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PubMed1991

Subcellular localization of glutamate dehydrogenases and alanine aminotransferase in epimastigotes of Trypanosoma cruzi.

The subcellular localization of NAD- and NADP-linked glutamate dehydrogenases (GDH-NAD and GDH-NADP), alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) in epimastigotes of Trypanosoma cruzi was studied by digitonin extraction from whole cells, subcellular fractionation by differential centrifugation and isopycnic ultracentrifugation. All enzymes presented both a cytosolic and a mitochondrial form; in addition, GDH-NADP seems to have a third, still undefined, localization. The results are compatible with the existence of two pathways for the production of L-alanine linked to the reoxidation of glycolytic NADH, one operative in the mitochondrion and the other in the cytosol, and perhaps responsible for the existence of the two alanine pools detected by 13C-nuclear magnetic resonance (B. Frydman et al., Eur. J. Biochem. 192 (1990) 363-368).

Alanine Transaminase↗

PubMed1988

Subcellular localization of a thromboxane A2/prostaglandin H2 receptor antagonist binding site in human platelets.

The subcellular localization of a binding site for the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist, 9,11-dimethylmethano-11,12-methano-16-(3-iodo-4- hydroxyphenyl)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranor TXA2 ([125I]-PTA-OH), was determined. Subcellular fractions of platelets were prepared by glycerol lysis or nitrogen cavitation, and were characterized by the use of enzymatic markers specific for plasma membranes, endoplasmic reticulum (dense tubular system), mitochondria, granules, and cytosolic constituents. The Kd and density of binding sites in the subcellular fractions were determined by Scatchard analysis of equilibrium binding data. The Kd and Bmax for [125I]-PTA-OH determined in the lysates were 49 +/- 11 nM and 4.1 +/- 1.7 pmol/mg protein respectively (N = 6). The Kd values were not significantly different in any of the fractions assayed. The binding sites were coenriched (4.5 +/- 0.66 fold) with the enzymatic markers for plasma membranes (3.7 +/- 0.5 fold) and dense tubular system (2.4 +/- 0.4 fold). The binding sites were not coenriched with markers for cytoplasmic constituents, mitochondria, or granules. The ability of the TXA2/PGH2 mimetic U46619 to compete with [125I]-PTA-OH for the binding site was also determined for the various subcellular fractions. The IC50 for U46619 was 5.4 +/- 1.2 microM in the lysate, and was not significantly different in the subcellular fractions. These data suggest that the binding site is the TXA2/PGH2 receptor described previously. These data are consistent with the notion that the putative TXA2/PGH2 receptor is localized in the plasma membranes and/or the dense tubular system.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed1979

Subcellular localization of three degeneration-associated phospholipids in cultured hamster fibroblasts (BHK21 cells).

Subcellular localization of bisphosphatidic acid, semilysobisphosphatidic acid and phosphatidyl-(N-acyl)ethanolamine was studied in normal and degenerating fibroblasts (BHK21 cells) by differential centrifugation. In the normal cells these lipids were highly enriched in the floating fraction consisting mainly of neutral lipid-rich lysosomes. They were also enriched in the mitochondrial fraction. In degenerating cells the high enrichment in the floating fraction was retained, but the other peak was displaced to the crude nuclear fraction. Subfractionation of the crude nuclear fraction indicated that these lipids were not enriched in the purified nuclei. Instead, their concentrations were relatively high in the other subfraction evidently enriched in the large secondary lysosomes characteristic for the degenerating cells. Neither in normal nor degenerating cells were these lipids enriched in the light mitochondrial fraction, where most of the smaller, and probably younger, lysosomes were found. On the basis of these results it is suggested that bisphosphatidic acid, semilysobisphosphatidic acid and phosphatidyl-(N-acyl)ethanolamine are lysosomal in origin. It appears possible that they are specifically associated with the organelles representing the later stages in the lysosomal lifespan.

Animals↗

PubMed1977

Cellular and subcellular localization of 3h-diethylstilboestrol in the central nervous system.

