INTRACELLULAR DISTRIBUTION OF PHOSPHOMONOESTERASES IN RAT LIVER HOMOGENATE

1954 ◽  
Vol 32 (1) ◽  
pp. 383-394 ◽  
Author(s):  
Claude Allard ◽  
Gaston de Lamirande ◽  
Hugo Faria ◽  
Antonio Cantero
Keyword(s):  
Acid Phosphatase ◽  
Rat Liver ◽  
Mouse Liver ◽  
Soluble Fraction ◽  
Liver Homogenate ◽  
Mitochondrial Fraction ◽  
Nuclear Fraction ◽  
Absolute Requirement ◽  
Rat Liver Cells ◽  
Liver Homogenates

Acid phosphatase or phosphomonoesterase II activity of rat and mouse liver homogenates, prepared in 0.25 M sucrose, was found mainly in the cytoplasmic granules. Since the small percentage of activity of the nuclear fraction activity could be explained by the presence of mitochondria (which were actually counted in this fraction) it is concluded that rat and mouse liver nuclei do not contain acid phosphatase activity.A rather broad range of acid phosphatase activity was observed in rat and mouse livers depending on the time elapsed between the preparation of homogenate and the activity determinations. However, a preincubation of the tissues or isolated fractions at 37° C. for 60 min. was sufficient to increase the activity to an optimal value, and thus eliminate variations due to the latency of this enzyme.Alkaline phosphatase or phosphomonoesterase I activity was also found to be latent in rat liver homogenates. The phenomenon was less apparent than for acid phosphatase and seemed to depend mostly on the nature of the buffer employed in the assay system.Some evidence for the presence of two forms of alkaline phosphatase in rat liver cells is presented. One form of the enzyme was found to have an absolute requirement of magnesium for activity and was present in the soluble fraction, whereas the other which was not activated by magnesium seemed firmly linked to the nuclei and microsomes and was absent in the soluble fraction. The activity in the mitochondrial fraction was small and seemed of doubtful significance.

INTRACELLULAR DISTRIBUTION OF PHOSPHOMONOESTERASES IN RAT LIVER HOMOGENATE

1954 ◽  
Vol 32 (4) ◽  
pp. 383-394 ◽  
Author(s):  
Claude Allard ◽  
Gaston de Lamirande ◽  
Hugo Faria ◽  
Antonio Cantero
Keyword(s):  
Acid Phosphatase ◽  
Rat Liver ◽  
Mouse Liver ◽  
Soluble Fraction ◽  
Liver Homogenate ◽  
Mitochondrial Fraction ◽  
Nuclear Fraction ◽  
Absolute Requirement ◽  
Rat Liver Cells ◽  
Liver Homogenates

Acid phosphatase or phosphomonoesterase II activity of rat and mouse liver homogenates, prepared in 0.25 M sucrose, was found mainly in the cytoplasmic granules. Since the small percentage of activity of the nuclear fraction activity could be explained by the presence of mitochondria (which were actually counted in this fraction) it is concluded that rat and mouse liver nuclei do not contain acid phosphatase activity.A rather broad range of acid phosphatase activity was observed in rat and mouse livers depending on the time elapsed between the preparation of homogenate and the activity determinations. However, a preincubation of the tissues or isolated fractions at 37° C. for 60 min. was sufficient to increase the activity to an optimal value, and thus eliminate variations due to the latency of this enzyme.Alkaline phosphatase or phosphomonoesterase I activity was also found to be latent in rat liver homogenates. The phenomenon was less apparent than for acid phosphatase and seemed to depend mostly on the nature of the buffer employed in the assay system.Some evidence for the presence of two forms of alkaline phosphatase in rat liver cells is presented. One form of the enzyme was found to have an absolute requirement of magnesium for activity and was present in the soluble fraction, whereas the other which was not activated by magnesium seemed firmly linked to the nuclei and microsomes and was absent in the soluble fraction. The activity in the mitochondrial fraction was small and seemed of doubtful significance.

