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PubMed1983

Acid hydrolases of the epidermis: subcellular localization and relationship to cornification.

Three lysosomal-type acid hydrolases were examined in subcellular fractions of the developing epidermis of fetal rats to assess the relationship of degradative enzymes to cornification. As the granular layer developed and cornified between 18 and 20 days (D) of gestation, epidermal acid phosphatase increased, acid phospholipase A remained constant, and beta-glucuronidase activity declined. The enzymes were present in 3,000, 17,000, and 100,000 g particulate fractions and soluble cytoplasm. However distribution differed: acid phosphatase and phospholipase A were more preferentially localized than was glucuronidase in the 17,000 g fraction which excluded mitochondria and ribosomes and was enriched in lamellar granules. The findings suggested that acid phosphatase and phospholipase were present in membrane-bound organelles (e.g., lamellar granules) in the granular layer. Particulate acid phosphatase increased with granular layers on days 19 and 20 while a 7-fold increase in soluble enzyme coincided with cornification on day 20. As shown by isoelectric focusing, the enzyme became more heterogeneous at day 20 than at day 18, suggesting increased glycosylation. The particulate fraction displayed lysosomal characteristics with respect to release of acid phosphatase, which was inhibited by hydrocortisone and enhanced by retinol. When fetal epidermis was allowed to cornify in organ cultures, similar increases in acid phosphatase occurred. The presence of hydrocortisone did not affect increase in total enzyme but a greater proportion remained in the particulate fraction. The findings suggest that particulate acid phosphatase and phospholipase are compartmentalized in organelles with lysosomal characteristics during development of granular cells and that release of phosphatase is coincident with cornification. This may reflect not only exocytosis of lamellar granules but also intracellular release of the hydrolytic enzyme.

Acid Phosphatase↗

PubMed1985

Subcellular localization and modification with ageing of glutathione, glutathione peroxidase and glutathione reductase activities in human fibroblasts.

Differential centrifugation and isopycnic equilibration in density gradients were used to localize glutathione (GSH), glutathione peroxidase and glutathione reductase in the subcellular organelles of WI-38 fibroblasts. GSH was present in all the subcellular fractions, whereas the glutathione peroxidase and reductase activities were restrained to the cytoplasm and the mitochondrial fractions. After equilibration in density gradients, the results showed the presence of GSH, glutathione peroxidase and glutathione reductase in both the cytoplasm and mitochondria. GSH was also located in plasma membranes and probably in peroxisomes, endoplasmic reticulum and lysosomal membranes. Evolution of GSH in ageing fibroblasts showed a sudden increase of its concentration just before cell death. The glutathione peroxidase activity already decreases in the early passages, while the decrease of the glutathione reductase activity was constant and reached a drastic low level at the end of the culture. In conclusion, GSH is probably involved in the cell degeneration associated with ageing but because of its multiple functions and its ubiquitous localization, it is difficult to assert to which extent this metabolite is implicated in the ageing process.

Cell Line↗

PubMed1996

Distribution and subcellular localization of a growth inhibitory factor in hamster liver and its intracellular partner(s).

The subcellular, intralobular distributions and intracellular partner(s) of a factor which inhibits the proliferation of cell growth (Hashimoto C. et al. (1994) Biochim. Biophys. Acta 1221, 107-117) were determined in hamster livers, using a combination of immunological and biochemical techniques. The IgG fraction from an antiserum raised against the growth inhibitory factor with 37 kDa was shown to be highly specific for the antigen. The nuclear and cytosolic fractions demonstrated inhibitory effects on cell growth and Western blot analysis revealed that both fractions contained the immunoreactive 37 kDa protein with the anti-inhibitory factor IgG but microsomal and mitochondrial fractions did not. The nuclear and cytoplasmic localization of the inhibitory factor were further confirmed by immunochemical staining mediated through the immune IgG and an avidin-biotinylated horseradish peroxidase complex, the parenchymal liver cells were clearly stained, but endothelial and connective tissue cells were not. Although some staining was evident throughout the liver parenchyma, the hepatocytes with most intensively stained nuclei were located in the periportal region. In the liver from hamsters 6 days old or the regenerating hamster livers 3 days after partial hepatectomy, the staining intensity was low and the number of hepatocytes with the inhibitory factor positive nuclei was very few compared with the adult hamster livers. In primary cultures of the isolated hepatocytes from adult hamster the inhibitory factor disappeared from nuclei after incubation for 24-48 h. The extracts of hepatic nuclei from adult hamsters were immunoprecipitated with either the anti-growth inhibitory factor IgG or a monoclonal antibody to the RM protein. The growth inhibitory factor and the RB protein coprecipitated in each case, implying that the proteins were complexed with each other in the nuclei. The RB protein family is composed of two sets of species, an un- or underphosphorylated species and a hyperphosphorylated one. It was suggested that the factor bound preferentially to the un- or underphosphorylated member of the family.

