Biomedical subjects

W Reutter

Publications and source records attributed to W Reutter.

At least 145 records · Page 8Linked to original sources

PubMed1988

Incorporation of non-acetylated hexosamines into plasma membrane glycoproteins of liver cells after galactosamine injection.

The following procedure for the detection of non-acetylated amino sugars in the plasma membrane was established: i) derivatization of free amino groups with dansyl-chloride, ii) hydrolysis with 3 M HCl (for 4 h at 105 degrees C) to liberate the dansylated carbohydrate moieties from the plasma membrane, iii) purification of the dansylated amino sugars by paper chromatography and subsequent analysis by thin-layer chromatography. Using this procedure, plasma membranes from rat liver were analysed after injection of D-[14C]galactosamine. For this purpose, rats were divided into three groups: the first received D-galactosamine.HCl at a dose of 2 mg/kg b.w., the second at a dose of 75 mg/kg b.w. and the third at a hepatitis-inducing dose of 260 mg/kg b.w.. In all three groups the majority of the protein-bound radioactivity in the plasma membrane was not dansylated, thus representing N-acetylated amino sugars. At a dose of 2 mg/kg, only 0.34% of the protein-bound radioactivity in the plasma membrane reacted with dansyl-chloride. At a dose of 70 mg/kg this value increased to 1.9%. At 260 mg/kg the value was 3.6%. These results indicate that the incorporation of non-acetylated amino sugar into the plasma membrane was dose-dependent and reached 90 pmol per mg plasma membrane protein during galactosamine injury. However, this incorporation of non-acetylated amino sugars into the plasma membrane did not represent a pathological mechanism responsible for the onset of the galactosamine-induced liver injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗

PubMed1988

Isolation of immunoglobulins and their use in immunoaffinity HPLC.

For the isolation of monoclonal and polyclonal antibodies different high performance liquid chromatography (HPLC) and high performance affinity chromatography (HPAC) methods were investigated. Specially designed "mixed-bed" ion-exchange and hydroxylapatite columns as well as hydrophobic interaction columns were efficiently applied to the isolation of monoclonal antibodies. When these methods are used for the isolation of polyclonal antibodies from antiserum, the sample has to be pre-treated, e.g. by removal of serum albumin. Protein A HPAC is an easy method and quick to handle, especially for the preparative isolation of antibodies. The antibodies that do not bind to protein A, can be purified by protein G HPAC. If this method cannot be used because of the rather extreme elution conditions, hydroxylapatite, ion-exchange or hydrophobic interaction HPLC have to be considered as alternatives. We further concentrate on immunoaffinity HPLC with immobilized antibodies. This method has proved to be very effective for one-step isolation of antigens, even from very complex samples such as plasma membrane extracts. The problem with immunoaffinity HPLC is the quick deterioration of the columns, caused by increasing denaturing of the immobilized antibodies during elution. In order to solve this problem, an indirect method is recommended for analytical immunoaffinity HPLC. For this purpose, the antibodies are bound to a protein A HPAC column. The solution containing the antigens is then applied. After washing, the antigen-antibody complex is eluted from the column.

Animals↗

PubMed1988

Vitamin A excess alters membrane flow in rat liver.

Pulse-chase methodology with [35S]methionine as label was employed to determine flow kinetics through the endoplasmic reticulum-Golgi apparatus-(lysosome-) secretory vesicle-plasma membrane export route in livers of animals receiving vitamin A excess by gavage. Overall fraction composition determined by morphometry and by analyses of marker enzymes was unchanged by vitamin administration. The vitamin modified the pattern of flow of proteins through the Golgi apparatus to the cell surface and to lysosomes. Altered flux was evidenced by a markedly reduced rate of labeling of lysosomes and a slightly increased rate of labeling of both total membrane proteins of the plasma membrane and of a specific membrane glycoprotein GP80. Also reduced was overall labeling of the Golgi apparatus. Differences in the rate or routes of trafficking of glycoproteins through the Golgi apparatus together with altered opportunities for processing might account for some of the alterations in glycoconjugate glycosylation associated with excess vitamin A administration.

Animals↗

PubMed1987

High-performance liquid affinity chromatography of liver plasma membrane proteins.

Plasma membrane proteins from liver were analysed by concanavalin A affinity and immunoaffinity high-performance liquid chromatography. In the method, four peptide bands with apparent molecular weights of 140,000, 120,000, 80,000 and 60,000 could be isolated. In the second method, with two immobilized monoclonal antibodies, two corresponding antigens--the membrane proteins dipeptidyl-peptidase IV and GP 110--could be highly purified from plasma membrane extract with good yield in only one step.

Animals↗

PubMed1987

Rapid purification of dipeptidyl peptidase IV from rat liver plasma membrane.

