scholarly journals Differences in distribution of esterase between cell fractions of rat liver homogenates prepared in various media. Relevance to the lysosomal location of the enzyme in the intact cell

1971 ◽  
Vol 125 (2) ◽  
pp. 545-555 ◽  
Author(s):  
Patience C. Barrow ◽  
S. J. Holt
Keyword(s):  
Acid Phosphatase ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Intact Cell ◽  
Cellular Damage ◽  
Phase System ◽  
Two Phase ◽  
Liver Homogenates ◽  
Cell Fractions ◽  
Standard Fractionation

The distribution of esterase in subcellular fractions of rat liver homogenates was compared with that of the lysosomal enzyme acid phosphatase and the microsomal enzyme glucose 6-phosphatase. Most of the esterase from sucrose homogenate sediments with glucose 6-phosphatase and about 8% is recovered in the supernatant. However, up to 53% of the esterase can be washed from microtome sections of unfixed liver, in which less cellular damage would be expected than that caused by homogenization. About 40% of both esterase and acid phosphatase are recovered in the soluble fraction after homogenization in aqueous glycerol or in a two-phase system (Arcton 113–0.25m-sucrose), although glucose 6-phosphatase is still recovered in the microsomal fraction of such homogenates. The esterase of the microsomal fraction prepared from a sucrose homogenate is much more readily released by treatment with 0.26% deoxycholate than are other constituents of this fraction. The release of esterase from the microsomal fraction by the detergent and its concomitant release with acid phosphatase after homogenization in glycerol or the two-phase system suggests that a greater proportion of esterase may be present in lysosomes of the intact cell than is indicated by the results of standard fractionation procedures.

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.

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.

scholarly journals Heterogeneity of smooth endoplasmic reticulum from rat liver studied by two-phase partitioning

1989 ◽  
Vol 262 (1) ◽  
pp. 55-61 ◽  
Author(s):  
P Gierow ◽  
B Jergil
Keyword(s):  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Phase System ◽  
Marker Enzymes ◽  
Two Phase ◽  
Sucrose Density Gradient Centrifugation ◽  
Phase Partitioning ◽  
Microsomal Membranes

Smooth microsomal membranes, prepared from rat liver by sucrose-density-gradient centrifugation, were subfractionated by counter-current distribution in an aqueous two-phase system consisting of poly(ethylene glycol) and Dextran T500. A comparison of the distribution curves of marker enzymes, together with theoretically calculated curves, indicated the presence of at least five membrane subfractions, differing in the ratios of the marker enzymes. Glucose-6-phosphatase and arylesterase distributed in one manner, and NADPH-cytochrome c reductase and NADH-ferricyanide reductase in another. Evidence for further heterogeneities in the distribution of marker enzymes in smooth microsomes was obtained by analysing the membrane domain structure using a recently described method [Albertsson (1988) Q. Rev. Biophys. 21, 61-98]. Phenobarbital treatment did not influence the behaviour of the marker enzymes.

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 Assay and properties of digitonin-activated bilirubin uridine diphosphate glucuronyltransferase from rat liver

1972 ◽  
Vol 129 (3) ◽  
pp. 605-618 ◽  
Author(s):  
K. P. M. Heirwegh ◽  
M. Van De Vijver ◽  
J. Fevery
Keyword(s):  
Rat Liver ◽  
Metal Ion ◽  
Glucuronic Acid ◽  
Uridine Diphosphate ◽  
Bivalent Metal ◽  
Bivalent Metal Ions ◽  
Liver Homogenates ◽  
Cell Fractions ◽  
Biliary Excretion Rate ◽  
Substrate Concentrations

