Subcellular localization of adenylate cyclase in thymocytes

In almost all cell types, adenylate cyclase is located in the plasma membrane. In lymphocytes, however, this enzyme has been claimed to be largely present in intracellular compartments. In this study, the distribution of adenylate cyclase activity in subcellular fractions of calf thymocytes was reinvestigated by a balance sheet approach. When subcellular fractionation was performed in the absence of ATP and dithiothreitol, less than a half of the homogenate basal activity could be recovered in the fractions, and this amount was distributed almost equally in three main compartments: the plasma membrane fraction, the microsomal and mitochondrial fractions and the nuclear fraction. However, if enzyme activity in the above fractions was measured in the presence of the stimulatory agents NaF, guanylylimidophosphate or guanosine 5'-O-(3-thio)triphosphate, or if the subcellular fractionation was performed in media containing ATP and dithiothreitol, the overall recovered activity greatly increased (up to 90%) and the distribution was shifted in favour of the plasma membrane fraction (up to 65% of the recovered activity). The adenylate cyclase properties were similar in all fractions. The ionophore alamethicin did not alter the subcellular distribution of the enzyme. The localization of adenylate cyclase in thymocytes thus appears to be primarily, if not uniquely, in the plasma membrane, as generally found in other cell types.     

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.     

Subcellular localization and binding of 125I-triiodothyronine in calf thyroid

The subcellular distribution of 125I-T3 was studied in calf thyroid slices, under the same experimental conditions where T3 inhibits protein and RNA synthesis, labelled hormone was found mainly in the 20,000 X g supernatant. The specificity of each subcellular localization was determined by incubating the slices with 10(-5)M T3. Only in the purified nuclei a significant decrease was found, indicating a specific localization of the labelled hormone. When slices were incubated with 125I both labelled T3 and T4 were found in purified nuclei, indicating that endogenously synthesized hormones can reach thyroid nuclei. Purified thyroid nuclei were incubated with labelled T3 and increasing amounts of cold hormone. Specific binding reached a plateau after 90 min of incubation at 20 degrees C. When the displacement curves were analysed by a Scatchard plot a binding site with a Ka of 5.2 X 10(7) M-1 and a capacity of 3.0 X 10(-15) moles/microgram DNA was observed. Digestion of nuclei with trypsin and protease abolished completely the binding of 125I-T3 thus indicating the protein nature of the receptor. The hormone-receptor complex could be extracted with 0.4M KCI and eluted in the void volume after Sephadex G-25 column chromatography, similar to peripheral tissues nuclear T3 receptors. The present studies provide the first evidence for the existence of nuclear receptors for T3 in the thyroid, an event probably related to the autoregulatory mechanism.     

Subcellular localization and possible functions of gamma-glutamyltransferase in the radish (Raphanus sativus L.) plant

Previously we reported the purification of soluble gamma-glutamyltransferases (GGTs) from radish cotyledon. Subcellular fractionation of radish cells revealed that soluble GGT is a vacuolar enzyme. Acivicin, a GGT inhibitor, mediated the in vivo catabolism inhibition of the glutathione S-conjugate generated from endogenous glutathione and exogenously supplied monochlorobimane. Thus soluble GGT is possibly involved in the catabolism of glutathione S-conjugates.     

Subcellular localization of CIAPIN1.

Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) is a newly identified anti-apoptotic molecule. Our previous studies have demonstrated that CIAPIN1 is ubiquitously expressed in normal fetal and adult human tissues and confers multidrug resistance in gastric cancer cells, possibly by upregulating the expression of multidrug resistance gene 1 and multidrug resistance-related protein 1. However, fundamental biological functions of CIAPIN1 have not been elucidated. In this study, we first predicted the subcellular localization of CIAPIN1 with bioinformatic approaches and then characterized the intracellular localization of CIAPIN1 in both human and mouse cells by a combination of techniques including (a)immunohistochemistry and immunofluorescence, (b) His-tagged CIAPIN1 expression, and (c)subcellular fractionation and analysis of CIAPIN1 in the fractions by Western blotting. All methods produced consistent results; CIAPIN1 was localized in both the cytoplasm and the nucleus and was accumulated in the nucleolus. Bioinformatic prediction disclosed a putative nuclear localization signal and a putative nuclear export signal within both human and mouse CIAPIN1. These findings suggest that CIAPIN1 may undergo a cytoplasm-nucleus-nucleolus translocation.     

Shedding of gangliosides from calf thymocytes

In the course of our work on calf thymus gangliosides [Dyatlovitskaya, Zablotskaya, Azizov & Bergelson (1980) Eur. J. Biochem. 110, 475-483] we studied the gangliosides exfoliated from the cell surface of thymocytes. It was shown that calf thymocytes shed gangliosides both in vivo and in vitro. Various gangliosides were found to be present in high amounts both in extracellular plasma membrane vesicles and in the 64000 X g supernatant. The extracellular membrane fragments were comparatively higher in disialosyllactosylceramide and the 64000 X g supernatant was higher in sialosyllactosylceramide than the cells. Comparison of the ganglioside composition of extracellular membrane fragments, thymocytes and lymphocytes led us to suggest that the shedding of gangliosides from the surface of thymocytes may be involved in the transformation of immunologically incompetent cortical thymocytes into immunocompetent virgin T-cells.     

