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Results for “Subcellular localization”

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PubMed1988

Subcellular localization of functionally differentiated microtubules in squid neurons: regional distribution of microtubule-associated proteins and beta-tubulin isotypes.

The subcellular localization of microtubule proteins in the neurons of squid (Doryteuthis bleekeri) was immunologically studied using monoclonal antibodies against the microtubule proteins. We found that (1) the squid neurons contained three kinds of high-molecular-weight microtubule-associated proteins [MAP A of approximately 300 kilodaltons (kD), MAP B of 260 kD, and axolinin of 260 kD] and two kinds of beta-tubulin isotypes (beta 1 and beta 2); (2) the cell body of the squid giant neuron contained MAP A, MAP B, and the two beta-tubulin isotypes (beta 1 and beta 2); (3) axolinin and the beta 1 isotype were present exclusively in the peripheral axoplasm of the giant axon; and (4) a small amount of axolinin, MAP A, and the beta 1 isotype was found in the insoluble aspect of the central axoplasm, whereas the soluble aspect of the central axoplasm contained an abundant amount of MAP A along with the modified form of the beta 1 isotype. The regional difference of the distribution of the microtubule protein components may explain the differences in stability among axonal microtubules. Microtubules in the soluble aspect of the central axoplasm are sensitive to any treatment with colchicine, cold temperature, and high ionic strength but those both in the insoluble aspect of the central axoplasm and in the peripheral axoplasm are highly insensitive to the treatment.

Animals↗

PubMed1978

Lipolytic enzymes in bovine thyroid tissue. I. Subcellular localization, purification and characterization of acid phospholipase A1.

In mammalian cells the catabolism of membrane phosphoglycerides proceeds probably entirely through a deacylation pathway catalysed by phospholipase A and lysophospholipase (Wise & Elwyn, 1965). In the initial attack of diacylphosphoglycerides by phospholipase A two enzymatic activities with different positional specificities have been distinguished: phospholipase A1 (phosphatidate 1-acyl hydrolase EN 3.1.1.32) and phospholipase A2 (phosphatidate 2-acyl hydrolase EN 3.1.1.4) (Van Deenen & De Haas, 1966). Studies on these intracellular phospholipases were mainly concerned with their subcellular localization. Only occasionally more detailed enzymatic investigations have been conducted on them, in contrast to export phospholipases e.g. from snake venom, bee venom and porcine pancreas, which have been extensively investigated (Brockerhoff & Jensen 1974a). In a previous paper (De Wolf et al., 1976a), the presence of phospholipase A1 and phospholipase A2 activities in bovine thyroid was demonstrated, using 1-[9, 10-3H] stearoyl-2-[1-14C] linoleyl-sn-glycero-3-phosphocholine as a substrate. Optimal activity was observed in both instances at pH 4. Addition of the anionic detergent sodium taurocholate increased the A2 type activity and decreased the A1 type activity suggesting the presence of different enzymes. The lack of influence of Ca2+-ions and EDTA and the acid pH optima could suggest lysosomal localization. In this paper the subcellular distribution of both acid phospholipase activities is described as well as a purification scheme for phospholipase A1. Some characteristics of the purified enzyme preparation are discussed.

Animals↗

PubMed1989

Changes in activity and subcellular localization of alpha-like DNA polymerase during cell cycle of Physarum polycephalum.

Regulation of DNA replication was given attention by examining the subcellular localization of alpha-like DNA polymerase in Physarum polycephalum. The activity per plasmodium increased by 2-fold in the cytoplasm during the G2-phase, and decreased with a concomitant increase in nuclei prior to DNA replication. This would suggest that the enzyme translocates from the cytoplasm to the nucleus, co-ordinately with DNA replication. In nuclei, the nuclear matrix-bound activity increased during the S-phase, but changes did not always parallel the rate of DNA replication. On the other hand, the activity measured without exogenous templates did change with the rate, although the activity was low. It is, therefore, suggested that part of the enzyme binding with the matrix participates in DNA replication.

Binding Sites↗

PubMed1975

Subcellular localization of gamma-aminobutyrate transaminase and glutamate dehydrogenase in adult rat brain. Evidence for at least two small glutamate compartments in brain.

