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PubMed2001

Differential induction of peroxisomal populations in subcellular fractions of rat liver.

In rat liver, peroxisome proliferators induce profound changes in the number and protein composition of peroxisomes, which upon subcellular fractionation is reflected in heterogeneity in sedimentation properties of peroxisome populations. In this study we have investigated the time course of induction of the peroxisomal proteins catalase, acyl-CoA oxidase (ACO) and the 70 kDa peroxisomal membrane protein (PMP70) in different subcellular fractions. Rats were fed a di(2-ethylhexyl)phthalate (DEHP) containing diet for 8 days and livers were removed at different time-points, fractionated by differential centrifugation into nuclear, heavy and light mitochondrial, microsomal and soluble fractions, and organelle marker enzymes were measured. Catalase was enriched mainly in the light mitochondrial and soluble fractions, while ACO was enriched in the nuclear fraction (about 30%) and in the soluble fraction. PMP70 was found in all fractions except the soluble fraction. DEHP treatment induced ACO, catalase and PMP70 activity and immunoreactive protein, but the time course and extent of induction was markedly different in the various subcellular fractions. All three proteins were induced more rapidly in the nuclear fraction than in the light mitochondrial or microsomal fractions, with catalase and PMP70 being maximally induced in the nuclear fraction already at 2 days of treatment. Refeeding a normal diet quickly normalized most parameters. These results suggest that induction of a heavy peroxisomal compartment is an early event and that induction of 'small peroxisomes', containing PMP70 and ACO, is a late event. These data are compatible with a model where peroxisomes initially proliferate by growth of a heavy, possibly reticular-like, structure rather than formation of peroxisomes by division of pre-existing organelles into small peroxisomes that subsequently grow. The various peroxisome populations that can be separated by subcellular fractionation may represent peroxisomes at different stages of biogenesis.

ATP-Binding Cassette Transporters↗
PubMed1981

Newly synthesized cholecystokinin in subcellular fractions of the rat brain.

The subcellular localization of in vivo synthesized cholecystokinin (CCK) in different parts of the rat brain was studied after intracisternal pulse injections of [35S]methionine. The rats were decapitated 1 h after the injection, and the brain was divided into cortex, hippocampus and remainder. Subcellular fractions were obtained according to Whittaker's method. De novo synthesized CCK in the crude mitochondrial-synaptosomal fraction, P2, and in the purified synaptosomal fraction was demonstrated by affinity chromatography, using antibodies specific for the COOH-terminal sequence of CCK. By subsequent gel chromatography two molecular forms of labelled CCK occurred, with elution constants, Kav, of 1.1 (corresponding to the COOH-terminal octapeptide) and of 1.40 (a component which may correspond to the COOH-terminal tetrapeptide amide, CCK-4). The findings support the idea that the small molecular forms are the transmitter forms of CCK.

Animals↗
PubMed1965

STUDIES ON THE TISSUE ACTIVATOR OF PLASMINOGEN. DISTRIBUTION OF ACTIVATOR AND PROTEOLYTIC ACTIVITY IN THE SUBCELLULAR FRACTIONS OF RABBIT KIDNEY.

1. On subcellular fractionation of rabbit kidney by differential and density-gradient centrifugation, a high proportion of the tissue activator of plasminogen activity was found to be particulate and displayed sedimentation properties associated with the lysosome-rich fraction as judged biochemically by the acid-phosphatase activity. 2. The activator activity is closely associated with a latent protease whose activity is enhanced in the presence of Triton X-100 or sodium deoxycholate in the neutral pH range. Besides hydrolysing casein this protease is also capable of attacking fibrinogen at pH7.4. 3. The pH optimum for activator activity and its inhibition by in-hexanoic acid (in-aminocaproic acid) point to its possible similarity to urokinase, an activator of plasminogen present in the urine of most mammals.

Acid Phosphatase↗
PubMed2004

Localization of endoplasmic reticulum and plasma membrane Ca2+-ATPases in subcellular fractions and sections of pig cerebellum.

