the stability in various detergents of transferrin-transferrin receptor complexes from reticulocyte plasma membranes

Transferrin-membrane protein complexes were solubilized either with 0.4% sodium dodecyl sulfate (SDS), 1% Triton X-100 or 0.5% sulfobetaine 3-14 from the plasma membranes of rabbit reticulocytes previously labeled with 125I and then incubated with 131-labeled transferrin. When the solubilized membranes were analyzed by gel filtration fractionation, marked variation in the preservation of transferrin-transferrin receptor interaction was noted between the three detergents. After SDS solubilization, more than 80% of the 131I-labeled transferrin remained associated with membrane proteins with apparent molecular weight of the transferrin-receptor complexes of 1400 000 and 240 000. In contrast, after Triton X-100 solubilization only 40% of the transferrin was still complexed to membrane proteins with an apparent molecular weight of the complex of 450 000. Dissociation of transferrin from its receptor was most marked following sulfobetaine solubilization, with less than 30% of the transferrin still complexed. Following gel filtration 131I-labeled transferrin-125I-labeled membrane protein complexes were immunoprecipitated with goat specific anti-rabbit transferrin antibodies. The immunoprecipitates were analyzed under stringent dissociating conditions by two SDS-polyacrylamide gel electrophoretic techniques. In a linear 5-25% polyacrylamide gradient the 125I-labeled receptor obtained after membrane solubilization with all three detergents had an apparent molecular weight of 80 000. In contrast, in a different system using 10% polyacrylamide gel two 125I-labeled receptor components were detected wih apparent molecular weights of 90 000 and 80 000. These results demonstrate that estimates of the molecular weight of the transferrin receptor depended on the conditions of electrophoresis and suggest that the transferrin receptor is partially modified, perhaps by glycosylation.     

Characterization of polyclonal antibodies to cell-surface antigens from ovine adipose tissue.

High titres of polyclonal antibodies to specific proteins of ovine adipose tissue plasma membranes were raised in horses and chickens following repeated injections of purified plasma membranes. Horse antiserum was highly species specific, reacting only weakly with rat adipose tissue plasma membranes. A protein of molecular weight 68,000 was most antigenic in that it was readily precipitated; however proteins of 25,000, 82,000 and 94,000 were also precipitated when the reaction was performed for longer with a higher antiserum concentration. Chicken egg yolk IgY reacted strongly with ovine adipose tissue plasma membranes as did those preparations from horse, but IgY was ineffective in immunoprecipitating solubilized membrane proteins and exhibited no cytotoxic reaction when incubated with intact ovine adipocytes. However, horse antiserum produced a strong complement-dependent cytotoxic reaction with ovine adipocytes, as measured by leakage of lactate dehydrogenase. This work suggests that the membrane protein of molecular weight 68,000 is likely to be an important antigenic marker for ovine adipocytes.     

Fate of the transferrin receptor during maturation of sheep reticulocytes in vitro: Selective externalization of the receptor

The fate of the transferrin receptor during in vitro maturation of sheep reticulocytes has been followed using FITC- and ¹²⁵l-labeled anti-transferrin-receptor antibodies. Vesicles containing peptides that comigrate with the transferrin receptor on polyacrylamide gels are released during incubation of sheep reticulocytes, tagged with anti-transferrin-receptor anti-bodies. Vesicle formation does not require the presence of the anti-transferrin-receptor antibodies. Using ¹²⁵l-surface-labeled reticulocytes, it can be shown that the ¹²⁵l-labeled material which is released is retained by an immunoaffinity column of the anti-transferrin-receptor antibody. Using reticulocytes tagged with ¹²⁵l-labeled anti-transferrin-receptor antibodies to follow the formation of vesicles, it can be shown that at 0°C or in phosphate-buffered saline the rate of vesicle release is less than that at 37°C in culture medium. There is selective externalization of the antibody-receptor complex since few other membrane proteins are found in the externalized vesicles. The anti-transferrin-receptor antibodies cause redistribution of the receptor into patches that do not appear to be required for vesicle formation.     

Lymphocyte plasma membranes. VI. Plasma membrane glycoproteins of thymic and splenic lymphocytes from inbred rats.