The subcellular localizations of gamma-aminobutyrate transaminase (EC 2.6.1.19) and glutamate dehydrogenase (EC 1.4.1.2) in brain tissue of adult rats were compared with each other and with those of NAD+-isocitrate dehydrogenase (EC 1.1.41) and monoamine oxidase (EC 1.4.3.4; kynuramine as substrate). Crude mitochondrial fractions from brain tissue were centrifuged in continuous sucrose density gradients. gamma-Aminobutyrate transaminase and glutamate dehydrogenase were always found at a higher density than NAD+-isocitrate dehydrogenase and monoamine oxidase. When centrifuged for 1 h at 53 000gav., there was a slight difference between the distribution profiles of glutamate dehydrogenase and gamma-aminobutyrate transaminase. This difference was larger when the centrifugation time was only 15 min. It is concluded that there are subpopulations of brain mitochondria with differing proportions of gamma-aminobutyrate transaminase and glutamate dehydrogenase. The results are discussed in relation to evidence obtained with labelled precursors in vivo that there are at least two small glutamate compartments in adult brain.

4-Aminobutyrate Transaminase↗

PubMed1975

Short-chain fatty acid synthesis in brain. Subcellular localization and changes during development.

Acetyl-CoA synthase (EC 6.2.1.1), Propionyl-CoA synthase (EC 6.2.1.-) and butyryl-CoA synthase (EC 6.2.1.2) were measured in subcellular fractions prepared by primary and density-gradient fractionation from adult rat brain by a method resulting in recoveries close to 100%. Most of the activity of the three enzymes was recovered in the crude mitochondrial fraction. On subfractionation of this crude mitochondrial fraction with continuous sucrose density gradients, most of the activity of the three enzymes was found at a higher density than NAD+-isocitrate dehydrogenase and at about the same density as glutamate dehydrogenase, confirming earlier reported data for acetyl-CoA synthase. The finding that propionyl-CoA synthase and butyryl-CoA synthase had about the same distribution in the gradients as acetyl-CoA synthase adds support to the hypothesis that mitochondria involved in the metabolism of these short-chain fatty acids (all three of which have been shown to result in a rapid and high labelling of glutamine in vivo) form a distinct subpopulation of the total mitochondrial population. The three synthase activities were found to differ from each other in their rate of change and their subcellular localization during rat brain development. This, in combination with the observation that in gradients of adult brain preparations the three activities did not completely overlap, suggests that the three synthase activities are not present in the same proportion to each other in the same subpopulation (s) of mitochondria in the brain.

Acetate-CoA Ligase↗

PubMed1975

Cell proliferation and subcellular localization of alkaline phosphatase activity in rat liver parenchyma during azo dye carcinogenesis.

A combined method of phosphatase histochemistry and (3H)thymidine radioautography was devised to study the subcellular localization of alkaline phosphatase (AP) activiity with changing pattern of cell proliferation in precancerous livers of rats fed dimethylaminoazobenzene. After 50 hr of continuous infusion of (3H)thymidine into the rats, labeled liver tissue were fixed in glutaraldehyde. Sections were incubated for AP activity in a lead citrate medium (pH 9.4) with beta-glycerophosphate as substrate. Light and electron microscopic examinations of radioautographs revealed that focal groups of 3H-labeled hepatocytes within hyperplastic nodules were coincident to hyperbasophilic foci and distinguishable from the surrounding parenchyma, which was sparsely labeled. Proliferative hepatocytes in the foci exhibited enzyme reaction product indicative of AP activity along the entire surface membranes. The surface AP tography was in contrast to that of the surrounding hyperplastic parencyma, in which regenerative hepatocytes showed a normal localization of AP activity at the bile canalicular membranes. The L-phenylalanine-sensitive snd heat-resistant activity of hyperbasophilic hepatocytes was different from that of normal hepatocytes. The surface enzyme differentiation was accompanied by a decrease of cytoplasmic AP. Golgi elements apparently function in the mobilization of AP into the surface membranes. The phenomena of AP alterations might be related to the abnormal control of cell proliferation and cytodifferentiation leading to malignant growth.

Alkaline Phosphatase↗

PubMed1991

Subcellular localization of blood group A substance produced by pancreatic adenocarcinoma induced in hamsters by N-nitrosobis(2-oxopropyl)amine (BOP) and by its cell line (PC-1).