Animals↗

PubMed1994

Cellular and subcellular localization of the mGluR5a metabotropic glutamate receptor in rat spinal cord.

The cellular and subcellular distribution of the mGluR5a metabotropic glutamate receptor was studied in the spinal cord of the rat using an antibody raised against a mGluR5a-specific carboxy-terminal peptide. Strong mGluR5a-immunoreactivity (mGluR5a-ir) was found in the laminae I-II of the dorsal horn, which gradually decreased towards the deeper layers. At the electron microscopical level, mGluR5a-ir was present exclusively in neuronal somata and dendrites. Immunometal labelling revealed that mGluR5a-ir is concentrated at the periphery of postsynaptic densities of asymmetrical synapses or localized extrasynaptically at dendritic and somatic membranes. The mGluR5a-immunoreactive dendritic profiles were often targeted by synaptic boutons with the morphological characteristics of C-fibre terminals. These observations provide evidence for mGluR5a being involved in the nociceptive transmission at the dorsal horn.

Amino Acid Sequence↗

PubMed1993

The specific subcellular localization of two isoforms of cytochrome b5 suggests novel targeting pathways.

Two forms of cytochrome b5 are present in rat tissues, with a sequence identity of approximately 60% in the cytoplasmically exposed, tryptic fragments (Lederer, F., Ghrir, R., Guiard, B., Cortial, S., and Ito, A. (1983) Eur. J. Biochem. 132, 95-102). It has been suggested that the two isoforms have partially overlapping subcellular distributions, with each form localized to some extent on both endoplasmic reticulum and outer mitochondrial membranes (Ito, A. (1980) J. Biochem. (Tokyo) 87, 73-80). To investigate the degree of specificity of the localization of cytochrome b5 isoforms, we studied their subcellular distributions with antipeptide antibodies, one specific for microsomal cytochrome b5, one specific for outer membrane cytochrome b, and one against a sequence common to the two cytochromes. We first identified outer membrane Cyt b as a tightly bound, Triton X-114-extractable, 23-kDa polypeptide. We then analyzed biochemically characterized rat liver subcellular fractions by Western blotting and found that outer mitochondrial membrane cytochrome b was not present on endoplasmic reticulum membranes. Conversely, microsomal cytochrome b5 was present on outer mitochondrial membranes in extremely low concentration, at a level < 5% of that on endoplasmic reticulum membranes. Thus, the subcellular distribution of microsomal cytochrome b5 is more restricted than previously thought, suggesting that novel posttranslational targeting mechanisms direct it to the endoplasmic reticulum.

Amino Acid Sequence↗

PubMed2001

Expression patterns and subcellular localization of a 52 kDa sucrose-binding protein homologue of Vicia faba (VfSBPL) suggest different functions during development.

A cDNA coding for a 54 kDa signal sequence containing protein has been isolated from a faba bean cotyledonary library and characterized. The deduced protein is designated Vicia faba SBP-like protein (VfSBPL) since it shares 58% homology to a 62 kDa soybean (Glycine max) protein (GmSBP) which has been described as a sucrose-binding and sucrose-transporting protein (SBP). VfSBPL as well as GmSBP are outgroup members of the large vicilin storage protein family. We were unable to measure any sucrose transport activity in mutant yeast cells expressing VfSBPL. During seed maturation in late (stage VII) cotyledons mRNA was localized by in situ hybridization in the storage parenchyma cells. At the subcellular level, immunolocalization studies proved VfSBPL accumulation in storage protein vacuoles. However, mRNA localization in stage VI cotyledons during the prestorage/storage transition phase was untypical for a storage protein in that, in addition to storage parenchyma cell labelling, strong labelling was found over seed coat vascular strands and the embryo epidermal transfer cell layer reminiscent of sucrose transporter localization. The VfSBPL gene is composed of 6 exons and 5 introns with introns located at the same sites as in a Vicia faba 50 kDa vicilin storage protein gene. The time pattern of expression as revealed by northern blotting and the GUS accumulation pattern caused by a VfSBPL-promoter/GUS construct in transgenic tobacco seeds was similar to a seed protein gene with increasing expression during seed maturation. Our data suggest different functions of VfSBPL during seed development.

