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At least 1,207 records · Page 67Linked to original sources
PubMed1976

A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Subcellular localization of the protease in porcine skeletal muscle.

A study was done to determine whether the Ca2+-activated muscle protease (CAF) that removes Z disks from myofibrils in the presence of Ca2+ is located in a sedimentable subcellular organelle. Porcine skeletal muscle cells were diced finely with a scalpel and were suspended in 0.25 M sucrose, 4 mM EDTA with a VIRTIS homogenizer. Filtration of the suspended muscle through four layers of cheesecloth removed most of the myofibrils and stromal protein. Nuclear (1,000 gavg for 15 min), mitochondrial-microsomal (50,000 gavg for 60 min), and supernatant fractions were assayed for succinic dehydrogenase, acid ribonuclease, cathepsin D, and CAF activities. Approximately 96% of total succinic dehydrogenase activity, 81% of cathepsin D activity, and 45% of acid ribonuclease activity, but only 14% of total CAF activity, were found in the nuclear and mitochondrial-microsomal fractions. Cathepsin D activity in the nuclear and mitochondrial-microsomal fractions was decreased if assays were done without prior treatment to rupture membranous structures; hence, our cell rupture and homogenization procedures preserved some intact lysosomal organelles. The results indicate that the small amount of CAF activity in the nuclear and mitochondrial-microsomal fractions was due to contamination by supernate and that CAF is not located in a membrane-bounded subcellular particle. Because CAF is active at the intracellular pH and temperature of living skeletal muscle cells and is in direct contact with the cytoplasm of muscle cells, its activity must be regulated by intracellular cellular Ca2+ concentration to prevent continuous and indiscriminate degradation of myofibrils.

Animals↗
PubMed1996

Mitotic regulation of TFIID: inhibition of activator-dependent transcription and changes in subcellular localization.

Mitosis in higher eukaryotes is accompanied by a general inhibition of transcription. To begin to understand the mechanisms underlying this inhibition we have examined the behavior of the general transcription factor TFIID during mitosis. Immunocytochemistry and subcellular fractionation studies indicate that the majority of TFIID is displaced from the disassembling prophase nucleus to the mitotic cytoplasm around the time of nuclear envelope breakdown. However, a subpopulation of TFIID remains associated tightly with the condensed mitotic chromosomes. Metabolic labeling of mitotic cells revealed that several subunits of TFIID undergo mitosis-specific phosphorylation, but in spite of these changes, the TFIID complex remains intact. Functional analysis of purified TFIID from mitotic cells shows that phosphorylated forms are unable to direct activator-dependent transcription, but that this activity is restored upon dephosphorylation. These results demonstrate that TFIID regulation by phosphorylation is likely to have an important role in mitotic inhibition of RNA polymerase II transcription. In addition, they suggest a mechanism for regulating gene expression through the selective disruption of polymerase II promoter structures during mitosis.

Cell Fractionation↗
PubMed1992

Diacylglycerol kinase from suspension cultured plant cells : characterization and subcellular localization.

Diacylglycerol kinase (adenosine 5'-triphosphate:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107), purified from suspension cultured Catharanthus roseus cells (J Wissing, S Heim, KG Wagner [1989] Plant Physiol 90: 1546-1551), was further characterized and its subcellular location was investigated. The enzyme revealed a complex dependency on lipids and surfactants; its activity was stimulated by certain phospholipids, with phosphatidylinositol and phosphatidylglycerol as the most effective species, and by deoxycholate. In the presence of Triton X-100, used for its purification, a biphasic dependency upon diacylglycerol was observed and the apparent Michaelis constant values for diacylglycerol decreased with decreasing Triton concentration. The enzyme accepted both adenosine 5'-triphosphate and guanosine 5'-triphosphate as substrate and showed rather low apparent inhibition constant values for all nucleoside diphosphates tested. Diacylglycerol kinase is an intrinsic membrane protein and no activity was found in the cytosol. An investigation of different cellular membrane fractions confirmed its location in the plasma membrane.