The subcellular localization and biochemical characteristics of blood group A antigen were studied by immunogold methods and by SDS-PAGE and Western blotting procedures in N-nitrosobis)2-oxopropyl)amine (BOP)-induced pancreatic cancer (PC) in Syrian hamsters, in the pancreatic cancer cell line (PC-1) derived from a primary induced pancreatic cancer, and in intrapancreatic and subcutaneous transplants of PC-1 cells. Normal hamster duodenal epithelial cells expressing A antigen were compared with the normal hamster pancreas (lacking A antigen), human PC tissues from patients with blood group A and human PC cell lines. Blood group A antigen was present on the membrane of hamster duodenal cells, but was absent in the normal pancreatic cells. A antigen was localized mainly on the cell membrane of the hamster cancer cells both in vivo and in vitro. Glycoproteins with blood group A specificity were observed by SDS-PAGE and Western blotting procedures in the membrane fraction of PC-1 cells, with a major component of molecular mass of approximately 120 kd. Similar migration patterns were observed in the primary induced PC and in subcutaneous and intrapancreatic transplants of PC-1 cells. Membrane preparations from cell lines derived from two primary pancreatic cancers from patients of blood group A and from human pancreatic cell lines, CD11 and CD18, showed a major A reactive component with a molecular mass similar to that found in the hamster PC cells. These findings suggest that: (i) both the hamster and human PC cells in vitro produce glycoproteins with blood group A specificity of similar molecular masses; (ii) differences exist in the structure of the glycoprotein immunoreactive with the anti-A antigen between the normal and cancerous cells; and (iii) differences exist in the molecular mass of the anti-A reactive substance between hamsters and human PC cells and between tissues in vivo and in vitro.

ABO Blood-Group System↗

PubMed1992

Presence and subcellular localization of tyrosine aminotransferase and p-hydroxyphenyllactate dehydrogenase in epimastigotes of Trypanosoma cruzi.

Cell-free extracts of epimastigotes of Trypanosoma cruzi contain tyrosine aminotransferase (TAT) and p-hydroxyphenyllactate dehydrogenase (pHPLDH). The TAT activity could be separated from aspartate aminotransferase (ASAT) by polyacrylamide gel electrophoresis or DEAE-cellulose chromatography; the latter procedure also allowed complete separation of pHPLDH. The subcellular localization of both T. cruzi enzymes, as determined by digitonin extraction, subcellular fractionation by differential centrifugation, and isopycnic ultracentrifugation in sucrose gradients, was mainly cytosolic, with low mitochondrial activities.

Animals↗

PubMed1985

Subcellular localization of vasopressin-like material in platelets.

It has recently been reported that 90% of circulating vasopressin in humans appears to be in platelets. We have confirmed these results and studied the subcellular localization and secretion of vasopressin from human platelets. Sucrose density gradient analysis showed highest relative specific activity for vasopressin in the membrane fractions. Stirring of platelet-rich plasma with a number of different platelet-aggregating agents including epinephrine, norepinephrine, arachidonic acid, collagen, adenosine diphosphate, A23187, and platelet-activating factor failed to release significant amounts of immunoreactive vasopressin, despite the expected aggregation and secretion of platelet factor 4 by these agents. Thus vasopressin appears to be primarily a platelet membrane component and is not secreted in the usual way by agents modulating platelet function.

Adenosine Diphosphate↗

PubMed1989

Subcellular localization of sterol carrier protein-2 in rat hepatocytes: its primary localization to peroxisomes.

Sterol carrier protein-2 (SCP-2) is a nonenzymatic protein of 13.5 kD which has been shown in in vitro experiments to be required for several stages in cholesterol utilization and biosynthesis. The subcellular localization of SCP-2 has not been definitively established. Using affinity-purified rabbit polyclonal antibodies against electrophoretically pure SCP-2 from rat liver, we demonstrate by immunoelectron microscopic labeling of ultrathin frozen sections of rat liver that the largest concentration of SCP-2 is inside peroxisomes. In addition the immunolabeling indicates that there are significant concentrations of SCP-2 inside mitochondria, and associated with the endoplasmic reticulum and the cytosol, but not inside the Golgi apparatus, lysosomes, or the nucleus. These results were confirmed by immunoblotting experiments with proteins from purified subcellular fractions of the rat liver cells carried out with the anti-SCP-2 antibodies. The large concentration of SCP-2 inside peroxisomes strongly supports the proposal that peroxisomes are critical sites of cholesterol utilization and biosynthesis. The presence of SCP-2 inside peroxisomes and mitochondria raises questions about the mechanisms involved in the differential targeting of SCP-2 to these organelles.

Animals↗

PubMed1992

Subcellular localization of prostaglandin E2 receptors in the gastric mucosa.