Subcellular fractions and sections of the cerebellum were analysed to evaluate the relative activity and distribution of organellar and plasma membrane Ca2+-ATPases (SERCA and PMCA). Western blot analysis of the fractions with IID8 or Y/1F4 SERCA-specific antibodies or else with 5F10 or pbPMCA antibodies, specific to PMCA pump, revealed a major content of SERCA protein in microsomes and of PMCA protein in plasma membrane vesicles. The Ca2+-ATPase activity of microsomes was more sensitive to thapsigargin, a SERCA-specific inhibitor, whereas the activity of the plasma membrane vesicle fraction was inhibited more by vanadate, a blocker of PMCA activity. The SERCA and PMCA distribution analysed in cerebellar sections revealed IID8 antibody reactions in Purkinje cell cytoplasm, granule cells and cerebellar glomeruli. Y/1F4 gave immunostaining in Purkinje cells, molecular layer interneurons (basket and stellate cells) and glomeruli, but granule cells were not labelled. The 5F10 antibody reacted with Purkinje cells, including their dendritic spines, as well as cerebellar glomeruli, whereas the pbPMCA antibody labelled several processes in all three layers and some synaptic interaction sites. The differential content and localization of the two types of Ca2+ pumps in specific neuronal areas of pig cerebellum indicate precise Ca2+ requirements of specific cellular regions.

Animals↗
PubMed1980

The acylation of lysophosphatidylcholine by subcellular fractions of guinea-pig cerebral cortex.

The acylation of lysophosphatidylcholine by isolated subcellular fractions of guinea-pig cerebral cortex has been determined. The microsomal fraction contained the highest acylation activity, in terms of both specific and total activity. In all particulate fractions, including synaptic plasma membrane and mitochondria, there was a high correlation (correlation coefficient r = 0.90; P less than 0.001) between acylation and the activity of the microsomal enzyme, NADPH-cytochrome c reductase. No correlation existed between acylation and the activities of (Na+ + K+)-ATPase, acetylcholinesterase or succinate dehydrogenase. Acyl-CoA synthetase and lysophosphatidylcholine/acyltransferase, the individual enzymes responsible for acylation were enriched in the microsomal fraction. The activities of both enzymes in subcellular fractions correlated well with those of NADPH-cytochrome c reductase, with the exception that acyl-CoA synthetase activity in the mitochondrial fraction was largely independent of endoplasmic reticulum. Neither synaptic plasma membranes nor mitochondria appeared to possess significant amounts of acyltransferase activity. The results indicate that the acylation of lysophosphatidylcholine is confined to the endoplasmic reticulum, and that activity present in the synaptic plasma membrane or mitochondrial fraction is attributable to microsomal contamination.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1991

Characterization of the protein contents of rhoptries and dense granules of Toxoplasma gondii tachyzoites by subcellular fractionation and monoclonal antibodies.

A subcellular fractionation procedure has been established to isolate the rhoptries of Toxoplasma gondii tachyzoites on a self-generating Percoll gradient. The rhoptry fraction also contains dense granules. Monoclonal antibodies have been raised against the fraction and used to identify major proteins in the organelles by immunoelectron microscopy and Western blotting. Six major rhoptry proteins (60.5 kDa, Pi 5.8; 55, 57, 59, 60 kDa, all of Pi over 8; 42 kDa, Pi 4.8) and 3 dense granule proteins (30 kDa; 28kDa, Pi 7.5; 27 kDa, Pi 4.5) together with 5 other proteins of 57, 90, 120, 168, 220 kDa that have been located in the rhoptry area by indirect immunofluorescence have been identified.

Animals↗
PubMed1990

Immunogenicity of subcellular fractions of Brucella abortus: measurement by in vitro lymphocyte proliferative responses.

Five groups of heifers were immunized with various subcellular fractions of Brucella abortus and tested for their responsiveness in lymphocyte proliferative responses in vitro. The five subcellular fractions used as immunogens were: (1) a mixture of recombinant outer membrane proteins fused to Escherichia coli beta-galactosidase, (2) a mixture of outer membrane proteins BaomI, BaomIIB1, and BaomIII1, (3) a mixture of outer membrane proteins 7.5 kDa and 8.8 kDa, (4) a complex of smooth lipopolysaccharide and proteins, and (5) a complex of outer membranes and peptidoglycan (OM-PG complex) from a rough strain. All immunogens were emulsified in adjuvant and administered twice at a 61-day interval. Two other groups of cows were included; one immunized with strain 19 and the other with adjuvant only. Strain 19 and the rough OM-PG complex induced responsiveness in lymphocyte proliferation assays in a high percentage of immunized cows. The smooth lipopolysaccharide-protein complex induced responsiveness in fewer cows. The lowest frequencies of responding cows were found in groups that received either recombinant proteins or purified protein mixtures. Based on these results, we concluded: (1) cellular immunity, as measured by in vitro lymphocyte proliferative responses, can be induced with subcellular fractions of B. abortus and (2) the more complex the immunogen, the greater the frequency of responding cows.

Analysis of Variance↗
PubMed1987

Increased activity of the respiratory burst in cord blood neutrophils: kinetics of the NADPH oxidase enzyme system in subcellular fractions.

