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At least 1,279 records · Page 71Linked to original sources
PubMed1981

Subcellular localization of aldosterone-induced proteins in toad urinary bladders.

Paired toad urinary hemibladders were incubated with [35S]methionine in the presence (experimental) or absence (control) of aldosterone. Short-circuit current was used to monitor aldosterone-induced Na+ transport. Protein synthesis in epithelial cell subcellular fractions (cytosolic, microsomal, mitochondrial) was evaluated by gradient polyacrylamide gel electrophoresis and autoradiography. Aldosterone-induced proteins were identified in the cytosolic and microsomal fractions (70 000 and 15 000 daltons, respectively). These results represent the first demonstration of aldosterone-induced proteins in subcellular fractions of epithelial cells derived from single toad urinary hemibladders.

Aldosterone↗
PubMed1980

Hyphal tip growth in Achlya. II. Subcellular localization of cellulase and associated enzymes.

The isolation and characterization of cellulase-containing membranes of Achlya ambisexualis Raper was attempted by differential and density gradient centrifugations. Maximum cellulase activity was found at an isopycnic density of 1.19 g/cm3, although some activity was found at other densities. A similar distribution of activity was shown by IDPase, ATPase, UDPG transferase, and by sedimentable carbohydrate. The coequilibration and steady enrichment of these activities during purification suggests their presence in a single type of subcellular particle. It was not possible to identify clearly the particle(s) in question from isolated fractions by electron microscopy, but when compared with the cytochemical localization of carbohydrate and IDPase in intact hypha, cytoplasmic vesicles with 150-mm diameters seem to be likely candidates.

Acid Anhydride Hydrolases↗
PubMed1988

Phospholipase A2 activity in resting and activated human neutrophils. Substrate specificity, pH dependence, and subcellular localization.

We have studied the phospholipase A2 activity in fractionated human neutrophils, employing labeled phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine as exogenous substrates. We used these phospholipid substrates labeled in the sn-1 position and measured the resulting labeled lysophospholipid forms in order to ascertain the phospholipase A2 specificity. In postnuclear supernatants from resting and A23187-activated cells, the phospholipase A2 activity showed a similar pH dependence curve with two pH optima at 5.5 and 7.5. Extracts from activated cells showed a 3-6-fold increase in enzyme activity. The subcellular distribution of phospholipase A2 activity in resting and A23187-treated human neutrophils was investigated by fractionation of postnuclear supernatants on continuous sucrose gradients. The neutral phospholipase A2 behaved as a membrane-bound enzyme and was mainly localized in the plasma membrane, the azurophilic granule, and in an ill-defined region of the gradient between the specific granules and mitochondria. The phospholipase A2 located in this undefined region showed a higher degree of activation than that located in other subcellular particulates in A23187-treated cells. This specific activation of an intracellular phospholipase A2 activity during cell stimulation indicates that cell compartmentalization may play a role in the formation of cell-activating and/or signal-transducing agents through the generation of arachidonate metabolites. Phosphatidylinositol was a better substrate for the plasma membrane enzyme, whereas phosphatidylcholine and phosphatidylethanolamine behaved as better substrates for intracellular organelle phospholipase A2 activities. The phospholipase A2 with maximal activity at pH 5.5 behaved as a soluble enzyme, and was almost completely localized in the azurophilic granules. Upon cell activation this acid enzyme activity was released in a similar way to beta-glucuronidase, a marker of azurophilic granules. These results demonstrate the different molecular properties of the phospholipase A2 activity, on the basis of its cellular location.

Calcimycin↗
PubMed2004

GLUT4 overexpression or deficiency in adipocytes of transgenic mice alters the composition of GLUT4 vesicles and the subcellular localization of GLUT4 and insulin-responsive aminopeptidase.