scholarly journals BIOCHEMICAL HETEROGENEITY OF THE CYTOPLASMIC PARTICLES ISOLATED FROM RAT LIVER HOMOGENATE

1953 ◽  
Vol 1 (1) ◽  
pp. 27-46 ◽  
Author(s):  
ALEX B. NOVIKOFF ◽  
ESTELLE PODBER ◽  
JEAN RYAN ◽  
ELSIE NOE
Keyword(s):  
Acid Phosphatase ◽  
Nucleic Acid ◽  
Rat Liver ◽  
Liver Homogenate ◽  
Mitochondrial Fraction ◽  
Nuclear Fraction ◽  
Supernatant Fluid ◽  
Cytoplasmic Granules ◽  
Isolated Mitochondria ◽  
Low Levels

By separating the cytoplasmic granules of 0.88 M sucrose homogenates of rat liver into eight fractions it has been possible to demonstrate marked chemical and enzymatic heterogeneity among the isolated microsomes and less pronounced heterogeneity among the isolated mitochondria. The more readily sedimented microsomes are rich in ribose nucleic acid and show high esterase, adenosine-5’-phosphatase, acid phosphatase and uricase activities, while the less readily sedimented microsomes, although rich in ribose nucleic acid esterase and adenosine-5’-phosphatase have low levels of acid phosphatase and uricase activities. The very small mitochondria differ from the larger ones in the levels of activities of all enzymes studied, with the exception of adenosine-5’-phosphatase; the most striking differences were found in the cases of acid phosphatase and uricase. A centrifugation schedule is given to isolate a "nuclear fraction," a mitochondrial fraction, a mixed fraction of smallest mitochondria and microsomes (of two types differing in optical density), a fraction of optically less dense microsomes, and a "supernatant fluid."

scholarly journals Localization of Cytosolic NADP-dependent Isocitrate Dehydrogenase in the Peroxisomes of Rat Liver Cells

2001 ◽  
Vol 49 (9) ◽  
pp. 1123-1131 ◽  
Author(s):  
Takashi Yoshihara ◽  
Tatsuhiko Hamamoto ◽  
Ryo Munakata ◽  
Ryosuke Tajiri ◽  
Mariko Ohsumi ◽  
...  
Keyword(s):  
Rat Liver ◽  
Gradient Centrifugation ◽  
Liver Homogenate ◽  
Rat Hepatocytes ◽  
Mitochondrial Fraction ◽  
Peroxisomal Enzyme ◽  
Antigenic Sites ◽  
Level Of Activity ◽  
Rat Liver Cells ◽  
Targeting Signal

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123–1131, 2001)

INHIBITION OF CHOLESTEROL FORMATION BY RAT LIVER HOMOGENATES

1955 ◽  
Vol 33 (2) ◽  
pp. 135-138 ◽  
Author(s):  
B. B. Migicovsky
Keyword(s):  
Vitamin A ◽  
Rat Liver ◽  
Cholesterol Synthesis ◽  
Liver Homogenate ◽  
Inhibitory Factor ◽  
Normal Rat ◽  
Liver Homogenates

The inability of liver homogenates, from starved and vitamin A deficient rats, to synthesize cholesterol is illustrated. A possible reason for this phenomenon is that these preparations inhibit cholesterol synthesis when added to a liver homogenate from a normal rat. The inhibitory factor or factors are present in both the supernate and residue portions of the homogenate, although the residue matter is more inhibitory.

scholarly journals Aqueous Extracts of Teucrium polium Possess Remarkable Antioxidant Activity In Vitro

2006 ◽  
Vol 3 (3) ◽  
pp. 329-338 ◽  
Author(s):  
Predrag Ljubuncic ◽  
Suha Dakwar ◽  
Irina Portnaya ◽  
Uri Cogan ◽  
Hassan Azaizeh ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Antioxidant Activity ◽  
Rat Liver ◽  
Antioxidant Properties ◽  
Liver Homogenate ◽  
Plasma Oxidation ◽  
Teucrium Polium ◽  
Liver Homogenates ◽  
Β Carotene

