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Results for “Subcellular localization”

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PubMed1991

Subcellular localization of porphyrins using confocal laser scanning microscopy.

The in vitro subcellular distribution patterns of 10 porphyrins, varying in hydrophobicity and charge, were studied using confocal laser scanning microscopy on two cell lines (V79 and C6 glioma cells) for incubation times up to 24 h. All of the porphyrins were taken up rapidly by both cell lines and distinct classes of subcellular distribution patterns were observed: general cytoplasmic staining; localization in lysosomes (usually associated with general cytoplasmic staining); localization in mitochondria (and general cytoplasmic staining); localization in mitochondria with subsequent uptake into lysosomes. Structure-localization relationships which have emerged are that porphyrins with dominantly cationic side chains localize in mitochondria, whereas those with a more anionic character tend to localize in lysosomes.

Animals↗

PubMed1981

Subcellular localization and properties of alkaline inorganic pyrophosphatase in human polymorphonuclear leucocytes.

A new rapid assay for inorganic pyrophosphatase has been developed and the procedure optimised for measurement of the enzyme in human neutrophils. Kinetic studies showed that the activity was optimal at pH 8.0 and was activated by Mg2+. No neutral or acid pyrophosphatase was detected. Neutrophils were homogenised in isotonic sucrose and, after low speed centrifugation the intracellular localization of pyrophosphatase was determined by analytical subcellular fractionation with sucrose density gradient centrifugation. Pyrophosphatase was shown to have a dual localization to mitochondria and cytosol. No activity could be attributed to either the endoplasmic reticulum or alkaline phosphatase-containing granules (phosphasomes). Inhibitor studies clearly show that the cytosolic and mitochondrial pyrophosphatases are due to distinct enzymes. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and subjects in the third trimester of pregnancy. The specific activity (mU/mg protein) of pyrophosphatase, in contrast to that of alkaline phosphatase was similar in the three groups. Levamisole, a potent inhibitor of alkaline phosphatase had no effect on pyrophosphatase activity, confirming that this activity is not attributable to neutrophil alkaline phosphatase.

Cytosol↗

PubMed2004

Subcellular localization and targeting of N-acetylglucosaminyl phosphatidylinositol de-N-acetylase, the second enzyme in the glycosylphosphatidylinositol biosynthetic pathway.

The second step in glycosylphosphatidylinositol biosynthesis is the de-N-acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) catalyzed by N-acetylglucosaminylphosphatidylinositol deacetylase (PIG-L). Previous studies of mouse thymoma cells showed that GlcNAc-PI de-N-acetylase activity is localized to the endoplasmic reticulum (ER) but enriched in a mitochondria-associated ER membrane (MAM) domain. Because PIG-L has no readily identifiable ER sorting determinants, we were interested in learning how PIG-L is localized to the ER and possibly enriched in MAM. We used HeLa cells transiently or stably expressing epitope-tagged PIG-L variants or chimeric constructs composed of elements of PIG-L fused to Tac antigen, a cell surface protein. We first analyzed the subcellular distribution of PIG-L and Glc-NAc-PI-de-N-acetylase activity and then studied the localization of Tac-PIG-L chimeras to identify sequence elements in PIG-L responsible for its subcellular localization. We show that human PIG-L is a type I membrane protein with a large cytoplasmic domain and that, unlike the result with mouse thymoma cells, both PIG-L and GlcNAc-PI-de-N-acetylase activity are uniformly distributed between ER and MAM in HeLa cells. Analyses of a series of Tac-PIG-L chimeras indicated that PIG-L contains two ER localization signals, an independent retention signal located between residues 60 and 88 of its cytoplasmic domain and another weak signal in the luminal and transmembrane domains that functions autonomously in the presence of membrane proximal residues of the cytoplasmic domain that themselves lack any retention information. We conclude that PIG-L, like a number of other ER membrane proteins, is retained in the ER through a multi-component localization signal rather than a discrete sorting motif.

Acetylglucosamine↗

PubMed1983

Subcellular localization of human heart atypical creatine kinase.

Human heart subcellular fractions were obtained using method A described by Scholte et al [7,8]. It was found that 3.5 +/- 2.5%, 3.4 +/- 1.9%, 8.5 +/- 2.9%, 2.5 +/- 1.3%, and 88 +/- 21% of the total creatine kinase activity was associated with the myofibrillar, nuclear, mitochondrial, microsomal and particle-free soluble fractions respectively. Using DEAE-sephacel chromatography it was determined that the atypical creatine kinase (CK-Z) accounted for approximately 90% of the creatine kinase activity associated with the mitochondrial fraction.

