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PubMed1982

Subcellular distribution of iodothyronine 5'-deiodinase in cerebral cortex from hypothyroid rats.

We have examined iodothyronine deiodination in subcellular fractions of cerebral cortex obtained from hypothyroid rats. Enzymatic activities were measured at 37 degrees C in the presence of 20 mM dithiothreitol with 125I-labeled T4 and 125I-labeled rT3 as substrates for 5'-deiodination and 131I-labeled T3 as the substrate for the 5-deiodinase. Reaction products were separated by descending paper and/or ion-exchange chromatography. Cerebral cortex subcellular fractions were also characterized by marker enzyme analysis and electron microscopy. Under optimal reaction conditions more than 80% of the 5'-deiodinase was recovered after fractionation. Both 5'-deiodinase and (Na+ + K+)-ATPase showed similar subcellular distributions and were enriched approx. 3-fold in the easily sedimenting membrane fraction and nerve terminal plasma membranes. Crude microsomal membranes (6 X 10(6) g X min pellet) also showed 2-fold enrichment of these enzymes. Nuclei and isolated mitochondria were devoid of deiodinating activity. T4 and T3 5-deiodinating activity was absent in the easily sedimenting membranes and present but not enriched in particulate fractions containing microsomal membranes. These data suggest that iodothyronine 5'-deiodinase is associated with plasma membrane fractions in the cerebral cortex.

Animals↗

PubMed1994

Effect of alcohol on the activity and subcellular distribution of phosphatidate phosphohydrolase in rat liver.

The activity and subcellular distribution, before and after translocation with oleic acid, of hepatic phosphatidate phosphohydrolase (PAH: E.C.3.1.3.4) was measured following acute and chronic administration of ethanol with either 18% or 4.4% total calories as fat, to male Wistar rats. Acute injection produced a significant increase in PAH activity, without affecting the subcellular distribution or translocation of activity from the cytosol into the membraneous compartments induced by oleic acid. PAH activity, subcellular distribution and the translocation of activity by oleic acid was similar in all groups following chronic liquid feeding. Chronic ethanol feeding was associated with significantly increased plasma corticosterone in the ethanol-fed rats compared with controls, independent of the fat content of the diet. Plasma insulin was similar in chronic alcohol fed rats and controls. This suggests that enhanced PAH activity may contribute to triacylglycerol accumulation following acute alcohol injection, but activity normalises as hepatic lipid accumulation progresses and this effect is not due to attenuation of the hormonal effects of ethanol.

Animals↗

PubMed1993

Differential properties of phosphatidate phosphohydrolase and diacylglyceride lipase activities in retinal subcellular fractions and rod outer segments.

1. The effect of magnesium and dl-propranolol on phosphatidate phosphohydrolase (PAPase) and diacylglycerol lipase (DGL) activities in isolated rod outer segments (ROS) and of the former on subcellular fractions from bovine retina was investigated. 2. Mg(2+)-independent PAPase activity was found in ROS, whereas in the other subcellular fractions PAPase activities both dependent on and independent of Mg2+ were detected. 3. The membrane-bound PAPase activity was stimulated at low concentrations of Mg2+ and inhibited at higher concentrations. The soluble activity was always stimulated by the ion. 4. dl-Propranolol (1000 microM) exerted a slight stimulatory effect on PAPase in ROS whereas total PAPase activity of microsomal fraction was not affected. 5. Mg2+ (0.2 mM) stimulated DGL activity (30%) whereas it was inhibited at higher concentration. 6. DGL lipase activities, both dependent on and independent of Mg2+, were detected in subcellular fractions of bovine retina. 7. DGL properties in ROS are also described.

Animals↗

PubMed1992

Regional and subcellular distribution of [125I]endothelin binding sites in rat brain.

