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PubMed1993

Characterization of phosphatidic acid phosphohydrolase in neutrophil subcellular fractions.

This investigation was designed to confirm the presence of PA phosphohydrolase in human neutrophils and to determine the distribution and characteristics of the enzyme in soluble and particulate subcellular fractions of disrupted neutrophils. Enzyme activity was detected in unseparated extracts of sonicated neutrophils. The majority of the recovered activity was recovered in a particulate fraction rich in neutrophil plasma-membrane markers; moderate levels (20%) of the total activity were recovered in the cytosol. While Mg2+ markedly potentiated the cytosolic but not the particulate activity, Ca2+ moderately inhibited both the cytosolic and particulate enzymes. The plasma-membrane-associated activity was absolutely dependent on detergent (0.5% Triton X-100) and displayed an apparent Km of 62 microM for phosphatidic acid. Enzyme activity was markedly inhibited by NaF, not influenced by excess glycerophosphate and slightly attenuated by propranolol, an inhibitor of PA phosphohydrolase in other systems. Preincubation of plasma membranes with N-ethylmaleimide at concentrations up to 25 mM had little effect on enzyme activity. However, activity in cytosolic and microsomal fractions of neutrophils were completely abolished by preincubation with N-ethylmaleimide at concentrations of less than 5 mM. We conclude that neutrophils possess a potent PA phosphohydrolase localized in their plasma membranes. Metabolism of cellular second-messengers by this enzyme may exert a profound effect on the functions of stimulated neutrophils.

Calcium↗

PubMed1961

Autoradiographic analysis on agar plates of antigens from subcellular fractions of rat liver slices.

Slices of rat livers were incubated with 14C amino acids, homogenized, and subjected to differential centrifugation. The microsomes were further extracted with the non-ionic detergent Lubrol W and with EDTA. These extracts and the microsome free "cell sap," freed from the pH 5 precipitable fraction, were subsequently reacted with antisera using agar diffusion techniques. The antisera employed were obtained from rabbits injected with different subcellular fractions of rat liver or with rat serum proteins. When the agar diffusion plates were autoradiographed it was found that some of the precipitates were radioactive while others were not. Control experiments indicated that this labeling was due to the specific incorporation of (14)C amino acids into various rat liver antigens during incubation of the slices rather than to a non-specific adsorption of radioactive material to the immunological precipitates. When the slices were incubated with the isotope for up to 30 minutes, the serum proteins which could be extracted from the microsomes with the detergent were strongly labeled, as were a number of additional microsomal antigens of unknown significance. In contrast, the serum proteins present in the cell sap were only weakly labeled. Most of the typical cell sap proteins, both those precipitable and those soluble at pH 5, seemed to remain unlabeled. No consistently reproducible results were obtained with the EDTA extracts of the ribosomal residues remaining after extraction of the microsomes with the detergent. Incubation of the liver slices for longer periods (up to 120 minutes) led to a strong labeling of the serum proteins in the cell sap as well as to the appearance of labeling in additional cell sap proteins. The results are discussed with regard to the subcellular site of synthesis and the metabolism of the different antigens.

Agar↗

PubMedDate not supplied

[Distribution of radioactivity in subcellular fractions of genital tract tissue and skeletal muscles of guinea pig embryos after administration of 1 alpha,2 alpha-3H(n) testosterone in vitro].

The dynamics of radioactivity in subcellular fractions of the reproductive system and muscle has been studied in the guinea pig embryos from the 21st day of development till birth after the preincubation in a medium containing 1 alpha,2 alpha-3H(n) testosterone (T-3H). The total uptake of T-3H per mg tissue is higher in the reproductive system than in the muscle and attains the highest values during the period of differentiation of target organs by the male type (30-33 days). The nucleocytoplasmic ratio of radioactivity is shifted towards its increase in the nucleus with maximum on the 41st-45th days and by the moment of birth. Radioactivity in the microsomal fraction is higher than in the cytosol (soluble proteins). From the 27th day of development on, when the hormone uptake becomes selective, the radioactivity in the cytosol from the reproductive organs is higher than in the muscle tissue. The data obtained suggest the importance of processes in microsomes for differentiation and organogenesis of reproductive system.