Animals↗

PubMed1988

Subcellular localization of rat brain insulin binding sites.

Fractions and subcellular structures were prepared from rat brain homogenate and their purity was assessed using enzyme markers, gamma-aminobutyric acid binding, DNA content, and electron microscopy. Insulin binding was highest on the plasma membrane preparations and approximately 50% less so on brain homogenate crude mitochondrial (P2), myelinated axon, and synaptosome preparations. Very low levels of binding were found on mitochondria and nuclei. Differences in binding between fractions were due to numbers of binding sites, and not variable binding affinity. There was a close relationship between insulin binding and the activity of Na/K ATPase (E.C. 3.6.1.4) in all fractions (r = 0.98). Insulin binding to the P2 was compared with plasma membrane fractions in seven brain regions, and the results demonstrated the same close relationship between insulin binding and plasma membrane content in all regions except hypothalamus. Plasma membrane insulin binding was well represented by the binding on P2 membranes in all regions except hypothalamus and brainstem. It was concluded that insulin binding is distributed evenly over the surface of brain cells and is not increased on nerve endings.

Animals↗

PubMed1981

Late nonstructural 100,000- and 33,000-dalton proteins of adenovirus type 2. I. Subcellular localization during the course of infection.

We analyzed the subcellular locations of the late adenovirus type 2 nonstructural 100,000-dalton (100K) and 33K proteins in adenovirus type 2-infected HeLa cells both by biochemical cell fractionation and by immunofluorescence microscopy, using specific antisera against purified sodium dodecyl sulfate-denatured 100K and 33K polypeptides. Both methods showed that the 100K protein was present in the cytoplasm as well as in the nuclei of infected cells and that it accumulated in the nuclei during the course of infection. Phosphorylated 100K protein also was found both in the cytoplasm and in nuclei. However, the nuclear 100K protein pool was phosphorylated to a higher degree than the cytoplasmic pool. In all experiments the 33K protein, which also is a phosphoprotein, was present exclusively in the nuclei of infected cells. The 100K and 33K proteins were associated with different nuclear substructures; this was demonstrated serologically by an analysis of infected cells in which double color immunofluorescence microscopy was used. In these experiments antibodies against the 100K protein decorated different nuclear structures than antibodies against the 33K protein.

Adenoviruses, Human↗

PubMed1999

Subcellular localization of fructosyl amino acid oxidases in peroxisomes of Aspergillus terreus and Penicillium janthinellum.

Fructosyl amino acid oxidase (FAOD) is the enzyme catalyzing the oxidative deglycation of Amadori compounds, such as fructosyl amino acids, yielding the corresponding amino acids, glucosone, and H(2)O(2). In a previous report, we determined the primary structures of cDNAs coding for FAODs from two fungal strains Aspergillus terreus AP1 and Penicillium janthinellum and we found that both fungal FAODs included the putative peroxisome targeting signal 1 (PTS1) at the carboxyl terminal (Yoshida, N. et al., Eur. J. Biochem., 242, 499-505, 1996). In this study, we determined the intracellular localization of FAODs in these two fungi. Subcellular fractionation experiments and immuno-electronmicroscopic observations, together with the previous findings indicated that the FAODs were localized in peroxisomes of A. terreus AP1 and P. janthinellum. These FAODs were also found to belong to a new member of "peroxisomal sarcosine oxidase family protein" in eucaryotic cells.

Journal Article↗

PubMed2002

Differences in the subcellular localization of alpha1-adrenoceptor subtypes can affect the subtype selectivity of drugs in a study with the fluorescent ligand BODIPY FL-prazosin.