Amino Acid Sequence↗

PubMed1995

Discrete subcellular localization of membrane-bound ATPase activity in marine angiosperms and marine algae.

The subcellular distribution of membrane-bound ATPases was compared among terrestrial plants, seagrasses and marine algae by cytochemical techniques. High ATPase activity was detected in the copiously invaginated plsma membrane that was characteristic of transfer cells but not in the tonoplast of epidermal cells in mature leaves of seagrasses. Magnesium- or Ca(2+)-dependent ATPase activity was induced together with the characteristics of transfer cells during the development of leaf tissues able to resist seawater. Northern hybridization revealed the effective induction of the synthesis of mRNA for plasma-membrane H(+)-ATPase during the development of leaves. Such high ATPase activity was not detected in the smooth plasma membranes of marine macro-algae but was found in the membranes of some cytoplasmic vesicles or microvacuoles, providing evidence of the excretion of salts by exocytosis. It appears, therefore, that two essentially different methods for excreting excess salts have developed separately in these two classes of marine plants. The evolution of mechanisms of salt tolerance in the plant kingdom is discussed in terms of the differential subcellular distribution of ATPase activity.

Adenosine Triphosphatases↗

PubMedDate not supplied

Subcellular localization of immunoreactive alpha-melanocyte stimulating hormone in human brain.

The regional and subcellular distribution of immunoreactive alpha-melanocyte stimulating hormone (alpha-MSHi) in the post mortem adult human brain was investigated. alpha-MSHi was highly concentrated in medial basal hypothalamic tissue (1.02 ng/mg protein). Lower levels of alpha-MSHi were present in the optic chiasm and mammillary bodies, 0.08 and 0.11 ng/mg protein, respectively. The concentrations of alpha-MSHi in cerebellum and frontal cerebral cortex were 1/1,000th that of the medial basal hypothalamus. When medial basal hypothalamic homogenates were subjected to discontinuous or continuous sucrose density gradients, alpha-MSHi was found to be associated primarily with subcellular particles which resembled isolated nerve terminals, i.e., synaptosomes. Low to undetectable amounts of alpha-MSHi were found in the cytosol or the myelin/microsome fraction of the gradients. The results of these studies are consistent with the view that alpha-MSH is a neuronal peptide in the human brain.

Brain↗

PubMed1980

Kininogenase activity in plasma membranes and cell organelles from rabbit kidney cortex: subcellular localization of renal kallikrein by free-flow electrophoresis and density-gradient fractionation.

Subcellular fractions were prepared from a rabbit kidney cortex homogenate by density gradient and free-flow electrophoresis techniques. After enzymatic and morphologic characterization, we determined the kininogenase activity in the different fractions. This activity was present in those plasma membranes that also contained high specific activities Na-K-ATPase and in lysosomal-like particles. No activity was found in the lumen, that is, the microvillous part of the proximal tubule cell. The kallikrein-like nature of this kininogenase activity was established by several methods.

Animals↗

PubMed1985

Subcellular localization of the pathway of de novo pyrimidine nucleotide biosynthesis in pea leaves.

The subcellular distribution of the enzymes of de novo pyrimidine nucleotide biosynthesis was investigated in pea (Pisum sativum L. cv Progress No. 9) leaves. Aspartate carbamoyltransferase, the committed step of the pathway, was found to be strictly confined to the chloroplasts. Dihydro-orotase, orotate phosphoribosyl transferase, and orotidine decarboxylase activities were also found only in the plastids. The remaining enzyme of the pathway, dihydroorotate dehydrogenase, was shown to be mitochondrial.

Journal Article↗

PubMed1997

Cellular and subcellular localization of nicotinamide adenine dinucleotide phosphate diaphorase activity.

