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Results for “Subcellular localization”

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PubMed1993

Subcellular localization and dynamics of Mac-1 (alpha m beta 2) in human neutrophils.

The subcellular localization of Mac-1 was determined in resting and stimulated human neutrophils after disruption by nitrogen cavitation and fractionation on two-layer Percoll density gradients. Light membranes were further separated by high voltage free flow electrophoresis. Mac-1 was determined by an ELISA with monoclonal antibodies that were specific for the alpha-chain (CD11b). In unstimulated neutrophils, 75% of Mac-1 colocalized with specific granules including gelatinase granules, 20% with secretory vesicles and the rest with plasma membranes. Stimulation with nanomolar concentrations of FMLP resulted in the translocation of Mac-1 from secretory vesicles to the plasma membrane, and only minimal translocation from specific granules and gelatinase granules. Stimulation with PMA or Ionomycin resulted in full translocation of Mac-1 from secretory vesicles and gelatinase granules to the plasma membrane, and partial translocation of Mac-1 from specific granules. These findings were corroborated by flow cytometry, which demonstrated a 6-10-fold increase in the surface membrane content of Mac-1 in response to stimulation with FMLP, granulocyte-macrophage colony stimulating factor, IL-8, leukotriene B4, platelet-activating factor, TNF-alpha, and zymosan-activated serum, and a 25-fold increase in response to Ionomycin. Thus, secretory vesicles constitute the most important reservoir of Mac-1 that is incorporated into the plasma membrane during stimulation with inflammatory mediators.

Cell Compartmentation↗

PubMed1990

Type II regulatory subunit dimerization determines the subcellular localization of the cAMP-dependent protein kinase.

The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of dimeric regulatory subunits (RII) to anchoring proteins. Cytoskeletal localization occurs through RII dimer interaction with the PKA substrate molecule microtubule-associated protein 2 (MAP2). RII alpha deletion mutants and RII alpha/endonexin chimeras retained MAP2 binding activity if they contained the first 79 residues of the molecule. Disruption of RII alpha dimerization always prevented MAP2 interaction because 1) RII delta 1-14 (an amino-terminal deletion mutant lacking residues 1-14) was unable to bind MAP2 or form dimers, and 2) a modified RII alpha monomer including residues 1-14 did not bind MAP2. Chimeric proteins containing the first 30 residues of RII alpha fused to endonexin II formed dimers but did not bind MAP2. This suggested other side-chains between residues 30-79 also participate in MAP2 interaction. Peptide studies indicate additional contact with MAP2 may occur through an acidic region (residues 68-82) close to the RII autoinhibitor domain. Therefore, anchored PKA holoenzyme topology may position the catalytic subunit and MAP2 as to allow its preferential phosphorylation upon kinase activation.

Amino Acid Sequence↗

PubMed1997

Subcellular localization of G-proteins in primary-cultured mouse preadipocytes and adipocytes.

The subcellular localization of G5 alpha, Gi alpha 1&2, Gi alpha 3, and G beta was studied in primary-cultured undifferentiated and differentiated, lipid replete, adipose cells. The results show a distinct distribution for each of these G-proteins and differences between differentiated and undifferentiated cells. All the G-proteins examined had a cytoplasmic localization; only Gi alpha 1 and 2 showed a significant colocalization with the plasma membrane and this only in differentiated cells. Most studies using cells in culture have reported an intracellular localization for G-proteins, whereas in tissue sections the localization has been reported to be largely with the plasma membrane, with some intracellular localization. The results suggest that the cell-cell interactions or the specific geometry imposed by culture conditions favor the intracellular compared to peripheral localization of G-proteins. Alternately, the posttranslational modifications necessary for G-protein insertion in the plasma membrane may be deficient in cultured cells.

Adenylyl Cyclases↗

PubMed2006

Subcellular localization and regulation of type-1C and type-5 phosphodiesterases.