Animals↗

PubMed1976

Distribution of protein kinase activities in subcellular fractions of rat brain.

The subcellular distribution of histone and phosvitin kinase activities in brain has been studied and the ability of the various fractions to catalyse the phosphorylation of their endogenous proteins (intrinsic protein kinase activity) also examined. Synaptosome membrane fragments have little or no histone or phosvitin kinase activity but contain the highest concentration of cyclic AMP-stimulated intrinsic protein kinase activity. Homogenisation of the membrane fragments in Triton X-100 increased the histone kinase activity but on centrifugation it was all recovered in the supernatant, while the insoluble material contained all the intrinsic protein kinase activity. These results indicate that the intrinsic protein kinase activity of cerebral membrane fragments is due to the presence of a kinase enzyme which is specific to certain membrane proteins. The intrinsic protein kinase activity of synaptosome membrane fragments is a rather slow reaction which takes several minutes to saturate all the acceptor proteins.

Animals↗

PubMed2000

Protein N-arginine methylation in subcellular fractions of lymphoblastoid cells.

Arginine methylation in RNA-binding proteins containing arginine- and glycine-rich RGG motifs is catalyzed by specific protein arginine N-methyltransferase in cells. We previously showed that lymphoblastoid cells grown in the presence of an indirect methyltransferase inhibitor, adenosine dialdehyde (AdOx), accumulated high level of hypomethylated protein substrates for the endogenous protein methyltransferases or recombinant yeast arginine methyltransferase [Li, C. et al. (1998) Arch. Biochem. Biophys. 351, 53-59]. In this study we fractionated the lymphoblastoid cells to locate the methyltransferases and the substrates in cells. Different sets of hypomethylated methyl-accepting polypeptides with wide range of molecular masses were present in cytosolic, ribosomal, and nucleus fractions. The methylated amino acid residues of the methyl-accepting proteins in these fractions were determined. In all three fractions, dimethylarginine was the most abundant methylated amino acid. The protein-arginine methyltransferase activities in the three fractions were analyzed using recombinant fibrillarin (a nucleolar RGG protein) as the methyl-accepting substrate. Fibrillarin methylation was strongest in the presence of the cytosolic fraction, followed by the ribosomal and then the nucleus fractions. The results demonstrated that protein-arginine methyltransferases as well as their methyl-accepting substrates were widely distributed in different subcellular fractions of lymphoblastoid cells.

Adenosine↗

PubMed1977

The effects of Ca2+ antagonists on Ca2+ accumulation by subcellular fractions of rat myometrium.

Several agents which antagonize or interact with Ca2+ in the excitation-contraction coupling of smooth muscle have been tested for their effects on Ca2+ accumulation by subcellular fractions of the rat uterus. Na+, but not K+, reduced the amount of Ca2+ that could be accumulated by the plasma membrane fraction (PM) and the endoplasmic reticular fraction (ER). Sr2+ considerably reduced Ca2+ accumulation by PM, ER, and the mitochondrial fraction; Ba2+ had a similar effect but was not as potent as Sr2+. La3+ at concentrations up to 10 mM was unable to block Ca2+ accumulation by PM and ER completely. The pharmacological agents D600, verapamil, and chlorpromazine all inhibited Ca+ accumulation at dose levels much higher than those required to inhibit contractility, and were ineffective at lower doses. The action of Ca2+ antagonists is seen to be complex, involving interactions at different sites and on different processes. For a fuller understanding of their mechanisms of action further studies upon passive binding and on the release of Ca2+ from subcellular sites are required.

Animals↗

PubMed1989

Effect of chronic ethanol consumption on the content of alpha-tocopherol in subcellular fractions of rat liver.

