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PubMed1981

Regional and subcellular localization in rat brain of the enzymes that can synthesize gamma-hydroxybutyric acid.

Rat brain contains two major NADPH-linked aldehyde reductases that can reduce succinate semialdehyde to 4-hydroxybutyrate. One of these enzymes appears to be fairly specific for succinate semialdehyde and is not significantly inhibited by classic aldehyde reductase inhibitors such as barbiturates. The other enzyme can reduce several aromatic aldehydes and is strongly inhibited by barbiturates and branched-chain fatty acids. Using one such inhibitor, it was possible to distinguish between and measure the two enzyme activities separately in various rat brain regions and in subcellular fractions. Both enzymes are mainly cytoplasmic but there is some activity in the synaptosomal fraction. The activity of the specific succinic semialdehyde reductase is highest in the cerebellum, where it represents 21% of the total activity, and lowest in the cortex, where it represents about 11% of the total activity.

Animals↗
PubMed2004

Secretion mode and subcellular localization of human interferon-beta exogenously expressed in porcine renal epithelial LLC-PK1 cells.

We have investigated the secretion polarity of human interferon-beta (HuIFN-beta) exogenously expressed in the porcine renal proximal tubule cell line, LLC-PK1. In these cells, stably expressed HuIFN-beta was secreted to the apical and basolateral sides. However, when transiently expressed by apical lipofection, HuIFN-beta was secreted to both cell sides, while basolateral-preferential secretion was seen for basal transfection. Confocal imaging using enhanced green fluorescent protein (EGFP)-tagged HuIFN-beta revealed no difference in the subcellular distribution in either of the chimeric protein-expressing cells examined. These results suggest that the secretion polarity of HuIFN-beta is regulated by a post trans-Golgi network in a cell type-dependent manner.

Animals↗
PubMed1976

Zonal rotor analysis of the subcellular localization of alpha-glycerphosphate dehydrogenase, alpha-naphthyl palmitate and beta-naphthyl laurate hydrolases in the mucosa of the guinea-pig small intestine.

Whole homogenate of guinea-pig small intestine mucosa was analysed by centrifugation in a zonal rotor. The results indicate that FAD-linked alpha-glycerophosphate dehydrogenase is localized in the mitochondria and that NAD-linked alpha-glycerophosphate dehydrogeanse is a soluble phase enzyme. An enzyme hydrolysing alpha-naphthyl palmitate at an acid pH was localized in the lysosomes and was activated by 0.1% Triton X-100 and by freezing and thawing. An alkaline hydrolase acting on beta-naphthyl laurate was localized in the 'microsomes'. The possibility of this enzyme being different from alpha-naphthyl acetate hydrolase is discussed.

Animals↗
PubMed1984

The subcellular localization of lipid in myelin-free axonal preparations.

Bovine myelin-free axonal preparations were subjected to a series of washes designed to partition membranes from other cellular components initially present in these preparations. These washes were composed entirely of membranous structures, essentially free of neurofilament protein subunits, and contained high specific activity of acetylcholinesterase, an axolemma-specific enzyme. The distribution of acetylcholinesterase in the washes paralleled the distribution of lipid and the lipid composition of these washes closely resembled that of bovine axolemma-enriched fractions. In addition, acetylcholinesterase, lipid and galactocerebroside were histo- and immunohistochemically localized on similar structures in the starting material. Our results demonstrate that some of the lipid in myelin-free axonal preparations may be accounted for by axolemma.

Acetylcholinesterase↗
PubMed1997

Movement and subcellular localization of a tobamovirus in Arabidopsis.

Tobamoviruses represent a well-characterized system used to examine viral infection, whereas Arabidopsis is a choice plant for most genetic experiments. It would be useful to combine both approaches into one experimental system for virus-plant interaction. Most tobamoviruses, however, are not pathogenic in Arabidopsis. Here, we describe infection of Arabidopsis by a recently discovered crucifer-infecting turnip vein clearing tobamovirus (TVCV). Using this system, we determined patterns and kinetics of viral local and systemic movement within Arabidopsis plants. Localization studies showed that the virus infects both vegetative and reproductive plant tissues. However, there may be a transport barrier between the seed coat and the embryo which virions cannot cross, preventing seed transmission of TVCV. The ability to move both locally and systemically in Arabidopsis, causing mild and fast-developing symptoms but allowing survival and fertility of the infected plants, distinguish TVCV infection of Arabidopsis as a model system to study virus-plant interaction.

Arabidopsis↗
PubMed1982

Density gradient fractionation of digitonin-treated rat liver plasma membranes and subcellular localization of NADH-oxidoreductase and B-type cytochromes.