The cellular and subcellular localization of radioactivity in the brain of immature female rats was determined by dry-mount autoradiography 2 h after iv injection of 1.0 microgram of (monethyl-3H) diethylstilboestrol per 100 g body weight. A specific topographic pattern of nuclear concentration of the synthetic oestrogen was obtained similar to that for 3H-oestradiol-17 beta in specific neurons of the basal hypothalamus, preoptic region and amygdala. In competition experiments, the nuclear concentration of radioactivity in all areas studied was inhibited by unlabeled oestradiol, while unlabeled testosterone had no effect. These data suggest that although oestradiol can bind to androgen receptors, the oestrogen receptor itself can account for the localization seen after the injection of 3H-oestradiol.

Amygdala↗

PubMed1990

Subcellular localization of blood group substances ABH in human salivary glands.

We investigated the subcellular localization of ABH antigens in human submandibular, sublingual, and buccal glands by applying a post-embedding immunogold method using monoclonal antibodies specific for A, B, and H antigens. In most glands the immunoreactivity was usually restricted to mucous cells, in which only secretory granules and sometimes Golgi cisternae were specifically labeled. A and B antigens were demonstrated only in the glands of type A, B, and AB subjects, while H antigen was visualized in glands from individuals of all blood types. Moreover, differences were observed in the relative distribution of ABH antigens, depending on the type of gland.

ABO Blood-Group System↗

PubMed1990

Type II regulatory subunit dimerization determines the subcellular localization of the cAMP-dependent protein kinase.

The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of dimeric regulatory subunits (RII) to anchoring proteins. Cytoskeletal localization occurs through RII dimer interaction with the PKA substrate molecule microtubule-associated protein 2 (MAP2). RII alpha deletion mutants and RII alpha/endonexin chimeras retained MAP2 binding activity if they contained the first 79 residues of the molecule. Disruption of RII alpha dimerization always prevented MAP2 interaction because 1) RII delta 1-14 (an amino-terminal deletion mutant lacking residues 1-14) was unable to bind MAP2 or form dimers, and 2) a modified RII alpha monomer including residues 1-14 did not bind MAP2. Chimeric proteins containing the first 30 residues of RII alpha fused to endonexin II formed dimers but did not bind MAP2. This suggested other side-chains between residues 30-79 also participate in MAP2 interaction. Peptide studies indicate additional contact with MAP2 may occur through an acidic region (residues 68-82) close to the RII autoinhibitor domain. Therefore, anchored PKA holoenzyme topology may position the catalytic subunit and MAP2 as to allow its preferential phosphorylation upon kinase activation.

Amino Acid Sequence↗

PubMed1975

The subcellular localization of the erythrostimulant factors present in the kidney and spleen of the rabbit.

The subcellular localization of erythrostimulant factors contained in the kidney and spleen of the rabbit have been studied. This was done by subjecting homogenates to fractionation by centrifugation and assessing the erythrostimulant capacity of the hypotonic extracts prepared from the various fractions thus obtained. The results of these studies show that the erythrostimulant factors are present in the light mitochondrial and microsomal fractions of the normal kidney. In conditions of anaemia they are to be found not only in the above-mentioned fractions but also in the mitochondrial fraction. By contrast, in the spleen such factors are present only in the mitochondrial fraction. It is likely that the different localization of the erythrostimulant factors in the kidney and in the spleen of the rabbit may be closely connected with the different functions performed by these organs in the humoral regulation of erythropoiesis.

Animals↗

PubMed1992

Regional and subcellular localization of Li+ and other cations in the rat brain following long-term lithium administration.

Rats were given LiCl in their diet (40 mmol/kg dry weight) for at least 3 months to elucidate the regional and subcellular localization of Li+ in the brain as well as the effect of chronic lithium administration on the distribution of other cations. At steady-state the mean concentrations of Li+ were 0.66 mmol/kg wet weight in the whole brain and 0.52 mM in plasma. The tissue/plasma concentration ratio exceeded unity in all anatomical regions. No region showed excessive accumulation of Li+. Whole brain or regional contents of Na+ or K+ were unaffected by lithium treatment. Subcellular Li+ localization was demonstrated in nuclear, crude mitochondrial, and microsomal fractions of whole brain homogenate. Subfractionation of the crude mitochondrial fraction revealed energy-independent intrasynaptosomal and intramitochondrial Li+ and K+ localization at 0-4 degrees C. Li+ administered in vivo disappeared within 10 min from synaptosomes incubated at 37 degrees C. Li+ added in vitro at 1 mM attained a synaptosomal steady-state concentration within 30 min at 37 degrees C. In control rats, synaptosomal concentrations and synaptosomal/medium concentration gradients of cations paralleled their respective in vivo concentrations and gradients. Lithium treatment caused synaptosomal depletion of K+ and Mg2+ and hence probably partial membrane depolarization. Addition of 1 mM Li+ in vitro also caused synaptosomal Mg2+ depletion. The results indicate that Li+ is "accumulated" in brain sediments and synaptosomes following its long-term treatment. The estimated intracellular and intrasynaptosomal Li+ concentrations are lower than predicted by passive distribution according to the Nernst equation, evidencing active extrusion of Li+.