The effects of long-term administration of ethanol (35% of total energy for 6-8 weeks) on the distribution and concentration of alpha-tocopherol in subcellular fractions of rat liver have been studied. Marker enzymes were measured in all fractions. The highest concentration of alpha-tocopherol was found in the light mitochondrial fraction both in ethanol-fed and control rats, 754 +/- 104 and 1127 +/- 126 pmol/mg protein, respectively. The microsomal, heavy mitochondrial, and nuclear fractions also had high concentrations of alpha-tocopherol, whereas the cytosolic fraction contained minor amounts. In the light mitochondrial fraction we found the highest concentration of alpha-tocopherol in lysosomes, whereas small amounts were detected in peroxisomes. In the microsomal fraction the highest concentration was found in the Golgi apparatus. The content of alpha-tocopherol in the light mitochondrial fraction was reduced by 33% (p less than 0.02) in the ethanol-fed group as compared to the controls. In the other fractions no significant differences between the two groups were observed. Long term administration of ethanol promoted, however, a further enrichment of alpha-tocopherol (178% higher than controls) in the Golgi apparatus, possibly due to reduced secretion of very low density lipoprotein-associated alpha-tocopherol.

Animals↗

PubMed1985

Relation between Ca metabolism and ATPase activities in the subcellular fractions from duck eggshell gland mucosa after DDE administration during different stages of eggshell formation.

The ATP-dependent Ca2+ uptake by and the Ca2+-Mg2+-activated ATPase activity in the subcellular fractions from the eggshell gland of two varieties of ducks, Indian Runner ducks (IRD) and the cross-breed of Swedish and Rouen ducks (SRD), showed functional changes during eggshell formation in relation to the values in the resting state. DDE inhibited the Ca2+ uptake (Ca) but increased the Ca2+-Mg2+-ATPase activity (P) of fractions from active but not of those from resting glands. DDE reduced the Ca/P ratio of several fractions from active glands. DDE inhibited the functional increase in Ca2+-Mg2+-ATPase in the IRD homogenate, however. The effect of DDE on the eggshell index of mature eggshell was greater in IRD than in SRD, the reason for this being that the effect was exerted during a shorter time period in the latter variety.

Animals↗

PubMed1983

Analytical subcellular fractionation of cultivated mouse resident peritoneal macrophages.

Resident peritoneal macrophages of the mouse, cultivated for 3 d, have been studied by quantitative subcellular fractionation using differential centrifugation and density equilibration in linear gradients of sucrose. Density equilibration experiments were carried out on untreated cytoplasmic extracts, on cytoplasmic extracts treated with digitonin or sodium pyrophosphate, and on cytoplasmic extracts derived from cells cultivated for 24 h in the presence of Triton WR-1339. The enzyme distributions obtained distinguished six typical behaviors characteristic of distinct subcellular entities. Acid alpha-galactosidase and other acid hydrolases displayed the highest average velocity of sedimentation and equilibrium density. Culturing in a medium that contained Triton WR-1339 markedly decreased their density, most likely as a result of Triton WR-1339 accumulation within lysosomes. Cytochrome c oxidase and the sedimentable activity of malate dehydrogenase showed a narrow density distribution centered around 1.17, very similar under all the experimental situations; their rate of sedimentation fell within the range expected for mitochondria. Catalase was particle-bound and exhibited structure-linked latency (80 percent); it was released in soluble and fully active form by digitonin, but this required a much higher concentration than in the case of lysosomal enzymes. Differences relative to all the other enzymes studied suggest the existence of a particular species of organelles, distinctly smaller than mitochondria, and possibly related to peroxisomes. Many enzymes were microsomal in the sense that the specific activities, but not the yields, were greater in microsomes than in other fractions obtained by differential centrifugation. These enzymes were distinguished in three groups by their properties in density equilibration experiments. NAD glycohydrolase, alkaline phosphodiesterase I, and 5'-nucleotidase had low equilibrium densities but became noticeably more dense after addition of digitonin. The other microsomal enzymes were not shifted by digitonin, in particular N-acetylglucosaminyltransferase and galactosyltransferase, which otherwise equilibrated at the same position in the gradient. We assign the digitonin-sensitive enzymes to plasma membranes and possibly to related endomembranes of the cells, and the two glycosyltransferases to elements derived from the Golgi apparatus. Finally, alpha-glucosidase, sulphatase C, NADH cytochrome c reductase, NADPH cytochrome c reductase, and mannosyltransferase, equilibrated at a relatively high density but were shifted to lower density values after addition of sodium pyrophosphate. These properties support their association with elements derived from the endoplasmic reticulum.

