiolabeled with 125I, and analyzed by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to the three structural polypeptides previously reported, a fourth polypeptide with a molecular weight of 14,000 was detected and shown to be a component of hepatitis A virus by immune precipitation techniques. Intact virions were also shown to sediment at 160S on sucrose gradients. These findings are consistent with hepatitis A virus being an enterovirus within the family Picornaviridae. (+info)
Differences in the subpopulations of the structural proteins of polyoma virions and capsids: biological functions of the multiple VP1 species.
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The structural proteins of polyoma virions and capsids were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyoma virion VP1 was found to be composed of six distinct species which had pI's between pH 6.75 and 5.75. Polyoma capsid VP1 was found to contain four species with pI's between pH 6.60 and 5.75. The different forms of virion and capsid VP1 appeared to be generated by modifications (phosphorylation and acetylation) of the initial translation product. The most basic of the virion VP1 species (pI, pH 6.75) was absent in capsids and was found to be exclusively associated with the viral nucleoprotein complex. Three of the virion VP1 species and three of the capsid VP1 species were found in capsomere preparations enriched for hexon subunits. Two VP1 species were specifically immune precipitated from virions with hemagglutination-inhibiting antibodies. These two VP1 species were common to both virions and capsids. Polyoma virions, but not capsids, possessed a single VP1 species which was immune precipitated with neutralizing antibodies. Both virion and capsid VP2 were found to have pI's of approximately pH 5.50. Virion VP3 had a pI of approximately pH 7.00, whereas capsid VP3 had a pI of approximately pH 6.50. (+info)
Dynamic nature of the association of large tumor antigen and p53 cellular protein with the surfaces of simian virus 40-transformed cells.
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A molecular complex of simian virus 40 large tumor antigen (T-Ag) and p53 cellular protein is present on the surface of simian virus 40-transformed mouse cells. The stability of the association of the two proteins with the cell surface was characterized. Cells were either surface iodinated by the lactoperoxidase technique or metabolically labeled with [35S]methionine, and surface antigens were detected by differential immunoprecipitation with specific antibodies immediately after labeling or after incubation at 37 degrees C. A rapid, concomitant disappearance of T-Ag and p53 from the cell surface was observed. The half-life of iodinated surface T-Ag was less than 30 min, whereas that of [35S]methionine-labeled surface T-Ag was 1 to 2 h. Although T-Ag and p53 were rapidly lost, both were also rapidly replaced on the cell surface, since newly exposed molecules could be detected when cells were reiodinated after a 2-h chase period. Control experiments established that the loss of the surface molecules was not induced by the iodination reaction. The appearance of surface T-Ag was prevented when cellular protein synthesis was inhibited with cycloheximide. The disappearance and replacement of T-Ag and p53 appeared to be energy-independent processes, as neither was inhibited by sodium azide or 2,4-dinitrophenol. Incubation of iodinated cells at 4 degrees C did block the loss of T-Ag and p53. These observations suggest that T-Ag and p53 are coordinately turned over in the plasma membrane. The nature of the association of the T-Ag-p53 complex with the cell surface can best be described as highly dynamic. (+info)
Most iodinatable fibroblast surface proteins accompany the cytoplast membrane during cytochalasin B-mediated enucleation of chick embryo fibroblasts.
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Six different proteins are found to be reproducibly exposed on the cell surface of chicken embryo fibroblasts (CEF) by the criterion of lactoperoxidase-catalyzed iodination (250,000, 185,000, 130,000, 100,000, 87,000, and 75,000 daltons). We wondered whether cell enucleation might lead to a differential partition of these surface proteins with the karyoplast or cytoplast membrane. We found that there is a marked enrichment of most iodinatable cell surface proteins in the cytoplast after cytochalasin-mediated enucleation of cell monolayers. Nearly all the iodinatable fibronectin remains with the cytoplast. Of the six labeled proteins, the karyoplast membrane contains a small amount of the 130 kdalton protein as well as trace levels of the 100-, 85-, and 75-kdalton proteins. Proteolysis or selective shedding of membrane proteins were not significant factors in the relative exclusion of iodinatable membrane proteins from the karyoplast. The cytoplast could replace some exposed membrane proteins after removal by trypsinization; however, fibronectin was not detectable within 10 h. That the karyoplast was not capable of membrane protein synthesis and/or insertion was suggested by the lack of any change in the labeling pattern of karyoplasts up to 8-h incubation after enucleation. A variety of control studies indicated that the surface proteins identified in this report were cell-derived and not adsorbed serum components. That some of the iodinatable proteins are intrinsic membrane proteins was suggested by their resistance to removal by conditions thought to extract extrinsic membrane proteins (i.e., low salt, high salt, and NaOH washes). lack of effect of cytoskeletal disrupting agents (preliminary evidence) suggests the nonrandom partition of membrane proteins may depend on anchoring of membrane proteins by a system(s) in the cytoplast other than intact microtubules and microfilaments. (+info)
Proteins iodinated by the chloramine-T method appear to be degraded at an abnormally rapid rate after endocytosis.