The majority of GLUT4 is sequestered in unique intracellular vesicles in the absence of insulin. Upon insulin stimulation GLUT4 vesicles translocate to, and fuse with, the plasma membrane. To determine the effect of GLUT4 content on the distribution and subcellular trafficking of GLUT4 and other vesicle proteins, adipocytes of adipose-specific, GLUT4-deficient (aP2-GLUT4-/-) mice and adipose-specific, GLUT4-overexpressing (aP2-GLUT4-Tg) mice were studied. GLUT4 amount was reduced by 80-95% in aP2-GLUT4-/- adipocytes and increased approximately 10-fold in aP2-GLUT4-Tg adipocytes compared with controls. Insulin-responsive aminopeptidase (IRAP) protein amount was decreased 35% in aP2-GLUT4-/- adipocytes and increased 45% in aP2-GLUT4-Tg adipocytes. VAMP2 protein was also decreased by 60% in aP2-GLUT4-/- adipocytes and increased 2-fold in aP2-GLUT4-Tg adipocytes. IRAP and VAMP2 mRNA levels were unaffected in aP2-GLUT4-Tg, suggesting that overexpression of GLUT4 affects IRAP and VAMP2 protein stability. The amount and subcellular distribution of syntaxin4, SNAP23, Munc-18c, and GLUT1 were unchanged in either aP2-GLUT4-/- or aP2-GLUT4-Tg adipocytes, but transferrin receptor was partially redistributed to the plasma membrane in aP2-GLUT4-Tg adipocytes. Immunogold electron microscopy revealed that overexpression of GLUT4 in adipocytes increased the number of GLUT4 molecules per vesicle nearly 2-fold and the number of GLUT4 and IRAP-containing vesicles per cell 3-fold. In addition, the proportion of cellular GLUT4 and IRAP at the plasma membrane in unstimulated aP2-GLUT4-Tg adipocytes was increased 4- and 2-fold, respectively, suggesting that sequestration of GLUT4 and IRAP is saturable. Our results show that GLUT4 overexpression or deficiency affects the amount of other GLUT4-vesicle proteins including IRAP and VAMP2 and that GLUT4 sequestration is saturable.

Adipocytes↗
PubMed1996

Subcellular localization of diacylglycerol kinase activity in stimulated and unstimulated human peripheral blood lymphocytes and neutrophils.

The diacylglycerol (DAG) kinase activity was determined in crude cytosol and membrane fractions in human peripheral blood lymphocytes and neutrophils. In neutrophils the basal lipid kinase activity was identical in the two subcellular fractions whereas in lymphocytes the basal enzyme activity was 1.6-fold higher in the membrane fraction. In general, the DAG kinase activity in lymphocyte fractions was 10- to 20-fold higher than in neutrophil fractions. When lymphocytes were stimulated with phytohaemagglutinin for 4 hours, a significant decrease in the DAG kinase activity in the membrane fraction was detected. By contrast, a 30-min activation with N-formyl-methionyl-leucyl-phenylalanine markedly increased the lipid kinase activity in both subcellular fractions in neutrophils. This activity was partially inhibited by the compound R59022. These results suggest that the DAG kinase is widely distributed in human leukocytes, but the enzyme's activity and regulation may vary in different leukocyte types.

Cell Membrane↗
PubMed1996

Immunocytochemical evidence for differential subcellular localization of 5 alpha-reductase isoenzymes in human tissues.

To contrast the previously described distribution of 5 alpha-reductase isoenzyme 2 in human organs we have raised synthetic C-terminal peptides of human 5 alpha-reductase isoenzyme 1 (h 5 alpha r1) and isoenzyme 2 (h 5 alpha r2) respectively, and studied the cellular and subcellular distribution of both isoforms of this key enzyme of testosterone metabolism using the human prostate gland as a reference. h 5 alpha r1, which in Western blots of human prostatic proteins had an apparent molecular weight of 23 kDa, was localized immunohistochemically in the nucleus of prostatic epithelial and stromal cells. Ultrastructurally, it was closely associated with the nuclear matrix. The apparent molecular weight of h 5 alpha r2 was 26 kDa in Western blotting of human prostatic proteins. In immunohistochemically processed sections, it was seen in the cytoplasm of prostatic epithelial as well as stromal cells. An organ screening with genital and extragenital tissues of males and females was performed and to elucidate the distribution of the isoenzymes. Presence of both isoenzymes was demonstrated for a number of tissues, including the central nervous system, the urogenital tract, the respiratory tract, the gastrointestinal tract, skin, and the endocrine system. The divergent localization of 5 alpha-reductase isoenzymes points to potentially different functions in various organs. In view of the nuclear localization of isoenzyme 1, its close spatial relationship with the androgen receptor is presumed to indicate a close association with the receptor mechanism.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1994

Subcellular localization of CFTR to endosomes in a ductal epithelium.