Animals↗

PubMed1979

Subcellular localization of thyroxine and reverse triiodothyronine outer ring monodeiodinating activities.

In order to examine the subcellular localization of outer ring T4- and rT3-monodeiodinating activities, nuclear, mitochondrial, microsomal, cytosol, and plasma membrane fractions of rat liver homogenate were incubated with either T4 or rT3 in phosphate buffer (pH 7.35) in the presence of 2 mM dithiothreitol for 15 min at 37 C, and the amount of product (T3 in the case of T4 and 3,3'-diiodothyronine in the case of rT3) was measured by specific RIA. The various tissue fractions were also examined for the relative concentration of various marker enzymes. T4 and rT3 monodeiodinating activities correlated better with enzyme markers of plasma membranes than of any other subcellular fraction in most tissue fractions. A fraction could be isolated, however, in which the monodeiodinating activities correlated better with the enzyme markers of microsomes than of plasma membranes. The various data suggest that plasma membranes and microsomes are two main sites of T4- and rT3-monodeiodinating activities. The location of T4 to T3 converting activity in the plasma membranes may serve to modulate the delivery of the more potent thyroid hormone, i.e. T3, into the cells.

Animals↗

PubMed1988

Subcellular localization of DNA polymerase gamma and changes in its activity in sea urchin embryos.

1. Subcellular localization and changes in the activity of DNA polymerase gamma were examined in sea urchin eggs and embryos. 2. The enzyme was shown to be localized predominantly in mitochondria by differential and isopycnic centrifugation. 3. During embryogenesis, the enzyme activity per embryo remained constant until blastula stage, and thereafter increased. 4. Similarly mitochondrial DNA per embryo increased, indicating that mitochondrial DNA replication starts during embryogenesis. 5. The gamma-activity per mitochondrial DNA remained constant during embryogenesis. 6. These results suggest that mitochondria contain a constant amount of replicative enzyme (DNA polymerase gamma) regardless of mitochondrial DNA replication, which differs from the case of nuclear DNA replication.

Animals↗

PubMed1976

Subcellular localizations of guanylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in sea urchin sperm.

The subcellular localizations of guanylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in sea urchin sperm were examined. Both the specific and total activities of these two enzymes were much higher in sperm flagella (tails) than in the heads. In addition to the observation that guanylate cyclase in the flagella was particulate-bound and solubilized by Triton X-100, more than 80% of the cyclase activity in the flagella was found in the plasma membrane fraction, whereas the activity of cyclic nucleotide phosphodiesterase was observed in both the axonemal and plasma membrane fractions. The observations indicated that the cyclase in the flagella appeared to be associated with the plasma membrane. Cyclic nucleotide phosphodiesterase in the plasma membrane fraction as well as the axonemal fraction hydrolyzed both cyclic GMP and cyclic AMP; however, the rates of hydrolysis for cyclic GMP were obviously higher than those for cyclic AMP. The enzymic properties of guanylate cyclase and cyclic nucleotide phosphodiesterase in sperm flagella were also briefly described.

Animals↗

PubMed1989

The subcellular localization of the neu protein in human normal and neoplastic cells.