We investigated the subcellular localization of PDE5 in in vitro human myometrial cells. We demonstrated for the first time that PDE5 is localized in discrete cytoplasmic foci and vesicular compartments corresponding to centrosomes. We also found that PDE5 intracellular localization is not cell- or species-specific, as it is conserved in different animal and human cells. PDE5 protein levels are strongly regulated by the mitotic activity of the smooth muscle cells (SMCs), as they were increased in quiescent, contractile myometrial cultures, and conditions in which proliferation was inhibited. In contrast, PDE1C levels decreased in all conditions that inhibited proliferation. This mirrored the enzymatic activity of both PDE5 and PDE1C. Increasing cGMP intracellular levels by dbcGMP or sildenafil treatments did not block proliferation, while dbcAMP inhibited myometrial cell proliferation. Together, these results suggest that PDE5 regulation of cGMP intracellular levels is not involved in the control of SMC cycle progression, but may represent one of the markers of the contractile phenotype.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2002

Subcellular localization of pentachlorophenol 4-monooxygenase in Sphingobium chlorophenolicum ATCC 39723.

We have studied the subcellular localization of pentachlorophenol 4-monooxygenase (PCP4MO) in Sphingobium chlorophenolicum ATCC 39723 during induction by pentachlorophenol (PCP). Using a monoclonal antibody CL6 specific to the native and recombinant PCP4MO, the enzyme was primarily found soluble as determined by immunoblot and ELISA analyses of cellular fractions. However, the enzyme was observed both in the soluble and membrane-bound forms during induction for 2-4 h, suggesting its translocation out from the cytoplasm. Electron microscopy confirmed that PCP4MO was predominantly present in the cytoplasm at 1 h, whereas at 4 h significant amount was detected also in the membrane and periplasm. After 6 h, the majority of PCP4MO was in the periplasm and only small amount was bound to the inner membrane or present in the cytoplasm. The results indicate that after biosynthesis PCP4MO in S. chlorophenolicum is exported via the inner membrane to the final location in the periplasm.

Alphaproteobacteria↗

PubMed1988

Subcellular Localization of Proteases in Developing Leaves of Oats (Avena sativa L.).

The distribution and subcellular localization of the two major proteases present in oat (Avena sativa L. cv Victory) leaves was investigated. Both the acidic protease, active at pH 4.5, and the neutral protease, active at pH 7.5, are soluble enzymes; a few percent of the enzyme activity was ionically bound or loosely associated with organellar structures sedimenting at 1000g. On the average, 16% of the acidic protease could be washed out of the intercellular space of the leaf. Since isolated protoplasts contained correspondingly lower activities as compared to crude leaf extracts, part of the acidic activity is associated with cell walls. No neutral protease activity was recovered in intercellular washing fluid. Of the activities present in protoplasts, the acidic protease was localized in the vacuole, whereas the neutral protease was not. The localization of the acidic protease in vacuoles did not change during leaf development up to an advanced stage of senescence, when more than 50% of the leaf protein had been degraded. These observations indicate that protein degradation during leaf senescence is not due to a redistribution of acidic protease activity from the vacuole to the cytoplasm.

Journal Article↗

PubMed1997

Subcellular localization of dihydropyrimidine dehydrogenase.

Although dihydropyrimidine dehydrogenase (DPD) has been purified and characterized from liver tissues of various mammals conflicting data exist on its subcellular localization. To determine the localization of DPD we prepared crude subcellular fractions of a rat liver homogenate by means of differential centrifugation. In the fractions obtained (heavy mitochondrial, light mitochondrial, microsomal and cytosolic) the activities of different marker enzymes were measured as well as the activity of DPD. These results showed that almost all of the activity of DPD was located in the cytosolic fraction. To exclude any particulate-associated DPD, a light mitochondrial fraction was subsequently subjected to equilibrium density gradient centrifugation. The distribution profile of the activity of DPD and the various marker enzymes indicated that DPD from rat liver was exclusively located in the cytosol since no significant activity of DPD could be detected in any subcellular organelle.

Animals↗

PubMed1983

Subcellular localization of the receptor for gonadotropin-releasing hormone in pituitary and ovarian tissue.