Journal Article↗
PubMed1996

alpha-Oxidation of 3-methyl-substituted fatty acids in rat liver. Production of formic acid instead of CO2, cofactor requirements, subcellular localization and formation of a 2-hydroxy-3-methylacyl-CoA intermediate.

alpha-Oxidation of 3-methyl-substituted fatty acids in rat liver was studied in intact and permeabilized rat hepatocytes, and in homogenates and subcellular fractions. The experiments revealed that the primary end product of alpha-oxidation is formic acid, which is then converted to CO2. Rates of alpha-oxidation identical to those observed in intact hepatocytes were obtained in the permeabilized hepatocytes and liver homogenates when ATP, Mg2+ and CoA, and Fe2+, 2-oxoglutarate and ascorbate were added, suggesting that alpha-oxidation involves a fatty acid activation reaction and a dioxygenase reaction. Subcellular fractionation by differential and density gradient centrifugation demonstrated that alpha-oxidation is confined to peroxisomes, which produce formic acid that is converted to CO2, mainly in the cytosol. alpha-Oxidation in broken cell systems went hand in hand with the formation of a 2-hydroxy-3-methylacyl-CoA ester. Formation of the metabolite was strictly dependent on the presence of the above-mentioned cofactors, was confined to peroxisomes and was inhibited by fenoprofen and propyl gallate, inhibitors of alpha-oxidation in intact cells, indicating that the 2-hydroxyacyl-CoA ester is a bona fide intermediate of alpha-oxidation. Selective omission of cofactors from the reaction mixture and analysis of the incubation mixtures for 3-methyl fatty acids, 3-methyl fatty acyl-CoAs and their respective 2-hydroxy derivatives revealed that the activation reaction precedes the dioxygenase (hydroxylase) reaction. Our experiments demonstrate that alpha-oxidation is a peroxisomal process that consists of at least three reactions: fatty acid activation, hydroxylation and the reaction(s) involved in the release of formic acid.

Acyl Coenzyme A↗
PubMed1987

Subcellular localization of muscarinic effects on enzymes of cyclic nucleotide metabolism in cultured corneal epithelial cells of the rabbit.

Activities of adenylate and guanylate cyclases and cAMP and cGMP phosphodiesterases (cAPDE, cGPDE) were assayed in cell homogenates and subcellular fractions of cultured rabbit corneal epithelium, and effects of carbamylcholine on enzyme activities in each fraction were evaluated. Activity of cyclases and phosphodiesterases was detectable in control incubations of homogenates, nuclei, the mitochondrial/lysosomal fraction, microsomes, and cytosol, although microsomal guanylate cyclase represented a very small proportion of the total cellular activity. In homogenates, carbamylcholine significantly elevated guanylate cyclase and cAPDE and reduced cGPDE activity. In mitochondria/lysosomes, guanylate cyclase was elevated and cGPDE reduced, but the drug did not alter cAPDE activity. In microsomes, carbamylcholine enhanced cAPDE but did not alter guanylate cyclase of cGPDE activity. In the soluble cytoplasmic fraction the drug reduced guanylate cyclase activity. The purified nuclear fraction exhibited substantial activity of cyclases and phosphodiesterases. Carbamylcholine significantly elevated activity of nuclear guanylate cyclase and cAPDE and significantly reduced nuclear cGPDE activity. The drug did not significantly alter adenylate cyclase in homogenates or in any cell fraction. The presence of activity of enzymes of cyclic nucleotide metabolism in the cell nucleus and the sensitivity of nuclear guanylate cyclase, cAPDE and cGPDE to carbamylcholine, which in the same concentration range enhances activity of DNA and RNA polymerases, suggested the hypothesis that effects on cyclic nucleotide-dependent phosphorylation of nuclear proteins might be among regulatory mechanisms by which the drug alters rates of replication and transcription in corneal epithelial cells.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2000

Subcellular localization of galactose-1-phosphate uridylyltransferase in the yeast Saccharomyces cerevisiae.

The enzyme galactose-1-phosphate uridylyltransferase (GALT) catalyzes the second step of the Leloir pathway of galactose metabolism, following galactokinase (GALK) and preceding UDP-galactose-4-epimerase (GALE). Impairment of GALT in humans results in the potentially lethal disorder classic galactosemia. Standard lysis protocols of bacteria, yeast, or mammalian cells release all three Leloir enzymes in the soluble fraction, leading to the historical assumption that all three function as free cytosolic enzymes. We have tested this assumption with regard to GALT in vivo using the yeast Saccharomyces cerevisiae, by linking a GFP-tag onto the amino terminus of Gal7p, the endogenous yeast GALT. We find clear evidence of localization of the fusion protein to discrete spots in the cytoplasm of the majority of cells expressing all three Leloir enzymes, although GFP alone appears freely cytosolic. In contrast, yeast expressing GFP-Gal7p but lacking Gal1p (GALK), Gal10p (GALE), or both do not demonstrate spots in the majority of cells, implicating a role, either direct or indirect, for these other Leloir proteins in the Gal7p localization process. Preliminary truncation experiments reveal that amino acids 1-134 of Gal7p are sufficient to drive localization of the fusion protein, while amino acids 1-66 are not. Finally, GFP-tagged human GALT expressed in yeast also localizes to spots, demonstrating that at least some of the intrinsic determinants of localization have been conserved. These observations raise the intriguing possibility that GALT may function in a sequestered rather than a freely diffusible state, and that this subcellular organization may have been conserved through evolution.