We have examined the subcellular localization of the neu protein by immunohistochemistry and immuno-electron microscopy, associated with immunoblotting of normal and neoplastic tissues with 2 monoclonal antibodies (MAbs). Immunoelectron microscopy clearly reveals that neu protein resides only on the lateral plasma membrane of the simple epithelium of the breast and on the plasma membrane of malignant breast cells. It is also found on the membranes of the microvilli and the apical vacuoles of the cells of the proximal convoluted tubule of the kidney. In the cytoplasm, the only immunoreactivity detected with both antibodies was on the membrane of the mitochondrial cristae of normal and malignant cells. Immunoblotting reveals that the molecular weight of the membrane protein is 185 and 155 kDa for the mitochondrial protein. The cell membrane staining pattern can be revealed by light microscopic immunohistochemistry only in malignant cells and is therefore specific for malignancy. The membrane expression in normal cells cannot be visualized in this way. The mitochondrial reactivity appears as a cytoplasmic granular staining when examined under the light microscope. Similar cytoplasmic staining has been described previously in other studies with other antibodies against the neu protein and has lead to speculation about its function in normal and malignant cells. However, it is demonstrated in this study that it is not the known neu-oncogene product.

Antibodies, Monoclonal↗

PubMed1989

Determination of the inorganic pyrophosphate level and its subcellular localization in Chara corallina.

In order to determine the concentration of pyrophosphate (PPi) and its subcellular distribution in Chara corallina, a new method to concentrate PPi from cell extracts was developed. PPi was extracted and concentrated as Ca2P2O7 under alkaline conditions. The amount of PPi in the precipitate was measured using an enzyme system containing pyrophosphate:fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) coupled to NADH oxidation in the presence of [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid. The subcellular localization of PPi and inorganic phosphate (Pi) was studied using the intracellular perfusion technique. The relative volumes of the cytoplasm (6.4%) and the vacuole (93.6%) were determined by perfusing Lucifer Yellow CH into the vacuole and by assuming that the Lucifer Yellow CH dead space represented the cytoplasmic volume. The volume of the chloroplast layer was determined microscopically, and it was found that it occupied 10% of the Chara cytoplasm. PPi was present predominantly in the cytosol at a level of 193 microM, while it existed in the vacuole at a level of only 2.20 microM and less than 1 microM in chloroplasts. By contrast, Pi was distributed almost equally in the cytosol (12.0 mM), chloroplasts (16.2 mM), and the vacuole (6.70 mM). The electrochemical potential gradient across the tonoplast for H+ (delta mu H+ = -11.6 to -18.0 KJ/mol) was nearly equal to the free energy release from the hydrolysis of PPi in cytoplasm (delta Gpp = -18.9 KJ/mol), indicating that the H+-translocating inorganic pyrophosphatase can work as a H+ pump in C. corallina.

Chemical Precipitation↗

PubMed1991

Subcellular localization of rat gastric phospholipase A2.

In the present study, we have performed experiments to gain some insight into the subcellular localization and biochemical properties of gastric mucosal phospholipase A2. After classical subcellular fractionation of whole glandular stomach mucosa, we found that gastric phospholipase A2 was essentially enriched in the 105,000 x g pellet that contains microsomes and plasma membranes. Except for the cytosol, all the subcellular fractions exhibited similar phospholipase A2 activity (i.e., optimum of pH, calcium dependence, apparent Km and positional specificity). The high-speed pellet was further characterized by ultracentrifugation on a sucrose gradient. Data showed that the sedimentation profile of phospholipase A2 was quite similar to those of plasma membrane markers and more specifically to an apical membrane marker. These results, taken together, showed that a gastric phospholipase A2 is distributed among the various subcellular fractions (as a result of cross-contamination) together with the membrane fraction on which it is associated. It is proposed that this fraction is the apical plasma membrane which would be the main site of phospholipase A2 action for arachidonic acid release. Lysophospholipase showed the same sedimentation profile as phospholipase A2, whereas acyl CoA-lysophosphatidylcholine: acyltransferase mainly sedimented with heavy microsomes. The substrate specificity of the enzyme was assessed by endogenous hydrolysis of gastric mucosal phospholipids. We were able to show that the enzyme acts at nearly the same rate on two major gastric membrane phospholipids, namely phosphatidylcholine and phosphatidylethanolamine.

Animals↗

PubMed1990

Hepatic subcellular localization of cresylbenzodioxaphosphorin oxide (CBDP)-sensitive soman binding sites.

