scholarly journals INTRACELLULAR DISTRIBUTION OF ALKALINE PHOSPHATASE IN RAT LIVER CELLS

1955 ◽  
Vol 1 (4) ◽  
pp. 315-330 ◽  
Author(s):  
Arthur J. Emery ◽  
Alexander L. Dounce
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Liver ◽  
Nuclear Membrane ◽  
Aqueous Media ◽  
Isotonic Saline ◽  
Intracellular Distribution ◽  
Supernatant Fraction ◽  
Nuclear Fraction ◽  
Modified Method ◽  
Soluble Supernatant

1. Cytochemical studies of the intracellular distribution of alkaline phosphatase in rat liver have been made, using a fractionation procedure recently developed in this laboratory (8) and a similar but modified method not described previously. Aqueous media were used in both cases. 2. The alkaline phosphatase was found to consist of two forms, one of which is strongly activated by magnesium and one of which is not sensitive to this metal. 3. The form of the enzyme that is not activated by magnesium occurs mainly in the nuclear fraction, where it seems to be rather firmly bound. Some of this form of the enzyme is also found in the microsomes, but very little if any occurs in the soluble supernatant fraction. 4. The form of alkaline phosphatase which is activated by magnesium occurs mainly in the soluble supernatant fraction, but what is believed are significant amounts also occur in nuclei. A significant portion of this form of the enzyme can be extracted from the isolated nuclei with cold, isotonic saline solution. Some activity of this form of the enzyme is also found in the microsomal fraction. 5. Mitochondria appear to contain relatively little alkaline phosphatase of either kind. 6. The concept of a porous nuclear membrane has been invoked to explain some of the results obtained in this work. It is postulated that part at least of the form of the enzyme that is activated by magnesium is free to diffuse back and forth through pores in the nuclear membrane, whereas this is considered not to be possible for the form of the enzyme that is insensitive to magnesium as a result of the firm binding of the latter to nuclear substance.

scholarly journals SOLUBLE ENZYMES OF NUCLEI ISOLATED IN SUCROSE AND NON-AQUEOUS MEDIA

1953 ◽  
Vol 37 (2) ◽  
pp. 177-187 ◽  
Author(s):  
Herbert Stern ◽  
A. E. Mirsky
Keyword(s):  
Adenosine Deaminase ◽  
Organic Solvents ◽  
Rat Liver ◽  
Nuclear Membrane ◽  
Aqueous Media ◽  
Isolated Nuclei ◽  
Calf Thymus ◽  
Liver Nuclei ◽  
Soluble Enzymes ◽  
Soluble Components

Nuclei of calf thymus and liver and of rat liver were isolated in sucrose media and a number of their properties studied in relation to those of corresponding nuclei isolated in non-aqueous media with a view to determining their capacity to retain soluble components. The best preparations of sucrose nuclei were obtained from calf thymus. Cytochrome oxidase measurements and DNA/N ratios were far less sensitive than microscopic examination as indicators of purity when rat liver and calf thymus nuclei were compared. No satisfactory preparation of calf liver nuclei was obtained, contamination with whole cells having been appreciable; such preparations, nevertheless, could be used to advantage in the tests undertaken. DNA content of thymus nuclei isolated in sucrose was much the same as that of non-aqueous ones, pointing to a retention of soluble protein under aqueous conditions of isolation. That this net retention of protein was not due to the impermeability of the nuclear membrane was shown by the hydrolysis of the DNA upon addition of some crystalline DNAase to a sucrose suspension of nuclei. A comparative study of liver and thymus nuclei isolated in aqueous and non-aqueous media with respect to the soluble enzymes glucose-6-phosphate dehydrogenase, adenosine deaminase, and nucleoside phosphorylase yielded the following results: 1. Lyophilization of sucrose-isolated nuclei and their extraction with the organic solvents used in the non-aqueous procedure did not inactivate any of the enzymes tested. In the case of thymus the reverse was true, there being a marked increase in activity of all the enzymes studied. 2. In thymus, nucleoside phosphorylase and adenosine deaminase were active to approximately the same extent in nuclei isolated by either procedure. Glucose phosphate dehydrogenase alone was more active in sucrose-isolated nuclei, pointing to the possibility of an adsorption of this enzyme. 3. In rat liver nuclei isolated in sucrose, lyophilization and treatment with organic solvents revealed only the presence of some dehydrogenase. 4. The washing out of soluble enzymes was most markedly demonstrated in the case of calf liver. Only traces of the nucleoside enzymes were found in the sucrose-isolated nuclei, and in the case of the dehydrogenase only a half of that present in the non-aqueous nucleus remained. The main conclusions drawn were as follows:— 1. In sucrose media the nuclear membrane is ineffectual in preventing the inward or outward diffusion of protein. 2. The extent to which soluble proteins are retained by a nucleus isolated in sucrose appears to depend upon internal structural factors, such as the concentration of DNA in the nucleus. 3. With respect to determining the composition of nuclei in terms of soluble components, the sucrose isolation procedure is considered to be of indifferent merit and hence invalid for such a type of analysis.