Plasma membranes of splenic and thymic lymphocytes from ACI rats were analyzed for their protein and glycoprotein components by surface radioiodination with 125I and SDS-polyacrylamide gel electrophoresis. The glycoproteins were extracted with lithium diiodosalicylate, characterized and assayed with antisera to thymic antigen. Plasma membranes of both cell types showed more than 25 proteins of which 10--15 were glycoproteins. Both cells showed five major glycoproteins but their apparent molecular weights or intensities differed. Surface radioiodination showed a 120 000 daltons component, common to both cell types, and a 27 000 daltons thymus-specific component as the most exposed surface glycoproteins. Lithium diiodosalicylate extracts of the plasma membranes contained almost all of the glycoprotein components and comprised 5-6 percent of the total membrane protein and 40-50 percent of the total membrane carbohydrate, with sialic acid content in thymus twice that of the spleen cells. About 1 percent of the total plasma membrane protein and 7 percent of the total isolated glycoproteins from thymocytes were reactive with rabbit anti-rat thymocyte antiserum and the immune precipitates showed two components with apparent molecular weights of 72 000 and 27 000.     

Analysis of hepatocyte cell membrane antigens in regenerating rat liver]

Antigens of plasma membranes in hepatocytes from regenerating rat liver were studied. Immunochemical investigation with polyvalent rabbits antiserum against plasma membrane proteins in hepatocytes from regenerating and normal rat liver have shown that liver regeneration processes are accompanied by the increase of proteins number with molecular weight of--80 kDa, 62 kDa, 40 kDa and 27 kDa. It is not excluded that protein with molecular weight of 27 kDa is the tissue-specific peripheral protein. The influence of antibodies against proteins of hepatocytes plasmatic membranes on histostructure of pathologically changed liver tissue has been studied. The data obtained testify to a possibility of participation of the above mentioned proteins in the regulation of rat liver regeneration processes.     

In vitro acylation of the transferrin receptor

In vitro fatty acylation of the transferrin receptor with [3H]tetradecanoate or [3H]tetradecanoyl-CoA has been demonstrated for isolated sheep reticulocyte plasma membranes. Although less than 5% of the receptor was labeled in vitro, the acylated protein could be readily observed after sodium dodecyl sulfate-gel electrophoresis. The acylated transferrin receptor in the reticulocyte membrane was specifically precipitated with a monoclonal antibody and was absent from mature red cell membranes. Incorporation of fatty acid was dependent on ATP, and fatty acid was 5-10 times less effective as an acyl donor than the acyl-CoA derivative, pointing out the strong potential of this reagent for in vitro acylation of membrane proteins. During in vitro maturation of reticulocytes, the receptor is released in vesicles into the incubation medium. Using reticulocytes labeled with [3H]tetradecanoate, it can be shown that the 3H-labeled receptor is transferred from the cells to the vesicles without loss of acyl groups, suggesting that the vesiculation process does not involve deacylation.     

p180, a novel recycling transmembrane glycoprotein with restricted cell type expression.

A 180-kilodalton (kDa) protein (p180) was identified among the antigens for a panel of monoclonal antibodies raised against human fibroblast cell surface proteins. Binding studies with 125I-Fab' fragments of an anti-p180 monoclonal antibody demonstrated that 10 to 30% of p180 was located on the plasma membrane and that the remaining 70 to 90% was on intracellular membranes. p180 was rapidly internalized from the cell surface at 37 degrees C, and kinetic analyses indicated that this was a constitutive process followed by the recycling of p180 back to the plasma membrane. Morphological studies demonstrated that on the cell surface p180 was concentrated in coated pits, whereas inside the cell it was found in endosomes as suggested by its colocalization with the transferrin receptor. Immunoblot analysis with a polyclonal antiserum raised against purified human protein showed that p180 has a restricted distribution with expression at high levels in fibroblast cultures and in tissues containing cells of mesodermal origin. A biochemical characterization of p180 showed it to be a transmembrane glycoprotein with an extracellular domain, which consists of approximately 30 kDa of complex oligosaccharides attached to at least 45 kDa of the protein core. The cytoplasmic domain of p180 was found to contain a serine residue(s) that was phosphorylated both in vivo and in vitro by activated protein kinase C. p180 was purified by subjecting solubilized membrane proteins from a human osteosarcoma cell line to immunoaffinity chromatography and gel filtration. The N-terminal sequence information obtained from the purified protein showed no homology to other known proteins. It was concluded that p180 may be a novel recycling receptor which is highly restricted in its expression to fibroblastlike cells.     