The binding of [125I]endothelin-1 (125I-ET-1) to membranes from whole rat brain, from individual brain regions, and derived from subcellular fractionation of whole rat brain was investigated. 125I-ET-1 binding to whole rat brain membranes was rapid, concentration-dependent, saturable, and characterized as irreversible because it was not displaced by unlabeled endothelin-1 (ET-1) and different concentrations of ligand produced, with time, a similar magnitude of binding. The maximum binding site capacity and second-order forward rate association constant of binding were 1,946 +/- 147 fm/mg protein and 5.53 +/- 1.72 x 10(6) M-1 s-1. Removal of either extramembranal calcium or membrane-bound calcium and calcium binding proteins did not affect the binding of 125I-ET-1 to whole rat brain membranes. The brain stem and cerebellum contained the highest levels of 125I-ET-1 binding sites, whereas the cerebral cortex, striatum, and hippocampus contained binding site levels three- to fourfold less. Subcellular fractionation of whole rat brain and subsequent analyses of the distribution of 125I-ET-1 binding demonstrated a twofold enrichment of binding sites in the synaptosomal fraction compared to the homogenate. The myelin fraction contained a similar density of binding sites compared to the homogenate, while the mitochondrial and microsomal fractions contained considerably less binding sites. The ribosomal fraction did not contain any 125I-ET-1 binding sites. The subcellular distribution of 125I-ET-1 binding sites did not correlate with the distribution of 5'-nucleotidase, cytochrome-C oxidase, phosphodiesterase, and alkaline phosphatase. Depletion of extracellular calcium increased 125I-ET-1 binding in the synaptosomal fraction but not in the myelin and mitochondrial fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Analysis and subcellular localization of lipid in alcoholic liver disease.

The nature and subcellular distribution of lipids in alcoholic liver disease have been little studied. Micro-methods for lipid analysis were applied to needle biopsy homogenates and their subcellular fractions. Alcoholic fatty liver was accompanied by a major increase (up to 50 fold) in triglyceride and a smaller (2-3 fold) increase in cholesteryl ester: there was no significant change in the free cholesterol, free fatty acid or phospholipid content. Homogenates were fractionated into macro- and micro-droplets and membrane fractions by differential centrifugation. The subcellular location of the membrane lipids were determined by sucrose density gradient centrifugation in a vertical pocket reorientating rotor. In fatty liver, although there was a 2-3 fold increase in macro-droplet and micro-droplet (tentatively identified as VLDL) lipid, the major increase was in the membrane-bound triglyceride (8-10 fold). Sucrose density gradient centrifugation demonstrated that these membranes had an equilibrium density of 1.12 g/ml, clearly separated from droplet lipid, density less than 1.04 g/ml. The membrane fraction was tentatively identified as Golgi in origin and it is suggested that alcoholic fatty liver in man is due to impaired Golgi secretion of triglyceride-rich lipid complexes.

Biopsy, Needle↗

PubMed2004

Evaluation of GABA uptake in subcellular fractions of bovine frontal cortex and brainstem.

GABA uptake as well as the distribution of GAT-1, GAT-2 and GAT-3 were investigated in bovine brain membrane fractions. GABA uptake was characterised by kinetic constants and IC50-values for a series of known inhibitors in subcellular fractions of frontal cortex and brainstem obtained by subsequent centrifugations on sucrose gradients. Additionally, the immunoreactivity for rGAT-1, rGAT-2 and rGAT-3 antibodies was studied in these fractions. The pharmacological profile for GABA uptake inhibition as well as results from immunoblotting indicated that GABA uptake in a selected subcellular fraction of frontal cortex (P2B) is almost exclusively due to GAT-1 whereas GABA uptake performed with a selected subcellular fraction of brainstem (P2A) in the presence of NNC 711 is mainly attributable to GAT-3.

Animals↗

PubMed2004

A subcellular distribution of estrogen receptor-alpha is changed during artificially induced senescence of PC12 pheochromocytoma cells.

Although estrogen has been considered as a sex hormone for decades, recent reports suggest that estrogen might modulate the development and physiological function of the brain. In addition, the subcellular localization of estrogen receptors (ERs) has shown their presence within both the perinuclear cytoplasm and nuclei, suggesting that these ERs may differ functionally. We, therefore, assayed changes in the subcellular localization of ER-alpha immunoreactivity (IR) in rat pheochromocytoma PC12 cells during the artificial senescence induced by the telomerase inhibitor, 3'-azido-3'-deoxythymidine (AZT). After 2 months of culture with AZT, PC12 cells showed morphological and biochemical characteristics of cellular senescence. In the cells showing artificial senescence, the ER-alpha IR was mainly localized within the cytoplasm, whereas in control cells, ER-alpha IR was found only in the nuclei. Since senescence was induced by AZT, which inhibits the action of telomerase whenever the cells divide, the change in subcellular distribution of ER-alpha IR may be correlated with the length of the telomere.