Plasma membrane chloride transport by the cystic fibrosis transmembrane conductance regulator (CFTR) may be regulated by cellular processes that affect the cycling of CFTR with the plasma membrane. Testing this hypothesis requires cytochemical evidence for the presence of a subcellular compartment of CFTR. In this study, the subcellular distribution of CFTR in a normal epithelial cell population was characterized using immunofluorescence and immunoelectron microscopy. Two anti-CFTR antibodies, raised against different epitopes of the CFTR molecule, specifically labeled the apical pole of striated duct epithelial cells in tissue sections of rat submandibular gland. By use of electron microscopy, the CFTR immunoreactivity was associated with the apical plasma membrane and the membranes of many subapical vesicles. In this preparation, some of the CFTR-labeled vesicles were also labeled with antibodies against transferrin receptor and rab4, two markers of early endosomes and receptor-mediated endocytosis. These observations provide direct cytochemical evidence for the existence of peripherally located CFTR-expressing endosomes and support the hypothesis that membrane recycling may contribute to CFTR function.

Animals↗
PubMed1975

Subcellular localization of LH releasing activity in the rat hypothalamus.

Separation of particulate fractions associated with LH releasing activity has been effected by differential centrifugation followed by sucrose density gradient centrifugation. The fractions were monitored for LH releasing activity by an in vitro incubation with rat pituitaries and for subcellular organelles by electron microscopy. LH released into the incubation medium and contained in the pituitaries was measured by radioimmunoassay (RIA). After differential centrifugation only the "crude mitochondrial fraction" caused an increase of LH release accompanied by a depletion of pituitary LH and a rise in total LH. Further fractionation of this pellet on a discontinuous sucrose gradient resulted in three opaque bands and three clear areas which were removed as sic fractions. Only one fraction, not associated with myelin, synaptosomes, or mitochondria, caused an increase in LH release and total LH while pituitary LH remained unchanged. This fraction appears to be predominantly composed of electron dense vesicles of varying sizes. Also of interest is another fraction which decreased LH release and was again not associated with myelin, synaptosomes, or mitochondria. The decreased release was associated with an increased pituitary LH while total LH remained unchanged. Of the six subcellular fractions obtained from the "crude mitochondrial pellet" only these two caused significant changes in LH release. Two other neural tissues, the cerebellum and cerebral cortex, were similarly processed. None of these subcellular fractions significantly altered LH release and/or synthesis.

Animals↗
PubMed2004

Expression and subcellular localization of P9-ZFD protein in patients with myasthenia gravis.

OBJECTIVE: To express and purify the protein coded by the TRAF-type zinc finger domain of myasthenia gravis (MG)-related gene P9 (P9-ZFD) and to prepare P9-ZFD antiserum for detecting expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of patient with MG. METHODS: The cDNA encoding P9-ZFD was amplified by RT-PCR. The cloned P9-ZFD cDNA was ligated into pET24a, and the P9-ZFD recombinant protein was induced via E. coli. BL21 (DE3) and purified by histidine affinity chromatography. P9-ZFD antiserum was prepared and its titer and specificity were determined by ELISA and Western blot. Expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of MG and control were studied. RESULTS: The molecular weight of purified P9-ZFD protein was about 30 kD. Its purity was more than 95%. Antiserum specific for P9-ZFD was excellent. P9-ZFD protein is fully confined to the cytoplasm membrane of skeletal muscle cell of MG, obvious immunostaining was absent in the A, I, and Z bands of cytoplasm and no immunoreactivity was observed in the skeletal muscle cell of control. CONCLUSION: P9-ZFD protein is expressed as a cytoplasm membrane-bound protein and has obvious distribution difference in the skeletal muscle cells of patient with MG and normal control.

Adult↗