G protein-coupled receptor (GPCR) subtypes are differentially distributed in the cell; however, it remains unclear how this affects the subtype selectivity of particular drugs. In the present study, we used flow cytometry analysis with the fluorescent ligand, BODIPY FL-prazosin, to study the relationship between the subcellular distribution of subtype receptors and the subtype-selective character of ligands using alpha1a and alpha1b-adrenoceptors (ARs). Alpha1a-ARs predominantly localize inside the cell, while alpha1b-ARs on the cell surface. Flow cytometry analysis and confocal laser-scanning micrographs of living cells showed that BODIPY FL-prazosin can label not only alpha1-ARs on the cell surface, but also those localized inside the cell. Furthermore, flow cytometry analysis of alpha1A-AR-selective drug, KMD-3213, and alpha1B-AR-selective drug, CEC, revealed that the major determinant of the subtype selectivity of each drug is different. The alpha1A-AR selectivity of KMD-3213 can be explained by its much higher affinity for alpha1a-AR than alpha1b-AR (affinity-dependent selectivity), while the alpha1B-AR selectivity of the hydrophilic alkylating agent CEC is due to preferential inactivation of alpha1-ARs on the cell surface (receptor localization-dependent selectivity). This study illustrates that factors in addition to the affinity of the drug for the receptor, such as subcellular localization of the receptor, should be taken into account in assessing the subtype selectivity of a drug.

Adrenergic alpha-Antagonists↗

PubMed1984

Distribution and subcellular localization of a hepatic proliferation inhibitor in rat liver.

The subcellular and intralobular distributions of a protein which specifically inhibits the proliferation of normal liver cells were determined in rat liver, using a combination of immunological and biochemical techniques. The IgG fraction from an antiserum raised against the hepatic proliferation inhibitor was isolated by protein A-Sepharose CL-4B chromatography and shown to be highly specific for the antigen using electroimmunodiffusion and affinity chromatography. To determine the intracellular location of the inhibitor, subcellular fractions were prepared from adult rat livers by differential centrifugation. The cytoplasmic fraction contained the biologically active cytostatic inhibitor, whereas the nuclear and mitochondrial fractions were inactive. Cytoplasmic localization of the hepatic proliferation inhibitor was further confirmed by anion exchange high performance liquid chromatography and by double immunodiffusion with the anti-hepatic proliferation inhibitor IgG. When liver sections were subjected to histochemical staining mediated through the immune IgG and an avidin-biotinylated horseradish peroxidase complex, the parenchymal liver cells were stained, but endothelial and connective tissue cells were not. Although some staining was evident throughout the liver parenchyma, the most intensely stained cells were located in the centrilobular region. Moreover, an age-dependent increase in the staining intensity and/or in the number of cells containing the proliferation inhibitor was observed. Preliminary experiments showed that little, if any, staining occurred in hepatocellular carcinoma cells. This highly specific IgG can be used to monitor alterations in the content and location of hepatic proliferation inhibitor in proliferative disorders of the liver.

Animals↗

PubMed1980

Subcellular localization of serotonin and motilin in the human jejunum.

1. Analytical subcellular fractionation techniques have been applied to human jejunal homogenates to study the localization of serotonin and motilin. 2. Serotonin, detected by a fluorimetric assay, showed two distinct particulate distributions in the density gradients with modal densities of 1.14 and 1.20. Motilin, whether detected by N-terminal or C-terminal specific antibodies showed similar density distributions with a modal density of 1.20, coinciding with the denser peak of serotonin. 3. These results lend support to the histochemical suggestions that motilin is located in a a serotonin-containing granule.

Centrifugation, Isopycnic↗

PubMed1985

Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells.

To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22 h in the presence of D-[1-3H]galactose or [3H]GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipid-sialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellular membrane fractions studied was recovered from plasma membrane and only 10-15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous [3H]GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid.

Animals↗

PubMed2004

Subcellular localization of adenine and xanthine phosphoribosyltransferases in Leishmania donovani.

The subcellular location of a protein is a critical factor in its physiological function and an important consideration in therapeutic paradigms that target the protein. Because Leishmania donovani cannot synthesize purine nucleotides de novo, they rely predominantly upon therapeutically germane phosphoribosyltransferase (PRT) enzymes, hypoxanthine-guanine PRT (HGPRT), adenine PRT (APRT), and xanthine PRT (XPRT), for purine acquisition from the host. Previous studies have shown that the L. donovani HGPRT is localized to the glycosome, a fuel-metabolizing microbody that is unique to kinetoplastid parasites [J. Biol. Chem. 273 (1998) 1534]. The sequences of the other two PRTs indicate that XPRT, but not APRT, possesses a COOH-terminal tripeptide that mediates protein targeting to the glycosome. To determine definitively the intracellular milieu of APRT and XPRT, polyclonal antibodies were raised to each recombinant protein. APRT and XPRT were then shown by immunofluorescence to be localized to the cytosol and glycosome, respectively. The glycosomal milieu for XPRT was also verified by immunoelectron microscopy. Amputation of the glycosomal targeting signal from XPRT resulted in protein mislocalization to the cytosol, but the cytosolic xprt was still functional with respect to purine salvage. These studies establish that APRT is cytosolic and XPRT, like the homologous HGPRT, is glycosomal and demonstrate that a mutant xprt protein that mislocalizes to the cytosol is still functional and supports parasite viability.