Base Sequence↗
PubMed1993

Dual subcellular localization of the 3 beta-hydroxysteroid dehydrogenase isomerase: characterization of the mitochondrial enzyme in the bovine adrenal cortex.

The enzyme 3 beta-hydroxysteroid dehydrogenase isomerase (3 beta-HSD/I) is an essential step in the biosynthesis of steroid such as progesterone, mineralo- and gluco-corticoids, estrogens and androgens in steroidogenic tissues. It is considered to be mainly localized in microsomes; however, 3 beta-HSD/I activity has also been described to be associated with mitochondrial preparations. In this study, we examined the subcellular distribution of 3 beta-HSD/I in bovine adrenocortical tissue and we characterized the catalytic properties of the enzyme present in the various cell compartments. About 30% of the total 3 beta-HSD/I activity was found to remain tightly associated with the purified mitochondrial pellet. The 3 beta-HSD/I and 3-ketoreductase activities were found in microsomes as well as in mitochondria. The 3 beta-HSD/I associated with the mitochondrial fraction did not require addition of exogenous NAD+. When the pyridine nucleotide was reduced following addition of substrates of the tricarboxylic acids cycle, the mitochondrial 3 beta-HSD/I activity decreased, suggesting that the enzyme utilizes NAD+ available from the matrix space. By contrast, the microsomal enzyme was inactive in the absence of exogenous NAD+. Submitochondrial fractionation disclosed that 3 beta-HSD/I was associated (i) with the inner membrane and (ii) with a particulate fraction sedimenting in a density gradient between inner and outer membranes. This fraction was characterized as contact sites between the two membranes. 3 beta-HSD/I specific activity was much higher in this fraction than in the inner mitochondrial membrane. Altogether, these observations suggest that these mitochondrial intermembrane contact sites may represent a special organization of functional significance, facilitating both the access of cholesterol to the inner membrane where cytochrome P-450scc is located and the rapid transformation of its product, pregnenolone, to progesterone, through 3 beta-HSD/I activity.

3-Hydroxysteroid Dehydrogenases↗
PubMed1993

Differential expression and subcellular localization of protein kinase C alpha, beta, gamma, delta, and epsilon isoforms in SH-SY5Y neuroblastoma cells: modifications during differentiation.

A decrease in protein kinase C activity caused either by treatment with inhibitors, such as staurosporine or H-7, or by prolonged exposure to phorbol diesters has been proposed to be involved in the early events of SH-SY5Y neuroblastoma cell differentiation. Because eight distinct isoforms of protein kinase C with discrete subcellular and tissue distributions have been described, we determined which isoforms are present in SH-SY5Y cells and studied their modifications during differentiation. The alpha, beta 1, delta, and epsilon isoforms were present in SH-SY5Y cells, as well as in rat brain. Protein kinase C-alpha and -beta 1 were the most abundant isoforms in SH-SY5Y cells, and immunoreactive protein kinase C-delta and -epsilon were present in much smaller amounts than in rat brain. Subcellular fractionation and immunocytochemistry demonstrated that all four isoforms are distributed bimodally in the cytoplasm and the membranes. Immunocytochemical analysis showed that the alpha isoform is associated predominantly with the plasma membrane and the processes extended during treatment with 12-tetradecanoyl-13-acetyl-beta-phorbol or staurosporine, and that protein kinase C-epsilon is predominantly membrane-bound. Its localization did not change during differentiation. Western blots of total SH-SY5Y cell extracts and of subcellular fractions probed with isoform-specific polyclonal antibodies showed that when SH-SY5Y cells acquired a morphologically differentiated phenotype, protein kinase C-alpha and -epsilon decreased, and protein kinase C-beta 1 did not change. These data suggest distinct roles for the different protein kinase C isoforms during neuronal differentiation, as well as possible involvement of protein kinase alpha and epsilon in neuritogenesis.

