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PubMed1975

The measurement of iron in rat liver tissue and subcellular fractions using atomic absorption spectroscopy.

To permit the measurement of iron in small amounts of liver tissue or subcellular fractions, a procedure based on wet ashing and atomic absorption spectroscopy has been developed. This procedure, which can detect iron down to a concentration of 0.1 mug/ml, has been used with ferric chloride and Jectofer (iron-citric acid-sorbitol complex) solutions as well as liver homogenates and subcellular fractions. Interference from constituents of liver tissue has not been observed and measurements on fractions appear to be quantitative. Individual determinations had a variability of approximately 10%.

Animals↗

PubMed1992

Brain cytochrome P450 and testosterone metabolism by rat brain subcellular fractions: presence of cytochrome P450 3A immunoreactive protein in rat brain mitochondria.

The hydroxylation of testosterone by rat brain subcellular fractions has been studied using an HPLC method with an enhanced resolution for the separation of testosterone and its monohydroxy derivatives. Although the analysis time is longer than that reported for earlier methods, a baseline separation was obtained between all hydroxytestosterones, excepting 6 alpha-hydroxytestosterone and 15 beta-hydroxytestosterone, which were separated using a second chromatography system. This separation was important as rat brain microsomes metabolized testosterone to 15 alpha-, 6 beta-, 15 beta-, 16 beta-, 2 beta-, 1 beta-hydroxytestosterone and androstenedione. Testosterone metabolism was found to be linear with time and protein concentration. The rat brain mitochondrial fraction metabolized testosterone to androstenedione. Small amounts of immunoreactive bands comigrating with purified cytochromes P450j, P450b, and P450p were detected by Western blot analysis in rat brain microsomes, while only an immunoreactive protein related to cytochrome P450p was found in the mitochondrial fractions. Immunoinhibition studies showed that BEA33, a monoclonal antibody to cytochrome P450b and simultaneously recognizing cytochromes P450e and P450a, was able to inhibit the metabolism of testosterone to the 1 beta-, 15 alpha-, 2 beta-, and 6 alpha-hydroxylated metabolites, whereas polyclonal anti-cytochrome P450p did not inhibit the formation of the 6 beta-hydroxytestosterone by rat brain microsomes. The metabolism of testosterone by rat brain microsomal or mitochondrial fractions was refractory to induction by 3-methylcholanthrene or pregnenolone-16 alpha-carbonitrile. Thus, in the brain multiple isozymes of cytochrome P450 are constitutively expressed in different subcellular fractions, which suggests that brain cytochrome P450 may play an important role in the metabolism of endogenous compounds. The significance and role of cytochrome P450p-related protein in the rat brain mitochondrial fraction are yet to be determined.

Animals↗

PubMedDate not supplied

[Metabolism of riboflavin and its nucleotides in rat liver subcellular fractions in alloxan diabetes].

In alloxan-induced diabetes the content of riboflavin and FMN goes up in all of the subcellular fractions and that of FAD in the nuclear liver fraction alone. While administration of insulin to control animals reduces the riboflavin and FMN level in all of the subcellular fractions of the liver, in the diabetic animals such a decline is observed in the nuclei alone. Insulin brings down the FAD level in the nuclear liver fraction of both the control and test animals. An additional introduction of riboflavin depresses and that of FMN - raises the FAD content in diabetic animals as compared to controls.

Animals↗

PubMed1977

Binding of mercury and selenium in subcellular fractions of rat liver and kidneys following separate and joint administration.

The distribution of mercury and selenium has been examined in subcellular fractions of rat liver and kidneys in prolonged exposure to HgCl2 and Na2SeO3 administered separately and simultaneously. The molar ratio of mercury and selenium concentrations in subcellular fractions of the organs examined varied considerably. Selenium displaced mercury from the soluble kidney fraction bound mainly with metallothionein to the nonhistone protein fraction of liver nuclei. The Hg-stimulated biosynthesis of metallothionein has been eliminated under the influence of selenium.

Animals↗

PubMed1976

Preparation of subcellular fractions from rat liver: comparison of the Polytron with the Dounce homogenizer.