Plasma membranes, contaminated very little by endoplasmic reticulum elements, were isolated from rat liver. Activities of NADH-oxidoreductase and b-type cytochromes were higher than expected from this contamination. Between 50 and 80% of these activities were endogenous to plasma membrane. A similar result was obtained after refractionation of digitonin-treated plasma membranes on a sucrose density gradient. This is further evidence for the localization of NADH-ferricyanide reductase, NADH-cytochrome c reductase, and cytochromes b5, P450 and P420 in liver cell membranes.

Animals↗
PubMed1985

[Specific proteases which degrade prolactin in the mammary glands; subcellular localization and inhibitory analysis].

The intracellular fractions were shown to contain neutral and acidic proteases hydrolyzing (125I) prolactin with pH optima at pH 7.6 and 3.0. Neutral proteases are predominantly localized in the mitochondrial and nuclear fractions, while the acidic ones--in the lysosomes. Mitochondrial and lysosomal proteases are specific towards rat prolactin. The rate of rat prolactin proteolysis in these fractions is 2.7 (P less than 0.0027) and 1.6 (P less than 0.05) times higher than that of sheep prolactin or that of the luteinizing and follicle-stimulating hormones. Neutral proteases, which split of [125I] insulin, are distributed in the subcellular fractions in quite a different way. It was demonstrated that the prolactin-hydrolyzing activity of the nuclear and mitochondrial fractions is due to the presence of metal-dependent, serine and sulfhydryl proteases in these organelles.

Animals↗
PubMed1983

Pyruvate dehydrogenase phosphate (PDHb) phosphatase in brain: activity, properties, and subcellular localization.

The activity of pyruvate dehydrogenase phosphate (PDHb) phosphatase in rat brain mitochondria and homogenate was determined by measuring the rate of activation of purified, phosphorylated (i.e., inactive) pyruvate dehydrogenase complex (PDHC), which had been purified from bovine kidney and inactivated by phosphorylation with Mg . ATP. The PDHb phosphatase activity in purified mitochondria showed saturable kinetics with respect to its substrate, the phospho-PDHC. It had a pH optimum between 7.0 and 7.4, depended on Mg and Ca, and was inhibited by NaF and K-phosphate. These properties are consistent with those of the highly purified enzyme from beef heart. On subcellular fractionation, PDHb phosphatase copurified with mitochondrial marker enzymes (fumarase and PDHC) and separated from a cytosolic marker enzyme (lactate dehydrogenase) and a membrane marker enzyme (acetylcholinesterase), suggesting that it, like its substrate, is located in mitochondria. PDHb phosphatase had similar kinetic properties in purified mitochondria and in homogenate: dependence on Mg and Ca, independence of dichloroacetate, and inhibition by NaF and K-phosphate. These results are consistent with there being only one type of PDHb phosphatase in rat brain preparations. They support the validity of the measurements of the activity of this enzyme in brain homogenates.

Animals↗
PubMed1982

Biochemical studies of the renal radiopharmaceutical compound dimercaptosuccinate. II. Subcellular localization of 99Tc-DMS complex in the rat kidney in vivo.

In order to extend and confirm our previous studies of in vivo behaviour of 99mTc-DMS in rat kidney cells, the intracellular localization of 99Tc-DMS complex in rat kidney tissue was examined. Animals were injected IV with 99Tc-DMS solution (37 kbq in 1.0 ml) 2 h before killing. Dissected kidneys were homogenized and submitted to differential centrifugation to obtain organelles. The following radioactivity distribution in relation to the total radioactivity of homogenate was obtained in nine experiments: nuclei 3.30% (+/-1.27), mitochondria 9.48% (+/-2.17), microsomes 7.03% (+/-2.34) and cytosol 57.45% (+/-8.01). The subcellular distribution pattern of 99Tc-DMS was in very good agreement with the pattern of 99Tc-DMS binding to kidney cell organelles obtained by the same procedure. Soluble cytoplasmic proteins binding 99Tc-DMS were excluded from a Sephadex G-25 column with the void volume. Their electrophoretic mobility after agarose gel electrophoresis corresponded to beta-lc- and alpha-2-macroglobulins. The results obtained show unambiguously that the investigated complex penetrates into the kidney cells where it binds mostly to soluble cytoplasmic proteins and mitochondria, and to a lesser extent to both microsomes and nuclei. In another series of six experiments, isolated kidney cortical tissue was used, the same procedure was repeated and similar results were obtained.

Animals↗
PubMed1980

Secretion, subcellular localization and metabolic status of inorganic pyrophosphate in human platelets. A major constituent of the amine-storing granules.