1. The bilirubin UDP-glucuronyltransferase assay described by Van Roy & Heirwegh (1968) has been improved. 2. Extraction of final azo-derivatives is rendered more simple and efficient by thorough emulsification and by cooling. 3. Pretreatment of homogenates and cell fractions with digitonin increases the sensitivity of the assays and gives less variable results than those with untreated preparations. The activation procedure is flexible. 4. Blank values (obtained from incubation mixtures from which activating bivalent metal ion and UDP-glucuronic acid were omitted) are low. No endogenous conjugate formation could be detected except with untreated, fresh liver homogenates. Control incubation mixtures containing the latter preparations are preferably kept at 0°C. 5. With activated microsomal preparations, rates of breakdown of UDP-glucuronic acid (as monitored by release of Pi) were low. Little if any increase in enzyme activity was found when UDP-N-acetylglucosamine was included in the incubation mixtures. 6. Slight deviation from Michaelis–Menten kinetics with respect to bilirubin observed at low substrate concentrations is probably related to the use of binding protein in the assay mixtures. Michaelis–Menten kinetics were followed with respect to UDP-glucuronic acid. Part of the enzyme in microsomal preparations from rat liver functioned independently of added bivalent metal ions. Mn2+ was slightly more, and Ca2+ somewhat less, stimulatory than Mg2+. The Mg2+-dependent fraction showed Michaelis–Menten kinetics with respect to the added Mg2+. 7. The enzyme activities found were higher than values reported in the literature for untreated or purified preparations from rat liver. They were above reported values of the maximal biliary excretion rate of bilirubin.

scholarly journals GOLGI FRACTIONS PREPARED FROM RAT LIVER HOMOGENATES

1973 ◽  
Vol 59 (1) ◽  
pp. 73-88 ◽  
Author(s):  
J. J. M. Bergeron ◽  
J. H. Ehrenreich ◽  
P. Siekevitz ◽  
G. E. Palade
Keyword(s):  
Endoplasmic Reticulum ◽  
Cytochrome C ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Companion Paper ◽  
Transferase Activity ◽  
Ethanol Treatment ◽  
Liver Homogenates ◽  
Cytochrome P 450 ◽  
Nadph Cytochrome C Reductase

The three Golgi fractions isolated from rat liver homogenates by the procedure given in the companion paper account for 6–7% of the protein of the total microsomal fraction used as starting preparation. The lightest, most homogeneous Golgi fraction (GF1) lacks typical "microsomal" activities, e.g., glucose-6-phosphatase, NADPH-cytochrome c-reductase, and cytochrome P-450. The heaviest, most heterogeneous fraction (GF3) is contaminated by endoplasmic reticulum membranes to the extent of ∼15% of its protein. The three fractions taken together account for nearly all the UDP-galactose: N-acetyl-glucosamine galactosyltransferase of the parent microsomal fraction, and for ∼70% of the activity of the original homogenate. Omission of the ethanol treatment of the animals reduces the recovery by half. The transferase activity is associated with the membranes of the Golgi elements, not with their content. Galactose is transferred not only to N-acetyl-glucosamine but also to an unidentified lipid-soluble component.

THE EFFECT OF FASTING ON ESTERIFICATION OF PALMITATE BY RAT LIVER IN VITRO

1966 ◽  
Vol 44 (1) ◽  
pp. 129-140 ◽  
Author(s):  
Bernard Rubenstein ◽  
David Rubinstein
Keyword(s):  
Rat Liver ◽  
Incubation Medium ◽  
Intact Cell ◽  
Glycerol Kinase ◽  
Glycerophosphate Dehydrogenase ◽  
Liver Slices ◽  
Liver Homogenates

The ability of liver slices from rats fasted 48 hours to esterify palmitate-1-14C is about half of that of slices from fed animals. The same effect can be observed in homogenates of liver freed of nuclei, either with or without mitochondria. In both preparations, the synthesis of triglycerides is chiefly affected. The decrease in esterification by homogenate supernatants containing microsomes from fasted animals can be overcome by using increased amounts of ATP or an ATP generator and NaF in the incubation medium. The ATPase responsible for the higher ATP requirement in liver homogenates from fasted animals is Mg++-dependent. Higher concentrations of ATP inhibit esterification by mitochondrial preparations. Incorporation of both palmitate-9-10-3H and of glycerol-1-3-14C is reduced to the same extent for each compound in slices from fasting animals. Glycerol kinase and α-glycerophosphate dehydrogenase are both unaffected by 48 hours' fasting. It is concluded that the decrease in ATP from the activation of ATPase is responsible for the decreased esterification in the intact cell.