Dipeptidyl peptidase IV (EC 3.4.14.5) was solubilized from rat liver plasma membranes with sulphobetaine 14 and purified by successive affinity chromatography on Con A-Sepharose, wheat germ lectin-Sepharose and arginine-Sepharose columns. The specific activity of the final preparation was 49.4 mumol Gly-Pro p-nitroanilide/min per mg protein, representing a 1098-fold purification of the homogenate. SDS-polyacrylamide gel electrophoresis of the arginine-Sepharose eluate showed a single protein band with a molecular weight of 105,000. The isoelectric point was determined to be 3.9 under non-denaturing conditions with sulphobetaine 14. The preparation was free of post-proline cleaving enzyme. The content of aminopeptidase M was 0.2% of the total protein.

Animals↗

PubMed1987

Increased expression of a high molecular weight matrix component in rat hepatocellular carcinoma.

The urea extract of the glycoproteins from the extracellular matrix of rat liver has been compared with that of Morris hepatoma 7777. A high molecular weight glycoprotein present in Morris hepatoma 7777 was not found in the extract of liver matrix. Under reducing conditions in SDS-gel electrophoresis this component gave two glycoprotein bands with Mr 53 k and 56 k. The indirect immunofluorescence staining with a monospecific antiserum directed against the component showed its abundant presence in Morris hepatoma 7777 as well as in the less malignant Morris hepatoma 9121 in form of extracellular network structures. The antigen also densely filled some cumuli of cells. In contrast the liver tissue showed only very weak staining of the extracellular areas. The overall distribution of the component could be correlated with the distribution of several hydrolases in the tumor matrix, notably beta-D-glucuronidase.

Animals↗

PubMed1987

Role of membrane glycoproteins in mediating trophic responses.

During growth and differentiation the plasma membrane has a key role not only in the reception and transmission of extracellular signals such as hormones and growth factors, but also in communicating cellular response to the cellular microenvironment. Cellular response to trophic stimuli includes alterations of cell shape and cell surface antigenicity, of cell-cell recognition and cellular adhesion, of cell matrix binding and the adaptation of cell surface receptors. The plasma membrane is therefore regarded as a 'central agency' for the integration of a single cell into the complex system of a tissue or of an organism. The numerous functions of the plasma membrane are mainly mediated by membrane integrated glycoproteins or glycolipids both sharing the common feature of covalently bound oligosaccharide side chains. Specific alterations of oligosaccharide structure and metabolism associated with growth, differentiation and various pathologic conditions suggest a specific role for the oligosaccharide moieties in the regulation of cell surface functions (Table 1). This review intends to focus on the role of plasma membrane glycoproteins describing briefly principles of glycoprotein structure and function, and characteristics of their biosynthesis and degradation.

Animals↗

PubMed1987

Half-lives of L-[35S]methionine and L-[3H]fucose of transferrin in the serum of rats.

The half-lives of 35S-labelled L-methionine and of 3H-labelled L-fucose of serum transferrin of rats were measured in pulse-chase experiments in vivo. Both L-[35S]methionine and L-[6-3H]fucose disappeared from transferrin with nearly the same half-lives of 33.8 h and 36.5 h, respectively. The data show that in this major serum glycoprotein peripheral carbohydrates and the protein moiety are degraded as a unit.

Animals↗

PubMed1987

Golgi apparatus cisternae of monensin-treated cells accumulate in the cytoplasm of liver slices.

Protein transport via the endoplasmic reticulum Golgi apparatus-cell surface export route was blocked when slices (6-15 cells thick) of livers of 10-day-old rats were incubated with 1 microM monensin. Production of secretory vesicles by Golgi apparatus was reduced or eliminated and, in their place, swollen cisternae accumulated in the cytoplasm at the trans Golgi apparatus face. The swelling response was restricted to the six external cell layers of the liver slices, and the number of cells showing the response was little increased by either a greater concentration of monensin or by longer times of incubation. When monensin was added post-chase to the slices, flux of radioactive proteins to the cell surface was inhibited by about 80% as determined from standard pulse-chase analyses with isolated cell fractions. Radioactive proteins accumulated in both endoplasmic reticulum and Golgi apparatus and in a fraction that may contain monensin-blocked Golgi apparatus cisternae released from the stack. The latter fraction was characterized by galactosyltransferase/thiamine pyrophosphatase ratios similar to those of Golgi apparatus from control slices. The use of monensin with the tissue slice system may provide an opportunity for the cells to accumulate monensin-blocked Golgi apparatus cisternae in sufficient quantities to permit their isolation and purification by conventional cell fractionation methods.

Animals↗

PubMed1986

Ion-exchange and hydrophobic-interaction high-performance liquid chromatography of proteins. A practical study.