Animals↗

PubMed1982

[De-etherification of estradiol 3-propyl ether by subcellular fractions of rat liver].

The incubation of PE2 ((6,7-3H)-labelled 3-propyl ether of estra-1,3,5(10)triene-3, 17 beta-diol (estradiol)) with various subcellular fractions of rat liver indicated that the hepatic metabolism of this compound occurs mainly in the microsomal fraction. In addition to the formation of 3-propyl ethers of estra-1,3,5(10)triene-3-ol-17-one (estrone) and estra-1,3,5(10-triene-3, 16alpha, 17 beta-triol (estriol) directly deriving from PE2, the microsomal proteins carried out the deetherification of the propyl ether group leading to phenolic steroids; among them, estradiol, estrone, and estriol were characterized. Protein-bound and water-soluble metabolites were found; the effects of glutathione and of the incubation conditions were in agreement with the thioconjugation of these derivatives. The microsomal metabolism of PE2, and specially the deetherification reaction, required the presence of oxygen and of NADPH as cofactor, the optimum pH ranging from 7.4 to 8. The participation of cytochrome P450 in these metabolic pathways was shown by a partially inhibited catabolism with carbon monoxide and by a more active metabolism in males than in females and when animals were pretreated with phenobarbital. These results allowed us to conclude that the hepatic deetherification of PE2 is carried out by a microsomal oxidative system which is very similar to the system involved in the demethylation of methyl ethers of estrogens.

Animals↗

PubMed1992

Transferrin and iron distribution in subcellular fractions of K562 cells in the early stages of transferrin endocytosis.

Iron distribution in subcellular fractions was investigated at different times after a single cohort of 59Fe-125 I-labeled transferrin (Tf) endocytosis in K562 cells. Cell homogenates prepared by hypotonic lysis and deoxyribonuclease (DNAase) treatment were fractionated on Percoll density gradients. Iron-containing components in the postmitochondrial supernatant were further fractionated according to their molecular weight using gel chromatography and membrane filtration. In the initial phases of endocytosis, both iron and Tf were found in the light vesicular fraction. After 3 min the labels diverged, with iron appearing in the postmitochondrial supernatant and Tf in the heavy fraction containing mitochondria, lysosomes and nuclei. Iron released from Tf-containing vesicles appeared both in low- and high-molecular-weight fractions in the postmitochondrial supernatant. After 5 min of endocytosis 59Fe activity in the low-molecular-weight fraction remained constant and 59Fe accumulated in a high-molecular-weight fraction susceptible to desferrioxamine chelation. After 10 min, 59Fe radioactivity in this fraction decreased and a majority of cytosolic 59Fe was found in ferritin. These results do not support the concept of the cytosolic low-molecular-weight iron pool as a kinetic intermediate between transferrin and ferritin iron in K562 cells.

Cell Line, Transformed↗

PubMedDate not supplied

[Effect of L-thyroxine on content of carbohydrate components of glycoproteins in subcellular fractions of liver, thyroid gland and blood cells of rats].

Content of carbohydrate components of glycoproteins (sialic acids, hexosamines, hexoses bound with proteins and L-fucose), their correlation and normalization in subcellular fractions of liver and thyroid gland tissues as well as in blood cells were studied after administration of various doses of L-thyroxin. Various doses of the hormone caused a dissimilar effect on alterations of the carbohydrate components in the structural elements of the tissues studied. Within 24 days after the hormone administration its small doses increased generally the carbohydrate content, middle doses--decreased their content in mitochondria and microsomes but increased in homogenate, plasmatic, mitochondrial and microsomal membranes, high doses--decreased the carbohydrate components in all the preparations studied. The period of normalization in the level of carbohydrate-containing components of glycoproteins from rat liver tissue depended on the dose of L-thyroxin administered. Quantitative impairments in carbohydrate-containing components of glycoproteins are discussed.

Animals↗

PubMed1989

Intracellular transport and degradation of 125I-tyramine cellobiose-labelled low-density lipoprotein endocytosed in vivo in rat liver cells studied by means of subcellular fractionation.