Studies on the mechanisms involved in iron transfer across the isolated guinea pig placenta by means of bolus experiments

Placental binding and uptake of diferric transferrin as well as transplacental iron transfer has been studied in isolated, perfused guinea pig placenta. The process of binding and uptake of transferrin was saturable only on the maternal side. On the fetal side no specific binding occurred. This indicates an asymmetric distribution of transferrin receptors. No receptors are present for albumin, neither on maternal, nor fetal side. Most of the 125I-59Fe transferrin, administered with a single bolus, enters the trophoblast. A small part remains attached to the plasma membranes, as shown by cell fractionation and in transferrin exchange experiments. The majority transferrin, which was internalized, is unlikely to be bound to plasma membranes and may be bound to receptors dissociated from plasma membranes. Based on kinetics of 59Fe appearance and washout at the fetal side of the perfused placenta as a model for trans-placental iron transfer has been postulated. A central feature is the role played by a small compartment (0.14 mumol) to which iron is supplied by a very rapid process at the trophoblast receptor, without internalisation of transferrin. A second un-identified pathway is supposed to regulate the magnitude of the iron transfer pool.     

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.     

HeLa cell plasma membranes : IV. Iodination of membrane proteins in synchronized cells

Intact HeLa cells and isolated HeLa cell plasma membranes were subjected to lactoperoxidase-catalysed iodination. The ¹²⁵I-labelled proteins were separated by SDS-polyacrylamide gel electrophoresis. Six protein species with apparent molecular weights from 32 000 to 200 000 were accessible to labelling from the outer cell surface, while most of the proteins present in the plasma membrane were labelled when isolated plasma membranes were iodinated. Iodination of synchronized intact cells revealed that the labelling obtained was cell cycle dependent with maximal labelling at mitosis. No changes in the distribution of radioactivity among the labelled proteins were observed when cells from different phases were iodinated.     

Isolation and characterization of the plasma membrane of L-1210 cells. Iodination as a marker for the plasma membrane.

Mouse leukemia L-1210 cells were iodinated with 125I; this permitted the development of a method for the isolation of the plasma membranes. These show a 10- to 16-fold increase in the specific activity of 125I over that of the cell homogenate and a 20-fold increase in the specific activities of 5'-nucleotidase and alkaline phosphatase; 20-fold increase in the specific activities of 5'-nucleotidase and alkaline phosphatase; no mitochondrial or microsomal marker enzyme activities were detected. Sodium dodecyl sulfate gel electrophoresis of the plasma membranes shows approx. 40 peptides with molecular weights ranging from 10 000 to over 200 000; a polypeptide (Mr 50 000) predominates. Of 13 iodinated surface membrane proteins, the major radioactive peptide has a molecular weight of 85 000. The importance of the selection of the appropriate gel system for the analysis of membrane proteins is emphasized.     

Solubilization and characterization of the chicken oocyte vitellogenin receptor.

This paper describes the biochemical characterization of the chicken oocyte plasma-membrane receptor for one of the major lipid-carrying yolk proteins, vitellogenin (VTG). The receptor was extracted from oocyte membranes with the non-ionic detergent octyl-beta-D-glucoside and visualized by ligand blotting, with 125I-VTG as a protein with an apparent Mr of 96000, under non-reducing conditions. It exhibited high affinity for native chicken VTG (Kd 2 X 10(-7) M) but was unable to bind VTG with reductively methylated lysine residues or phosvitin (the phosphoserine-rich intracellular cleavage product of VTG). Polyclonal antibodies to the 96 kDa protein inhibited VTG binding to the receptor and were able to precipitate functional VTG-receptor activity from oocyte-membrane detergent extracts with a concomitant removal of the 96 kDa protein. Antibodies directed against the mammalian receptor for low-density lipoprotein showed cross-reactivity with the chicken oocyte VTG receptor, raising the possibility that lipoprotein receptors in birds are structurally related to those in mammalian species.     

Biosynthesis of plasma-membrane proteins during myogenesis of skeletal muscle in vitro.

