SEARCH · PubMed Health
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
The subcellular distribution of epoxide hydrolase activity towards TSO and HEOM in mouse liver and kidney was investigated using zonal rotor centrifugation. Epoxide hydrolase activity towards TSO was found predominantly in the soluble fraction with peroxisomes accounting for activity in the particulate fractions. Renal particulate activity towards HEOM was found predominantly in the microsomes.
Using nitrogen cavitation and Percoll density gradient centrifugation for subcellular centrifugation of human neutrophils, approximately 90% of the low potential b-cytochrome, unique for phagocytes, as well as 50% of the flavoproteins in normal neutrophils were found in a granule fraction which co-sedimented with the specific granules. Upon stimulation of the intact cells with phorbol myristate acetate, both the b-cytochrome and the flavoprotein translocated from this granule fraction to the fractions which contained the plasma membranes and the NADPH oxidase activity. In neutrophils from two patients with chronic granulomatous disease, both the b-cytochrome and the flavoprotein of the granules were absent, but flavoprotein was present in normal amounts in the membrane and cytosol fractions. Taken together, these findings suggest that the specific granules, or granules co-sedimenting with the specific granules, are important stores for the components of the NADPH oxidase, which is responsible for the respiratory burst. Analysis of the stoichiometry of CO2 generation, H+ secretion and O2 consumption by stimulated neutrophils indicated that the hexose monophosphate shunt is the source of both protons and electrons for the NADPH oxidase activity, as well as of the extra protons secreted during the respiratory burst.
The subcellular distribution of thiamine derivatives in rat brain was studied. Thiamine diphosphate content was highest in the mitochondrial and synaptosomal fractions, and lowest in microsomal, myelin and cytosolic fractions. Only 3-5% of total thiamine diphosphate was bound to transketolase, a cytosolic enzyme. Thiamine triphosphate was barely detectable in the microsomal and cytosolic fraction, but synaptosomes were slightly enriched in this compound compared to the crude homogenate. Both myelin and mitochondrial fractions contained significant amounts of thiamine triphosphate. In order to estimate the relative turnover rates of these compounds, the animals received an intraperitoneal injection of either [14C]thiamine or [14C]sulbutiamine (isobutyrylthiamine disulfide) 1 h before decapitation. The specific radioactivities of thiamine compounds found in the brain decreased in the order: thiamine > thiamine triphosphate > thiamine monophosphate > thiamine diphosphate. Incorporation of radioactivity into thiamine triphosphate was more marked with [14C]sulbutiamine than with [14C]thiamine. The highest specific radioactivity of thiamine diphosphate was found in the cytosolic fraction of the brain, though this pool represents less than 10% of total thiamine diphosphate. Cytosolic thiamine diphosphate had a twice higher specific radioactivity when [14C]sulbutiamine was used as precursor compared with thiamine though no significant differences were found in the other cellular compartments. Our results suggest the existence of two thiamine diphosphate pools: the bound cofactor pool is essentially mitochondrial and has a low turnover; a much smaller cytosolic pool (6-7% of total TDP) of high turnover is the likely precursor of thiamine triphosphate.
The subcellular distributions of glutamyl carboxypeptidase, folate specific activities, and radioactive metabolites of injected [3H] folic acid were studied in rat liver. The specific activity of glutamyl carboxypeptidase in the lysosomal fraction was near or greater than four times that in the other subcellular fractions. The specific activity of folates was highest in the soluble fraction (102 ng folate/mg protein) and lowest in the microsomal fraction (22 ng folate/mg protein). Nuclear, mitochondrial, and lysosomal folates were 95% folate polyglutamates, and microsomal and soluble folates were 85--90% folate polyglutamates. Injected [3H] folic acid was initially concentrated in the microsomal fraction, as measured by 3h cpm per ng folate. Initially, injected [3H] folic acid was found converted to folate penta- and hexaglutamates in all fractions to a similar extent except in the microsomes where the percentage conversion was much less, as measured by the percentage of total 3H cpm determined to be [3H] folate penta- and hexaglutamates. At 24 h, the conversion of [3H] folates to penta- and hexaglutamates in each fraction was less than that found for the endogenous folates. Injected [3H] folic acid after 2h was found to consist of 94% reduced folates in the soluble fraction, 56% in the mitochondrial, 55% in the nuclear, 20% in the lysosomal, and 15% in the microsomal fraction.
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.
