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Biomedical subjects

W Reutter

Publications and source records attributed to W Reutter.

At least 163 records · Page 9Linked to original sources
PubMed1986

Identification of a transformation-sensitive 110-kDa plasma membrane glycoprotein of rat hepatocytes.

Monoclonal antibodies were used to define cell surface antigens which are present on rat hepatocytes but are absent from hepatoma cells. One monoclonal antibody, referred to as Be 9.2, recognizes a major component of purified rat liver plasma membranes with a Mr of 110 000. This antigen (gp110) was not found in the transplantable Morris hepatoma 9121 and 7777 nor on two cultured hepatoma cell lines. Isoelectric focussing showed that gp110 is a very acidic membrane component with an isoelectric point of 3.6 to 3.8. Treatment with neuraminidase reduced the Mr to 95 000. Gp110 while bound to the membrane was resistant to trypsin, but sensitive to papain. The tissue distribution of gp110 was examined by indirect immunofluorescence in frozen sections. The antigen was found on the bile canalicular domain of hepatocytes, the microvillous zone of enterocytes of the small intestinal villi, the luminal plasma membrane of acinar cells in the submaxillary and extraorbital gland and of epithelial cells of the vesicular gland. Gp110 could not be detected in the stomach, pancreas, large intestine, kidney, thymus, spleen, heart, lung, muscle cells and fibers and in the brain. Identical results were obtained by the use of an antiserum raised against purified gp110. They confirm the transformation-sensitive character of this glycoprotein. A possible identity with dipeptidyl peptidase IV and aminopeptidase M, which have similar molecular weights and are also present in rat liver on the bile canalicular domains, could be excluded. The results suggest that the loss of gp110 might be regarded as a marker for transformation or dedifferentiation of hepatocytes.

Animals↗
PubMed1985

Hepatocyte adhesion on plastic. Different mechanisms for serum- and fibronectin-mediated adhesion.

Hepatocytes adhere well on plastic in the presence of serum or fibronectin and subsequent spreading is not prevented when protein synthesis was blocked by cycloheximide. Protein synthesis-independent spreading was also observed in cultures containing serum depleted of fibronectin by affinity chromatography. This indicates that serum-mediated adhesion is independent of fibronectin and suggests the existence of an adhesion factor other than fibronectin in serum. The involvement of different membrane components for fibronectin- and serum-mediated adhesion was demonstrated by experiments where the different adhesion-inhibiting activities of antisera raised against plasma membranes of rat liver and Morris hepatoma 7777 (Neumeier et al., FEBS lett 168 (1984) 241-244) were used. Whereas anti-liver antibodies inhibited both types of adhesion, anti-hepatoma antibodies were only able to prevent fibronectin-mediated adhesion. This indicates again that two different mechanisms are responsible for fibronectin- and serum-mediated adhesion. Fractionation of fetal calf serum (FCS) by size exclusion HPLC revealed that proteins of molecular weights of 60-80 kD promoted attachment and spreading of hepatocytes. Spreading was not perturbated by anti-hepatoma antibodies, indicating that an adhesion factor of 60-80 kD is responsible for serum-mediated adhesion. 'Serum-spreading factor', also called vitronectin, from human plasma has been described as having a similar molecular weight. The purified factor was found to mediate hepatocyte adhesion which was not inhibited by anti-hepatoma antibodies. This suggests that serum-mediated adhesion depends on an adhesion factor present in FCS, which is similar to or identical with vitronectin.

Animals↗
PubMed1985

Inhibitors of glycoprotein biosynthesis.

Altogether 30 different sugar analogues have been tested in a cell free system from rat liver or, in part, in freshly prepared hepatocytes. It is our aim to find suitable compounds which are able either to interfere with the metabolization of L-fucose, galactose and N-acetylmannosamine or, alternatively, to block the attachment of these sugars to the nascent oligosaccharide chain. 1-Methylfucoside inhibits the fucokinase by a competitive mode (Ki = 1.1 mmol/l). Both the fucokinase and fucose-1-phosphate pyrophosphorylase activity are impaired by Clobenoside, a chloro-containing glucofuranoside (Ki values between 5 to 10 mmol/l). In hepatocytes this inhibition leads to a drastic reduction of fucoprotein biosynthesis and secretion. 1-Methylenegalactose proved to be a promising competitive inhibitor of the galactokinase (Ki = 4.1 mmol/l), while the efficacy of 2-deoxy-2-fluoro-galactose and 6-deoxy-6-fluoro-galactose is less pronounced. Part of these sugar analogues could become a suitable tool in order to elucidate the biological significance of terminal and subterminal sugars.

Animals↗
PubMed1985

Involvement of plasma membrane dipeptidyl peptidase IV in fibronectin-mediated adhesion of cells on collagen.