Gastric mucosal PG E2 receptors are the common antisecretory working point of all prostanoid types and may also be involved in "protective" effects. We investigated the subcellular localization of these receptors, as measured by displaceable 3H-PG E2 binding, and identified different organelles by monitoring the activities of specific marker enzymes. Porcine mucosal homogenates were subdivided by differential centrifugation into fractions P1 (1000 x g), P2 (20,000 x g), P3 (300,000 x g) and the supernatant S1. P3 was further fractionated over a series of sucrose step gradients. Mitochondria and lysosomes were enriched in P2 (maximum specific activities of cytochrome-c-oxidase of beta-glucosidase, beta-glucuronidase, beta-galactosidase, respectively). Plasma membranes (alkaline phosphatase, gamma-glutamyl-transpeptidase, 5-nucleotidase), tubulovesicles (H+/K(+)-ATPase) and rough endoplasmic reticulum (NADPH-cytochrome-c-reductase) were mainly found in P3, which also contained the majority of 3H-PG E2 binding sites. In contrast, prostanoid binding was barely detectable in S1. Density fractionation of P3 revealed that 3H-PG E2 binding sites shared a similar sedimentation profile with plasma membranes and tubulovesicular markers. No or negative correlation was found with lysosomes, rough endoplasmic reticulum and mitochondria. We conclude that mucosal PG E2 receptors are predominantly located at the cell surface. This supports the view that prostanoids inhibit gastric secretion through membrane receptors, but gives no clue for intracellular "protective" working points.

Animals↗

PubMed1987

Modification, processing, and subcellular localization in Escherichia coli of the pCloDF13-encoded bacteriocin release protein fused to the mature portion of beta-lactamase.

A fusion between the pCloDF13-derived bacteriocin release protein and beta-lactamase was constructed to investigate the subcellular localization and posttranslational modification of the bacteriocin release protein in Escherichia coli. The signal sequence and 25 of the 28 amino acid residues of the mature bacteriocin release protein were fused to the mature portion of beta-lactamase. The hybrid protein (Mr, 31,588) was expressed in minicells and whole cells and possessed full beta-lactamase activity. Immunoblotting of subcellular fractions revealed that the hybrid protein is present in both the cytoplasmic and outer membranes of E. coli. Radioactive labeling experiments in the presence or absence of globomycin showed that the hybrid protein is modified with a diglyceride and fatty acids and is processed by signal peptidase II, as is the murein lipoprotein. The results indicated that the pCloDF13-encoded bacteriocin release protein is a lipoprotein which is associated with both membranes of E. coli cells.

Bacterial Proteins↗

PubMed1979

Re-examination of the subcellular localization of thyroxine 5'-deiodination in rat liver.

We describe the existence of at least two thyroxine 5'-deiodinases in rat liver. They co-fractionate with NADPH-cytochrome c reductase, the marker enzyme for membranes of the endoplasmic reticulum. Subcellular-localization studies of the most active microsomal thyroxine 5'-deiodinase were performed under substrate saturation and at optimal pH 6.8. This enzyme was a Km(app.) of about 3 microM-thyroxine and a Vmax. of about 8 ng of tri-iodothyronine/min per mg of protein. Our study confirms in part the earlier reports of microsomal localization of thyroxine 5'-deiodination. However, this process is not mediated by only a single enzyme.

Animals↗

PubMed1992

Identification and subcellular localization of the Q gene product of visna virus.

The genome of the sheep visna lentivirus contains an open reading frame, Q, which has a coding potential of 230 amino acid residues. This paper reports the identification and the subcellular localization of the Q ORF-encoded protein detected in lysates of visna virus-infected sheep choroid plexus cells. Sera from sheep either experimentally or naturally infected with visna virus reacted with the bacterially synthesized Q protein indicating that the in vivo expressed Q product is immunogenic. Antibodies raised against a synthetic N-terminal peptide, reacted with either the bacterial Q or the in vitro translated Q protein as well as with the Q protein expressed during cellular infection. This 29 kDa protein is detectable late in the lytic viral cycle, i.e., 72 hr postinfection, and this expression correlates with the late transcription of its 4.8-kb mRNA. These results provide evidence for the first time that the Q ORF is a late gene of visna virus and that the Q protein is located in the cytosol compartment, without evidence of accumulation at the cell membrane, or in cell-free virion particles.

Animals↗

PubMed1989

Subcellular localization of iodinated thyroid tubulin.