A polyclonal antibody was raised against a Toxoplasma gondii 14-3-3-gluthatione S-transferase fusion protein obtained by cloning a 14-3-3 cDNA sequence determined from the T. gondii database. This antibody specifically recognized T. gondii 14-3-3 without any cross-reaction with mammalian proteins. Immunofluorescence microscopy studies of the tachyzoites or the T. gondii-infected cells suggested cytosolic and membranous localizations of 14-3-3 protein. Different subcellular fractions were prepared for electrophoresis analysis and immunodetection. 14-3-3 proteins were found in the cytosol, the membrane fraction and Triton X-100-resistant membranes. Two 14-3-3 isoforms were detected. The major one was mainly cytoplasmic and to a lesser extent membrane-associated, whereas the minor isoform was associated with the detergent-resistant lipid rafts.
Branched chain amino-acid aminotransferase (BCAT) activity is present in fetal liver but the developmental pattern of mitochondrial BCAT (BCATm) expression in rat liver has not been studied. The aim of this study was to determine the activity, protein and mRNA concentration of BCATm in fetal and postnatal rat liver, and to localize this enzyme at the cellular and subcellular levels at both developmental stages. Maximal BCAT activity and BCATm mRNA expression occurred at 17 days' gestation in fetal rat liver and then declined significantly immediately after birth. This pattern was observed only in liver; rat heart showed a different developmental pattern. Fetal liver showed intense immunostaining to BCATm in the nuclei and mitochondria of hepatic cells and blood cell precursors; in contrast, adult liver showed mild immunoreactivity located only in the mitochondria of hepatocytes. BCAT activity in isolated fetal liver nuclei was 0.64 mU x mg(-1) protein whereas it was undetectable in adult liver nuclei. By Western blot analysis the BCATm antibody recognized a 41-kDa protein in fetal liver nuclei, and proteins of 41 and 43 kDa in fetal liver supernatant. In adult rat liver supernatant, the BCATm antibody recognized only a 43-kDa protein; however, neither protein was detected in adult rat liver nuclei. The appearance of the 41-kDa protein was associated with the presence of the highly active form of BCATm. These results suggest the existence of active and inactive forms of BCAT in rat liver.
We have investigated the subcellular location of the Trypanosoma cruzi surface glycoprotein, Gp72, by introducing epitope-tagged copies of gp72 null-mutant cells. A tagged Gp72, containing three tandemly repeated copies of a human influenza hemagglutinin nonapeptide (HA) adjacent to the mature Gp72 amino terminus, was able to complement the null mutant phenotype, as well as being recognized in Western blots by both anti-HA antibody and the carbohydrate-specific monoclonal antibody WIC29.26. Integration of this epitope-tagged gp72 into the chromosomal gp72 locus produced a clonal cell line, 72HAN3.1G7, which was used for studies of the subcellular location of the epitope-tagged Gp72. Indirect immunofluorescence microscopy of fixed 72HAN3.1G7 epimastigotes showed that GP72 was evenly distributed over the cell body and somewhat concentrated in the proximal region of the flagellum. No fluorescence could be detected in the distal tip of the flagellum. Immunoelectron microscopy of fixed 72HAN3.1G7 epimastigotes revealed that Gp72 was predominantly membrane-associated and located on the cell surface. Indirect immunofluorescence microscopy of live 72HAN3.1G7 epimastigote cells showed a similar pattern of fluorescence on the flagellum, but no fluorescence was detected on the cell body, which was attributed to masking by other cell-surface components. Indirect immunofluorescence microscopy of fixed 72HAN3.1G7 amastigotes revealed that Gp72, which has long been considered to be expressed only in epimastigotes and metacyclic trypomastigotes, can be expressed in amastigotes, but it no longer contains the WIC29.26 carbohydrate epitope.
Eucaryotic mRNAs are generally localized in the cell body, where most protein synthesis occurs. We have found that mRNAs encoding the glial intermediate filament protein are spatially distributed in the glial cell cytoplasm close to the location of the glial filaments. Whereas the glial filament protein mRNA was located predominantly in the distal process, actin mRNA was found almost exclusively in the apical portion of the glial cell. This pattern of mRNA localization might provide a mechanism for synthesis of proteins in specific subcellular compartments by mRNA translation locally.
The catalase gene, vktA, of the hydrogen peroxide-resistant bacterium Vibrio rumoiensis S-1 has been isolated and sequenced. Plasmid pBSsa1 was obtained by genome library screening and it complemented a catalase-less mutant, Escherichia coli strain UM2, indicating that pBSsa1 contained the catalase gene (vktA). The vktA gene consisted of an open reading frame of 1530 bp encoding a 508 amino-acid protein with a calculated size of 57657.79 Da. The deduced amino acid sequence showed high homology with that of bacterial group III catalases. Investigation of the subcellular catalase localization using an immuno-electron microscopical technique revealed that a larger amount of VktA catalase is present in the periplasmic space than in the cytoplasm. The periplasmic space of this bacterium can therefore be regarded as a reservoir of VktA catalase in V. rumoiensis S-1.