Subcellular localization of oestrogen-induced uterine peroxidase.

The distribution of oestrogen-induced peroxidase in the resuspended 8000g pellet of rat uterine homogenates was examined by centrifugation in a sucrose density gradient. Within 10h of treatment with oestradiol, peroxidase activity was found in a region devoid of catalase or urate oxidase (peroxisomal markers) which did not overlap the fractions containing succinate dehydrogenase (mitochondrial marker) or acid phosphatase (lysosomal marker). The induced uterine enzyme was localized in reticular membrane-bound vesicles with isopycnic density of 1.28g/ml from which it could be released by treatment with detergent.     

Subcellular localization of superoxide dismutase in rat liver.

The subcellular localization of superoxide dismutase was investigated in rat liver homogenates. Most of the superoxide dismutase activity is present in the soluble fraction (84%), the rest being associated with mitochondria. No indications for the occurrence of superoxide dismutase in other subcellular structures, particularly in peroxisomes, was found. Mitochondrial activity is not due to adsorption, since the sedimentable activity is essentially latent. Subfractionation of mitochondria by hypo-osmotic shock and sonication shows that half of the mitochondrial superoxide dismutase activity is localized in the intermembrane space, the rest of the enzyme being a component of the matrix space. In non-ionic media the matrix enzyme is, however, adsorbed to the inner membrane, from which it can be desorbed by low (0.04M) concentration of KCl. Superoxide dismutase activity was found in all rat organs investigated. Maximal activity of the enzyme is observed in liver, adrenals and kidney. In adrenals, the highest specific activity is associated with the medulla.     

Subcellular localization of transglutaminase. Effect of collagen.

1. The subcellular distribution of transglutaminase was investigated by using the analytical approach of differential and isopycnic centrifugation as applied to three organs of the rat: liver, kidney and lung. After differential centrifugation by the method of de Duve, Pressman, Gianetto, Wattiaux & Appelmans [(1955) Biochem. J. 63, 604-617], transglutaminase is mostly recovered in the unsedimentable fraction S and the nuclear fraction N. After isopycnic centrifugation of the N fraction in a sucrose density gradient, a high proportion of the enzyme remains at the top of the gradient; a second but minor peak of activity is present in high-density regions, where a small proportion of 5'-nucleotidase, a plasma-membrane marker, is present together with a large proportion of collagen recovered in that fraction. 2. Fractions where a peak of transglutaminase was apparent in the sucrose gradient were examined by electron microscopy. The main components are large membrane sheets with extracellular matrix and free collagen fibers. 3. As these results seem to indicate that some correlation exists between particulate transglutaminase distribution and those of collagen and plasma membranes, the possible binding of transglutaminase by collagen (type I) and by purified rat liver plasma membrane was investigated. 4. The binding studies indicated that collagen is able to bind transglutaminase and to make complexes with plasma-membrane fragments whose density is higher than that of plasma-membrane fragments alone. Transglutaminase cannot be removed from such complexes by 1% Triton X-100, but can be to a relatively large extent by 0.5 M-KCl and by 50% (w/v) glycerol. 5. Such results suggest that the apparent association of transglutaminase with plasma membrane originates from binding in vitro of the cytosolic enzyme to plasma membrane bound to collagen, which takes place during homogenization of the tissue, when the soluble enzyme and extracellular components are brought together.     

The isolation and characterization of plasma membranes from calf thymocytes.

A simple method is described for the isolation and characterization of plasma membranes from calf thymocytes. The procedure involves extraction of thymocytes in a hypotonic medium containing borate and EDTA. Membrane ghosts, obtained by centrifugation of the cell lysate, are purified by passage through a column containing glass beads. The purity of plasma membranes was checked by chemical analysis, by assay of marker enzymes and also by electron microscopy. Polyacrylamide gel electrophoresis of the calf thymocyte plasma membrane produced a number of protein bands as well as a major band which stained for carbohydrate. The method is rapid and could be applied to isolate plasma membranes from nucleated cells of various types in large quantities.     

Protease of adenovirus type 2. Subcellular localization.

The subcellular localization of the adenovirus type 2 core polypeptide specific protease activity was investigated using an in vitro assay system. The protease activity was recovered exclusively from infected cell nuclei and was insoluble, sedimenting with the membrane fraction. Endogenous activity could be demonstrated in young virions which contain precursor PVII molecules. This protease activity only became sensitive to L-1-tosylamide-2-phenylethylchloromethyl ketone- or phenylmethylsulfonyl fluoride-mediated inhibition after disruption of the virus particles by sonication, suggesting that the enzyme was internally located. The putative precursors to virus particles, referred to as top components, which do not contain a full complement of viral DNA, did not contain protease activity. The protease released from sonicated virions converted exogenous PVII substrate molecules to polypeptide VII. The noninfectious H2ts1 virus particles synthesized at the nonpermissive temperature phenotypically resemble young virions, but unlike their wild type counterparts, were devoid of protease activity. The results show that the protease enters the precursor particles concurrently with the viral chromosome and that its presence is a prerequisite for the processing and subsequent maturation of infectious adenovirions.     