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Proteins labeled with either (3)H by reductive methylation or (125)I by the chloramine-T method were incubated with Xenopus laevis oocytes; the incorporation and acid precipitability of the proteins were then studied. The uptake rates of both specifically incorporated (vitellogenin) and nonspecifically incorporated proteins (bovine serum albumin and X. laevis serum proteins lacking albumin) were not influenced by the method of labeling. However, (125)I-labeled proteins were apparently degraded at rates far exceeding their (3)H-labeled counterparts, based on the generation of acid-soluble radioactivity. Thus, after a 3-hr incubation, 3-5 times more (125)I-labeled bovine serum albumin and X. laevis serum proteins lacking albumin were degraded than the corresponding (3)H-labeled proteins (95% compared to 30% and 75% compared to 15%, respectively), whereas after a 24-hr incubation, the degradation of (125)I-labeled vitellogenin was 15 times greater than that of [(3)H]vitellogenin labeled in vivo (60% compared to 4%). Moreover, examination of the relative amounts of (3)H- compared to (125)I-labeled bovine serum albumin deposited into the exogenously derived yolk platelet compartment of the oocyte revealed 7 times more acid-precipitable (3)H-labeled protein, indicating that the observed discrepancies were not due to reincorporation of the (3)H-labeled ligands. Passage of dissolved oocytes previously exposed to (125)I-labeled bovine serum albumin (chloramine-T method) over a column of Bio-Gel P-10 revealed some breakdown of bovine serum albumin to intermediate molecular weight components and the presence of a large amount ( approximately 90%) of labeled low molecular weight compounds, which analysis showed to be 72% free iodine. The evolution of either iodotyrosine or free iodine would nevertheless be perceived as protein degradation by most analytical procedures (e.g., acid precipitation or autoradiography). We conclude, therefore, that apparent degradation rates observed for endocytotically incorporated proteins may vary depending on the method used to label the protein and caution should be exercised when interpreting results obtained with labeled, particularly chloramine-T labeled, proteins. (+info)
Plasma membrane polypeptides of resident and activated mouse peritoneal macrophages.
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With the lactoperoxidase/glucose oxidase-catalyzed iodination method, we have identified at least 19 exteriorly disposed plasma membrane polypeptides on mouse peritoneal macrophages, with molecular weights ranging from 12,000 to 290,000. Resident and inflammatory macrophages could be distinguished by qualitative and quantitative differences in the display of selected polypeptides, although the overall banding patterns were similar. Some of the labeled polypeptides were identified by immunoprecipitation. (+info)
Degradation of proteins microinjected into IMR-90 human diploid fibroblasts.
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Erythrocyte ghosts loaded with 125I-labeled proteins were fused with confluent monolayers of IMR-90 fibroblasts using polyethylene glycol. Erythrocyte-mediated microinjection of 125I-proteins did not seriously perturb the metabolism of the recipient fibroblasts as assessed by measurements of rates of protein synthesis, rates of protein degradation, or rates of cellular growth after addition of fresh serum. A mixture of cytosolic proteins was degraded after microinjection according to expected characteristics established for catabolism of endogenous cytosolic proteins. Furthermore, withdrawal of serum, insulin, fibroblast growth factor, and dexamethasone from the culture medium increased the degradative rates of microinjected cytosolic proteins, and catabolism of long-lived proteins was preferentially enhanced with little or no effect on degradation of short-lived proteins. Six specific polypeptides were degraded after microinjection with markedly different half-lives ranging from 20 to 320 h. Degradative rates of certain purified proteins (but not others) were also increased in the absence of serum, insulin, fibroblast growth factor, and dexamethasone. The results suggest that erythrocyte-mediated microinjection is a valid approach for analysis of intracellular protein degradation. However, one potential limitation is that some microinjected proteins are structurally altered by the procedures required for labeling proteins to high specific radioactivities. Of the four purified proteins examined in this regard, only ribonuclease A consistently showed unaltered enzymatic activity and unaltered susceptibility to proteolytic attack in vitro after iodination. (+info)
A new mechanism for the reabsorption of thyroid iodoproteins: selective fluid pinocytosis.
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Rat thyroid hemilobes were incubated in presence of exogeneous, heterologous and homologous thyroglobulins. The median density of the thyroglobulin molecules originally added to the medium was compared with that of the molecules remaining at the end of a three-hour incubation period (37 degrees C). A certain degree of specificity in the reabsorption process of thyroglobulin was found: the uptake of homologous molecules (rat) was higher than heterologous (hog) molecules. The median density of the iodoproteins remaining after the incubation did not change for the heterologous whereas it shifted towards lower density for the homologous thyroglobulins (equilibrium labelled, 35 iodine atoms/molecule). In addition, rat follicular cells display selectivity in the endocytosis of homologous thyroglobulin. Among the rat molecules, the normally iodinated are taken up more actively than the lowly iodinated or newly synthesized ones. Dissimilarity in the median density of the thyroglobulin molecules before and after endocytosis was only evident for the equilibrium-labelled, normally iodinated rat preparation: the disappearance from the medium of 25-30% of exogenous iodoproteins was sufficient to lower significantly the median density of the remaining molecules. This means that the thyroglobulin molecules having higher density are taken up preferentially by the tissue. A mechanism involving specific interactions between the iodoproteins and the surface of thyroid cell is suggested. (+info)