1. Surface labelling of plasma-membrane proteins with 125I, catalysed by lactoperoxidase, and radioactive l-fucose incorporation into glycoprotein were used as plasma-membrane markers for skeletal-muscle cells in culture. 2. Plasma membranes were prepared at various stages of myogenesis in vitro and rates of synthesis and accumulation of proteins in the membranes were compared. 3. Increased synthesis and accumulation of a protein of apparent mol.wt. 70000 occurred in the plasma-membrane fraction concomitant with the onset of myoblast fusion. 4. In cultures in which fusion of myoblasts was inhibited by 5'-bromo-2-deoxyuridine, synthesis and accumulation of the protein of apparent mol.wt. 70000 was selectively inhibited. 5. It is suggested the protein of apparent mol.wt. 70000 may be involved in the process of myoblast fusion.     

Biosynthesis of the insulin receptor in rat adipose cells. Intracellular processing of the Mr-190 000 pro-receptor.

We investigated the biosynthesis of the insulin receptor in primary cultures of isolated rat adipose cells. Cells were pulse-chase-labelled with [3H]mannose, and at intervals samples were homogenized. Three subcellular membrane fractions were prepared by differential centrifugation: high-density microsomal (endoplasmic-reticulum-enriched), low-density microsomal (Golgi-enriched), and plasma membranes. After detergent solubilization, the insulin receptors were immunoprecipitated with anti-receptor antibodies and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography. After a 30 min pulse-label [3H]mannose first appeared in a band of Mr 190 000. More than 80% of the Mr-190 000 component was recovered in the microsomal fractions. Its intensity reached a maximum at 1 h in the high-density microsomal fraction and at 2 h in the low-density microsomal fraction, and thereafter declined rapidly (t 1/2 approx. 3 h) in both fractions. In the plasma-membrane fraction, the radioactivity in the major receptor subunits, of Mr 135 000 (alpha) and 95 000 (beta), rose steadily during the chase and reached a maximum at 6 h. The Mr-190 000 precursor could also be detected in the high-density microsomal fraction by affinity cross-linking to 125I-insulin. In the presence of monensin, a cationic ionophore that interferes with intracellular transport within the Golgi complex, the processing of the Mr-190 000 precursor into the alpha and beta subunits was completely inhibited. Our results suggest that the Mr-190 000 pro-receptor originates in the endoplasmic reticulum and is subsequently transferred to the Golgi complex. Maturation of the pro-receptor does not seem to be necessary for the expression of the insulin-binding site. Processing of the precursor into the mature receptor subunits appears to occur during the transfer of the pro-receptor from the Golgi complex to the plasma membrane.     

Characterization and localization of human placental ferritin.

Ferritin has been purified from normal full-term human placentae and its antigenic and molecular characteristics compared with adult liver ferritin. Placental ferritin is composed predominantly of a single subunit type, co-migrating with a liver ferritin standard on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Comparison of dose-response curves in an immunoradiometric assay indicated some tissue-specific antigenicity for placental ferritin. This was supported by immunofluorescence studies on cryostat sections of human placentae by using antibodies to placental and spleen ferritin. Specific staining for placental ferritin was demonstrated within placental syncytiotrophoblast, particularly localized towards the microvillus plasma membrane. Ferritin has also been shown by electrophoretic and antigenic analysis to be present in protein fractions solubilized from isolated human syncytiotrophoblast microvillus plasma-membrane preparations, suggesting that ferritin may play an active role in the transfer of iron from maternal transferrin across the syncytiotrophoblast plasma membrane.     

Similarities between the transferrin receptor proteins on human reticulocytes and human placentae

The transferrin receptor of the human reticulocyte was isolated by two different immunoaffinity procedures. These included indirect immunoprecipitation with a transferrin/anti-transferrin complex and direct immunoprecipitation with antiserum to purified transferrin receptor from placentae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the receptor isolated from reticulocytes reveals a polypeptide at Mr = 94,000 identical in molecular weight with that of the placenta. A radioimmunoassay using purified 125I-labeled transferrin receptor from placentae and antiserum to transferrin receptor fails to distinguish any immunological differences between the reticulocyte and placental forms of the protein. In addition, proteolytic digests of both of these polypeptides with Staphylococcus aureus protease show identical proteolytic patterns, indicating similar sequences.     