Subcellular localization of low-molecular-weight RNA components in rat liver.

The subcellular localization of the four major low-molecular-weight RNA components, D, C, A and L, was studied in rat liver cells. The cells were fractionated by a non-aqueous technique into a nuclear and a cytoplasmic fraction. The cytoplasm contained 43% of component D, 57% of component C and more than 80% of component L.     

Subcellular localization of glucose-6-phosphatase in animal tissues.

1. Glucose-6-phosphatase (EC 3.1.3.9 D-glucose-6-phosphate phosphohydrolase) was found to be localized mainly in the endoplasmic reticulum (microsomal fraction) of all species of vertebrate liver tissue examined. 2. Hepatopancreas tissue from gastropod molluscs was found to be unique in showing the localization of glucose-6-phosphatase in the cytosol (soluble fraction).     

Subcellular localization of aldehyde reductase activities in ox brain.

The distribution of the two principal isoenzymes of aldehyde reductase (EC 1.1.1.2) has been studied in ox brain. The more active of these, which has been termed the high-Km enzyme, has been shown to be located in the cytosol and the less abundant low-Km form has a similar localization. p-Nitrobenzaldehyde, which has been used as a substrate in previous studies, caused the reduction of NADH in the presence of the mitochondrial fraction, but mixed substrate experiments with 1,3-dinitrobenzene and the effects of pH on the activity indicate that this is due to the presence of a nitro reductase activity which has been recently described (Köchli, Wermuth & von Wartburg (1980) Biochim. Biophys. Acta 616, 133-142] rather than to the low-Km aldehyde reductase activity. Fractionation of the mitochondria indicated this activity to be largely confined to the mitochondrial inner membrane.     

Subcellular localization of alpha-melanocyte-stimulating hormone in the rat hypothalamus.

Homogenates of male rat hypothalami were fractionated by means of differential centrifugation, and α-melanocyte-stimulating hormone (α-MSH) in the various fractions was quantified by radioimmunoassay. Of the total quantity of α-MSH in the homogenate, 36% was recovered in the 11,500 g pellet and 31% sedimented between 11,500 and 105,000 g. α-MSH was not detected in the 105,000 g supernatant fluid. When the 900 g supernatant fluid was fractionated on continuous sucrose density gradients at non-equilibrium conditions, two populations of particles containing α-MSH were observed. When fractionated at equilibrium conditions, the two populations were recovered in a single band. These sedimentation characteristics indicate that the particles that contain α-MSH differ in size but are similar in density. After hypo-osmotic shock, the large particles containing α-MSH were not demonstrable, whereas the small particles appeared to be resistant to such treatment. In their sedimentation, the particles containing α-MSH were indistinguishable from particles containing thyrotropin releasing hormone (TRH) but were separable from those that contained luteinizing hormone releasing hormone (LHRH). It is suggested that the large particles containing α-MSH are synaptosomes.     

Subcellular localization of renin and kallikrein in rat kidney.

The subcellular localization of renin and kallikrein in rat kidney cortex homogenate was investigated using both differential and density gradient centrifugation techniques. Highest specific activity of renin was found in the heavy mitochondrial fraction. Mitochondrial localization of renin was further supported by the behaviour of succinic dehydrogenase. By differential centrifugation, highest specific activity of kallikrein was found in the light mitochondrial fraction, while by density gradient centrifugation kallikrein was almost completely recovered in the lysosomal fraction. Lysosomal localization of kallikrein is further supported by the behaviour of acid phosphatase. The different subcellular localizations of renin and kallikrein are confirmed and the suggestion that kallikrein is located in the lysosomes is advanced.     

Subcellular localization of aldolase B

The localization of the aldolase B isozyme was determined immunohistochemically in rat kidney and liver using a polyclonal antibody. Aldolase B was preferentially localized in a nuclear region of hepatocytes from the periportal region and was absent in those from the perivenous region. Aldolase B was also preferentially localized in the proximal tubules and was absent in other structures of the renal cortex as well as in the renal medulla. Using reflection confocal microscopy, the enzyme was preferentially localized in a nuclear position in liver and renal cells, which was similar to the cellular and intracellular location found for the gluconeogenic enzyme fructose-1,6-bisphosphatase (Sáez et al. [1996] J. Cell. Biochem. 63:453-462). Subcellular fractionation studies followed by enzyme activity assays revealed that aldolase activity was associated with subcellular particulate structures. Overall, the data suggest that different aldolase isoenzymes are needed in the glycolytic and gluconeogenic pathways.