Subcellular fractions enriched in mitochondria, plasma membranes, microsomes and Golgi apparatus were obtained from thyroid glands of rats injected with I125. Autoradiography of SDS-polyacrylamide gels revealed the presence of a number of radiolabelled proteins in each membrane fraction. One polypeptide, with the same electrophoretic mobility as brain tubulin, was found in all fractions except the plasma membranes and was immunoprecipitated with commercial anti-tubulin monoclonal antibodies. Hydrolysis of Asp-Pro linkages of I125 labelled tubulin with formic acid indicated that there were iodination sites in both the carboxy terminal one third and the amino terminal two thirds of the molecule. These results, together with the absence of iodinated tubulin from the cytosolic fraction, are consistent with the idea that a population of thyroid membrane tubulin is iodinated at multiple sites either just before or after insertion into intracellular membranes where it may act as an anchorage point for microtubule-membrane interactions.

Animals↗

PubMed1990

Subcellular localization of GABAA/benzodiazepine receptor-like immunoreactivity in the superficial gray layer of the rat superior colliculus.

The monoclonal antibody bd-17, which recognizes the beta 2 and beta 3-subunits of GABAA/benzodiazepine receptors, was used to determine the cellular and subcellular localization of receptor-like immunoreactivity in the superficial gray layer of the rat superior colliculus. In numerous dendrites, very strong immunostaining was present in the cytoplasm and on the postsynaptic dendritic membrane of synaptic junctions. The extrasynaptic portion of the dendritic membrane also very often showed [beta 2 + beta 3]-like immunoreactivity. However, due to methodological limitations, it could not be stated with certainty whether presynaptic beta 2- and beta 3-subunits of GABAA/benzodiazepine receptors actually occur in this mesencephalic visual structure. In conclusion, these results strongly suggest that synaptic and non-synaptic GABAA/benzodiazepine receptors are present in the superficial gray layer of the rat superior colliculus. These receptors may modulate neuronal cell activity in different ways, depending on their location.

Animals↗

PubMed1990

Developmental changes of synapsin I subcellular localization in rat cerebellar neurons.

Synapsin I, one of the major synaptic proteins, is thought to associate with synaptic vesicles and to play a regulatory role in neurotransmitter release. In mature neurons, it is concentrated almost exclusively in presynaptic nerve endings. Here, we studied the subcellular localization of synapsin I during the development of rat cerebellar cortices by immunocytochemistry, using anti-synapsin I antibodies and found that during the development of rat cerebellar cortices it tentatively exists in the dendritic growth cones of immature internal granule cells and in the axonal growth cones of mossy fibers as well as mature presynaptic endings. Also, we found that synapsin I, in the axonal and dendritic growth cones does not necessarily associate with vesicles, but rather with fuzzy filamentous structures in the cytoplasm. In search of the structure of synapsin I in vivo, we employed the quick-freeze, deep-etch technique after immunogold labeling. Synapsin I seems to thereby connect synaptic vesicles or anchor them to cytoskeletons in presynaptic endings.

Animals↗

PubMed1987

Influence of conversion of penicillin G into a basic derivative on its accumulation and subcellular localization in cultured macrophages.

beta-Lactam antibiotics do not accumulate in phagocytes, probably because of their acidic character. We therefore synthesized a basic derivative of penicillin G, namely, 14C-labeled N-(3-dimethylamino-propyl)benzylpenicillinamide (ABP), and studied its uptake and subcellular localization in J774 macrophages compared with that of 14C-labeled penicillin G. Whereas the intracellular concentration (Ci) of penicillin G remained lower than its extracellular concentration (Ce), ABP reached a Ci/Ce ratio of 4 to 5. Moreover, approximately 50% of intracellular ABP was found associated with lysosomes after isopycnic centrifugation of cell homogenates in isoosmotic Percoll or hyperosmotic sucrose gradients. The behavior of ABP was thus partly consistent with the model of de Duve et al. (C. de Duve, T. de Barsy, B. Poole, A. Trovet, P. Tulkens, and A. Van Hoof, Biochem. Pharmacol. 23:2495-2531, 1974), in which they described the intralysosomal accumulation of weak organic bases in lysosomes. Although ABP is microbiologically inactive, our results show that beta-lactam antibiotics can be driven into cells by appropriate modification. Further efforts therefore may be warranted in the design of active compounds or prodrugs that may prove useful in the chemotherapy of intracellular infections.

Animals↗