Differentiation of the Nucleotide Pyrophosphatases of Rat‐Liver Plasma Membrane and Endoplasmic Reticulum by Enzymic Iodination

Bischoff, Erwin; Wilkening, J.; Tran‐Thi, Thuy‐Anh; Decker, Karl

doi:10.1111/j.1432-1033.1976.tb10158.x

Cited by 51 publications

(16 citation statements)

References 23 publications

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“…Suspensions of rat hepatocytes were prepared as previously described by Bischoff et al [29]. After washing the cells with Krebs-Henseleit buffer they were suspended in a modified Waymouth medium [30] containing 10% fetal calf serum, 50 U/ml penicillin, 50 pg/ml streptomycin, 1 pM dexamethasone, and 1 pM insulin.…”

Section: Preparation Of Rat Hepatocyte Monolayersmentioning

confidence: 99%

The Biosynthesis of Acute‐Phase Proteins in Primary Cultures of Rat Hepatocytes

Andus

Groß

Tran‐Thi

et al. 1983

European Journal of Biochemistry

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The biosynthesis and secretion of a2-macroglobulin, transferrin, al-acid glycoprotein and al-proteinase inhibitor were studied in rat hepatocyte primary cultures. After labeling with [35S]methionine, two forms, which can be separated electrophoretically differing by molecular weight, were found for each of the four glycoproteins. The following molecular weights were estimated for the intracellular precursors and the secreted forms : a2-macroglobulin, 176000 and 182000; transferrin, 84000 and 86000; a1 -acid glycoprotein, 39000 and 43000-60000; al-proteinase inhibitor, 49000 and 54000.Carbohydrate moieties could be removed from intracellular forms by treatment with endoglucosaminidase H indicating that their oligosaccharide chains were of the high-mannose type. The extracellular forms were sensitive to sialidase. They incorporated [3H]galactose and [3H]fucose showing that their oligosaccharide chains were of the complex type.Pulse-chase experiments revealed a precursor-product relationship for the high-mannose and the complex type glycoproteins. In the hepatocyte medium newly synthesized albumin was detected after 30 min and newly synthesized glycoproteins after 60 min.Unglycosylated a2-macroglobulin (162000), transferrin (79000), al-acid glycoprotein (23000), and al-proteinase inhibitor (41 000) were found in the cells as well as in the medium, when the transfer of oligosaccharide chains onto the polypeptide chains was blocked by tunicamycin. Tunicamycin led to a marked reduction of the secretion of a2-macroglobulin, al-acid glycoprotein and al-proteinase inhibitor, whereas the secretion of transferrin was less affected.There is a group of plasma proteins, designated acutephase proteins, whose concentrations in the plasma increase during inflammation caused by infections, necrosis, vaccination, burning and neoplastic growth [l -101. The majority, if not all, of those acute-phase proteins are glycoproteins synthesized in the liver. The biosynthesis of glycoproteins containing N-linked oligosaccharide chains of the complex type is a multi-step process. While the polypeptide chains are synthesized in the endoplasmic reticulum a concomitant glycosylation occurs resulting in high-mannose-type glycoproteins. The high-mannose-type glycoproteins can be processed to complex-type glycoproteins by enzymes located in the Golgi apparatus (for recent reviews see [ l l -151).The biosynthesis of a2-macroglobulin [16], transferrin [17-191, El-acid glycoprotein [18-221 and El-proteinase inhibitor [23 -281 has been studied in several laboratories.In the present paper their glycosylation and secretion were studied in primary cultures of rat hepatocytes. The existence of a high-mannose and a complex-type form is demonstrated for each of the four proteins, the high-mannosetype being the main intracellular form, whereas the complextype is secreted into the medium. chased from Amersham International. ~-[6-~H]Galactose (15 Ci/mmol) and Protosol were obtained from New England Nuclear (Boston). Endoglucosaminidase H from Streptomyces gris...

