scholarly journals The content of glutathione and glutathione S-transferases and the glutathione peroxidase activity in rat liver nuclei determined by a non-aqueous technique of cell fractionation

1995 ◽  
Vol 311 (3) ◽  
pp. 889-894 ◽  
Author(s):  
S Soboll ◽  
S Gründel ◽  
J Harris ◽  
V Kolb-Bachofen ◽  
B Ketterer ◽  
...  
Keyword(s):  
Glutathione Peroxidase ◽  
Rat Liver ◽  
Water Soluble ◽  
Small Scale ◽  
Nuclear Fraction ◽  
Cell Fractionation ◽  
Glutathione S Transferases ◽  
Scale Preparation ◽  
Liver Nuclei

Hepatocellular nuclei require glutathione, glutathione S-transferases (GSTs) and Se-dependent glutathione peroxidase (GPx) for intranuclear protection against damage from electrophiles or products of active oxygen. Data so far available from the literature on nuclei isolated in aqueous systems range from glutathione, GSTs and GPx either being absent altogether to being present in quantities in excess of those in the cytoplasm. This paper describes a small-scale preparation of a nuclear fraction from rat liver by a non-aqueous technique, designed to retain nuclear water-soluble molecules in situ, since low-molecular-mass compounds can diffuse freely into other compartments during aqueous separation. This non-aqueous procedure shows the nucleus to contain glutathione at 8.4 mM and soluble GSTs at 38 micrograms/mg of protein, the enrichment over the homogenate being 1.2-1.4-fold. Se-dependent GPx activity was also present in the nucleus (56 m-units/mg), although with slightly lower activity than in the homogenate (0.7-fold).

scholarly journals Identification and subcellular localization of sphinganine-phosphatases in rat liver

1995 ◽  
Vol 311 (1) ◽  
pp. 139-146 ◽  
Author(s):  
P De Ceuster ◽  
G P Mannaerts ◽  
P P Van Veldhoven
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Water Soluble ◽  
Nuclear Fraction ◽  
Cell Fractionation ◽  
Acidic Ph ◽  
Ph Optimum ◽  
High Affinity ◽  
Neuronal Tissues

One of the primary products of [4,5-3H]sphinganine phosphate, added to fibroblast cultures, is sphinganine [Van Veldhoven and Mannaerts (1994) Biochem. J. 299, 597-601], implicating the physiological action of (a) hitherto unknown phosphatase(s). We have now further characterized this activity in rat liver. In homogenates, the dephosphorylation appeared to be catalysed by multiple enzymes. A low-affinity system was active at acidic pH, whereas at physiological pH values hydrolysis was carried out by a high-affinity enzyme. The latter was sensitive to Zn2+ and detergents and possessed a pH optimum of 7.5. Upon cell fractionation the major portion of the high-affinity activity was recovered in the nuclear and microsomal fractions. Further separation of the microsomal fraction showed an association predominantly with vesicles derived from the plasma membrane. Likewise, when plasma membranes were prepared from the nuclear fraction, the high-affinity phosphatase co-purified with the plasma membrane markers. From the differential effects of bivalent cations, chelators, water-soluble and amphiphilic phosphate esters, detergents and other compounds, it could be concluded that the plasma membrane-associated sphinganine-phosphatase activity is not due to alkaline phosphatase, dolichol-phosphatase, the N-ethylmaleimide-insensitive phosphatidate phosphatase or ceramide-phosphatase. The dephosphorylation observed at acidic pH in homogenates appeared also to be enriched in purified plasma membranes and might represent a side-activity of ceramide-phosphatase. We speculate that the high-affinity phosphatase, which is especially active in neuronal tissues, plays a role in the attenuation of bioactive phosphorylated sphingoid bases such as sphingenine phosphate, and propose to name it sphingosine-phosphatase.

scholarly journals ISOLATION OF TWO DISTINCT CLASSES OF POLYSOMES FROM A NUCLEAR FRACTION OF RAT LIVER

1968 ◽  
Vol 37 (1) ◽  
pp. 163-181 ◽  
Author(s):  
Paul D. Sadowski ◽  
Janet Alcock Howden
Keyword(s):  
Outer Membrane ◽  
Rat Liver ◽  
Orotic Acid ◽  
Specific Activity ◽  
Nuclear Fraction ◽  
Differential Centrifugation ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Triton X