Subcellular fractionation by differential centrifugation was used to study protocollagen proline hydroxylase (EC 1.14.11.2) localization from chick embryo liver. The fractions have been characterized by marker enzymes and electron microscopy. By these methods, it was observed that procollagen-proline-hydroxylase is concentrated in the microsomal fraction which is sedimented at 145 000 X g in 250 mM sucrose.
The subcellular distribution of the superoxide (O2-)-forming enzyme in human neutrophils was investigated. Cells were activated by phorbolmyristate acetate or by opsonized zymosan, and were then fractionated by zonal-rate sedimentation at two different speeds. At high speed, the specific granules were resolved from the azurophils and the membrane fraction, while at low speed, the azurophil granules could be separated from fast-sedimenting particle aggregates. Under both conditions, the major portion of the O-2--forming activity (60--70% of the total) was found to be associated with the membrane fraction which was characterized by the presence of alkaline phosphatase, alkaline phosphodiesterase I, and acid aryl phosphatase. No significant O-2--forming activity was found in either specific or azurophil granules. Some activity was present in the fastest sedimenting fractions which, as shown by electron microscopy, were heterogeneous and contained aggregated material which included membrane fragments. These fractionation results provide strong additional support for the current view that the activable O-2--forming system is localized in the plasma membrane of human neutrophils.
The distribution of LDL receptors within subcellular compartments of isolated rat adipose cells and the effects of insulin on their expression have been assessed. By immunoblotting with specific anti-rat LDL receptor antibodies, LDL receptors were 2.3- and 4.5-fold enriched in endoplasmic reticulum-rich high-density microsomes (HDM) and Golgi complex-rich low-density microsomes (LDM), respectively, compared to plasma membranes (PM). This distribution was similar in cultured cells in which total receptors were increased 2.5-fold compared to freshly isolated cells. After correction for enzyme recoveries, LDL receptors were distributed approximately 4% in HDM, approximately 73% in LDM, and approximately 23% in PM. Insulin decreased total LDL receptors in adipose cells approximately 44%, with a 48% and 49% decrease in HDM and LDM, respectively, without any changes in PM. In contrast, insulin caused an increase of glucose transporters in PM while also decreasing glucose transporters in LDM. When adipose cells were depleted of potassium to inhibit receptor-mediated endocytosis, insulin again caused a decrease of LDL receptors in LDM but now increased LDL receptors in PM. Insulin increased the rate of LDL receptor synthesis approximately 24%, but decreased their half life approximately 40%. Thus, in isolated adipose cells the majority of LDL receptors appear to be located in an intracellular compartment that co-sediments with the Golgi complex rather than located in the PM. The LDL receptors localized in intracellular compartments seem to be functionally regulated as insulin acutely diminishes the number of receptors by apparently accelerating their rate of degradation through, as yet, incompletely determined mechanisms.
DYRK1 is a dual specificity protein kinase presumably involved in brain development. Here we show that the kinase belongs to a new family of protein kinases comprising at least seven mammalian isoforms (DYRK1A, DYRK1B, DYRK1C, DYRK2, DYRK3, DYRK4A, and DYRK4B), the yeast homolog Yak1p, and the Drosophila kinase minibrain (MNB). In rat tissues, DYRK1A is expressed ubiquitously, whereas transcripts for DYRK1B, DYRK2, DYRK3, and DYRK4 were detected predominantly in testes of adult but not prepuberal rats. By fluorescence microscopy and subcellular fractionation, a green fluorescent protein (GFP) fusion protein of DYRK1A was found to accumulate in the nucleus of transfected COS-7 and HEK293 cells, whereas GFP-DYRK2 was predominantly detected in the cytoplasm. DYRK1A exhibited a punctate pattern of GFP fluorescence inside the nucleus and was co-purified with the nuclear matrix. Analysis of GFP-DYRK1A deletion constructs showed that the nuclear localization of DYRK1A was mediated by its nuclear targeting signal (amino acids 105-139) but that its characteristic subnuclear distribution depended on additional N-terminal elements (amino acids 1-104). When expressed in Escherichia coli, DYRK1A, DYRK2, DYRK3, MNB, and Yak1p catalyzed their autophosphorylation on tyrosine residues. The kinases differed in their substrate specificity in that DYRK2 and DYRK3, but not DYRK1A and MNB, catalyzed phosphorylation of histone H2B. The heterogeneity of their subcellular localization and substrate specificity suggests that the kinases are involved in different cellular functions.