The toxicity of the organophosphorus poison soman (pinacolylmethylphosphonofluoridate) is attributable to its irreversible inhibition of the enzyme acetylcholinesterase. In addition, soman binds irreversibly to a number of noncholinesterase tissue binding sites which appear to be its major means of in vivo detoxification. This study was conducted to determine the hepatic subcellular localization of these sites. Subcellular fractions of liver from male Sprague-Dawley rats (200-250 g) were prepared by differential and isopycnic density gradient centrifugation. The binding of [14C]soman to these subcellular fractions was determined in the presence and absence of cresylbenzodioxaphosphorin oxide (CBDP), a compound that binds irreversibly to the noncholinesterase soman binding sites. Crude fractionation of liver homogenates into nuclear, mitochondrial, microsomal, and soluble fractions revealed that 78% of the total CBDP-sensitive binding activity was localized in the nuclear and microsomal fractions. Further purification of these fractions indicated that all of the homogenate binding activity could be accounted for in the purified microsomal fraction. When purified liver microsomes were solubilized and fractionated on linear sucrose gradients, 90% of the CBDP-sensitive soman binding activity cosedimented with carboxylesterase activity which suggests that these binding sites are carboxylesterase.

Animals↗

PubMed1977

Subcellular localization of the alterations in phosphatidylinositol metabolism following glucose-induced insulin release from rat pancreatic islets.

The subcellular localization of the incorporation of 2-(3H)-myoinositol into lipids has been studied in isolated pancreatic islets of the rat. The recovery of lipid-bound myoinositol increased with time in the nuclear, mitochondrial, microsomal, and secretory granule fractions. The utilization of a filtration technique for the more complete separation of mitochondrial and secretory granule elements permitted us to show that the recovery of lipid-bound 2-(3H)-myoinositol increased most rapidly in the secretory granule fraction. A 30-minute exposure of prelabeled islets to a stimulatory concentration of D-glucose (3.0 mg./ml.) resulted in a statistically significant decrease in the amount of lipid-bound 2-(3H)-myoinositol that was recovered from the secretory granule fraction (p less than 0.001). In contrast, exposure of islets to the elevated glucose concentration had no statistically significant effect on the recovery of lipid-bound radioactivity from other subcellular fractions. Since the majority of lipid-bound radioactivity associated with the secretory granule fraction could be recovered with the presumptive secretory granule membranes, these data suggest that the hydrolysis of phosphatidylinositol that accompanies glucose-induced insulin secretion from the rat pancreatic islet may be localized to the beta granule and, in particular, to its limiting membrane.

Animals↗

PubMed1976

Esterolytic activities of rat intestinal mucosa. 1. Characterization, cellular distribution and subcellular localization of a glycerol-ester hydrolase.

The preferential cellular distribution in the villus tip and the subcellular localization in the endoplasmic reticulum of an intestinal glycerol-ester hydrolase from rat mucosa are described. The enzyme is shown not to be from either pancreatic or bacterial origin; it catalyzes the hydrolysis of short- and medium chain triglycerides and of p-nitrophenylacetate. Contrarily to the specificity found for the pig intestinal lipase (Serrero, Négrel and Ailhaud, 1975), no activity is detectable against acylCoA; a thiolester hydrolase different from the glycerol-ester hydrolase was demonstrated after differential solubilization and chromatographic separation. A high proportion of glycerol-ester hydrolase is present in the intestinal lumen; its possible complementary role in lipid degradation is discussed.

Aging↗

PubMed1988

Different subcellular localization of neurotensin-receptor and neurotensin-acceptor sites in the rat brain dopaminergic system.

The subcellular localization of neurotensin-receptor sites (NT2 sites) and neurotensin-acceptor sites (NT1 sites) was studied in rat caudate-putamen by isopycnic centrifugation in sucrose density gradients. [3H]Neurotensin binding to NT2 sites occurred as a major peak at higher sucrose densities, colocalized with [3H]dopamine uptake, and as a small peak at a lower density; whereas binding to NT1 sites occurred as a single large peak at an intermediate density. 6-Hydroxydopamine lesions of the median forebrain bundle resulted in a total loss of NT2 sites in the caudate-putamen but did not affect NT2 sites in the nucleus accumbens and the olfactory tubercle. NT1 sites were not affected. Kainic acid injections into the rat caudate-putamen led to a partial decrease of NT1 sites in this region 5 days later. After a few weeks they returned to normal. Therefore NT2 sites are probably associated with presynaptic nigrostriatal dopaminergic terminals in the caudate-putamen but not in the nucleus accumbens and the olfactory tubercle. A possible association of NT1 sites with glial cells is suggested.

Animals↗