The intracellular transport and degradation of in vivo endocytosed 125I-tyramine cellobiose-labelled low density lipoprotein (125I-TC-LDL) in rat liver cells were studied by means of subcellular fractionation in Nycodenz, sucrose and Percoll density gradients, as well as by means of analytical differential centrifugation. Initially, labelled LDL was located in endocytic vesicles of low densities. Subsequently, acid-soluble and acid-precipitable radioactivities were found in organelles with buoyant densities distinctly lower than that of the main peaks of the lysosomal marker enzymes acid phosphatase and N-acetyl-beta-glucosaminidase. These prelysosomal organelles may represent multivesicular bodies (MVBs). Finally, 6 h after injection and onwards, the acid-soluble radioactivity cosegregated completely with the two lysosomal marker enzymes, suggesting that the degradation products were in secondary lysosomes. The rate of intracellular processing of LDL was very slow compared to that of asialoglycoproteins, suggesting that LDL followed a unique intracellular pathway, that may be specific for this type of ligand.

Animals↗

PubMed2005

Selective determination of the doxorubicin content of individual acidic organelles in impure subcellular fractions.

Since organelle preparations often contain more than one organelle type (e.g., acidic organelles and mitochondria), techniques that measure the properties of a given organelle type while avoiding biases caused by ancillary subcellular compartments are highly desirable. We report here the use of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) dual-channel detection to identify acidic organelles containing doxorubicin (DOX) in crude subcellular fractions from CCRF-CEM and CEM/C2 cell lines. As confirmed by confocal microscopy, acidic organelles are identified by their accumulation of fluorescently labeled nanospheres. Using CE-LIF analysis, individually detected organelles are classified into three kinds: acidic organelles containing only nanospheres, acidic organelles containing nanospheres and DOX, and other organelles containing DOX (e.g., mitochondria) with no detectable nanospheres. Electrophoretic mobility, DOX fluorescence intensity, and nanosphere fluorescence intensity distributions of individual acidic organelles and other organelles containing DOX are determined in the same CE-LIF run. The acidic organelle mobilities range from (-0.7 to -2.0) x 10(-4) cm(2) V(-1) s(-1) while those of the other organelles spread from (-0.6 to -3.5) x 10(-4) cm(2) V(-1) s(-1). In addition, by calibrating the detector response, DOX content in individual acidic organelles and other organelles can be estimated. The average amounts of DOX per acidic organelle in CEM/C2 and CCRF-CEM cells are 11.1 +/- 0.5 and 10.6 +/- 0.4 zmol, respectively. This first report of an analysis of the accumulation of DOX in individual acidic organelles presents a procedure for analyzing the accumulation of fluorescent compounds in acidic organelles that could be useful for investigating acidic organelle maturation and the role of these organelles in drug resistance.

Antibiotics, Antineoplastic↗

PubMedDate not supplied

[Energy metabolism in subcellular fractions of normal and acute ischemic kidneys].

The level of oxidative phosphorylation, activity of phosphofructokinase, fructose-1,6-diphosphate aldolase, ketose-1-phosphate aldolase, glucose-6-phosphatase and lactate dehydrogenase are determined in subcellular fractions in the kidney cortex layer of rabbits which have suffered from acute ischemia (for 15, 30, 60, 120 min). Ischemia inhibits the oxidative processes in mitochondria which is proportional to the duration of the effect. An increase in the activity of glycolytic chain enzymes in microsomes and soluble fraction for 15-30 min of ischemia evidences for a compensation of the energy losses at the expense of glycolysis with short periods of ischemia. Glycolysis is inhibited with a more prolonged effect. It is established that the anti-ischemic protection of the organ viability is to be conducted not only with allo- but also with auto-transplantation of the kidney in case of short acute ischemia.

Animals↗

PubMed1984

Proteolytic degradation and modification of rat prolactin by subcellular fractions of the rat ventral prostate gland.