The intracellular localization of acid lipase was examined by several subcellular fractionation techniques. Upon differential centrifugation, acid lipase was detected mainly in the lysosomal fraction. When linear sucrose density gradient centrifugation was used, the mean density of acid lipase was similar to that of lysosomal marker enzymes. After Triton-treatment of rats, however, acid lipase was recovered at a density of 1.10-1.16, which is more than that of tritosomes, when tissues were homogenized with a Potter-Elvehjem homogenizer. But by homogenizing the liver from these rats by pressing it through a stainless steel net, acid lipase was mainly recovered at the same density as that of tritosomes. These results indicate that acid lipase is localized mainly in lysosomes in rat liver.
Protein serine/threonine phosphatase 5 (PP5) plays an important role in signal transduction in animal cells, but in plants, knowledge about PP5 is scarce. Here, we describe the isolation of a full-length cDNA encoding tomato (Lycopersicon esculentum) PP5 (LePP5) and its expression in Escherichia coli. Biochemical characterization showed that recombinant LePP5 has a low intrinsic protein phosphatase activity. This activity was increased 6- to 10-fold by either removal of the N-terminal tetratricopeptide repeat domain or by addition of fatty acids, indicating that biochemical features specific for PP5 homologs from other species are conserved in tomato. The single-copy LePP5 gene was cloned and shown to encode two mRNA species that arise by alternative pre-mRNA splicing. Similarly, Arabidopsis was found to express two PP5 transcripts, suggesting that alternative splicing of PP5 pre-mRNA is not specific for tomato. Alternative splicing results in a larger transcript containing an additional exon encoding two putative transmembrane domains and, hence, in a larger PP5 isoform. Subcellular fractionation studies on tomato protein lysates indicated that the majority of the 55-kD LePP5 isoform is soluble, whereas the 62-kD isoform is an integral membrane protein. Production of yellow fluorescent protein-PP5 chimeras in plant cells indicated that the 55-kD isoform is localized in both the nucleus and the cytoplasm, whereas the 62-kD isoform is targeted to the endoplasmic reticulum, including the nuclear envelope. Our findings show that alternative splicing generates two LePP5 isoforms with a different subcellular localization.
Endothelial cells (EC) contain a constitutive Ca2+/calmodulin-dependent nitric oxide (NO) synthase (cNOS) which plays an important role in the local control of vascular tone. We compared the subcellular distribution of this enzyme in cultured and freshly isolated pig EC by determination of specific cNOS activity and immunoblot analysis. Similar studies were also performed with cultured and freshly isolated bovine and cultured human EC. Enzyme activity was predominantly (> 70%) associated with the particulate fraction of all EC types tested and was highest in freshly isolated porcine EC. Both specific cNOS activity and immunoreactivity were substantially higher (> 3-fold) in the microsomal as compared with the soluble fraction of all EC types tested. In freshly isolated pig EC, these two fractions also differed in terms of their Ca(2+)-dependency, pH optimum and inhibitor specificity. EC may thus contain either two different cNOS isoenzymes or a single enzyme, the conformation of which differs between the soluble and membrane-bound state. Moreover, detailed subcellular fractionation of freshly isolated pig EC revealed that the distribution of cNOS activity closely resembled that of the plasma membrane marker 5'-nucleotidase, suggesting that most, if not all, of the cNOS activity in these cells is associated with the plasma membrane. This localization might render the enzyme more susceptible to activation by physical stimuli, such as a shear stress-induced change in the fluidity of the plasma membrane. Moreover, the continuous exposure to shear stress in vivo may also upregulate cNOS expression in EC, since specific enzyme activity, immunoreactivity and basal NO release were significantly higher in freshly isolated EC as compared with cultured EC.
Subcellular distribution of hexokinase (HK) isoenzymes in 22 human breast cancers (21 primary cancers and 1 axillary metastatic growth) and 7 non-pathological human mammary gland tissue samples was studied with starch gel electrophoresis on isolated cell fractions obtained by differential centrifugation. Fractions used were cytosol, mitochondria and microsomes. A comparison of two methods for detecting HK activity was made, yielding different results regarding HK II and HK III. A method based on the ability of NADPH to fluoresce in UV gave a constant pattern of HK isoenzymes. In non-pathological breast tissue, only HK I was seen, i.e. in the cytosol and the microsomal fractions. HK I was also seen in all fractions of the cancers, but another more anodal band of HK I, as well as HK II and HK III, consistently appeared in the cytosol fractions. The more commonly used staining technique with tetrazolium dye revealed HK II in 45% and HK III in 50% of the samples. The pattern of HK isoenzymes in the cancers was the same irrespective of estrogen and progesterone cytosolic receptor contents and the histology of the tumors. The fluorescence method is, therefore, much more sensitive than the tetrazolium technique for detecting HK activity after electrophoresis and could explain difference in results obtained by various laboratories.