Previous studies with neutrophils from newborn infants compared to neutrophils from healthy adults have documented increased respiratory burst activity including enhanced superoxide anion (O2-) production, nitroblue tetrazolium dye reduction, and hexose monophosphate shunt activity. To investigate the biochemical basis for these observations, we examined oxidative metabolism in membrane-rich fractions of neutrophils. Neutrophils from cord blood of vaginally delivered term infants or healthy adults were disrupted by nitrogen cavitation and subcellular fractions collected on discontinuous sucrose density gradients. Subcellular fractions of newborn neutrophils separated in a fashion identical with samples from healthy adults. Activity of alkaline phosphatase, a plasma membrane marker, was increased 4- to 5-fold in disrupted cells free from nuclei (postnuclear supernatant) as well as plasma membrane fractions from newborn samples compared to those from healthy adults. Content of lactoferrin, a specific granule marker, was decreased in postnuclear supernatants but equivalent in specific granule fractions of newborn cells compared to those from adults. No differences were noted in myeloperoxidase content of postnuclear supernatants or any other subcellular fraction. Plasma membrane fractions from phorbol myristate acetate-stimulated cord blood neutrophils made significantly more O2- than samples from adults (newborn 32.9 +/- 8.1 nmol O2-/min/mg protein mean +/- SEM, n = 3 versus adult 10.8 +/- 4.2, n = 3; p less than 0.05). Plasma membrane-rich fractions were also collected by the technique of differential centrifugation and kinetic parameters of the NADPH-dependent oxidase enzyme(s) were measured for vaginally delivered newborn and adult samples.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1984

Topology of asialoglycoprotein receptor in rat liver subcellular fractions: a ferritin immunoelectron microscopic study.

Direct ferritin immunoelectron microscopy was used to visualize the asialoglycoprotein receptor in various rat liver subcellular fractions. The cytoplasmic surfaces of cytoplasmic organelles such as the rough and smooth microsomes, Golgi cisternae and lysosomes showed hardly any ferritin label exception for the slight labeling of secretory granules found mainly in the light Golgi fraction (GF1). Occasionally, however, open membrane sheet structures, smooth vesicular or tubular structures heavily labeled with ferritin, were present in all these subcellular fractions. These structures probably correspond to fragmented sinusoidal or lateral hepatocyte plasma membranes recovered to these subcellular fractions. When the limiting membranes of the secretion granules were partially broken by mechanical force, a number of ferritin particles frequently were seen attached in large clusters to the luminal surface of the membrane, the cytoplasmic surface of the corresponding domain being slightly labeled. These observations are strong evidence that the receptor protein is never translocated vertically throughout the intracellular transport from ER to plasma membrane via Golgi apparatus and from plasma membrane back to trans-Golgi elements and also in lysosomes, always exposing the major antigenic sites to the luminal or extracellular surface and the minor counterparts to the cytoplasmic surface of the membranes. The receptor protein also is suggested to be concentrated in clusters on the luminal surface of secretion granules when they form on the trans-side of the Golgi apparatus.

Animals↗
PubMed1979

Isolation of synaptic junction-enriched fraction from the forebrain of day-old chickens. Preparation and characterization of chick forebrain subcellular fractions.

Synaptosomal plasma membrane (SPM) and other subcellular fractions were isolated from the forebrain of 1-day-old chickens by a procedure based on that of Davis and Bloom (16) and Cotman and Taylor (13). The procedure involves the centrifugation through a discontinuous sucrose gradient of a crude synaptosomal-mitochondrial fraction which has been lysed and weighed with iodonitrotetrazolium. SPM isolated by this method contains only small amounts of lysosomal or mitochondrial membranes and is practically devoid of contaminating microsomal membranes, as estimated by enzyme marker assays. The purity of chick-brain SPM prepared by this method is compared to the purity of chick-brain fractions obtained by two other laboratories, using different methods (4, 59). The SPM were extracted with Triton X-100 and all fractions solubilized in sodium dodecyl sulfate (sds). The delipidated proteins of all fractions were subjected to SDS-polyacrylamide electrophoresis on slab gels and stained for protein. A distinct difference was observed between the patterns given by the Triton-soluble and -insoluble fractions. Electron microscopy of the synaptic junction fraction showed numerous junctional complexes.

Animals↗
PubMed1996

Agonist-induced desensitization and down-regulation of delta opioid receptors alter the levels of their 125I-beta-endorphin cross-linked products in subcellular fractions from NG108-15 cells.