We previously showed that at pH 7.4, the cytosol and a low speed pellet (3300 X g) of rat ventral prostate degraded rat PRL, whereas a high speed pellet (25,000 X g) was inactive. The current study further explores PRL degradation by ventral prostatic tissue. Enzyme markers indicated that the low speed pellet was mitochondria enriched (cytochrome c oxidase), and the high speed pellet was lysosome enriched (acid phosphatase), although there was considerable cross-contamination between the two fractions. Proteolysis of hormone was examined by incubating 125I-labeled rat iodo-PRL with tissue fractions, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and radioautography to identify products and quantify the extent of PRL degradation. Acidification of the incubation medium (pH 4-5) to optimize lysosomal protease activities inhibited PRL degradation in cytosol and activated the process in both pellets consistent with the distribution of acid phosphatase. Under acidic incubation conditions, both low and high speed sediments produced the same electrophoretic pattern of PRL degradation, indicating the generation of peptide fragments from the hormone with a molecular mass of approximately 16,000-8,000 daltons. Cytosol (pH 7.4) also produced peptide fragments in this weight range, although at much lower rates and in relatively lower proportions. Peptide fragment formation from PRL by the low speed pellet (pH 7.4) was minimal. On a protein basis, PRL degradation by the high speed sediment (pH 4.5) was 3-10 times that by the low speed sediment (pH 7.4). Enzyme inhibitor analysis indicated differences in each of the cell fractions with regard to the types of proteases involved in PRL degradation. When compared with other tissues of the male rat, ventral prostate was the most active in degrading PRL and generating peptide fragments. In addition, kidney, spleen, and lung were among the more active tissues, whereas liver, was deferens, and dorsolateral prostate were relatively less active in degrading hormone. Our studies indicate that qualitatively different processes degraded PRL in each of the active subcellular fractions and that the generation of peptide fragments from PRL predominates in a lysosome-rich fraction of ventral prostate. The possible significance of subcellular variations in PRL processing and the generation of peptide fragments of the hormone by peripheral tissue are discussed.

Animals↗

PubMed1989

Monoclonal antibody production against a subcellular fraction of vegetal pole cytoplasm containing the germ plasm of Xenopus 2-cell eggs.

In attempts to understand the molecular nature of substances localized in the germ plasm (GP), monoclonal antibodies against a subcellular fraction of vegetal pole cytoplasm containing the GP of Xenopus 2-cell eggs were produced. The monoclonal antibody produced by the hybridoma cell line (designated as no 48) reacted specifically with the GP of embryos at the cleavage stages. Beyond the gastrula stage, the antibody reacted with not only the GP of germ line cells but also the cytoplasm of somatic cells. By immunoblotting analysis with two-dimensional (2-D) gel the antibody was shown to react with two protein spots of approx. 40 kDa, with a pI of 6.0-6.5.

Animals↗

PubMed1989

Alterations in the protein-synthesis, -degradation and/or -secretion rates in hepatic subcellular fractions of selenium-deficient mice.

Single mouse livers were subfractionated by differential centrifugation and isopycnic centrifugation on sucrose or metrizamide gradients and separated into subcellular compartments. The fractionation procedure was highly reproducible and yielded essentially similar results in different preparations of livers from selenium-adequate (Se+) and selenium-deficient (Se-) mice that were fed on a Torula-yeast-based diet containing less than 10 parts per 10(9) of selenium for at least 16 weeks. Mice of both dietary groups were injected intraperitoneally with 370 kBq of L-[U-14C]leucine, and 48 h later 1.85 MBq of L-[4,5-3H]leucine was injected intraportally. After another 1 h, the livers were removed and subjected to subcellular fractionation. Incorporation of the 3H label into proteins of the subcellular fractions was taken as a measure of relative protein-synthesis rate. The ratio of the 3H to the 14C protein-bound label of the same fractions was used as an estimate of relative protein-degradation and/or -secretion rate. The results showed a statistically significant 180% increase in protein-synthesis rate in the endoplasmic reticulum and a 80% increase in relative protein-degradation and/or -secretion rate in the same compartment. A significant decrease in the 3H/14C ratio, by 40 and 30% respectively, was observed in the Golgi fraction and in liver homogenate. The observed changes suggest a highly regulated hepatic response to selenium deficiency.

Animals↗