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Section: Preparation Of Rat Hepatocyte Monolayersmentioning

confidence: 99%

The Biosynthesis of Acute‐Phase Proteins in Primary Cultures of Rat Hepatocytes

Andus

Groß

Tran‐Thi

et al. 1983

European Journal of Biochemistry

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“…Primary rat hepatocytes were prepared by peffu~,on of the hver as described [15]. Hepatocyte~ were cultured in M 199 medium ~upple-rnented w,th 4% FCS, streptomycin (100 rng/l), pemelllm (61 rag/I).…”

Section: Timp =1mentioning

confidence: 99%

Synthesis of tissue inhibitor of metalloproteinase‐1 (TIMP‐1) in human hepatoma cells (HepG2) Up‐regulation by interleukin‐6 and transforming growth factor β1

et al. 1992

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Metalioprotemases a,ld their specific mhibltors, believed to play a role in extraeellular matrix metabolism, are regulated by inflammatory cytokmes. Here we have addressed the question of whether liver, the major site of ~yntltests of plasma pl'oteinase mh~b~tors, is al~o capable ,~f synthesxzing the tissue inhibitor of metalloprotentase-I (TIMP.I) We show at mRNA and protein levels that TIMP-I 15 expres~d in differentiated human hepatoma cells (HepG2) and that its syntltesi.~ is up-regulated by interleakin-6 (IL-6), transforming growth factor ,81 and phorbol 12-mynstate 13-acetate. The physiological role of this phenomenon is underhned by the fact that hpopoly~aeehartde admmistmtmn into rat~ in vlvo. a~ well as IL-6-stmmlation of mt hepatocytes in primary culture, also leads to an merea~e ofTIMP-I mRNA in liver cells

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“…Suspensions of rat hepatocytes were prepared as described previously by Bischoff et al [20]. After washing the cells with Krebs-Henseleit buffer, they were suspended in a modified Waymouth medium [21] containing 10% fetal calf serum, SO U/ml penicillin, 50 pg/ml streptomycin, 1 pM dexametha-…”

Section: Preparation Of Rat Hepatocyte Monolayersmentioning

confidence: 99%

Cultured Ito cells of rat liver express the α₂‐macroglobulin gene

Andus

Ramadori

Heinrich

et al. 1987

European Journal of Biochemistry

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Ito cells were isolated from rat liver and kept in culture for up to 13 days. The capability of the Ito cells to synthesize a2-macroglobulin was analyzed at different times after isolation and by pulse-chase experiments. Newly synthesized a2-macroglobulin was determined by immunoprecipitation followed by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis and fluorography. a2-Macroglobulin synthesis was hardly detectable in Ito cells and their media 3 days after plating. However, 5 -11 days after the isolation of the cells, increasing amounts of a2-macroglobulin were synthesized. The results of pulse-chase experiments performed on day 7 showed that radioactively labeled a2-macroglobulin decreased in the intracellular compartment and increased in the culture medium. a2-Macroglobulin was identified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions. Furthermore, when unlabeled a2-macroglobulin was added during the immunoprecipitation, a competition was observed. Incubation of pancreatic elastase with culture medium of rat Ito cells or rat hepatocytes led to the same cleavage products as found with a2-macroglobulin. a2-Macroglobulin-specific mRNA could be demonstrated by Northern blot analysis of total RNA extracted from rat Ito cells.Under the conditions where a2-macroglobulin was synthesized in Ito cells, no synthesis of al-macroglobulin, al-inhibitor 3, a,-proteinase inhibitor, al-acid glycoprotein, al-acute-phase globulin (T-kininogen) and albumin could be demonstrated. It is concluded that a2-macroglobulin is a true secretory protein of rat Ito cells in culture. This could be of importance for collagen metabolism in liver diseases.

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Differentiation of the Nucleotide Pyrophosphatases of Rat‐Liver Plasma Membrane and Endoplasmic Reticulum by Enzymic Iodination

Cited by 51 publications

References 23 publications

The Biosynthesis of Acute‐Phase Proteins in Primary Cultures of Rat Hepatocytes

The Biosynthesis of Acute‐Phase Proteins in Primary Cultures of Rat Hepatocytes

Synthesis of tissue inhibitor of metalloproteinase‐1 (TIMP‐1) in human hepatoma cells (HepG2) Up‐regulation by interleukin‐6 and transforming growth factor β1

Cultured Ito cells of rat liver express the α₂‐macroglobulin gene

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Differentiation of the Nucleotide Pyrophosphatases of Rat‐Liver Plasma Membrane and Endoplasmic Reticulum by Enzymic Iodination

Cited by 51 publications

References 23 publications

The Biosynthesis of Acute‐Phase Proteins in Primary Cultures of Rat Hepatocytes

The Biosynthesis of Acute‐Phase Proteins in Primary Cultures of Rat Hepatocytes

Synthesis of tissue inhibitor of metalloproteinase‐1 (TIMP‐1) in human hepatoma cells (HepG2) Up‐regulation by interleukin‐6 and transforming growth factor β1

Cultured Ito cells of rat liver express the α2‐macroglobulin gene

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Cultured Ito cells of rat liver express the α₂‐macroglobulin gene