The delta opioid binding sites in subcellular fractions from NG108-15 cells were characterized with respect to their relative molecular size and levels under conditions of receptor adaptation. 125I-beta-Endorphin was cross-linked to preparations enriched in plasma membranes (P20), nuclear membranes or nuclear matrices. Five cross-linked bands appear in all subcellular fractions. The largest molecular size reaction product in nuclear matrix preparations (approximately 72 kDa) differed from that in the other two fractions-(approximately 83 kDa). Immunoblot analyses with an antibody to the delta opioid receptor gave a P20 band pattern similar to that for the corresponding cross-linked products. To determine which cross-linked products in P20 are glycoproteins, labeled membranes were solubilized and purified by wheat germ agglutinin chromatography. The absence of a approximately 36 kDa band after purification suggests that this product is not a glycoprotein. The remaining four bands were present in N-acetyl-D-glucosamine eluates, although their % distribution changes in favor of the largest molecular size band (approximately 83 kDa). Immunoblotting of the eluate gave a single diffuse band at approximately 73 kDa, suggesting the native glycoprotein has a molecular size in the 70-80 kDa range. Etorphine-induced desensitization of cell surface receptors increased the amount of some cross-linked products associated with nuclear membranes. The same treatment did not affect the relative density of the four larger molecular size bands in P20, but increased the density of the approximately 26 kDa product two fold. Etorphine-induced down-regulation evoked an elevation of cross-linked products in nuclear matrix preparations, while all band densities of P20 were diminished. These results suggest that nuclear matrix associated opioid binding sites represent internalized, truncated forms of the glycosylated delta opioid receptor found in P20.

Cell Line↗
PubMed1987

Profile of phospholipase A2 activity in subcellular fractions and lamellar bodies of developing, neonatal and adult rabbit lung. Correlation with intracellular levels of disaturated phosphatidylcholine.

Phospholipase A2 activity was determined in subcellular fractions and lamellar bodies of fetal, neonatal and adult rabbit lungs. Specific activity in most fractions decreased from the 24th to the 28th day of gestation. All fractions except the mitochondrial and the nuclear fractions exhibited a sharp increase in activity in the newborn lung. Specific activity in the adult lung generally declined in comparison to neonatal values. During gestation total enzyme activity per gram of lung was concentrated in the cytosolic fraction. With the exception of the lamellar body fraction, the total content of phospholipase A2 activity increased dramatically in all fractions from the neonatal lung. The lamellar body fractions displayed both low specific activity and low total enzyme activity during gestation. Specific activity increased dramatically in the neonatal and adult lung but still accounted for only a small fraction of the activity in comparison to the other subcellular fractions. The subcellular content of disaturated phosphatidylcholine (PC) appeared to correlate well with the activity of phospholipase A2 in the neonatal mitochondrial, microsomal and cytosolic fractions. Since decreasing prenatal enzyme levels are associated with increasing disaturated PC content, the alkaline and calcium-dependent phospholipase A2 may not be directly involved in disaturated PC synthesis in the fetus. However, postnatally, the correlation between the pattern of production of disaturated PC and the activity of the phospholipase A2 indicates a role for this enzyme in surfactant-related disaturated PC synthesis.

Animals↗
PubMedDate not supplied

[Distribution of gamma-hexachlorcyclohexane in subcellular fractions of the liver and brain].

The character of the gamma-HCCH distribution in the subcellular fractions of the liver and brain of albino rats with peroral threefold introduction of the compound in doses of 34 mg/kg (1/5 LD50) and in a dose of 1.7 mg/kg (1/100 LD50) for a space of 1, 3 and 6 months was investigated. It was found that gamma-HCCH penetrates the cells of the liver and brain and is non-uniformly distributed among subcellular fractions, viz. nuclear, mitochondrial and supernatant. Both in acute and chronic experiments the greatest amounts of the preparation were found in the nuclear fraction of the cell. The established features distinguishing the accumulation and distribution of the preparation in the subcellular fractions of the liver and brain explain the previously elicited biochemical and physiological shifts occurring in the organism after entrance of gamma-HCCH thereinto.

Animals↗
PubMed1993

Oxidation of alpha-tocopherol in subcellular fractions from rat brain and its possible involvement in nerve function.