Adenine Phosphoribosyltransferase↗

PubMed2006

Activity and subcellular localization of glucose-6-phosphate dehydrogenase in peach fruits.

The subcellular distribution and activity of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) were studied in developing peach (Prunus persica L. Batsch cv. Zaoyu) fruit. Fruit tissues were separated by differential centrifugation at 15,000g into plastidic and cytosolic fractions. There was no serious loss of enzyme activity (or activation) during the preparation of fractions. G6PDH activity was found in both the plastidic and cytosolic compartments. Moreover, DTT had no effect on the plastidic G6PDH activities, that is, the redox regulatory mechanism did not play an important role in the peach fleshy tissue. Results from the immunogold electron-microscope localization revealed that G6PDH isoenzymes were mainly present in the cytosol, the secondary wall and plastids (chloroplasts and chromoplasts), but scarcely found in the starch granules or the cell wall. In addition to a decrease in fruit firmness, the G6PDH activity in the cytotolic and plastidic fractions increased, and anthocyanin started to accumulate during fruit maturation. These results suggest that G6PDH, by providing precursors for metabolic processes, might be associated with the red coloration that occurs in peach fruit.

Anthocyanins↗

PubMed1994

Subcellular localization and chaperone activities of Borrelia burgdorferi Hsp60 and Hsp70.

Subcellular locations and chaperone functions of Hsp60 and Hsp70 with flagellin were investigated in Borrelia burgdorferi. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis of fractionated cells showed Hsp60 to be present in the soluble fractions and the Triton X-100 detergent-soluble membrane fraction at growth temperatures ranging from 20 to 37 degrees C. The relative amount of Hsp60 associated with the membrane increased with growth temperature. Hsp70 was found in soluble fractions at growth temperatures between 28 and 37 degrees C, but at 20 degrees C it was also present in the Triton X-100-insoluble membrane fraction. Immunoelectron microscopy revealed that the majority of Hsp60 was localized in the cytoplasm but a detectable fraction (approximately 30%) was associated with the cell envelope. The chaperone functions of Hsp60 and Hsp70 were analyzed by immunoprecipitation of [35S]methionine-labeled cell lysates under nondenaturing conditions in the presence or absence of ATP. Hsp70 was found to bind flagellin at all temperatures tested between 33 and 41 degrees C. This association could be decreased with ATP when cells had been incubated at 41 degrees C during radioactive labeling but not at lower temperatures. Both flagellin and Hsp70 were found to associate with Hsp60, forming a complex of the three proteins. Hsp70 association with this complex could be decreased with ATP, but flagellin binding to Hsp60 was ATP independent at all temperatures studied. Both Hsp70 and flagellin were inaccessible to monoclonal antibodies against them when bound to Hsp60. These studies suggest that in B. burgdorferi, a major function of Hsp60 and Hsp70 is in the molecular processing of flagellin.

Adenosine Triphosphate↗

PubMed1999

Subcellular localization of enzyme activities involved in the metabolism of platelet-activating factor in rainbow trout leukocytes.

The subcellular distribution of an alkyllyso-GPC: acetyl-CoA acetyltransferase (EC 2.3.1.67) and transacylase, two important enzyme activities involved in the remodeling pathway for the biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) have been examined in leukocytes isolated from the pronephros of the rainbow trout, Oncorhynchus mykiss. Contrary to mammalian systems, in which the acetyltransferase is localized to intracellular membranes, the subcellular distribution of an acetyltransferase activity in rainbow trout leukocytes was localized to the plasma membrane. Analysis of the acetyltransferase products by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) confirmed synthesis of two subclasses of PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine. The transacylase activity in this study was detected in membrane fractions in two domains of the intermediate density region which also contained the NADH dehydrogenase activity, a marker enzyme for the endoplasmic reticulum. Acylation of lysoPAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) exhibited approximately 95% specificity for omega-3 fatty acids. Acylation patterns were not significantly different in either domain of the endoplasmic reticulum. A model is proposed herein for the metabolism of PAF in rainbow trout leukocytes.