Cell Differentiation↗
PubMed1995

Subcellular localization of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid binding sites in Lewis lung carcinoma cells.

12(S)-Hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) stimulates both gene expression and cell surface expression of the heterodimeric integrin alpha IIb beta 3 in Lewis lung carcinoma cells. These cells contain high affinity binding sites which are specific for 12(S)-HETE. Analyses of the subcellular distribution and molecular size of these sites showed that cytosol was the fraction exhibiting the largest specific binding. On gel permeation chromatography the cytosolic 12(S)-HETE-binding component appeared to be slightly smaller than thyroglobulin (M(r) 669,000). The sedimentation coefficient (20.5 S, determined by sucrose density gradient centrifugation), on the other hand was 1 S unit higher than that of thyroglobulin. The radioactive material bound to the macromolecule was found to be unaltered 12(S)-HETE. Proteinase treatment disrupted the ligand/macromolecule complex, suggesting that a polypeptide component is essential. In addition to cytosol, mitochondria and nuclei also contained significant but lower amounts of specifically bound 12(S)-HETE. The biological significance of this is not clear, but the results are in agreement with observations that 12(S)-HETE exerts effects at several subcellular sites. Our results, to our knowledge for the first time, demonstrate a predominantly cytosolic localization of a recognition molecule for 12(S)-HETE. This localization is different from that of other eicosanoid receptors which are G-protein coupled plasma membrane proteins.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1983

Concentration and subcellular localization of zinc in the hippocampal formation, cerebellum, and whole brain during the postnatal development of the rat.

Zinc was determined by means of atomic absorption techniques in the hippocampal formation, the cerebellum, the whole brain, and their subcellular fractions during the postnatal development of rats. Large quantities of zinc were found already in newborn animals. In the whole brain, the zinc amounts increased between the birth and the 15th day of age, whereas in the hippocampal formation and cerebellum the zinc concentration did not change remarkably during the postnatal maturation. The zinc content of the subcellular fractions revealed a shift to the synaptosomes and mitochondria during the postnatal development. No significant difference of zinc amounts was shown between the CA3 and hilus fasciae dentatae region and the whole hippocampal formation. Concluding from the present results there are obvious discrepancies between the quantitative and histochemical data of the zinc distribution in the rat brain.

Aging↗
PubMed2006

Differential targets and subcellular localization of antitumor alkyl-lysophospholipid in leukemic versus solid tumor cells.

Synthetic alkyl-lysophospholipids represent a family of promising anticancer drugs that induce apoptosis in a variety of tumor cells. Here we have found a differential subcellular distribution of the alkyl-lysophospholipid edelfosine in leukemic and solid tumor cells that leads to distinct anticancer responses. Edelfosine induced rapid apoptosis in human leukemic cells, including acute T-cell leukemia Jurkat and Peer cells, but promoted a late apoptotic response, preceded by G(2)/M arrest, in human solid tumor cells such as cervix epitheloid carcinoma HeLa cells and lung carcinoma A549 cells. c-Jun amino-terminal kinase (JNK) and caspase-3 were accordingly activated at earlier times in edelfosine-treated Jurkat cells as compared with drug-treated HeLa cells. Both leukemic and solid tumor cells took up this alkyl-lysophospholipid and expressed the two putative edelfosine targets, namely cell surface Fas death receptor (also known as APO-1 or CD95) and endoplasmic reticulum CTP: phosphocholine cytidylyltransferase. However, edelfosine was mainly located to plasma membrane lipid rafts in Jurkat and Peer leukemic cells and to endoplasmic reticulum in solid tumor HeLa and A549 cells. Edelfosine induced translocation of Fas, Fas-associated death domain-containing protein, and JNK into membrane rafts in Jurkat cells, but not in HeLa cells. In contrast, edelfosine inhibited phosphatidylcholine biosynthesis in both HeLa and A549 cells, but not in Jurkat or Peer leukemic cells, before the triggering of apoptosis. These data indicate that edelfosine targets two different subcellular structures in a cell type-dependent manner, namely cell surface lipid rafts in leukemic cells and endoplasmic reticulum in solid tumor cells.

Antineoplastic Agents↗
PubMed1995

Alpha-fetoprotein signal sequences: a proposed mechanism for subcellular localization and organelle targeting.