Dipeptidyl peptidase IV is an exopeptidase found in the serum and in plasma membranes of most animal tissues. The role of this enzyme in cell-matrix interaction of BHK cells and hepatocytes grown on collagen-coated surfaces was investigated by three different approaches. 1) Glass surfaces were derivatized with bovine serum albumin which resulted in a cell-repulsing substratum. When it was further modified with Gly-Pro-Ala tripeptide, which is a substrate for dipeptidyl peptidase IV, BHK fibroblasts spread on it rapidly. The spreading could be inhibited by addition of free Gly-Pro-Ala or other substrates of the enzyme as well as by an inhibitor peptide Val-Pro-Leu. It was not influenced by tripeptides which were neither substrates nor inhibitors of dipeptidyl peptidase IV. 2) The addition of Gly-Pro-Ala to seeded cells slowed down the initial process of cell spreading on denatured collagen in the presence of fibronectin. The presence of both collagen and fibronectin was a necessary precondition for the spreading of cells in a manner sensitive to Gly-Pro-Ala. 3) Antiserum raised against mouse liver dipeptidyl peptidase IV added to the medium delayed the spreading of rat hepatocytes on denatured collagen in the presence of fibronectin in a manner similar to when Gly-Pro-Ala was added to the medium. These observations lead to the conclusion that plasma membrane dipeptidyl peptidase IV may be involved in the initial phase of fibronectin-mediated cell spreading on collagen.

Animals↗
PubMed1984

Automatic amino acid and sugar analysis of glycoproteins.

Amino acid analyzers were used for complete analyses of glycoproteins. The methods of analysis used were cation-exchange chromatography for amino acids, phosphoamino acids and amino sugars with ninhydrin as reagent, and anion-exchange chromatography for neutral sugars with copper Bicinchoninate as reagent. A separate program was developed for the analysis of charged sugar components, N-acetylneuraminic acid and amino sugars in one chromatogram. The possibility of detection of radioactively labelled components is also demonstrated. The detection limits and possibilities for column and reactor switches are discussed.

Amino Acids↗
PubMed1984

Size-exclusion high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins: a comparison.

When size-exclusion HPLC (SE-HPLC) is applied for the separation of hydrophilic and hydrophobic proteins, numerous problems can be encountered, which may present considerable difficulties. A major source of such complications is interaction between column packing and sample, especially on the so-called "Diol" and "Polyol" columns. In many cases interaction can be reduced only by adding detergents. Calibration proteins and hydrophobic membrane proteins of liver are separated by SE-HPLC. The influence of detergents on association and dissociation of protein subunits and protein configuration is shown. These factors can affect the elution volume during chromatography. Furthermore it is shown that a direct comparison can be drawn between the protein separation by SE-HPLC on the one hand and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on the other hand. As an example the separation of the delta-subunit of the acetylcholine receptor was shown under reducing and nonreducing conditions.

Animals↗
PubMed1984

Different adhesion inhibiting activities of antisera against plasma membranes of liver and Morris hepatoma 7777.

Cell-substratum adhesion of rat hepatocytes was inhibited by antisera raised against plasma membranes of liver (anti-liver antiserum) and Morris hepatoma 7777 (anti-hepatoma antiserum). Similar concentrations of both antisera inhibited adhesion on collagen. Anti-liver antiserum also inhibited the adhesion of hepatocytes on plastic, whereas anti-hepatoma antiserum was only able to inhibit the adhesion on collagen completely. These results suggest the existence of at least two different adhesion-involved molecules. Cells adhere to plastic by means of both molecules, whereas adhesion on collagen is mediated by only one of them. The results further suggest that hepatoma cells lost the molecule involved in adhesion on plastic.

Animals↗
PubMed1984

Modification of the intramolecular turnover of terminal carbohydrates of dipeptidylaminopeptidase IV isolated from rat-liver plasma membrane during liver regeneration.

An intramolecular turnover of the terminal carbohydrates L-fucose, N-acetylneuraminic acid and D-galactose is a characteristic property of several liver plasma membrane glycoproteins, first demonstrated for dipeptidylaminopeptidase IV (EC 3.4.14.5., DPP IV). The core carbohydrates D-mannose and N-acetyl-D-glucosamine turn over like the polypeptide chain. The ratio of apparent half-lives of L-fucose and L-methionine of DPP IV is shifted from 0.17 in normal liver to 0.60 in regenerating liver. The ratio of half-lives of N-acetylneuraminic acid and L-methionine is only slightly changed from 0.43 in normal liver to 0.61 in regenerating liver. The ratio of apparent half-lives of D-mannose and L-methionine amounts to 0.80 in normal liver and 0.71 after partial hepatectomy. From this a drastic reduction of the intramolecular turnover of L-fucose on plasma membrane DPP IV in regenerating liver can be derived. The intramolecular N-acetylneuraminic acid turnover is affected to only a minor extent. D-Mannose turns over like the polypeptide in both normal and regenerating liver. The intramolecular L-fucose turnover may be involved in membrane glycoprotein recycling, which presumably is altered in regenerating liver. Additionally, L-fucose could regulate the rate of degradation of DPP IV, since core-fucosylated glycoproteins appear to be resistant to mammalian endo-N-acetylglucosaminidase.