OBJECTIVE: To determine the cellular and subcellular distribution of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) in the human placenta and thereby to shed light on the possible role of nitric oxide (NO) and NO synthase in this tissue. METHODS: NADPH-d activity in 10 term placenta and in 10 first-trimester placentas was examined by enzyme histochemistry and by both light microscopy and electron microscopy. RESULTS: Light microscopy revealed that NADPH-d activity was marked in the syncytiotrophoblasts, moderate in the endothelium of cord vessels, and weak in the amniotic epithelia and chorionic trophoblasts. At the electron-microscopic level, NADPH-d activity was apparent as small patches distributed throughout the cytoplasm of the syncytiotrophoblasts. CONCLUSION: NO might be generated in the syncytiotrophoblast of the human placenta and might play a role in the regulation of placental hemodynamics or in the signaling between the syncytium and the villous mesenchyme.

Amnion↗

PubMed1984

Subcellular localization of alkaline phosphatase in Bacillus licheniformis 749/C by immunoelectron microscopy with colloidal gold.

Subcellular distribution of the alkaline phosphatase of Bacillus licheniformis 749/C was determined by an immunoelectron microscopy method. Anti-alkaline phosphatase antibody labeled with 15- to 18-nm colloidal gold particles (gold-immunoglobulin G [IgG] complex) were used for the study. Both the plasma membrane and cytoplasmic material were labeled with the gold-IgG particles. These particles formed clusters in association with the plasma membrane; in contrast, in the cytoplasm the particles were largely dispersed, and only a few clusters were found. The gold-IgG binding was quantitatively estimated by stereological analysis of labeled, frozen thin sections. This estimation of a variety of control samples showed that the labeling was specific for the alkaline phosphatase. Cluster formation of the gold-IgG particles in association with the plasma membrane suggests that existence of specific alkaline phosphatase binding sites (receptors) in the plasma membrane of B. licheniformis 749/C.

Alkaline Phosphatase↗

PubMed1983

Subcellular localization and properties of the NAD(P)H oxidase from equine polymorphonuclear leukocytes.

The subcellular distribution of the superoxide-forming enzyme in horse polymorphonuclear leukocytes was investigated. After activation of the cells with sodium oleate, a relatively stable and NAD(P)H-dependent oxygen consumption and superoxide production was found in association with the plasma membranes. The pH dependence displayed an optimum near neutrality. The apparent Km values were 38 x 10(-6) mol/l for NADPH and 1,560 x 10(-6) mol/l for NADH, suggesting that NADPH is the physiological donor. The rates of oxygen uptake, O2- production, and NADP consumption were consistent with the stoichiometry: 2 O2 + NADPH leads to 2 O2- + NADP. The failure to demonstrate an increase of NAD(P)H-dependent oxidative activity in the cellular fractions that the investigated NADPH oxidase is identical with the enzyme responsible for the respiratory burst in phagocytizing leukocytes.

Animals↗

PubMed1981

Subcellular localization of the enzymes involved in the late stage of ergosterol biosynthesis in yeast.

The subcellular distribution of enzymes involved in the reaction sequence from zymosterol to ergosterol was studied. The spheroplasts obtained from cells aerobically grown on ethanol were gently disrupted and the homogenate was fractionated into the subcellular organelles by differential centrifugation. Inspection of the distribution of several marker enzymes revealed that the fractionation was reasonably effected. As a result of experiments, delta 8-delta 7-sterol isomerase, S-adenosylmethionine: delta 24-sterol methyltransferase and the enzyme involved in the reaction sequence from episterol to ergosterol were localized in microsomes. These results suggest the localization of enzymes involved in the late stage of ergosterol synthesis in microsomes.

Cholestadienols↗

PubMed1994

Changes in subcellular localization and surface expression of L-selectin, alkaline phosphatase, and Mac-1 in human neutrophils during stimulation with inflammatory mediators.