The subcellular localization of GnRH receptors in weanling rat anterior pituitary and ovarian tissue was determined by radioligand binding in biochemically defined fractions prepared by differential and density centrifugation. Morphological identification of membrane organelles or fragments, visualized by electron microscopy, confirmed the biochemical characterization. The greatest amount of specific ligand binding in both tissues was measured in the crude membrane fraction, while small amounts were detected in other fractions. The distribution of pituitary binding sites in sucrose gradient subfractionation of crude membranes correlated with the distribution of plasma membrane (assessed by 5'-nucleotidase activity and electron microscopy), but not that of secretory granules (identified by immunoreactive LH) or lysosomes (acid phosphatase activity). These data suggest that the GnRH-binding sites detected by radioligand binding assay in pituitaries of rats not previously exposed to GnRH are localized almost exclusively in the plasma membrane fraction.

5'-Nucleotidase↗

PubMed2000

Distinct subcellular localization of transiently expressed types 1 and 2 iodothyronine deiodinases as determined by immunofluorescence confocal microscopy.

We compared the subcellular localization of FLAG-epitope tagged Types 1 and 2 deiodinases (D1 and D2) transiently expressed in human embryonic kidney (HEK-293) and mouse neuroblastoma (NB2A) cells. D2 is an integral membrane protein based on resistance to extraction at pH 11 with the NH2 terminus in the endoplasmic reticulum (ER). Immunofluorescence confocal microscopy using anti-FLAG and anti-GRP78/BiP antibodies showed the FLAG-D1 signal was found in the periphery of the cells and not co-localized with the ER specific marker GRP78/BiP. On the other hand, FLAG-D2 protein was found in the ER co-localized with the GRP78/BiP protein. These differential distribution patterns indicate subcellular sorting of D1 and D2 is determined by intrinsic protein sequence and can explain the ready access of D2-generated T3 to the nucleus.

Animals↗

PubMed1975

The subcellular localization of the erythrostimulant factors present in the kidney and spleen of the rabbit.

The subcellular localization of erythrostimulant factors contained in the kidney and spleen of the rabbit have been studied. This was done by subjecting homogenates to fractionation by centrifugation and assessing the erythrostimulant capacity of the hypotonic extracts prepared from the various fractions thus obtained. The results of these studies show that the erythrostimulant factors are present in the light mitochondrial and microsomal fractions of the normal kidney. In conditions of anaemia they are to be found not only in the above-mentioned fractions but also in the mitochondrial fraction. By contrast, in the spleen such factors are present only in the mitochondrial fraction. It is likely that the different localization of the erythrostimulant factors in the kidney and in the spleen of the rabbit may be closely connected with the different functions performed by these organs in the humoral regulation of erythropoiesis.

Animals↗

PubMed1982

Subcellular localization of the methylcitric-acid-cycle enzymes in propionate metabolism of Yarrowia lipolytica.

The subcellular localization of the four characteristic enzymes of the methylcitric acid cycle was studied with glucose-grown as well as n-alkane-grown cells of Yarrowia lipolytica. Microsomes and peroxisomes showed no cycle enzyme activities. The four cycle enzymes were constitutively localized in mitochondria, with the exception of the dual localization of the fourth enzyme, 2-methylisocitrate lyase, in mitochondria and cytoplasm, where the lyase may function to supply pyruvate (the end-product of the catabolism of the propionate residue) to various reactions.

Ascomycota↗

PubMed1992

Regional and subcellular localization of Li+ and other cations in the rat brain following long-term lithium administration.