Animals↗

PubMed2005

Subcellular distribution of membrane-bound aminopeptidases in the human and rat brain.

We evaluated the subcellular distribution of four membrane-bound aminopeptidases in the human and rat brain cortex. The particulate enzymes under study--puromycin-sensitive aminopeptidase (PSA), aminopeptidase N (APN), pyroglutamyl-peptidase I (PG I) and aspartyl-aminopeptidase (Asp-AP)--were fluorometrically measured using beta-naphthylamide derivatives. Membrane-bound aminopeptidase activity was found in all the studied subcellular fractions (myelinic, synaptosomal, mitochondrial, microsomal and nuclear fractions), although not homogenously. Human PSA showed highest activity in the microsomal fraction. APN was significantly higher in the nuclear fraction of both species, while PG I showed highest activity in the synaptosomal and myelinic fractions of the human and rat brain. The present results suggest that in addition to inactivating neuropeptides at the synaptic cleft, these enzymes may participate in other physiological processes. Moreover, these peptidases may play specific roles depending on their activity levels at the different subcellular structures where they are localized.

Aminopeptidases↗

PubMed2005

Subcellular localization and trafficking of kainate receptors.

Glutamate receptors of the kainate type have been identified recently as key players in the modulation of neuronal-network activity. The role of kainate receptors depends on their precise subcellular localization in presynaptic, postsynaptic and extrasynaptic domains. Subcellular localization of kainate receptors has been inferred mainly from electrophysiological studies with the help of selective pharmacological tools and kainate receptor mutant mice. These studies, combined with recent ultrastructural data, highlight the diversity of subcellular localizations of kainate receptors. It is important to understand the molecular mechanisms that underlie the polarized trafficking of kainate receptors in distinct neuronal domains. In this article, we review recent data that shed light on the trafficking and membrane delivery of kainate receptor isoforms, and on the identification of proteins that interact with kainate receptors and might regulate this trafficking.

Animals↗

PubMedDate not supplied

Evolutionary conservation of biotinidase: implications for the enzyme's structure and subcellular localization.

Biotinidases from various species ranging from fungi and insects to human have specific amino acids, and regions that are evolutionarily conserved. These specific amino acids and regions are further supported by their homology to a variety of amidases and nitrilases and by the location of missense mutations that cause biotinidase deficiency in humans. Glu-Lys-Cys residues from three of these regions are considered the catalytic triad involved in the catalysis of the amide linkage. The last one-third of the biotinidase sequence is lacking in nitrilases-amidases, which do not bind biocytin or biotin, therefore, it is likely that the biocytin-biotin-binding site of biotinidase is within this portion of the molecule. Although there are many missense mutations at the far C-terminus of the enzyme, the function of this region is still unclear. Biotinidase may have different functions in different cells or in different subcellular compartments. Using computer programs that predict the subcellular localization of proteins based on their N-terminal signal peptides, microsomal localization resulting in secretion was predicted for biotinidase from all species, whereas there is little consistent support for mitochondrial or nuclear localization of the enzymes. Additional immunohistochemical studies of various human tissues at different stages of development are necessary to resolve the ambiguity of subcellular localization of biotinidase.

Amino Acid Sequence↗

PubMed1989

Uptake and subcellular distribution of cadmium in resting and mitogen-activated lymphocytes and its relationship to a metallothionein-like protein.

The effects of cadmium on the immune system have been extensively studied, although relatively little is known about the nature of the interaction between cadmium and lymphocytes. We describe studies that examine the uptake and subcellular distribution of cadmium in resting and mitogen-activated mouse splenic lymphocytes exposed to cadmium in vitro. Mitogen activation was associated with an increased uptake and altered subcellular distribution of cadmium, compared to resting (nonmitogen-activated) lymphocytes. In both resting and mitogen-activated lymphocytes the total uptake of cadmium was concentration-dependent. However, compared to resting lymphocytes, in mitogen-activated lymphocytes, cadmium tended to distribute out of the nuclear and into the cytoplasmic fraction. The Sephadex G-75 exclusion chromatography profile revealed the presence of a cadmium-binding protein eluting identically to metallothionein in mitogen-activated, but not in resting, lymphocytes. The presence of this protein in mitogen-activated lymphocytes is consistent with the differences in subcellular distribution of cadmium reported. Further, the difference between resting and mitogen-activated lymphocytes in the production of this protein, and the known genetic differences in the metallothionein inducibility, may explain some of the reported variability in the effects of cadmium on the immune system.