The localization of the adhesion protein L-selectin in human neutrophils was determined by subcellular fractionation and immunoelectron microscopy and compared with the localization of Mac-1 (alpha m beta 2) and alkaline phosphatase, the marker for secretory vesicles. L-selectin was found to be localized exclusively on the plasma membrane of unstimulated cells and also of stimulated cells, although markedly diminished. This was in contrast to Mac-1, which was also localized in secretory vesicles and in specific/gelatinase granules as shown previously [Sengeløv, H., et al. J. Clin. Invest. (1993) 92, 1467-1476]. Stimulation of neutrophils with inflammatory mediators such as tumor necrosis factor (TNF), platelet-activating factor (PAF), or f-Met-Leu-Phe (fMLP), induced parallel up-regulation of the surface membrane content of alkaline phosphatase and Mac-1 and down-regulation of L-selectin, as evidenced by flow cytometry. Preimbedding immunoelectron microscopy confirmed that L-selectin was present mainly on tips of microvilli in unstimulated cells and showed that alkaline phosphatase and Mac-1 were randomly distributed on the surface membrane of fMLP-stimulated cells. These studies indicate that the transition of neutrophils from L-selectin-presenting cells to Mac-1-presenting cells induced by inflammatory mediators is mediated by incorporation of secretory vesicle membrane, rich in Mac-1 and devoid of L-selectin, into the plasma membrane.

Alkaline Phosphatase↗

PubMed1993

Purification, partial characterization, and subcellular localization of a 38 kilodalton, calcium-regulated protein of Rhizobium fredii USDA208.

Calcium is essential for the growth of rhizobia and the formation of nitrogen-fixing root-nodules on legumes, but its precise role in these processes remains unknown. We have found that Rhizobium fredii USDA208 accumulates a major 38 kDa protein when grown in media supplemented with 0.3-2 microM Cacl2. We have purified this protein and raised polyclonal antibodies against it. The protein initially is synthesized as a 40 kDa precursor which subsequently undergoes calcium-dependent processing to give rise to the mature polypeptide. Subcellular and immunocytochemical localization studies indicate that the 38 kDa protein accumulates preferentially in the periplasmic space. Its N-terminal sequence, AETIKIGVAGPMTG, shows significant homology to the N-termini of amino acid binding proteins from the periplasm, including leucine-, isoleucine-, and valine-specific binding proteins of Pseudomonas aeruginosa and Escherichia coli and a leucine-specific binding protein of E. coli. The R. fredii protein does not, however, bind [3H]-leucine. The 38 kDa protein is encoded by the bacterial chromosome. It is absent in several rhizobia other than R. fredii, but antigenically related polypeptides are present in Escherichia coli and Erwinia carotovora subsp. carotovora.

Amino Acid Sequence↗

PubMed1981

Subcellular localization and isolation of gamma-glutamyltransferase from rat hepatoma cells.

Cultured rat hepatoma cells were homogenized and subjected to subcellular fractionation by analytical sucrose density centrifugation to determine the localization of gamma-glutamyltransferase ((5-glutamyl-)-peptide: amino acid 5-glutamyltransferase, EC 2.3.2.2). The activity was exclusively localized to the plasma membrane. Diazotized sulphanilic acid was used as a non-penetrant membrane reagent which inactivates ectoenzymes. With both intact and sonicated cells, only 70-75% inhibition of gamma-glutamyltransferase activity was observed. At least 12% of the total cell complement of gamma-glutamyltransferase activity is highly resistant to inactivation by diazotized sulphanilic acid even after Triton X-100 solubilization. The enzyme was purified from hepatoma cells and its properties compared with enzyme from normal liver. Apart from the striking increase in Vapp there were only minor differences between the enzymes from the two sources. In contrast to the complete abolition of transpeptidase activity of the purified hepatoma enzyme by diazotized sulphanilic acid, the hydrolytic activity of this preparation was only slightly inhibited.

Animals↗

PubMed2002

Subcellular localization of beta-catenin is regulated by cell density.

It is generally accepted that subcellular distribution of beta-catenin regulates its function. Membrane-bound beta-catenin mediates cell-cell adhesion, whereas elevation of the cytoplasmic and nuclear pool of the protein is associated with an oncogenic function. Although the role of beta-catenin in transformed cells is relatively well characterized, little is known about its importance in proliferation and cell-cycle control of nontransformed epithelial cells. Using different approaches we show that in human keratinocytes (HaCaT) beta-catenin is distributed throughout the cells in subconfluent, proliferating cultures. In contrast, beta-catenin is nearly exclusively located at the plasma membrane in confluent, contact-inhibited cells. Hence, we demonstrate for the first time that beta-catenin is translocated from the cytoplasm to the plasma membrane in response to high cell density. We conclude that beta-catenin plays an important role in proliferation and mediating contact-inhibition by changing intracellular localization.

Cadherins↗