Rats were given LiCl in their diet (40 mmol/kg dry weight) for at least 3 months to elucidate the regional and subcellular localization of Li+ in the brain as well as the effect of chronic lithium administration on the distribution of other cations. At steady-state the mean concentrations of Li+ were 0.66 mmol/kg wet weight in the whole brain and 0.52 mM in plasma. The tissue/plasma concentration ratio exceeded unity in all anatomical regions. No region showed excessive accumulation of Li+. Whole brain or regional contents of Na+ or K+ were unaffected by lithium treatment. Subcellular Li+ localization was demonstrated in nuclear, crude mitochondrial, and microsomal fractions of whole brain homogenate. Subfractionation of the crude mitochondrial fraction revealed energy-independent intrasynaptosomal and intramitochondrial Li+ and K+ localization at 0-4 degrees C. Li+ administered in vivo disappeared within 10 min from synaptosomes incubated at 37 degrees C. Li+ added in vitro at 1 mM attained a synaptosomal steady-state concentration within 30 min at 37 degrees C. In control rats, synaptosomal concentrations and synaptosomal/medium concentration gradients of cations paralleled their respective in vivo concentrations and gradients. Lithium treatment caused synaptosomal depletion of K+ and Mg2+ and hence probably partial membrane depolarization. Addition of 1 mM Li+ in vitro also caused synaptosomal Mg2+ depletion. The results indicate that Li+ is "accumulated" in brain sediments and synaptosomes following its long-term treatment. The estimated intracellular and intrasynaptosomal Li+ concentrations are lower than predicted by passive distribution according to the Nernst equation, evidencing active extrusion of Li+.

Animals↗

PubMed1979

Subcellular localization of thyroxine and reverse triiodothyronine outer ring monodeiodinating activities.

In order to examine the subcellular localization of outer ring T4- and rT3-monodeiodinating activities, nuclear, mitochondrial, microsomal, cytosol, and plasma membrane fractions of rat liver homogenate were incubated with either T4 or rT3 in phosphate buffer (pH 7.35) in the presence of 2 mM dithiothreitol for 15 min at 37 C, and the amount of product (T3 in the case of T4 and 3,3'-diiodothyronine in the case of rT3) was measured by specific RIA. The various tissue fractions were also examined for the relative concentration of various marker enzymes. T4 and rT3 monodeiodinating activities correlated better with enzyme markers of plasma membranes than of any other subcellular fraction in most tissue fractions. A fraction could be isolated, however, in which the monodeiodinating activities correlated better with the enzyme markers of microsomes than of plasma membranes. The various data suggest that plasma membranes and microsomes are two main sites of T4- and rT3-monodeiodinating activities. The location of T4 to T3 converting activity in the plasma membranes may serve to modulate the delivery of the more potent thyroid hormone, i.e. T3, into the cells.

Animals↗

PubMed1982

Subcellular localization and quantification of cholesterol in cultured human fibroblasts exposed to human low density lipoprotein.

Subcellular localization of nonesterified cholesterol has been determined in normal human fibroblasts from cultures incubated with human low density lipoprotein (LDL). Nonesterified and esterified cholesterol content of fibroblasts, grown initially in the absence of cholesterol, increased significantly after a 1-hour incubation with LDL. Digitonin was used to localize nonesterified cholesterol that was accumulated within multivesicular and lamellar lysosomal inclusions. This was observed only in fibroblasts from cultures incubated with LDL. Accumulation of LDL-derived nonesterified cholesterol within lysosomes is consistent with the suggestion of other investigators that LDL is metabolized within lysosomes.

Cell Membrane↗

PubMed2001

The effects of subcellular localization of N-(2-hydroxypropyl)methacrylamide copolymer-Mce(6) conjugates in a human ovarian carcinoma.

Photosensitizers, light-sensitive compounds, become activated upon illumination with a specific wavelength of light generating cytotoxic oxygen species. Due to the short half-life of singlet oxygen, the subcellular site of localization and excitation affects the type of cellular damage produced as well as cellular responses to different types of photodamage created within the cell. Here, we investigated the effects of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-mesochlorin e(6) monoethylenediamine (Mce(6)) conjugates localized to different subcellular compartments. Temperature was utilized to achieve subcellular localization of conjugates and subcellular fractionation was performed to confirm localization patterns of HPMA copolymer-Mce(6) conjugates. Cytotoxicity studies suggest plasma membrane and late endosomes were more sensitive to photodamage than lysosomal compartments as observed by an approximate 2-fold decrease in the IC(50) compared to lysosomally accumulated conjugate. Releasing Mce(6) from the polymer backbone within lysosomal compartments significantly lowered the IC(50) when compared to HPMA copolymer conjugates with Mce6 bound via a nondegradable linkage. These differences will prove useful in the future design of HPMA copolymer-Mce(6) conjugates for the treatment of ovarian cancer.