A number of commercially available columns for ion-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC) have been tested, ranging from very fast columns with low capacity to preparative columns. The experiments represent a selection of column applications likely to occur in a biochemical laboratory. The use of several ion-exchange columns for separation of serum and membrane proteins has been demonstrated. Columns with a silica gel matrix and with a polymer matrix have proved to be of equal value in experiments with water-soluble proteins. In experiments with hydrophobic membrane proteins the choice of the column depends on the protein to be isolated. A combination of a cation-exchange and an anion-exchange column as a tandem as well as a mixed-bed column, were applied for the separation of serum proteins and for the isolation of a 175,000-dalton membrane glycoprotein. One problem of hydrophobic-interaction HPLC is the poor solubility of some proteins, for example serum proteins at high salt concentrations. This difficulty can be overcome by the use of columns with higher hydrophobicity which require lower initial salt concentrations. Less hydrophobic columns have been shown to separate hydrophobic membrane proteins by a combination of salt and detergent gradients.

Animals↗

PubMed1986

Identification of a 110-kDa glycoprotein involved in cell-substratum adhesion.

Plasma membrane glycoproteins are involved in cell-matrix interactions. For identification of such glycoproteins a recently developed cell system was used. Two cell populations differing in their adhesion properties were selected from Morris hepatoma 7777. One population was able to grow as a monolayer, while the other proliferated in suspension. From both cell lines spontaneous revertants were selected. By using antibodies raised against plasma membranes of the hepatoma and of rat liver for sequential immunoprecipitation a glycoprotein of Mr 110000 was identified. This glycoprotein was only expressed in adherent hepatoma cells as well as in normal rat liver, but was absent in non-adherent hepatoma cells and in the in vivo growing Morris hepatoma 7777. This suggests that the glycoprotein is involved in cell-substratum adhesion of hepatocytes and adherent hepatoma cells.

Animals↗

PubMed1986

Increased activity of dipeptidyl peptidase IV in serum of hepatoma-bearing rats coincides with the loss of the enzyme from the hepatoma plasma membrane.

The specific activity of dipeptidyl peptidase IV (DPP IV E.C. 3.4.14.-) in the plasma membrane of Morris hepatoma 9121 or hepatoma 7777 was 3.5% and 2.9%, respectively, of that in the plasma membrane of rat liver. The enzyme activity in the serum of hepatoma-bearing rats was 141% (hepatoma 91219) and 162% (hepatoma 7777) of the normal value. Cytochemical investigation showed that the DPP IV activity was almost completely absent from the hepatoma cell plasma membrane and was not sequestered within these cells. Indirect immunofluorescence staining with a polyclonal antibody directed against DPP IV indicated that the loss of activity was due to the absence of DPP IV molecules in the plasma membrane. The possibility that the enzyme is transferred from the membrane into the serum as a result of structural alterations is discussed.

Animals↗

PubMed1986

Anion-exchange high-performance liquid chromatography of membrane proteins from liver and Morris hepatomas.

The separation of proteins from plasma membranes of liver and two Morris hepatomas with different grades of differentiation (Morris hepatomas 7777 and 9121) is described. Size-exclusion high-performance liquid chromatography under non-denaturing conditions provides only poor separation and, with detergent-soluble fractions, unsatisfactory protein recovery. Much better results are obtained by ion-exchange chromatography. The amount of a 175 K protein in the membranes decreased in parallel with the increase in tumour malignancy. This protein appears during ion-exchange chromatography in a broad concentration range, between 0.1 and 0.3 M sodium chloride. Possible explanations for this phenomenon are discussed.

Animals↗

PubMed1986

High-performance liquid chromatographic methods for antibodies, glycosidases and membrane proteins.

The broad range of applications of high-performance liquid chromatography (HPLC) in biochemistry and cell biology is demonstrated by the purification of antibodies, separation of glycosidases and isolation of a liver membrane protein with a molecular weight of 65 000-67 000 daltons. The advantage of HPLC over classical chromatographic methods is shown by the purification of the glycosidases from Streptococcus pneumoniae. These enzymes can be purified to a degree similar to what can be achieved by "classical" ion exchange, combined with affinity chromatography, but the time needed for the HPLC experiment is much shorter and the yield at least three to five times higher. Particular attention is directed to sample preparation before HPLC separation. For the best results, a combination of HPLC with other biochemical and immunochemical methods is necessary, as is also demonstrated.

Antibodies↗

PubMed1986

Different oligosaccharide processing of the membrane-integrated and the secretory form of gp 80 in rat liver.