Teucrium poliumL. (Lamiaceae) (RDC 1117) is a medicinal plant whose species have been used for over 2000 years in traditional medicine due to its diuretic, diaphoretic, tonic, antipyretic, antispasmodic and cholagogic properties. The therapeutic benefit of medicinal plants is often attributed to their antioxidant properties. We previously reported that an aqueous extract of the leaves and stems of this plant could inhibit iron-induced lipid peroxidation in rat liver homogenate at concentrations that were not toxic to cultured hepatic cells. Others have reported that organic extracts of the aerial components of this plant could inhibit oxidative processes. Against this background, we felt further investigation on the antioxidant action of the extract ofT. poliumprepared according to traditional Arab medicine was warranted. Accordingly, we assessed (i) its ability to inhibit (a) oxidation of β-carotene, (b) 2,2′-azobis(2-amidinopropan) dihydrochloride (AAPH)-induced plasma oxidation and (c) iron-induced lipid peroxidation in rat liver homogenates; (ii) to scavenge the superoxide ($${\hbox{ O }}_{2}^{\bullet -}$$) radical and the hydroxyl radical (OH•); (iii) its effects on the enzyme xanthine oxidase activity; (iv) its capacity to bind iron; and (v) its effect on cell glutathione (GSH) homeostasis in cultured Hep G2 cells. We found that the extract (i) inhibited (a) oxidation of β-carotene, (b) AAPH-induced plasma oxidation (c) Fe2+-induced lipid peroxidation in rat liver homogenates (IC50 = 7 ± 2 μg ml−1); (ii) scavenged $${\hbox{ O }}_{2}^{\bullet -}$$(IC50 = 12 ± 3 μg ml−1) and OH• (IC50 = 66 ± 20 μg ml−1); (iii) binds iron (IC50 = 79 ± 17 μg ml−1); and (iv) tended to increase intracellular GSH levels resulting in a decrease in the GSSG/GSH ratio. These results demonstrate that the extract prepared from theT. poliumpossesses antioxidant activityin vitro. Further investigations are needed to verify whether this antioxidant effect occursin vivo.

Diminished hepatic triiodothyronine production in Gunn rats

1986 ◽  
Vol 113 (2) ◽  
pp. 281-288 ◽  
Author(s):  
J. R. Saltzman ◽  
D. W. Clark ◽  
R. D. Utiger
Keyword(s):  
Thyroid Hormone ◽  
Rat Liver ◽  
Liver Homogenate ◽  
Unconjugated Bilirubin ◽  
Thyroid Hormone Metabolism ◽  
Gunn Rats ◽  
Deiodinase Activity ◽  
Sediment Fraction ◽  
Liver Homogenates

Abstract. The liver is a major site of conversion of thyroxine (T4) to the more active thyroid hormone 3,5,3'-triiodothyronine (T3). Hepatic T4 to T3 conversion is altered by a variety of pathological processes and pharmacological agents. We studied T4 to T3 conversion in glucuronyl transferase deficient homozygous Gunn rats because they have a hepatic enzyme abnormality which leads to hyperbilirubinaemia, and also because they have been reported to have alterations in thyroid hormone metabolism. An in vitro incubation system employing the 10 000 × g supernatant of liver homogenate was used, and T3 production was measured by radioimmunoassay. Experiments were done using substrate concentrations ranging from 0.56 to 20 μm, tissue protein in concentrations ranging from 0.625 to 20 mg and incubation times of 15 to 60 min. T3 production by liver homogenates from homozygous Gunn rats in these studies ranged from 29 to 70% of that produced by liver homogenates from phenotypically normal heterozygous Gunn rats. The deficit in hepatic T3 production by homozygous rats could not be overcome by increasing cofactor concentrations. After ultracentrifugation at 100 000 μ g, T4-5'-deiodinase activity was found primarily in the 100 000 × g sediment fraction. Homogygous rat liver 100 000 × g sediment T3 production was 55% of that of the heterozygous rat liver 100 000 × g sediment. Liver cytosol from both homozygous and heterozygous rats inhibited microsomal T4-5'-deiodinase activity similarly. Addition of unconjugated bilirubin to liver homogenates resulted in reduction of T3 production in livers from both homozygous and heterozygous rats. Thus the diminished capacity for hepatic conversion of T4 to T3 in homozygous Gunn rats may be due to inhibition of T4-5'-deiodinase activity by high endogenous levels of unconjugated bilirubin.