The platelet content of PPi is 1.90 +/- mumol/10(11) platelets (S.E.M., n = 19) or about 10.5 nmol/mg of protein, several hundred times that found for rat liver. Some 80% of this PPi is secreted by platelets treated with thrombin with a time course and dose-response relationship similar to secretion of ATP, ADP and 5-hydroxytryptamine (serotonin) from the platelet dense granules. During platelet aggregation induced by ADP and adrenaline, substantial amounts of PPi were secreted, but no release of acid hydrolases was observed. Subcellular-fractionation studies showed that the PPi is highly enriched in the same fraction that contains the storage organelles which store ATP, ADP, Ca2+ and 5-hydroxytryptamine. Inorganic pyrophosphatase was present mainly in the soluble fraction and in the mitochondria. Secretion studies done with platelets prelabelled with [32P]Pi showed that the sequestered PPi was relatively metabolically inactive, as is the ATP and ADP in the storage organelles. The possible participation of PPi in the formation of a bivalent-cation-nucleotide complex associated with amine storage is discussed.

Adenosine Diphosphate↗
PubMed2005

Disparate subcellular localization patterns of Pseudomonas aeruginosa Type IV pilus ATPases involved in twitching motility.

The opportunistic pathogen Pseudomonas aeruginosa expresses polar type IV pili (TFP), which are responsible for adhesion to various materials and twitching motility on surfaces. Twitching occurs by alternate extension and retraction of TFP, which arise from assembly and disassembly of pilin subunits at the base of the pilus. The ATPase PilB promotes pilin assembly, while the ATPase PilT or PilU or both promote pilin dissociation. Fluorescent fusions to two of the three ATPases (PilT and PilU) were functional, as shown by complementation of the corresponding mutants. PilB and PilT fusions localized to both poles, while PilU fusions localized only to the piliated pole. To identify the portion of the ATPases required for localization, sequential C-terminal deletions of PilT and PilU were generated. The conserved His and Walker B boxes were dispensable for polar localization but were required for twitching motility, showing that localization and function could be uncoupled. Truncated fusions that retained polar localization maintained their distinctive distribution patterns. To dissect the cellular factors involved in establishing polarity, fusion protein localization was monitored with a panel of TFP mutants. The localization of yellow fluorescent protein (YFP)-PilT and YFP-PilU was independent of the subunit PilA, other TFP ATPases, and TFP-associated proteins previously shown to be associated with the membrane or exhibiting polar localization. In contrast, YFP-PilB exhibited diffuse cytoplasmic localization in a pilC mutant, suggesting that PilC is required for polar localization of PilB. Finally, localization studies performed with fluorescent ATPase chimeras of PilT and PilU demonstrated that information responsible for the characteristic localization patterns of the ATPases likely resides in their N termini.

Adenosine Triphosphatases↗
PubMedDate not supplied

Subcellular localization of immunoreactive dynorphin and vasopressin in rat pituitary and hypothalamus.

Dynorphin is found mainly in the particulate fraction of rat pituitary gland and hypothalamus homogenates. Dynorphin-like immunoreactivity (DYN-LI) from neurointermediate lobe (NIL) homogenates migrates at the same rate as vasopressin-like immunoreactivity (AVP-LI), in sucrose density gradients, whereas DYN-LI from the hypothalamus appears to migrate principally in a less dense region of the gradient. This suggests that dynorphin and vasopressin from pituitary are present in organelles of similar size and density, while the bulk of the dynorphin in the hypothalamus appears to be stored in a different subcellular organelle. Anterior lobe (AL) dynorphin appears to migrate in two separate bands on density gradients: the less dense band (slower) migrates at a similar rate to that of dynorphin and vasopressin from NIL. When alpha-neo-endorphin was measured in sucrose gradients of NIL and hypothalamus, it was found to co-migrate with DYN-LI.

Animals↗
PubMed1994

Subcellular localization of a protein-tyrosine phosphatase: evidence for association with chromatin.

A non-transmembrane protein-tyrosine phosphatase, PTP-S, has been shown to bind to DNA in vitro through its C-terminal noncatalytic domain. The cellular PTP-S gene product was identified as 42-44 kDa polypeptides in the rat fibroblast cell lines Rat2 and F111, by immunoblotting using polyclonal and monoclonal antibodies. Indirect immunofluorescence-staining experiments showed that PTP-S was predominantly associated with the nuclear compartment in interphase cells, although some fluorescence was present in cytoplasm. This protein appeared to be uniformly distributed throughout the cell during mitosis. Subcellular fractionation followed by immunoblotting supports the immunofluorescence data showing that PTP-S was present in the nucleus as well as cytoplasm. PTP-S could be released from the isolated nuclei by moderate salt concentration (400 mM NaCl) or mild treatment with DNAase I, suggesting that at least part of this protein present in the nucleus was associated with chromatin. PTP-S extracted from nuclei of Rat2 cells binds to DNA, as determined by DNA affinity chromatography. The nuclear location of this phosphatase, and its association with chromatin, provide support to our previous suggestion that the DNA-binding property of this phosphatase may be physiologically relevant.