Rat liver synthesizes a glycoprotein with Mr of 80.000 (gp 80) which is partly inserted into the plasma membrane and partly secreted into the serum. The membrane-integrated and the secretory form of this glycoprotein have an identical peptide pattern, but different N-linked glycans. Whereas gp 80 from the serum is glycosylated with complex-type oligosaccharides, gp 80 from the plasma membrane has high mannose glycans. Phase separation with Triton X-114 showed that membrane-integrated gp 80 contains hydrophobic portions, whereas secretory gp 80 has hydrophilic properties. Intracellular transport and oligosaccharide processing of gp 80 were studied in vivo in the endoplasmic reticulum, the Golgi apparatus and plasma membranes of rat liver and in serum using pulse-chase labeling with L-[35S]methionine and immunoprecipitation. Peak labeling of gp 80 was reached in the endoplasmic reticulum 10 min after the pulse, in the Golgi apparatus 20 min later, and in the plasma membrane after 2 h; in the serum the specific radioactivity was steadily increasing during the experiment. Gp 80 of the endoplasmic reticulum was completely sensitive to endo-beta-N-glucosaminidase H (endo H), but simultaneously occurred in the Golgi apparatus in an endo H-sensitive and endo H-resistant form. The endo H-sensitive form was transported to the plasma membrane, the endo H-resistant species secreted into the serum. Conversion from the endo H-sensitive to the endo H-resistant form was completed within 10 min after transfer of gp 80 to the Golgi apparatus.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1986

Cell surface glycoproteins of hepatocytes and hepatoma cells identified by monoclonal antibodies.

Eight hybridoma cell lines secreting monoclonal antibodies (MABs) directed to cell surface components of rat hepatocytes were isolated. The antigens of seven MABs were identified as glycosylated plasma membrane proteins. The presence of these glycoproteins on normal hepatocytes and hepatocellular carcinoma cells was analyzed. A semi-quantitative enzyme-linked immunosorbent assay revealed that only two MABs (Be 8.7, Ne 11.3) recognized proteins which were expressed not only in normal liver but also in chemically induced transplantable Morris hepatomas and hepatoma-derived cell lines. The expression of six antigens was found to be sensitive to transformation. The domain specificity of the MABs was determined by indirect immunofluorescence on sections of liver tissue containing neoplastic nodules. Three MABs (Be 8.4, Ne 11.1, Ne 11.3) specifically bound to the sinusoidal domain and two MABs (Be 9.2, De 13.4) to the bile canalicular domain. These five antigens were transformation-sensitive except for the glycoprotein recognized by the MAB Ne 11.3. Three MABs (Be 8.7, Be 9.1, De 13.2) also showed intracellular immunofluorescence. Two of the antigens (Be 9.1, De 13.2) were not present in hepatomas. The relative molar masses (Mr) of the glycoproteins were determined after protein immunoblotting and immunoprecipitation. Four MABs (Be 8.7, Be 9.1, Be 9.2, De 13.4) recognized antigens with a Mr of 110 000 but did not mutually cross-react. The antigen recognized by MAB De 13.4 was identified as the ectoenzyme dipeptidyl peptidase IV (EC 3.4.14.-).

Animals↗

PubMed1986

Quantitative study of cell interaction with collagen and fibronectin.

The interaction of BHK-fibroblasts with collagen or fibronectin-collagen complex was investigated quantitatively. For that purpose an improved method for production of defined cell substrata was developed. The method permitted reproducible coupling of different ligands to glass via an amino or carboxyl group. BHK-cells grown on collagen required a minimum density of 15-20 ng collagen/cm2 for spreading. When grown on fibronectin adsorbed on collagen the cells were found to remove fibronectin from the substratum at a rate of 0.15 pg/(cell X h).

Animals↗

PubMed1986

Effect of chloroquine on the degradation of L-fucose and the polypeptide moiety of plasma membrane glycoproteins.

To evaluate the role of lysosomes in the breakdown of the carbohydrate and the polypeptide moiety of plasma membrane glycoproteins, degradation of the plasma membrane glycoprotein gp120 was studied in the liver of rats treated with the lysosomotropic amine chloroquine. Half-lives of degradation of the terminal sugar L-fucose and of L-methionine of gp120 were measured in isolated plasma membranes after pulse-chase experiments in vivo. Chloroquine extended the plasma membrane half-life of the polypeptide moiety of gp120 from 51 h to 143 h. By contrast, L-fucose of gp120 in the plasma membrane was not affected by chloroquine, but decayed with the same short half-lives of 22 h and 23 h in both controls and chloroquine-treated rats. The data suggest that the protein portion of gp120 is degraded within the lysosomes. Conversely, the terminal sugar L-fucose is removed from the glycoprotein independent from proteolysis before segregation of the glycoprotein into the lysosomal compartment.

Animals↗