Alpha-fetoprotein (AFP) is an oncofetal protein, classified in an "albuminoid" superfamily (with albumin and Vitamin-D binding (Gc) protein) comprising molecules with three characteristic globular domains. The cellular uptake and internalization of AFP and its subcellular compartmentalization has previously been reported in a multitude of cell types. Studies have also emerged which have detected and characterized binding proteins complexed to AFP in various cell membranes and intracellular compartments. However, the literature is devoid of any attempts to relate these binding proteins to possible intracellular trafficking interactions of AFP. Recombinant DNA mutation/deletion technology has provided a means to pinpoint the amino acid sequence location of organelle localization signals on various transcription factors and/or receptors. Several subdomain regions on AFP have been reported to mimic heptad dimerization regions of the steroid/thyroid nuclear receptors. In light of these transcription factor-like docking motifs reported for AFP, the present report purposes various subdomain regions which might constitute basic amino acid sequences resembling recognition signals for binding (dimerizing) proteins. AFP appears to possess multiple prototypic amino acid sequence cassettes on each domain which consist of (i) classical short, compact sequences found on both steroid and thyroid receptors; (ii) proto-signals resembling a steroid receptor type; and (iii) degenerative signal sequence similar to the thyroid/retinoic acid receptor type. The concepts identifying binding or escort proteins for AFP together with organelle signal sequences on AFP have been reconciled in a composite hypothesis to formulate a mechanism which could account for some of the growth-regulating properties described for AFP during the last decade.

Amino Acid Sequence↗
PubMed1999

Syntaxin 2 splice variants exhibit differential expression patterns, biochemical properties and subcellular localizations.

The syntaxins are a large protein family implicated in the targeting and fusion of intracellular transport vesicles. A subset of proteins of this family are the four syntaxin 2 splice variants, syntaxins 2A (2), 2B (2'), 2C (2") and 2D. Each syntaxin 2 variant contains an identical, or nearly identical, amino-terminal cytoplasmic domain followed by a distinct hydrophobic (syntaxins 2A and 2B) or hydrophilic (syntaxins 2C and 2D) carboxyl-terminal domain. To investigate whether the difference among the syntaxin 2 variants is functionally important, we have examined comparatively their RNA transcript and protein expression patterns, membrane associations, protein-protein interactions and intracellular localizations. Analysis of the RNA transcript and protein expression patterns demonstrated that syntaxins 2A, 2B and 2C are broadly, but not uniformly, expressed while syntaxin 2D expression is restricted to the brain. Subcellular fractionation studies showed that syntaxins 2A and 2B behave as integral membrane proteins while syntaxin 2C is only partially associated with membranes. In vitro biochemical assays demonstrated that the syntaxin 2 variants exhibit similar yet distinct interactions with other proteins implicated in vesicular trafficking, including SNAP-25, SNAP-23, VAMP-2 and n-sec1. In a variety of nonpolarized cell types, syntaxins 2A and 2B localized to both the plasma membrane and endosomal membranes. However, in two polarized epithelial cell lines, MDCK and Caco-2, syntaxin 2A localized predominantly to the apical plasma membrane while syntaxin 2B was associated with both the apical and the basolateral membranes. These observations indicate that the distinct carboxyl-terminal domains of the syntaxin 2 variants influence their biochemical and localization properties and may therefore confer upon these variants different functional roles in the regulation of intracellular membrane trafficking.

Alternative Splicing↗
PubMed1981

Subcellular localization and properties of adenosine diphosphatase activity in human polymorphonuclear leukocytes.

Adenosine diphosphatase (ADPase) activities were studied in human polymorphonuclear leukocytes with a recently developed radio-assay. The neutrophils were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation. The sucrose density gradient fractions were assayed for ADPase activity and for principal organelle marker enzymes. ADPase activity was distributed between the plasma membrane, specific granule and soluble fractions. The plasma membrane and specific granule activities had similar kinetic and inhibitor properties but the cytosolic enzyme was clearly different. Studies with the non-penetrating inhibitor diazotized sulphanilic acid and measurements of latent activity indicate that plasma membrane ADPase activity is located on the external aspect to the cell. Its possible role in inhibiting platelet aggregation is discussed. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (mU/mg protein) of ADPase activity, in contrast to those of alkaline phosphatase, were similar in all three groups. This result, together with fractionation experiments and inhibition studies strongly suggests that ADPase activity is not attributable to neutrophil alkaline phosphatase.

Alkaline Phosphatase↗