Animals↗

PubMed1999

Prediction of protein subcellular locations using Markov chain models.

A novel method was introduced to predict protein subcellular locations from sequences. Using sequence data, this method achieved a prediction accuracy higher than previous methods based on the amino acid composition. For three subcellular locations in a prokaryotic organism, the overall prediction accuracy reached 89.1%. For eukaryotic proteins, prediction accuracies of 73.0% and 78.7% were attained within four and three location categories, respectively. These results demonstrate the applicability of this relative simple method and possible improvement of prediction for the protein subcellular location.

Markov Chains↗

PubMed1984

Membrane fractionation of canine aortic smooth muscle: subcellular distribution of calcium transport activity.

Various subcellular membrane fractions were isolated from dog aortic smooth muscle by conventional differential centrifugation followed by isopycnic centrifugation on a sucrose density gradient. These subcellular fractions were characterized by membrane marker enzyme activities, morphological features and the electrophoretic patterns on a sodium dodecyl sulfate polyacrylamide gel. Our results showed that the microsomal membrane fraction isolated by differential centrifugation was very heterogeneous and contained substantial amount of plasma membranes which could be further enriched as a light density fraction on the sucrose density gradient. The subcellular distribution of Ca2+ binding in the absence of ATP and Ca2+ transport in the presence of ATP closely paralleled the distribution of plasma membrane markers. The ATP-supported Ca2+ transport was inhibited by several Ca2+ ionophores, enhanced by inorganic phosphate and oxalate ions and cosedimented toward higher density in a continuous source density gradient with plasma membrane marker enzyme activity in the presence of digitonin. Our present work strongly suggests that plasma membrane is the predominant component of microsomal fraction and responsible for most, if not all, of the azide-insensitive ATP-supported Ca2+ accumulation.

Adenosine Triphosphate↗

PubMed2003

Phosphorylation of the N-terminal domain regulates subcellular localization and DNA binding properties of the peptidyl-prolyl cis/trans isomerase hPar14.

Human parvulin 14 (hPar14) is a folding helper enzyme belonging to the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases). This enzyme is thought to play a role in cell-cycle and chromatin remodeling. Although hPar14 was nuclearly localized and bound to double-stranded DNA, the molecular basis of the subcellular localization and the functional regulation remained unknown. Here we show that subcellular localization and DNA-binding ability of hPar14 is regulated by posttranslational modification of its N-terminal domain. As proved by MALDI-TOF mass spectrometry and MS/MS fragmentation, hPar14 is phosphorylated at Ser19 in vitro and in vivo. In human HeLa cells the protein is most likely modified by casein kinase 2 (CK2). Phosphorylation of hPar14 is inhibited both in vitro and in vivo by 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), a specific inhibitor of CK2 activity. Mutation of Ser19 to Ala abolishes phosphorylation and alters the subcellular localization of hPar14 from predominantly nuclear to significantly cytoplasmic. Immunostaining shows that a Glu19 mutant of hPar14, which mimics the phosphorylated state of Ser19, is localized around the nuclear envelope, but does not penetrate into the nucleoplasm. In contrast to wild-type hPar14, the in vitro DNA-binding affinity of the Glu19 mutant is strongly reduced, suggesting that only the dephosphorylated protein is the active DNA-binding form of hPar14 in the nucleus.

Blotting, Western↗

PubMed1981

An improved method for the extraction of corticosterone from cell homogenates and subcellular fractions of the rat adrenal cortex.

An improved technique is described for the extraction and analysis of corticosterone (11 beta,21-dihydroxy-4-pregnene-3,20-dione) from homogenates and subcellular fractions of the rat adrenal cortex. Factors influencing complete extraction of corticosterone were the nature of the organic solvent system and the concentration of the tissue being extracted. The continued activity of steroidogenic enzymes during subcellular fractionation was presented by 0.1 mM 1-benzylimidazole. For optimum extraction, homogenates were diluted 1:12 (v/v) in 0.25 M sucrose, containing 0.1 M potassium hydroxide. Dilute homogenate was mixed with absolute ethanol (1:10, v/v) and extracted three times with diethyl ether (1:5, v/v). Following extraction, corticosterone in each sample was isolated by thin-layer chromatography (TLC), quantitated by radioimmunoassay (RIA), and corrected by measuring the recovery of added H3 corticosterone. With these procedures, 90-100% of corticosterone found in extracts of adrenal homogenates was recovered in extracts of subcellular fractions of the homogenates.

