scholarly journals Biosynthesis of microsomal nicotinamide–adenine dinucleotide phosphate–cytochrome c reductase by membrane-bound and free polysomes from rat liver

1969 ◽  
Vol 112 (2) ◽  
pp. 139-147 ◽  
Author(s):  
G. Ragnotti ◽  
G. R. Lawford ◽  
P. N. Campbell
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Specific Activity ◽  
Reductase Activity ◽  
Gel Filtration ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
High Specific Activity ◽  
Rat Liver Cells ◽  
Microsome Fraction ◽  
Membrane Bound

1. NADPH–ferricytochrome c oxidoreductase (EC 1.6.2.3) was purified from the endoplasmic reticulum of rat liver cells. The methods, which involved digestion of membrane with Steapsin, a crude pancreatic extract containing diastase and trypsin, gel filtration and preparative electrophoresis on polyacrylamide, provided an enzyme with a high specific activity in good yield. 2. The incorporation of 14C-labelled amino acids into the purified reductase by the incubation of various subcellular fractions was studied. The microsome fraction, bound polysomes, free polysomes and detergent-treated polysomes effected the synthesis of the enzyme. 3. The reductase that had been synthesized by the polysomes was tightly bound to preparations of smooth-surfaced endoplasmic reticulum that were added to the incubation medium. 4. Reductase activity could be detected on both free and detergent-treated polysomes. Evidence is presented to show that this activity was due, at least in part, to the presence on the ribosomes of nascent enzyme. The association of enzyme with detergent-treated polysomes did not appear to be due to contamination of the ribosomes with either membrane or cell sap but it is possible for such ribosomes to adsorb some enzyme. 5. The amount of reductase activity associated with the detergent-treated polysomes was increased when the rats from which the polysomes were derived had been previously injected with phenobarbitone. 6. The results are discussed with respect to their relevance for the question of the existence of two functionally different groups of polysomes in the liver and for current ideas on the biogenesis of membranes.

scholarly journals Release of calcium from the endoplasmic reticulum by bile acids in rat liver cells.

1988 ◽  
Vol 263 (5) ◽  
pp. 2299-2303 ◽  
Author(s):  
L Combettes ◽  
M Dumont ◽  
B Berthon ◽  
S Erlinger ◽  
M Claret
Keyword(s):  
Endoplasmic Reticulum ◽  
Bile Acids ◽  
Rat Liver ◽  
Liver Cells ◽  
Rat Liver Cells

scholarly journals Effects of taurolithocholate, a Ca2+-mobilizing agent, on cell Ca2+ in rat hepatocytes, human platelets and neuroblastoma NG108-15 cell line

1991 ◽  
Vol 273 (1) ◽  
pp. 153-160 ◽  
Author(s):  
J F Coquil ◽  
B Berthon ◽  
N Chomiki ◽  
L Combettes ◽  
P Jourdon ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Bile Acid ◽  
Cell Line ◽  
Rat Liver ◽  
Neuronal Cell ◽  
Rat Hepatocytes ◽  
Cell Types ◽  
Liver Cells ◽  
Human Platelets ◽  
Rat Liver Cells

The monohydroxy bile acid taurolithocholate permeabilizes the endoplasmic reticulum to Ca2+ in rat liver cells. To assess whether this action on the endoplasmic reticulum was restricted to this tissue, the effects of bile acid were investigated in two cell types quite unrelated to rat hepatocyte, namely human platelets and neuronal NG108-15 cell line. The results showed that taurolithocholate (3-100 microM) had no effect on free cytosolic [Ca2+] in human platelets and NG108-15 cells. whereas it increased it from 180 to 520 nM in rat hepatocytes. In contrast, in cells permeabilized by saponin, taurolithocholate initiated a profound release of the stored Ca2+ from the internal Ca2+ pools in the three cell types. The bile acid released 90% of the Ca2+ pools, with rate constants of about 5 min-1 and half-maximal effects at 15-30 microM. The results also showed that, in contrast with liver cells, which displayed an influx of [14C]taurolithocholate of 2 nmol/min per mg, human platelets and the neuronal cell line appeared to be resistant to [14C]taurolithocholate uptake. The influx measured in these latter cells was about 100-fold lower than in rat liver cells. Taken together, these data suggest that human platelets and NG108-15 cells do not possess the transport system for concentrating monohydroxy bile acids into cells. However, they show that human platelets and neuronal NG108-15 possess, in common with liver cells, the intracellular system responsible for taurolithocholate-mediated Ca2+ release from internal stores.

Biosynthesis of 1,2-3H and 4-14C steroid sulfates of high specific activity

1970 ◽  
Vol 48 (1) ◽  
pp. 148-150 ◽  
Author(s):  
J. Torday ◽  
G. Hall ◽  
M. Schweitzer ◽  
C. J. P. Giroud
Keyword(s):  
Rat Liver ◽  
Specific Activity ◽  
Liver Homogenate ◽  
High Specific Activity ◽  
Supernatant Fraction ◽  
Metabolic Studies

A supernatant fraction of rat liver homogenate enriched with ATP was used for the biosynthesis of the ester sulfates of several 3H and 14C steroids of the pregn-4-ene series. The method provides a simple means to prepare steroid sulfates of high specific activity for use in either metabolic studies or as reference compounds in the quantification of such conjugates by isotope assays.

Studies on the site of addition of sialic acid and glucosamine to rat α1-acid glycoprotein

1977 ◽  
Vol 55 (4) ◽  
pp. 408-414 ◽  
Author(s):  
J. C. Jamieson
Keyword(s):  
Endoplasmic Reticulum ◽  
Sialic Acid ◽  
Rat Liver ◽  
Golgi Complex ◽  
Smooth Endoplasmic Reticulum ◽  
Main Site ◽  
Acid Glycoprotein ◽  
Membrane Bound ◽  
Polypeptide Chains

Ultrasonic extracts of rough and smooth endoplasmic reticulum fractions and Golgi fractions from rat liver were examined by immunoelectrophoresis using antiserum to α1-acid glycoprotein. Rough endoplasmic reticulum fractions contained only sialic acid free α1-acid glycoprotein, whereas smooth endoplasmic reticulum and Golgi fractions also contained sialic acid containing α1-acid glycoprotein. Determination of the sialic acid contents of immune precipitates isolated from the extracts suggested that the Golgi complex was the main site of addition of sialic acid to α1-acid glycoprotein. Immunological studies on puromycin extracts of polyribosomes showed that polypeptide chains of α1-acid glycoprotein and albumin were assembled mainly on membrane-bound polyribosomes. Evidence is presented from incorporation studies with labelled leucine and glucosamine that initial glycosylation of α1-acid glycoprotein occurs mainly or entirely after release of nascent polypeptide from the ribosomal site.

Gel filtration of marker enzymes of plasma membrane and endoplasmic reticulum from rat liver

1969 ◽  
Vol 193 (2) ◽  
pp. 468-471 ◽  
Author(s):  
Kenji Nakai ◽  
Shigehide Takemitsu ◽  
Toshisuke Kawasaki ◽  
Ikuo Yamashina
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Gel Filtration ◽  
Marker Enzymes

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

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