Antineoplastic Agents↗

PubMed1996

Subcellular localization of growth inhibitory factor in rat brain: light and electron microscopic immunohistochemical studies.

The subcellular localization of growth inhibitory factor (GIF), a brain-specific member of the metallothionein family, was determined in the rat brain by electron microscopic immunohistochemistry using a rabbit antiserum against a synthetic polypeptide specific for rat GIF. The major cell type that expressed a high level of GIF immunoreactivity was the astrocytes. In these cells, dense labelling was observed throughout the soma and the fine processes, in association with the free ribosomes, rough endoplasmic reticulum, small vesicles, the outer membrane of the mitochondria and part of the plasma membrane. Astrocytic end-feet around blood vessels exhibited intense immunoreactivity. Another cell type exhibiting GIF immunolabelling was the neurons. However, this immunoreactivity was restricted to a subset of the neuronal population, and in contrast to the astrocytic pattern, the labelling was localized predominantly in the processes including axons and dendrites, in association with microtubules, ribosomes, the outer membrane of the mitochondria and the plasmalemma. Synaptic elements, including dendritic spines, also showed definite immunoreactivity in association with synaptic vesicles and post-synaptic densities. No labelling was observed in the oligodendrocytes or microglia. The present data suggest that GIF is expressed in both astrocytes and neurons, and plays rather specific roles in each phenotype.

Animals↗

PubMed1988

Subcellular localization of DNA polymerase gamma and changes in its activity in sea urchin embryos.

1. Subcellular localization and changes in the activity of DNA polymerase gamma were examined in sea urchin eggs and embryos. 2. The enzyme was shown to be localized predominantly in mitochondria by differential and isopycnic centrifugation. 3. During embryogenesis, the enzyme activity per embryo remained constant until blastula stage, and thereafter increased. 4. Similarly mitochondrial DNA per embryo increased, indicating that mitochondrial DNA replication starts during embryogenesis. 5. The gamma-activity per mitochondrial DNA remained constant during embryogenesis. 6. These results suggest that mitochondria contain a constant amount of replicative enzyme (DNA polymerase gamma) regardless of mitochondrial DNA replication, which differs from the case of nuclear DNA replication.

Animals↗

PubMed1998

Subcellular localization and cytotoxic activity of the GroEL-like protein isolated from Actinobacillus actinomycetemcomitans.

The subcellular locations, ultrastructure, and cytotoxic activity of the GroEL-like protein from Actinobacillus actinomycetemcomitans were investigated. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) clearly indicated that synthesis of the GroEL-like protein is substantially increased after a thermal shock. Analysis of the purified native GroEL-like protein by transmission electron microscopy revealed the typical 14-mer cylindrical molecule, which had a diameter of about 12 nm. A. actinomycetemcomitans cells grown at 35 degreesC and heat shocked at 43 degreesC were fractionated, and fractions were separated by SDS-PAGE and analyzed by Western immunoblotting using antibodies to GroEL- and DnaK-like proteins. The GroEL-like protein was found in both the soluble and membrane fractions, whereas the DnaK-like protein was mostly found in the cytoplasm. An increase in specific proteins, including the GroEL- and DnaK-like proteins, was found in heat-shocked cells. The subcellular localization of the GroEL-like protein was examined by immunoelectron microscopy of whole cells. More GroEL-like protein was detected in stressed cells than in unstressed cells, and most of it was found not directly associated with outer membranes but rather in extracellular material. The native GroEL-like protein was assessed for cytotoxic activities. The GroEL-like protein increased the proliferation of periodontal ligament epithelial cells at concentrations between 0.4 and 1.0 microgram/ml. The number of cells in the culture decreased significantly at higher concentrations. A cell viability assay using HaCaT epithelial cells indicated that the GroEL-like protein was strongly toxic for the cells. These studies suggest the extracellular nature of the GroEL-like protein and its putative role in disease initiation.

Aggregatibacter actinomycetemcomitans↗