Animals↗
PubMed2000

Subcellular localization and physiological role of alpha-methylacyl-CoA racemase.

alpha-Methylacyl-CoA racemase plays an important role in the beta-oxidation of branched-chain fatty acids and fatty acid derivatives because it catalyzes the conversion of several (2R)-methyl-branched-chain fatty acyl-CoAs to their (S)-stereoisomers. Only stereoisomers with the 2-methyl group in the (S)-configuration can be degraded via beta-oxidation. Patients with a deficiency of alpha-methylacyl-CoA racemase accumulate in their plasma pristanic acid and the bile acid intermediates di- and trihydroxycholestanoic acid, which are all substrates of the peroxisomal beta-oxidation system. Subcellular fractionation experiments, however, revealed that both in humans and rats alpha-methylacyl-CoA racemase is bimodally distributed to both the peroxisome and the mitochondrion. Our findings show that the peroxisomal and mitochondrial enzymes are produced from the same gene and that, as a consequence, the bimodal distribution pattern must be the result of differential targeting of the same gene product. In addition, we investigated the physiological role of the enzyme in the mitochondrion. Both in vitro studies with purified heterologously expressed protein and in vivo studies in fibroblasts of patients with an alpha-methylacyl-CoA racemase deficiency revealed that the mitochondrial enzyme plays a crucial role in the mitochondrial beta-oxidation of the breakdown products of pristanic acid byconverting (2R,6)-dimethylheptanoyl-CoA to its (S)-stereoisomer.

Animals↗
PubMed2002

Tetraspan vesicle membrane proteins: synthesis, subcellular localization, and functional properties.

Tetraspan vesicle membrane proteins (TVPs) are characterized by four transmembrane regions and cytoplasmically located end domains. They are ubiquitous and abundant components of vesicles in most, if not all, cells of multicellular organisms. TVP-containing vesicles shuttle between various membranous compartments and are localized in biosynthetic and endocytotic pathways. Based on gene organization and amino acid sequence similarities TVPs can be grouped into three distinct families that are referred to as physins, gyrins, and secretory carrier-associated membrane proteins (SCAMPs). In mammals synaptophysin, synaptoporin, pantophysin, and mitsugumin29 constitute the physins, synaptogyrin 1-4 the gyrins, and SCAMP1-5 the SCAMPs. Members of each family are cell-type-specifically synthesized resulting in unique patterns of TVP coexpression and subcellular colocalization. TVP orthologs have been identified in most multicellular organisms, including diverse animal and plant species, but have not been detected in unicellular organisms. They are subject to protein modification, most notably to phosphorylation, and are part of multimeric complexes. Experimental evidence is reviewed showing that TVPs contribute to vesicle trafficking and membrane morphogenesis.

Amino Acid Sequence↗
PubMed1992

Posttranslational modification and subcellular localization of the p12 capsid protein of herpes simplex virus type 1.

We have previously shown that the 12-kDa capsid protein (p12) of herpes simplex virus type 1 (HSV-1) is a gamma 2 (true late) gene product encoded by the UL35 open reading frame (D. S. McNabb and R. J. Courtney, J. Virol. 66:2653-2663, 1992). To extend the characterization of p12, we have investigated the posttranslational modifications and intracellular localization of the 12-kDa polypeptide. These studies have demonstrated that p12 is modified by phosphorylation at serine and threonine residues. In addition, analysis of p12 by acid-urea gel electrophoresis has indicated that the protein can be resolved into three components, designated p12a, p12b, and p12c. Using isotopic-labeling and alkaline phosphatase digestion experiments, we have determined that p12a and p12b are phosphorylated forms of the protein, and p12c is likely to represent the unphosphorylated polypeptide. The kinetics of phosphorylation was examined by pulse-chase radiolabeling, and these studies indicated that p12c can be completely converted into p12a and p12b following a 4-h chase. All three species of p12 were found to be associated with purified HSV-1 virions; however, p12b and p12c represented the most abundant forms of the protein within viral particles. We have also examined the intracellular localization of p12 by cell fractionation and indirect immunofluorescence techniques. These results indicated that p12 is predominantly localized in the nucleus of HSV-1-infected cells and appears to be restricted to specific regions within the nucleus.

Animals↗