scholarly journals Inhibition of microsomal lipid peroxidation by glutathione and glutathione transferases B and AA. Role of endogenous phospholipase A2

1984 ◽  
Vol 220 (1) ◽  
pp. 243-252 ◽  
Author(s):  
K H Tan ◽  
D J Meyer ◽  
J Belin ◽  
B Ketterer
Keyword(s):  
Lipid Peroxidation ◽  
Phospholipase A2 ◽  
Rat Liver ◽  
Inhibitory Activity ◽  
Liver Microsomes ◽  
Supernatant Fraction ◽  
Rat Liver Microsomes ◽  
Microsomal Lipid Peroxidation ◽  
Soluble Supernatant

Lipid peroxidation in vitro in rat liver microsomes (microsomal fractions) initiated by ADP-Fe3+ and NADPH was inhibited by the rat liver soluble supernatant fraction. When this fraction was subjected to frontal-elution chromatography, most, if not all, of its inhibitory activity could be accounted for by the combined effects of two fractions, one containing Se-dependent glutathione (GSH) peroxidase activity and the other the GSH transferases. In the latter fraction, GSH transferases B and AA, but not GSH transferases A and C, possessed inhibitory activity. GSH transferase B replaced the soluble supernatant fraction as an effective inhibitor of lipid peroxidation in vitro. If the microsomes were pretreated with the phospholipase A2 inhibitor p-bromophenacyl bromide, neither the soluble supernatant fraction nor GSH transferase B inhibited lipid peroxidation in vitro. Similarly, if all microsomal enzymes were heat-inactivated and lipid peroxidation was initiated with FeCl3/sodium ascorbate neither the soluble supernatant fraction nor GSH transferase B caused inhibition, but in both cases inhibition could be restored by the addition of porcine pancreatic phospholipase A2 to the incubation. It is concluded that the inhibition of microsomal lipid peroxidation in vitro requires the consecutive action of phospholipase A2, which releases fatty acyl hydroperoxides from peroxidized phospholipids, and GSH peroxidases, which reduce them. The GSH peroxidases involved are the Se-dependent GSH peroxidase and the Se-independent GSH peroxidases GSH transferases B and AA.

scholarly journals STUDIES OF TWO TYPES OF ALKALINE PHOSPHATASE IN NUCLEI ISOLATED FROM THE LIVERS OF FED AND FASTED RATS BY A MODIFICATION OF THE BEHRENS TECHNIQUE

1955 ◽  
Vol 1 (4) ◽  
pp. 331-338 ◽  
Author(s):  
Arthur J. Emery ◽  
Alexander L. Dounce
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Liver ◽  
Liver Cell ◽  
Aqueous Media ◽  
Slight Modification ◽  
Magnesium Ions ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
The One ◽  
Fasted Rats

1. Rat liver nuclei were isolated from normal rats and rats fasted for 36 hours by a slight modification of the Behrens technique. 2. The nucleus of the rat liver cell contains two types of alkaline phosphatase. This confirms the previous findings on rat liver nuclei isolated in aqueous media. 3. The one type of alkaline phosphatase is not activated by magnesium ions, and this enzyme is very strongly bound to structural material of the nucleus. The other type of alkaline phosphatase is activated by magnesium ions, and this enzyme is probably free to diffuse from cytoplasm to nucleus and vice versa through the nuclear membrane. 4. Fasting caused a pronounced decrease of protein in general and of the alkaline phosphatase which is activated by magnesium ions from the nucleus of the rat liver cell, while the alkaline phosphatase that is not activated by magnesium was less affected.

INTRACELLULAR DISTRIBUTION OF DESOXYRIBONUCLEO-DEPOLYMERASE IN NORMAL RAT LIVER, LIVER TUMOR, AND LIVER OF ANIMALS FED p-DIMETHYLAMINOAZOBENZENE

1954 ◽  
Vol 32 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Gaston de Lamirande ◽  
Claude Allard ◽  
Antonio Cantero
Keyword(s):  
Rat Liver ◽  
Liver Tumor ◽  
Cell Activity ◽  
Mitotic Rate ◽  
Intracellular Distribution ◽  
Nuclear Fraction ◽  
Normal Rat ◽  
High Level ◽  
Soluble Material

The intracellular distribution of desoxyribonucleodepolymerase (DNase) has been investigated in the liver of animals fed p-dimethylaminoazobenzene (DAB), in liver freed from tumor, and in DAB induced tumor. The method is based on the determination of acid soluble material containing phosphorus, liberated by the action of the enzyme upon highly polymerized DNA. Results indicated that the nuclear DNase particularly accounts for a very low percentage of the whole cell activity in normal rat liver, whereas in nuclei of liver of DAB fed rats and of tumor the activity is increased to a high level. These facts suggest a possible correlation between the activity of DNase in the nuclear fraction and the mitotic rate of the tissue.