Isolated rat liver nuclei were washed with Triton-X-100 in the presence of liver cell sap. This treatment liberated a fraction of polysomes which were isolated by differential centrifugation and were designated "outer membrane polysomes." The outer membrane polysomes synthesized protein in vivo. Shortly after injection of orotic acid-14C, the RNA of outer membrane polysomes had a higher specific activity than that of cytoplasmic polysomes. It was postulated that outer membrane polysomes may be an intermediate in the transfer of newly synthesized RNA from the nucleus to the cytoplasm. In other experiments, Triton-washed rat liver nuclei were lysed in the presence of deoxycholate and deoxyribonuclease. A ribonucleoprotein fraction was isolated from the lysate by differential centrifugation. This fraction contained "intranuclear ribosomes," which sedimented like partially degraded polysomes in sucrose gradients. This degradation could be partially prevented if intranuclear ribosomes were purified by sedimentation through heavy sucrose. The resulting pellets were termed "intranuclear polysomes" because they contained some undergraded polysomes. Intranuclear polysomes were highly radioactive after a brief pulse with orotic acid-14C, but did not appear to synthesize protein rapidly in vivo. Intranuclear polysomes may represent the initial stage of assembly of polyribosomes in the nucleus.

scholarly journals STUDIES ON THE BIOGENESIS OF SMOOTH ENDOPLASMIC RETICULUM MEMBRANES IN LIVERS OF PHENOBARBITAL-TREATED RATS

1972 ◽  
Vol 55 (2) ◽  
pp. 282-298 ◽  
Author(s):  
Joan A. Higgins ◽  
Russell J. Barrnett
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Specific Activity ◽  
Smooth Endoplasmic Reticulum ◽  
Normal Liver ◽  
Subsequent Treatment ◽  
Cell Fractionation ◽  
Acyltransferase Activity ◽  
Normal Rat

The localization of acyltransferases involved in acylation of α-glycerophosphate, during phenobarbital induced proliferation of smooth endoplasmic reticulum (ser) membranes, has been investigated using cytochemical and cell fractionation techniques. In cytochemical studies of normal rat liver, reaction product marking acyltransferase activity was associated to the greatest extent with the rough endoplasmic reticulum (rer) membranes and to a lesser extent with ser membranes. In liver from phenobarbital-treated rats, reaction product was largely restricted to ser membranes. The specific activity of the acyltransferases of rough microsomes from normal rat liver was higher than that of the smooth microsomes. On injection of phenobarbital, this fell rapidly after three injections to a low level, at which it remained during subsequent treatment. The specific activity of the smooth microsomes, on injection of phenobarbital, rose to a peak 12 hr after the first injection, after which it fell to a level at an activity above that of smooth microsomes of normal liver. A mechanism is postulated for the biogenesis of smooth membranes in which the phospholipid is synthesized in situ and the protein is synthesized in the rer and moves to the site of newly synthesized phospholipid, where it is inserted to produce a whole membrane.

scholarly journals Two immunologically distinct Yc-type subunits are present in rat lung glutathione S-transferases

1984 ◽  
Vol 224 (1) ◽  
pp. 335-338 ◽  
Author(s):  
S V Singh ◽  
Y C Awasthi
Keyword(s):  
Glutathione Peroxidase ◽  
Rat Liver ◽  
Peroxidase Activity ◽  
Gel Electrophoresis ◽  
Rat Lung ◽  
Glutathione Peroxidase Activity ◽  
Glutathione S Transferase ◽  
Two Dimensional Gel Electrophoresis ◽  
Glutathione S Transferases

Two types of 25 000-Mr subunits are present in rat lung glutathione S-transferase I (pI 8.8). These subunits, designated Yc and Yc', are immunologically and functionally distinct from each other. The homodimers YcYc (pI 10.4) and Yc'Yc' (pI 7.6) obtained by hybridization in vitro of the two subunits of glutathione S-transferase I (pI 8.8) were isolated and characterized. Results of these studies indicate that only the Yc subunits express glutathione peroxidase activity and cross-react with the antibodies raised against glutathione S-transferase B (YaYc) or rat liver. The Yc' subunits do not express glutathione peroxidase activity and do not cross-react with the antibodies raised against glutathione S-transferase B of rat liver. The amino acid compositions of these two subunits are also different. These two subunits can also be separated by the two-dimensional gel electrophoresis of glutathione S-transferase I (pI 8.8) of rat lung.

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