scholarly journals Study of fluxes at low concentrations of l-tri-iodothyronine with rat liver cells and their plasma-membrane vesicles. Evidence for the accumulation of the hormone against a gradient

1981 ◽  
Vol 198 (3) ◽  
pp. 457-466 ◽  
Author(s):  
Govind S. Rao ◽  
Marie Luise Rao ◽  
Astrid Thilmann ◽  
Hans D. Quednau
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Liver Homogenate ◽  
Membrane Vesicles ◽  
Liver Cells ◽  
Free Form ◽  
Plasma Membrane Vesicles ◽  
Cytosol Fraction ◽  
Low Concentrations ◽  
Rat Liver Cells

1. Influx and efflux of l-tri-[125I]iodothyronine with isolated rat liver parenchymal cells and their plasma-membrane vesicles were studied by a rapid centrifugation technique. 2. At 23°C and in the concentration range that included the concentration of free l-tri-iodothyronine in rat plasma (3–5pm) influx into cells was saturable; an apparent Kt value of 8.6±1.6pm was obtained. 3. At 5pm-l-tri-[125I]iodothyronine in the external medium the ratios of the concentrations inside to outside in cells and plasma-membrane vesicles were 38:1 and 366:1 respectively after 7s of incubation. At equilibrium (60s at 23°C) uptake of l-tri-[125I]iodothyronine by cells was linear with the hormone concentration, whereas that by plasma-membrane vesicles exhibited an apparent saturation with a Kd value of 6.1±1.3pm. 4. Efflux of l-tri-[125I]iodothyronine from cells equilibrated with the hormone (5–123pm) was constant up to 21 s; the amount that flowed out was 17.7±3.8% when cells were equilibrated with 5pm-hormone. When plasma-membrane vesicles were equilibrated with l-tri-[125I]iodothyronine (556–1226pm) 66.8±5.8% flowed out after 21 s. 5. From a consideration of the data on efflux from cells and binding of l-tri-[125I]iodothyronine to the liver homogenate, as studied by the charcoal-adsorption and equilibrium-dialysis methods, it appears that 18–22% of the hormone exists in the free form in the cell. 6. Vinblastine and colchicine diminished the uptake of l-tri-[125I]iodothyronine by cells but not by plasma-membrane vesicles; binding to the cytosol fraction was not affected. Phenylbutazone, 6-n-propyl-2-thiouracil, methimazole and corticosterone diminished the uptake by cells, plasma-membrane vesicles and binding to the cytosol fraction to different extents. 7. These results suggest that at low concentrations of l-tri-[125I]iodothyronine rat liver cells and their plasma-membrane vesicles accumulated the hormone against an apparent gradient by a membrane-mediated process. Contribution of cytoplasmic proteins to uptake by plasma-membrane vesicles was negligible. The amount of l-tri-[125I]iodothyronine required to achieve half-maximal uptake agrees with that occurring in the free form in the blood, conferring physiological importance to the transporting system in the plasma membrane of the liver cell.

scholarly journals PURIFICATION OF GLUTARALDEHYDE ITS SIGNIFICANCE FOR PRESERVATION OF ACID PHOSPHATASE ACTIVITY

1968 ◽  
Vol 16 (3) ◽  
pp. 199-204 ◽  
Author(s):  
H. DARIUSH FAHIMI ◽  
PIERRE DROCHMANS ◽  
A. POPOWSKI
Keyword(s):  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Enzymatic Activity ◽  
Phosphatase Activity ◽  
Rat Liver ◽  
Acid Phosphatase Activity ◽  
High Concentration ◽  
Liver Homogenates ◽  
Inorganic Phosphates

The inhibition of acid phosphatase activity in rat liver homogenates after fixation in different lots of commercial glutaraldehyde is determined and compared with the inhibition following fixation with a distilled product. It is shown that commercial glutaraldehydes inhibit more of the enzyme activity than the distilled product. The acidic products of oxidation of glutaraldehyde do not increase the inhibition of the enzymatic activity. The presence of high concentration of inorganic phosphates in different lots of commercial glutaraldehyde, as presented here, suggests that probably such impurities may be responsible for increased inhibition of phosphatase activity noted after fixation in commercial glutaraldehydes.

Sign in / Sign up

Export Citation Format

Share Document