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."

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 The Effect of α-Tocopheryl Succinate on Succinate Respiration in Rat Liver Mitochondria

2015 ◽  
pp. S609-S615 ◽  
Author(s):  
O. SOBOTKA ◽  
Z. DRAHOTA ◽  
O. KUČERA ◽  
R. ENDLICHER ◽  
H. RAUCHOVÁ ◽  
...  
Keyword(s):  
Concentration Dependence ◽  
Rat Liver ◽  
Liver Homogenate ◽  
Liver Mitochondria ◽  
Inhibitory Effect ◽  
Experimental Models ◽  
Rat Liver Mitochondria ◽  
Isolated Mitochondria ◽  
Significant Inhibitory Effect ◽  
Tocopheryl Succinate

We compared the effect of α-tocopheryl succinate (TOS) on succinate-dependent respiration in rat liver mitochondria, homogenate and permeabilized hepatocytes in both a coupled and uncoupled state. In isolated mitochondria, a significant inhibitory effect was observed at a concentration of 5 µM, in liver homogenate at 25 µM and in permeabilized hepatocytes at 50 µM. The inhibitory effect of TOS on succinate respiration in an uncoupled state was less pronounced than in a coupled state in all the experimental models tested. When the concentration dependence of the TOS inhibitory effect was tested, the most sensitive in both states were isolated mitochondria; the most resistant were permeabilized hepatocytes.

scholarly journals CONCENTRATION OF ACID PHOSPHATASE, RIBONUCLEASE, DESOXYRIBONUCLEASE, ß-GLUCURONIDASE, AND CATHEPSIN IN "DROPLETS" ISOLATED FROM THE KIDNEY CELLS OF NORMAL RATS

1956 ◽  
Vol 2 (5) ◽  
pp. 513-521 ◽  
Author(s):  
Werner Straus
Keyword(s):  
Acid Phosphatase ◽  
Mitochondrial Fraction ◽  
Liver Cells ◽  
Enzymatic Properties ◽  
Kidney Cells ◽  
Supernatant Fluid ◽  
Fraction I

1. Three fractions of "droplets" having diameters of 1 to 5 µ (fraction I), 0.5 to 1.5 µ (fraction II), and 0.1 to 1.0 µ (fraction III) were isolated from the kidney cells of normal rats. 2. All three "droplet" fractions showed 10 to 15 times higher activities of acid phosphatase, ß-glucuronidase, ribonuclease, desoxyribonuclease, and cathepsin than the total homogenate and the mitochondrial fraction. 3. After a rough fractionation of the total homogenate, approximately 50 per cent of the 5 enzymes was found in the fractions which contained the "droplets" and approximately 30 per cent in the supernatant fluid. 4. The similarities between the enzymatic properties of the "droplets" from kidney cells and of the fractions isolated from liver cells by other investigators have been discussed.

scholarly journals Subcellular distribution of cytochrome c in rat liver. Methods for its extraction and purification

1967 ◽  
Vol 105 (2) ◽  
pp. 427-442 ◽  
Author(s):  
N. F. González-Cadavid ◽  
P. N. Campbell
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Subcellular Distribution ◽  
Microsomal Fraction ◽  
Gradient Centrifugation ◽  
Ammonium Phosphate ◽  
Mitochondrial Fraction ◽  
Subcellular Fractions ◽  
Nuclear Fraction ◽  
Extraction And Purification

1. A method for the extraction and purification of cytochrome c from rat liver is described. The method depends on multiple chromatography on Amberlite IRC-50 with elution with ammonium phosphate buffers of differing ionic composition and pH, interspersed with gel filtration with Sephadex G-25. Conditions leading to denaturation are avoided and the product is chromatographically pure. 2. The method may be used for the quantitative analysis of cytochrome c either in unfractionated liver or in subcellular fractions. 3. Two pools of cytochrome c were detected, one extractable at pH4·0 with distilled water and the other extracted from the residues of the first extraction with 0·15m-sodium chloride. 4. For subcellular distribution studies the liver was homogenized in 0·3m-sucrose and a nuclear fraction (washed thoroughly to remove trapped mitochondria), a mitochondrial fraction, a heavy microsomal fraction, a standard microsomal fraction and the cell sap were isolated. The mitochondrial fraction was subfractionated further by density-gradient centrifugation. Each fraction was analysed for protein, RNA, DNA, succinate–neotetrazolium oxidoreductase and glucose 6-phosphatase. 5. A total of 123μg. of cytochrome c was obtained/g. wet wt. of rat liver. 6. Values for the percentage subcellular distribution of cytochrome c are: nuclear fraction, 24·4; mitochondrial fraction, 57·2; heavy microsomal fraction, 5·2; standard microsomal fraction, 10·6; cell sap, 2·7. 7. Three out of the eight mitochondrial subfractions separated by gradient centrifugation contained 76% of the cytochrome c and 85% of the succinate–neotetrazolium oxidoreductase present in the mitochondrial fraction. 8. In unfractionated liver 94% of the cytochrome c was extracted at pH4·0 with water whereas in most of the subcellular fractions the corresponding value was approx. 75–80%.

THE BINDING OF SULPHATASE BY RAT-LIVER PARTICLES AS COMPARED TO THAT OF ACID PHOSPHATASE

1955 ◽  
Vol 33 (5) ◽  
pp. 839-844 ◽  
Author(s):  
Renée Viala ◽  
R. Gianetto
Keyword(s):  
Acid Phosphatase ◽  
Rat Liver ◽  
Cytoplasmic Granules

One of the sulphatases of rat liver, like acid phosphatase, is enclosed within cytoplasmic granules. The two enzymes are released from these granules in similar proportions by various treatments.

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)

THE BINDING OF SULPHATASE BY RAT-LIVER PARTICLES AS COMPARED TO THAT OF ACID PHOSPHATASE

1955 ◽  
Vol 33 (1) ◽  
pp. 839-844 ◽  
Author(s):  
Renée Viala ◽  
R. Gianetto
Keyword(s):  
Acid Phosphatase ◽  
Rat Liver ◽  
Cytoplasmic Granules

One of the sulphatases of rat liver, like acid phosphatase, is enclosed within cytoplasmic granules. The two enzymes are released from these granules in similar proportions by various treatments.

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