The PolytronR and Dounce homogenizers have been evaluated for preparation of homogenates of rat liver prior to isolation of subcellular fractions by differential centrifugation. Marker enzymes used to evaluate the subcellular fractions included cytochrome oxidase, monoamine oxidase, D-amino acid oxidase, acid phosphatase, glucose-6-phosphatase, ethyl morphine demethylase, and lactate dehydrogenase. No significant difference in the distribution of enzymes (percent recovery or specific activity) was observed between the two methods of homogenization. In addition, there were no significant differences in the ultrastructural appearances and respiratory control ratios of the mitochondrial fractions prepared by the two methods of homogenization.

Animals↗

PubMed1977

Comparative studies on the influence of some radioprotectors on the postirradiation hydrolytic function of subcellular fractions of pancreas.

Comparative studies on the influence of the two radioprotectors Fenchlorfos and cysteamine on the hydroproteolytic activity of pancreatic subcellular fractions of rats were carried out. The animals were irradiated with the single dose of 800 R of X-rays. Biochemical estimation of lipase, beta-glucuronidase, acid phosphatase and catheptic (caseinolytic) activity were done 24 hours after exposition. The two radioprotective substances have a lot of common in their action against hydrolytic activity. They induce the decrease of some enzymes of subcellular fractions we checked both in control as well as in irradiated groups. The ambivalent influence of Trichlorfon on the lipase activity in granules and supernatant fraction seems to be of interest.

Animals↗

PubMed1979

Taurine effects on 45Ca2+ transport in retinal subcellular fractions.

The effect of taurine on 45Ca2+ transport by subcellular fractions from the chick retina was examined. An inhibitory action of taurine on 45Ca2+ uptake was observed in retinal fractions incubated for 1--5 min in a Krebs--bicarbonate medium, pH 7.4. In the crude nuclear fraction, 25 mM taurine produced a decrease of 50% in 45Ca2+ uptake; in the crude synaptosomal fraction, taurine reduced 45Ca2+ accumulation by 70%; the maximum inhibitory effect of taurine on 45Ca2+ uptake (80%) was observed in a fraction containing outer segments and pigment epithelium cells. Taurine effect was specific, dose-dependent and related to osmotically sensitive particles. The results suggest a role for taurine in the regulation of calcium fluxes in the retina.

Amino Acids↗

PubMed1976

Calcium accumulation and enzymatic activities of subcellular fractions from aortas and ventricles of genetically hypertensive rats.

Subcellular fractions were obtained from aortas and ventricles of 6-month-old spontaneously hypertensive and normotensive Wistar rats by the use of differential and sucrose density gradient centrifugation. These preparations were studied to determine what alterations in calcium accumulation and enzymatic activities might be associated with hypertension. The total amount of calcium accumulation (in the presence of ATP and 17 muM free calcium) by the plasma membrane-enriched fraction from hypertensive rat aortas significantly less than that from normotensive rats (11.3 +/- 0.4 vs 16.2 +/- 1.6 mumol of calcium/g of protein, n = 8). In contrast the specific activities of the plasma membrane marker enzymes, 5'-nucleotidase and phosphodiesterase I, were 80% and 40% greater, respectively, in the hypertensive than in the normotensive fractions. On the other hand, various fractions from ventricles of the two types of rats were generally similar in enzyme activities and calcium accumulation. The decreased rate of relaxation of aortas from spontaneously hypertensive rats may be caused by the decreased rate of calcium transport demonstrated in this study.

Animals↗

PubMed1975

Distribution and properties of cholinesterases in subcellular fractions from rat jejunum and heart tissue.

Homogenates of rat jejunum and heart tissue in 0.3 M sucrose have been separated into different subcellular fractions by centrifugation. The distribution of cholinesterases in these two tissues is different. Acetylthiocholine (Ac), propionylthiocholine (Pc), and butyrylthiocholine (Bc) iodides were used as substrates. Using 1 mmol/1 Ac in the presence of hexafluorenium, a local anaesthetic drug, inhibition percentages of these subcellular cholinesterases were found to be different. The differences in the effect of hexafluorenium were most marked at a concentration of 5 mumol/1. Four electrophoretically distinct subcomponents of cholinesterase have been identified in different subcellular fractions from both jejunum and heart tissues. The electrophoretic profile, as determined by polyacrylamide disc electrophoresis, of different subcellular cholinesterases in these two tissues also appears to be different.

Acetylthiocholine↗

PubMed1976

Calcium uptake by subcellular fractions of human umbilical artery.