Animals↗
PubMed1984

Integral membrane antigens involved in cell-substratum adhesion of hepatocytes and hepatoma cells.

Cell-substratum adhesion of rat hepatocytes was inhibited by antisera raised against purified plasma membranes of rat liver (anti-liver-antiserum) and Morris hepatoma 7777 (anti-hepatoma-antiserum). It is assumed that substances which block the adhesion-inhibiting activity of the antisera are involved in cell-substratum adhesion. Adhesion-involved molecules of rat liver monitored as 'blocking activity' were compared with those of Morris hepatoma 7777 and 9121. They were found to be integral membrane glycoproteins, which could be solubilized only by detergents. Fractionation of plasma membrane extracts by size exclusion HPLC revealed two blocking activity peaks representing molecules involved in the adhesion to plastic (P-AIM) and collagen (C-AIM). In rat liver both adhesion-involved molecules were found; yet P-AIM seemed to be the major type of adhesion-involved molecule. In the relatively well differentiated Morris hepatoma 9121 also both types were detected. In membrane extracts of the high malignant and poorly differentiated Morris hepatoma 7777, however, no P-AIM but only C-AIM were found. Estimation by size exclusion HPLC revealed molecular weights of 120 kD for C-AIM and approx. 105 kD for P-AIM. On SDS gel electrophoresis proteins in the region of 95 kD were found in C-AIM containing fractions, whereas proteins of 105 kD are likely candidates for P-AIM.

Animals↗
PubMed1984

Turnover of the protein and carbohydrate moieties of a 105kD glycoprotein of plasma membranes from mouse liver determined by immunoprecipitation.

The turnover of a mouse liver plasma membrane glycoprotein (Mr 105 k) has been studied by means of immunoprecipitation and radioactive labeling. The method of immunoprecipitation proved to be a fast and efficient way of isolating a single protein for measurement of its specific radioactivity. The half-life of the carbohydrate moiety determined using 3H-galactose was 41 h and using 3H-mannose was 23 h, whereas that of the protein moiety determined using 3H-leucine was 63 h. The implications of this differential degradation of the protein and carbohydrate moieties are discussed in terms of the recycling of glycoproteins between the plasma membrane and intracellular membranes.

Animals↗
PubMed1983

Heterogeneous turnover of terminal and core sugars within the carbohydrate chain of dipeptidylaminopeptidase IV isolated from rat liver plasma membrane.

Dipeptidylaminopeptidase IV, a plasma membrane-bound glycoprotein, is characterized by an intramolecular heterogeneous turnover of the protein backbone and carbohydrate chain. The faster turnover of the latter is restricted only to the outer sugars. The inner core sugars D-mannose and N-acetyl-D-glucosamine turn over at the same rate as the protein backbone.

Animals↗
PubMed1983

Intramolecular heterogeneity of degradation in plasma membrane glycoproteins: evidence for a general characteristic.

Five integral plasma membrane glycoproteins (60, 80, 120, 140, and 160 kilodaltons) were isolated to homogeneity from rat liver by a four-step procedure: (i) extraction of plasma membranes with lithium diiodosalicylate, (ii) solubilization of glycoproteins with Nonidet P-40, (iii) affinity chromatography on concanavalin A-Sepharose, and (iv) semipreparative NaDodSO4/polyacrylamide gel electrophoresis. The glycoproteins contained 48.5--51.5% hydrophobic amino acids. Carbohydrate moieties contained N-acetyl-D-glucosamine, D-mannose, D-galactose, L-fucose, and N-acetylneuraminic acid. N-Acetyl-D-galactosamine was not detectable. Half-lives of degradation of the carbohydrate and protein moieties of the five glycoproteins were measured by pulse-chase experiments in vivo. Protein moieties had half-lives ranging from 52 to 88 hr in the five glycoproteins, with a mean of 73 +/- 15 hr. Terminal sugars, L-fucose, and N-acetylneuraminic acid had significantly shorter half-lives, averaging 18 +/- 2 hr and 29 +/- 3 hr, respectively. The half-life of D-mannose varied between that of the terminal sugars and that of the protein moiety, depending on the type of the glycoprotein. The data show that the carbohydrate moieties are degraded faster than the protein portion of the glycoproteins. As this finding was obtained in each of the five glycoproteins, intramolecular heterogeneity of breakdown may be a general characteristic of plasma membrane glycoproteins in liver.

Amino Acids↗