Acyltransferases↗

PubMed1983

Subcellular localization of bioactive cholecystokinin-like peptides in the rat cerebral cortex.

The subcellular distribution of cholecystokinin (CCK), especially its exact localization within the synaptosome, was studied in the rat cerebral cortex. Highest CCK-like bioactivity was measured in the synaptic membrane fractions, paralleling the distribution of (Na+ + K+)-dependent ATPase. In the synaptic vesicles, which were characterized by high acetylcholine content and by the absence of (Na+ + K+)-ATPase, only minimal quantities of CCK were detected.

Animals↗

PubMed1998

Loss or altered subcellular localization of p27 in Barrett's associated adenocarcinoma.

The cyclin-dependent kinase inhibitor p27 is a negative regulator of the cell division cycle. It is expressed at the highest levels during the quiescent (G0) and prereplicative (G1) phases, and its degradation is required for entry into the S phase. Because lack of p27 is associated with aggressive behavior in a variety of tumors of epithelial and lymphoid origin, we used immunohistochemistry and in situ hybridization to evaluate the expression of p27 in metaplastic and dysplastic Barrett's epithelium and to assess its prognostic significance in Barrett's associated adenocarcinoma (BAA) of the esophagus. In metaplastic Barrett's epithelium, p27 protein and mRNA were restricted to the superficial third of glands in all cases and extended to the lower third in 4 cases. In contrast, expression of p27 message and protein was both increased and full-thickness, in the 23 cases with high-grade dysplasia adjacent to BAA and in carcinoma in situ. Although all invasive carcinomas had elevated levels of p27 mRNA, 45 (83%) of 54 invasive carcinomas had low p27 protein levels (<50% positive tumor cells). Low p27 protein correlated with higher histological grade (P < 0.0001), depth of invasion (P = 0.0120), presence of lymph node metastasis (P = 0.05), and survival (P = 0.0197). In addition to the nuclear staining, cytoplasmic staining of p27 was noted in 11 of 23 (48%) of cases of dysplasia and in 14 of 54 (26%) adenocarcinomas and confirmed, in a subset of cases, by subcellular fractionation of protein lysates obtained from fresh tumor tissues. Cytoplasmic localization of p27 was also associated with decreased survival (P = 0.0239). Loss of p27 conferred poor prognosis independently of proliferative index, as assessed by Ki-67 (MIB-1) immunostaining, which was not significantly different in survivors versus nonsurvivors. These results show that: (a) distribution of p27 message and protein parallel one another in metaplastic and dysplastic Barrett's epithelium, suggesting transcriptional regulation of the gene in the nonneoplastic setting; (b) p27 is inactivated in the majority of BAA as a result of either post-transcriptional modification or altered subcellular localization; and (c) loss of the cell cycle inhibitor p27 is associated with parameters of aggressive behavior and unfavorable outcome in BAA.

Adenocarcinoma↗

PubMed1994

Subcellular localization of metallothionein IIA in human bladder tumor cells using a novel epitope-specific antiserum.

Human metallothioneins (hMTs) are a family of highly homologous proteins thought to be critical in cellular protection against toxins. The comparative function of individual isoforms is, however, obscure. Antibodies to individual MT isoforms might help clarify this issue but their generation has been challenging. We now describe a strategy that has successfully produced an epitope-specific antiserum to a major human isoform, hMT IIA. The immunogen, a conjugated tridecyl amino acid synthetic peptide unique to residues 8-20 in hMT IIA (AAGDSCTCAGSCK), yielded an antiserum (B2) that reacted specifically with hMT IIA in a concentration- and titer-dependent manner and showed no reactivity with human or rabbit liver MT I or horse MTs. This antiserum recognized rabbit liver MT II and, surprisingly, also reacted weakly with chicken MT. MT amino acid sequence comparisons and peptide blocking studies suggested aspartate-11 and threonine-14 are important antigenic determinants for B2. Using confocal immunofluorescence microscopy and B2 antiserum, we observed nuclear localization of hMT IIA with human bladder T24 tumor cells in exponential growth but more cytoplasmic localization at confluence. These results suggest the subcellular location of hMT IIA is a function of cell density in T24 bladder carcinoma cells. The general approach of epitope-specific antibodies may be useful for the generation of antibodies to other MT isoforms and for studying the role of individual MT isoforms in biology and toxicology.

Amino Acid Sequence↗