Two different mechanisms for the active accumulation of Ca2+ by subcellular fractions of human umbilical artery are described. One, located in the mitochondrial fraction, was induced by exogenous ATP or respiratory substrates (ADP and succinate) and was inhibited by azide. The other, located in the microsomal fraction, was induced by ATP and potentiated by oxalate, but not inhibited by azide. Increasing ATP concentrations up to 4-5 mM increased microsomal Ca2+ accumulation, whereas increasing ATP concentration above 2-3 mM caused inhibition of mitochondrial Ca2+ uptake. Although changing pH from 7.4 to 7.2 had no effect on mitochondrial Ca2+ accumulation, it doubled microsomal uptake. Neither adenosine 3',5'-monophosphate nor guanosine 3',5'-monophosphate in the presence or absence of protein kinase and kinase modulator affected Ca2+ uptake by or phosphorylation of the subcellular fractions. Partially purified protein kinases from umbilical and beef skeletal muscle contained a component(s) distinguishable from the kinase on the basis of its heat stability that enhanced ATP-induced Ca2+ uptake by mitochondrial fractions from the umbilical artery. It is suggested that alterations in Ca2+ sequestration induced by changes in ATP concentration and intracellular pH in mitochondrial and microsomal fractions, respectively, could play a role in the control of arterial patency and closure with changes in PO2.

Adenosine Triphosphate↗

PubMed1975

Phosphatidate phosphohydrolase and palmitoyl coenzyme A hydrolase in cardiac subcellular fractions.

The characteristics of the two enzymes related to fatty acid esterifaction were studied in order to provide fundamental information leading to further understanding of the control of myocardial glyceride formation. Palmitoyl-CoA hydrolase and phosphatidate phosphohydrolase are both distributed unevenly among heart subcellular fractions. The activity of the latter enzyme in subcellular fractions changes independently in response to Mg2+ addition and in response to thyroid hormone treatment of animals.

Animals↗

PubMed1978

NADPH-oxidation activities in subcellular fractions isolated from resting or phagocytozing human polymorphonuclears.

Using a fluorometric assay for the determination of oxidized pyridine nucleotides (NAD[P]+), total and cyanide-resistant NADPH-oxidative activities have been measured in subcellular fractions isolated from resting and phagocytosing human polymorphonuclears. Enzymatic activies responsible for the oxidation of the NADPH have been recovered in the heavy particles (15,000g/15 min), the low-density particles (100,000g/30 min), and the cytosolic fraction. Stimulation of the cells with opsonized zymosan had a different effect on the NADPH-oxidative activities of these subcellular fractions, which suggests the involvement of various types of enzymatic systems in the oxidation of NADPH. The cytosolic fraction interacted strongly with the enzymatic activities occurring in the sedimentable fractions and is therefore thought to play a central role in the regulation of the activation of the oxidative metabolism associated with phagocytosis.

Cell Fractionation↗

PubMedDate not supplied

[Localization of retinylphosphatase activity in subcellular fractions of rat liver].

Enzymic hydrolysis of retinyl phosphate in subcellular fractions of the rat liver has been investigated. A quantitative method of measuring activity of retinyl phosphatase has been developed. The method is based on the isolation of labeled retinol, the substance formed from chemically synthesized labeled retinyl phosphate through enzymatic hydrolysis. It has been shown that the major quantity of retinyl phosphatase activity (75%) with a specific activity of 80 micrograms retinyl phosphate hydrolyzed for 30 min/mg protein at 30 degrees C occurs in plasmatic membranes.

Animals↗

PubMed1976

Control by TSH of protein turnover in thyroid subcellular fractions.

The action of TSH on protein turnover in various subcellular fractions has been investigated in dog thyroid slices incubated in vitro. The results suggest a general inhibition by TSH of protein catabolism. Using double labeline (3/ and 14C) of the proteins, an increase of the disappearance of some labeled material from the microsomal fraction in the presence of TSH has been observed. The protein nature of this material has been established by testing its susceptibility to hydrolysis by trypsin. The fact that the microsomal pellet had to be treated by triton X 100 before hydrolysis by trypsin could occur, suggests that the material is probably enclosed in, or protected by membrane vesicles. Its high molecular weight and its ability to be immunoprecipitated by an antithyroglobulin serum suggest that the microsomal protein, the disappearance of which is stimulated by TSH, is thyroglobulin or one of its subunits. It is suggested that our results reflect the acceleration by TSH of the vectorial transfer of thyroglobulin through the membranes of the endoplasmic reticulum to the colloid space.