scholarly journals The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5′-nucleotidase

1986 ◽  
Vol 239 (1) ◽  
pp. 185-190 ◽  
Author(s):  
P Fritzson ◽  
T B Haugen ◽  
H Tjernshaugen
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Liver ◽  
Specific Activity ◽  
Ph Dependence ◽  
Gel Permeation Chromatography ◽  
Total Activity ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Distinct Fraction ◽  
Membrane Bound

An alkaline 5′-nucleotidase with properties similar to those of membrane-bound 5′-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.

scholarly journals Detoxification of DNA hydroperoxide by glutathione transferases and the purification and characterization of glutathione transferases of the rat liver nucleus

1988 ◽  
Vol 254 (3) ◽  
pp. 841-845 ◽  
Author(s):  
K H Tan ◽  
D J Meyer ◽  
N Gillies ◽  
B Ketterer
Keyword(s):  
Rat Liver ◽  
Peroxidase Activity ◽  
Glutathione Transferases ◽  
Supernatant Fraction ◽  
Liver Nucleus ◽  
Purification And Characterization ◽  
Soluble Supernatant

DNA peroxidized by exposure to ionizing radiation in the presence of oxygen is a substrate for the Se-independent GSH peroxidase activity of several GSH transferases, GSH transferases 5-5, 3-3 and 4-4 being the most active in the rat liver soluble supernatant fraction (500, 35 and 20 nmol/min per mg of protein respectively) and GSH transferases mu and pi the most active, so far found, in the human liver soluble supernatant fraction (80 and 10 nmol/min per mg respectively). Although the GSH transferase content of the rat nucleus was found to be much lower than that of the soluble supernatant, nuclear GSH transferases are likely to be more important in the detoxification of DNA hydroperoxide produced in vivo. Two nuclear fractions were studied, one extracted with 0.075 M-saline/0.025 M-EDTA, pH 8.0, and the other extracted from the residue with 8.5 M-urea. The saline/EDTA fraction contained subunits 1, 2, 3, 4 and a novel subunit, similar but not identical to 5, provisionally referred to as 5*, in the proportions 40:25:5:5:25 respectively. The 8.5 M-urea-extracted fraction contained principally subunit 5* together with a small amount of subunit 6 in the proportion 95:5 respectively. GSH transferase 5*-5* purified from the 8.5 M-urea extract has the highest activity towards DNA hydroperoxide of any GSH transferase so far studied (1.5 mumol/min per mg). A Se-dependent GSH peroxidase fraction from rat liver was also active towards DNA hydroperoxide; however, since this enzyme accounts for only 14% of the GSH peroxidase activity detectable in the nucleus, GSH transferases may be the more important source of this activity. The possible role of GSH transferases, in particular GSH transferase 5*-5*, in DNA repair is discussed.

INTRACELLULAR DISTRIBUTION OF DESOXYRIBONUCLEO-DEPOLYMERASE IN NORMAL RAT LIVER, LIVER TUMOR, AND LIVER OF ANIMALS FED p-DIMETHYLAMINOAZOBENZENE

1954 ◽  
Vol 32 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Gaston de Lamirande ◽  
Claude Allard ◽  
Antonio Cantero
Keyword(s):  
Rat Liver ◽  
Liver Tumor ◽  
Cell Activity ◽  
Mitotic Rate ◽  
Intracellular Distribution ◽  
Nuclear Fraction ◽  
Normal Rat ◽  
High Level ◽  
Soluble Material

The intracellular distribution of desoxyribonucleodepolymerase (DNase) has been investigated in the liver of animals fed p-dimethylaminoazobenzene (DAB), in liver freed from tumor, and in DAB induced tumor. The method is based on the determination of acid soluble material containing phosphorus, liberated by the action of the enzyme upon highly polymerized DNA. Results indicated that the nuclear DNase particularly accounts for a very low percentage of the whole cell activity in normal rat liver, whereas in nuclei of liver of DAB fed rats and of tumor the activity is increased to a high level. These facts suggest a possible correlation between the activity of DNase in the nuclear fraction and the mitotic rate of the tissue.

scholarly journals INFLUENCE OF CATIONS ON THE INTRACELLULAR DISTRIBUTION OF RAT LIVER ARGINASE

1956 ◽  
Vol 223 (1) ◽  
pp. 469-478 ◽  
Author(s):  
Otto Rosenthal ◽  
Betty Gottlieb ◽  
John D. Gorry ◽  
Harry M. Vars
Keyword(s):  
Rat Liver ◽  
Intracellular Distribution

Intracellular distribution of 5′-ribonuclease and 5′-phosphodiesterase in rat liver

1967 ◽  
Vol 118 (2) ◽  
pp. 347-351 ◽  
Author(s):  
Gaston De Lamirande ◽  
Réjean Morais ◽  
Martin Blackstein
Keyword(s):  
Rat Liver ◽  
Intracellular Distribution
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