scholarly journals Heterogeneous distribution of enzymes among plasma-membrane fragments sedimenting with the microsomal fraction of rat liver

1974 ◽  
Vol 142 (3) ◽  
pp. 667-671 ◽  
Author(s):  
Kenneth A. Norris ◽  
Miloslav Dobrota ◽  
Faiz S. Issa ◽  
Richard H. Hinton ◽  
Eric Reid
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Heterogeneous Distribution ◽  
Similar Product ◽  
Liver Homogenates

Plasma-membrane fragments recovered in the microsomal fraction of rat liver homogenates were shown to be heterogeneous in density. It was demonstrated that 5′-nucleotidase, the most commonly used plasma-membrane marker, is concentrated in the lightest subfraction. Two of the published procedures for the isolation of plasma-membrane fragments from the microsomal fraction (Touster et al., 1970; Hinton et al., 1971) are shown to give products which are not representative of all the plasma-membrane fragments of microsomal size, and it is argued that a third procedure (House & Weidemann, 1970) is likely to give a similar product.

scholarly journals Subcellular localization of a rat liver enzyme converting thyroxine into tri-iodothyronine and possible involvement of essential thiol groups

1976 ◽  
Vol 157 (2) ◽  
pp. 479-482 ◽  
Author(s):  
T J Visser ◽  
I Does-Tobé ◽  
R Docter ◽  
G Hennemann
Keyword(s):  
Rat Liver ◽  
Essential Role ◽  
Microsomal Fraction ◽  
Thiol Group ◽  
Subcellular Fractionation ◽  
Enzymic Activity ◽  
Marker Enzyme ◽  
Converting Enzyme ◽  
Thiol Groups ◽  
Liver Homogenates

Experiments with rat liver homogenates showed that on subcellular fractionation the ability to catalyse the conversion of thyroxine into tri-iodothyronine was lost. The activity could in part be restored by addition of the cytosol to the microsomal fraction. Both components were found to be heat labile. The necessity of the presence of cytosol could be circumvented by incorporation of thiol-group-containing compounds in the medium. Optimal enzymic activity was observed in the presence of dithiothreitol and EDTA in medium of low osmolarity. By comparing the distribution of the converting enzyme over the subcellular fractions with a microsomal marker enzyme, glucose 6-phosphatase, it was demonstrated that the former is indeed of microsomal origin. Finally, it was shown that thiol groups play an essential role in the conversion of thyroxine into tri-iodothyronine.

scholarly journals Phosphatidate biosynthesis in mitochondrial subfractions of rat liver

1969 ◽  
Vol 113 (2) ◽  
pp. 429-440 ◽  
Author(s):  
Elizabeth H. Shephard ◽  
G. Hübscher
Keyword(s):  
Rat Liver ◽  
Mitochondrial Membrane ◽  
Microsomal Fraction ◽  
Mitochondrial Fraction ◽  
Urate Oxidase ◽  
Outer Mitochondrial Membrane ◽  
Short Term ◽  
Conventional Fractionation ◽  
Liver Homogenates ◽  
Nadh Cytochrome

1. After conventional fractionation of rat liver homogenates in 0·88m-sucrose the mitochondrial fraction was subjected to short-term water lysis followed by separation of the resulting membrane preparations. 2. Phosphatidate formation was measured in all subcellular fractions and subfractions and was compared with the distribution of succinate dehydrogenase, monoamine oxidase, rotenone-insensitive NADH cytochrome c reductase, arylsulphatase, urate oxidase, arylesterase and glucose 6-phosphatase. 3. The results obtained indicated that mitochondria were capable of synthesizing phosphatidate, though this activity was only about one-third of the total homogenate activity. 4. Mitochondrial phosphatidate formation was located predominantly in the outer mitochondrial membrane. Although this membrane preparation was found to be significantly contaminated by the microsomal fraction, this contamination was estimated to account for not more than about 20% of the total phosphatidate formation observed in preparations of outer mitochondrial membrane.

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