Adrenal Cortex↗

PubMed2003

Use of correspondence discriminant analysis to predict the subcellular location of bacterial proteins.

Correspondence discriminant analysis (CDA) is a multivariate statistical method derived from discriminant analysis which can be used on contingency tables. We have used CDA to separate Gram negative bacteria proteins according to their subcellular location. The high resolution of the discrimination obtained makes this method a good tool to predict subcellular location when this information is not known. The main advantage of this technique is its simplicity. Indeed, by computing two linear formulae on amino acid composition, it is possible to classify a protein into one of the three classes of subcellular location we have defined. The CDA itself can be computed with the ADE-4 software package that can be downloaded, as well as the data set used in this study, from the Pôle Bio-Informatique Lyonnais (PBIL) server at http://pbil.univ-lyon1.fr.

Bacterial Proteins↗

PubMed2001

Subcellular localization of presenilin 2 endoproteolytic C-terminal fragments.

Mutations in the genes that encode the presenilin 1 and 2 (PS1 and PS2) proteins cause the majority of familial Alzheimer's disease (FAD). Differential cleavage of the presenilins results in a generation of at least two C-terminal fragments (CTFs). An increase in the smaller of these two CTFs is one of the few changes in presenilin processing associated with FAD mutations in both PS1 and PS2. Interestingly, the phosphorylation of PS2 modulates the production of the smaller, caspase-derived PS2 CTF, which indicates that the generation of this fragment is a regulated, physiologic event. To date, there is no data concerning the subcellular distribution of the caspase-derived PS2 CTF. Because this fragment is normally present at levels that are difficult to detect, we have used cell lines in which the production of wild-type or N141I mutant PS2 is controlled by a tetracycline-regulated promoter in order to assess the subcellular localization of the caspase CTF in relation to the larger, constitutive PS2 CTF and to PS2 holoprotein. We have found that when levels of PS2 are low, the constitutive CTF colocalizes with markers consistent with localization in the early Golgi-ER-Golgi intermediate compartment (ERGIC) while the caspase CTF colocalizes with markers for the endoplasmic reticulum (ER). Following induction of wild-type or mutant PS2, when the levels of PS2 are high, the primary localization of the constitutive CTF appears to shift from the early Golgi-ERGIC in addition to the ER. Interestingly, while the induction of wild-type PS2 resulted in the localization of the caspase CTF primarily in the ER, the induction of mutant PS2 resulted in the localization of the caspase CTF to both the ER and the early Golgi-ERGIC. In summary, these data suggest that the two presenilin 2 CTFs have different patterns of subcellular localization and that the N141I PS2 mutation alters the localization pattern of the PS2 caspase fragment.

Alzheimer Disease↗

PubMed2003

Physical law not natural selection as the major determinant of biological complexity in the subcellular realm: new support for the pre-Darwinian conception of evolution by natural law.

Before Darwin many biologists considered organic forms to be immutable natural forms or types which like inorganic forms such as atoms or crystals are part of a changeless world order and determined by physical law. Adaptations were viewed as secondary modifications of these 'crystal like' abstract afunctional 'givens of physics.' We argue here that much of the emerging picture of biological order in the subcellular realm resembles closely the pre-Darwinian conception of nature. We point out that in the subcellular realm, between nano and micrometers, physical law necessarily plays a far more significant role in organizing matter than in the familiar 'Darwinian world' between millimeters and meters (where matter can be arranged into almost any contingent artifactual arrangement we choose, as witness Lego toys, watches or jumbo jets). Consequently, when deploying matter into complex structures in the subcellular realm the cell must necessarily make extensive use of natural forms-such as the protein and RNA folds, microtubular forms and tensegrity structures-which like atoms or crystals self-organize under the direction of physical law into what are essentially 'pre-Darwinian' afunctional abstract molecular architectures in which adaptations are trivial secondary modifications of what are evidently primary givens of physics.

Biological Evolution↗