Exosome formation during maturation of mammalian and avian reticulocytes: evidence that exosome release is a major route for externalization of obsolete membrane proteins

We have assessed whether exosome formation is a significant route for loss of plasma membrane functions during sheep reticulocyte maturation in vitro. Although the recovery of transferrin binding activity in exosomes is at best approximately 25-30% of the lost activity, recoveries of over 50% of the lost receptor can be obtained if 125I-labelled transferrin receptor is measured using an that receptor instability may contribute to the less than quantitative recovery of the transferrin receptor. Significantly higher (75-80%) levels of the nucleoside transporter can be recovered in exosomes during red cell maturation using 3H-nitrobenzylthioinosine binding to measure the nucleoside transporter. These data suggest that exosome formation is a major route for removal of plasma membrane proteins during reticulocyte maturation and plasma membrane remodelling. We have also shown that both in vivo and in vitro, embryonic chicken reticulocytes form exosomes which contain the transferrin receptor. Thus, exosome formation is not restricted to mammalian red cells, but also occurs in red cells, which retain organelles, such as nuclei and mitochondria, into the mature red cell stage.     

Insulin inhibits the cholera-toxin-catalysed ribosylation of a Mr-25000 protein in rat liver plasma membranes.

A method is described for preparing a plasma-membrane fraction from hepatocytes by a rapid, gentle, Percoll fractionation procedure. Cholera toxin elicited the ribosylation of a number of proteins in these membranes, including the components of the stimulatory guanine nucleotide regulatory protein, Ns. Insulin, however, inhibited the ability of cholera toxin to ribosylate a protein of Mr 25 000. The action was decreased in membranes from cells that had been pre-treated with glucagon. Ribosylation of both the components of Ns and the Mr-25 000 species occurred in whole cells treated with cholera toxin, because membranes from such treated cells exhibited decreased labelling when incubated with [32P]NAD+ and activated cholera toxin. The labelling of proteins, including the Mr-25 000 species, with [32P]NAD+ and cholera toxin in the plasma membranes was decreased by an inhibitor of ribosylation. Azido-GTP photoaffinity labelling identified several high-affinity GTP-binding proteins, including one of Mr 25 000. Cholera toxin failed to ribosylate the Mr-25 000 protein in membranes from cells that had been pre-treated with the tumour-promoting agent 12-O-tetradecanoylphorbol 13-acetate (TPA). In membranes from such treated cells, insulin actually allowed cholera toxin to label this species. As TPA activates protein kinase C, it is possible that the Mr-25 000 protein, or a species that interacts with it, is a substrate for phosphorylation. These observations may offer an explanation for some of the perturbing effects that TPA exerts on insulin's action. It is suggested that the insulin receptor interacts with the guanine nucleotide regulatory protein system in the liver, and that the Mr-25 000 species may be a component of Nin, a specific guanine nucleotide regulatory protein that has been proposed to mediate certain of the actions of insulin on target cells [Houslay & Heyworth (1983) Trends Biochem. Sci. 8, 449-452].     

Two-dimensional electrophoretic analysis of secretory-granule,granule-membrane,and plasma-membrane proteins of rat parotid cells

Secretory granules and plasma membranes were isolated from rat parotid cells and characterized enzymatically and by electron microscopy. The proteins of the secretory granule membranes, the secretory granules and the plasma membranes were characterized by two-dimensional polyacrylamide gel electrophoresis and visualized by silver staining. The granule membrane contains 166 polypeptides of which only 26 are also present in the granule contents. The membrane proteins have isoelectric points between 4.75 and 6.45 and apparent molecular weights of 17 000 to 190 000 Daltons. The granule content proteins are surprisingly complex and contain 122 polypeptides with molecular weights of 11 000 to 138 000 and isoelectric points of 4.8 to 6.55. Thirteen of these peptides are present as major species. The plasma membrane contains 172 polypeptide species with molecular weights from 17 000 to 200 000 Daltons and isoelectric points of 5.0 to 6.8. Thirty-five of the plasma membrane proteins are also present in the secretory granule membranes indicating that the two membranes have some enzymatic or structural properties in common. Thus, secretory granule membranes and plasma membranes from parotid cells have a more complex polypeptide composition than has previously been shown for membranes of this type. The systems developed are suitable for the analysis of regulatory events such as protein phosphorylation, proteolytic processing, and other types of post-translational modifications that may be important to the secretory mechanism.