The turnover rate of vitamin E is slow in nerve tissue. Therefore, we have developed in vitro techniques to study the biochemical reactions of this nutrient in brain. Subcellular fractions were isolated from the cerebral hemispheres of 4-month-old, male, Fisher 344 rats. Aliquots of fractions (500 micrograms protein) were suspended in 50 mM phosphate buffer at pH 7.4 and incubated at room temperature (20-22 degrees) or 37 degrees for 2 hr in the presence or absence of the following oxidizing agents: 1 mM tertiary butyl hydroperoxide, 10 microM linoleic acid hydroperoxide, 0.5 to 50 mM 2,2'-azobis (2-amidinopropane) dihydrochloride (ABAPH) or 0.1 to 2 mM 2,2'-azobis (2,4-dimethyl) valeronitrile (ABDVN). The latter two compounds generate free radicals upon heating. After oxidation, the subcellular fractions were sedimented, saponified and assayed for tocopherol by liquid chromatography. Linoleic acid hydroperoxide was the most potent oxidizing agent, suggesting that endogenous fatty acid peroxides (e.g. eicosanoid intermediates) are very powerful oxidizing agents. Vitamin E may play an important role in providing antioxidant protection for membranes against excessive oxidation induced by these peroxides. Tocopherol in mitochondria and microsomes was much more susceptible to oxidation than synaptosomal tocopherol. The possible reasons for this observation are: (a) mitochondria and microsomes may contain less of the other reducing agents such as sulfhydryl compounds than synaptosomes, and/or (b) the electron transport structures in the former two subcellular fractions may be facilitating oxidation of tocopherol induced by free radicals. A portion of tocopherol remained unoxidized in all subcellular fractions even at high concentrations of ABAPH, suggesting that tocopherol exists in labile and nonlabile biochemical compartments or complexes.

Amidines↗
PubMedDate not supplied

[The content and composition of gangliosides in brain subcellular fractions of the frog Rana temporaria].

Ganglioside distribution in various frog brain subcellular fractions (myelin, microsomes, mitochondria, synaptosomes, plasma membranes of nerve endings and synaptic vesicles) was investigated. The synaptosomes and plasma membranes of nerve endings were found to be the main places of ganglioside localization, ganglioside concentration being 2.42 and 1.79 times higher than that in homogenates. Gangliosides were shown to be present in synaptic vesicles. The characteristic features of gangliosides from frog brain and its subcellular fractions are the predominance of polysialogangliosides with 3-5 sialic acid residues (up to 57.4%), low content of monosialogangliosides (not more than 7%) and the presence of disialogangliosides with short carbohydrate chain. The increase of ganglioside content per one nerve cell during phylogenetic development of vertebrates is discussed.

Animals↗
PubMed1964

FATTY ACID SYNTHESIS BY SUBCELLULAR FRACTIONS OF LUNG TISSUE.

The mitochondria-rich fraction of the lung tissue is the most active subcellular fraction for the synthesis of long-chain fatty acids. This observation is contrary to what has been reported for the subcellular fractions of the liver. The high rate of fatty acid synthesis observed with the mitochondria-rich fraction of the lung may be related to the morphological transformation of mitochondria.

Acetates↗
PubMed1986

A one-step technique for the subcellular fractionation of total cell homogenates.

A procedure was developed for the rapid, analytical subcellular fractionation of entire homogenates from the Chinese hamster ovary and HeLa cell lines. The procedure avoids a nuclear sedimentation step and the losses that accompany such a step. A key to the development of this procedure was the addition to homogenates of either micrococcal nuclease or DNase I. Nuclease-treated homogenates were fractionated on self-forming Percoll gradients. The entire procedure from cell harvesting through collecting gradient fractions took only 2.5 h. The position of marker enzymes in the gradient fractions indicated clear resolution of plasma membranes, Golgi apparatus, endoplasmic reticulum, and lysosomes. This procedure should facilitate many studies requiring subcellular fractionation of cultured cells.

Animals↗
PubMed1990

Adrenal autoantibodies bind to adrenal subcellular fractions enriched in cytochrome-c reductase and 5'-nucleotidase.

A quantitative assay for human adrenal autoantibodies has been developed to aid in the detection and isolation of human adrenal antigens. To define the subcellular location(s) of the antigen(s) capable of binding with these antibodies, we have quantitated both antibody binding to various adrenal subcellular fractions and the adrenal autoantibody binding inhibition caused by each subcellular fraction. To further define the subcellular location of the autoantibody binding, each fraction was assayed for organelle-specific marker enzyme activities. Enzyme activities were correlated to adrenal autoantibody binding to each fraction by linear regression. Of the materials tested, both antibody binding and inhibition of binding were most highly correlated with adrenal subcellular fractions enriched with cytochrome-c reductase and 5'-nucleotidase (r = 0.98; P less than 0.05). Thus, our data support the localization of adrenal autoantigen(s) in the microsomes, plasma membrane, or both.

5'-Nucleotidase↗