Animals↗

PubMed1978

The distribution of dietary plant sterols in serum lipoproteins and liver subcellular fractions of rats.

Rats were fed plant sterols containing campesterol and beta-sitosterol in the differerent proportions, and their distribution in serum lipoproteins and in liver subcellular fractions was determined. In serum lipoproteins, the percentage as well as the concentration of plant sterols increased with the increase in the density of lipoproteins. Thus, high density lipoprotein (HDL) contained the highest and very low density lipoprotein (VLDL), the lowest. Also, there were distinct differences in the ratio of campesterol to sitosterol among lipoproteins, it was the highest in VLDL and lowest in HDL. Quantitatively, more than 75% of campesterol and 80% of sitosterol were carried in HDL; the values were significantly different from those of cholesterol (ca. 70%) in relation to total cholesterol. The distribution of plant sterols in liver subcellular fractions was virtually the same with that of cholesterol. Both nuclei and microsomes contained approximately 40% of total plant sterols.

Animals↗

PubMed1975

Effect of denervation on the phospholipid content in subcellular fractions of tonic and tetanic muscles.

Four weeks after denervation, various changes were observed in the phospholipid composition of the sarcolemmal and sarcoplasmic fractions of skeletal muscles with different functions. Neurotomy also affected the innervated contralateral muscles and produced opposite changes in the phospholipid content of subcellular fractions. The increase in the amount of phospholipids in the sarcolemmal fractions of the denervated muscles was only apparent. The difference between the denervated and contralateral muscles was also due to the decrease of phospholipids in the contralateral muscles. These changes were more pronounced in the tetanic (fast-twitch) than in the tonic (slow-twitch) muscles. In the sarcoplasmic fraction of the denervated tetanic muscle an increase, while in that of the tonic one a slight decrease of phospholipids appeared. In contrast, the phospholipid content in the sarcoplasmic fractions of contralateral muscles did not decrease, while it increased slightly in the tonic muscle. The amount of plasmalogens (fatty aldehyde: lipid phosphorus ratio) decreased only in the subcellular fractions of the denervated muscles while there was no change in those of the contralateral muscles.

Aldehydes↗

PubMed1977

Binding of mescaline with subcellular fractions upon incubation of brain cortex slices with [14C] mescaline.

Incubation of brain cortex slices in the presence of glucose resulted in the permeation of about 65% of [14C] mescaline into slices. Of this, about one-third radioactivity was bound with nuclei, mitochondria, microsomes, and ribosomes. Dialysis of subcellular fractions did not markedly reduce the amounts of radioactivity bound to the fractions. The permeation into slices and the binding of mescaline to subcellular fractions were fairly time-dependent, but were inhibited by the presence of potassium cyanide, or by the absence of glucose and by heating to 80 degrees C for 1 min.

Animals↗

PubMed1979

Lipid composition and microviscosity of subcellular fractions from rabbit thymocytes. Differences in the microviscosity of plasma membranes from subclasses of thymocytes.

There are indications from freeze-fracture experiments that subclasses of rabbit thymocytes show different mobilities of plasma membrane components. Consequently, one would expect differences in the fluidity of the plasma membrane. For this reason, rabbit thymocytes were separated on a Ficoll/Metrizoate gradient yielding three subclasses representing various levels of cell differentiation. These thymocyte subclasses did not show any significant differences in the degree of fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. The fluorescence polarization of the plasma membrane may be overshadowed by the contribution of all cellular lipids due to penetration of the fluorescent probe into the cell. Therefore, plasma membranes were isolated from rabbit thymocytes using a cell-disrupting pump, differential centrifugation, and sucrose density gradient centrifugation. As shown by biochemical and electron microscopical analyses, plasma membranes with a high degree of purity were obtained. As expected the plasma membrane fractions showed a higher microviscosity than the other subcellular fractions. This was attributed to a higher cholesterol to phospholipid molar ratio and a higher degree of saturation of phospholipid fatty acid chains. Subsequently, the microviscosity was measured of plasma membrane preparations obtained from two main subclasses of thymocytes representing mature and immature lymphocytes. The immature thymocytes yielded two plasma membrane fractions with higher microviscosity than the mature cells. These finding is in line with earlier observed differences in the glycerol-induced clustering of intramembranous particles. Furthermore, the results of this study support the view that the fluorescence polarization technique applied to whole cells does not exclusively monitor the plasma membrane.

Animals↗