Molecular distribution of cytoplasmic zinc in phytohemagglutinin-stimulated and unstimulated human lymphocytes

Human peripheral blood lymphocytes were incubated with [⁶⁵Zn] zinc transferrin and with and without phytohemagglutinin for 1, 2, 4, 10, 24, 48, 72, and 96h. Gel filtration of cytoplasmic fractions obtained from these lymphocytes was then performed to determine the molecular distribution of incorporated zinc as a function of time in culture. The data obtained indicated that: (1) transferrin-bound zinc incorporated by human lymphocytes is associated with a variety of soluble molecules whose molecular weights range from less than 5,000 to greater than 70,000 daltons; (2) there is a time-dependent change in the distribution of cytoplasmic zinc for both phytohemagglutinin-stimulated and unstimulated lymphocytes; and (3) for all times studied, there is a difference in the elution profiles obtained for phytohemagglutinin-stimulated and unstimulated lymphocytes. Furthermore, lymphocytes from a donor with untreated hairy cell leukemia exhibited a totally different pattern of cytoplasmic zinc distribution than did lymphocytes from apparently healthy donors.     

The metabolism of metals in rat placenta

The status and transfer of metals across the rat placenta were studied by subcellular and molecular fractionations of this organ at 2 and 24 h after iv injection of radiolabeled metals. The soluble and nuclear fractions showed higher contents of copper and zinc, whereas most of the nickel was associated with the soluble fraction. Cadmium was almost evenly distributed between the microsomal and nuclear fractions. Gel filtration of the soluble fractions showed nickel associated with an unknown low molecular weight form; zinc with high molecular weight proteins; copper with metallothionein, ceruloplasmin, and high molecular weight proteins; and cadmium with high molecular weight proteins and metallothionein.     

Nanomolar concentrations of untransformed glucocorticoid receptor in nuclei of intact cells

The subcellular distribution of untransformed glucocorticoid-receptor complex in vivo has been studied by chemical crosslinking of intact cells, and using a procedure adequate for correction of experimental errors due to redistribution of components between cytosolic and nuclear fractions. We found that in HeLa S₃ cells 85.4% of total glucocorticoid-receptor complexes are located in nuclei, and 14.6% are cytosolic. If measurements were performed with MCF-7 cells, we determined that the nuclear pool of glucocorticoid-receptor complexes accounts for 75.2% of the total cellular content, whereas the remaining 24.8% are cytosolic. When the subcellular distribution of estrogen-receptor complexes was determined, instead, we found that they are almost exclusively located in nuclei of MCF-7 cells, which contain 88.9% of the total. In order to estimate the molar concentration of receptors in cytosol and nuclei of intact cells, we determined the free water content of the two compartments. The volume of solvent was found to vary in the three cell lines we have studied, and our data showed that these variations are due to the cytosolic fractions, as the free water content of nuclei is essentially the same in those cells. When the free water content and the levels of glucocorticoid-receptor complexes we have measured were used to estimate the molar ncentrations of receptors, we found that these range between 0.4 and 18.9 nM in cytosols, and between 3.9 and 6.3 nM in nuclei of the three cell lines we have studied. We then concluded that the relative distribution of untransformed glucocorticoid-receptor complexes between cytosol and nuclei is cell-specific but their molar concentration in the nuclear compartment does not greatly vary among different cells.     

Variations in Zinc Content of Subcellular Fractions from Young and Old Roots, Stems and Leaves of Broad Beans (Vicia faba)

⁶⁵Zn labelled seeds of broad beans (Vicia faba L.) germinated and grown in a complete nutrient solution containing the radioisotope were used to investigate the change in the subcellular distribution of this trace element during subsequent development of the labelled seeds. Homogenates of leaves, stems and roots were fractionated into cell-wall debris, nuclei, chloroplasts, mitochondria, ribosomes and “soluble” fraction. The concentration of the radioisotope in these fractions, expressed as cpm/mg dry weight, was highest in the soluble fraction and in the ribosomes isolated from young tissues. However, as roots and leaves aged, a general decrease in the overall zinc concentration was followed by a similar drop in the concentration of the metal associated with the “soluble” fraction and ribosomes, causing a more or less uniform zinc distribution pattern. On the other hand, there was a general increase in the zinc concentration of the “soluble” and in the particulate fractions of relatively high sedimentation coefficient from older stem samples. Taking also into consideration the dry weight change per unit volume of the tissues under investigation, the relationship between these variations and the metabolism of the plant is discussed.     

INCORPORATION IN VIVO OF RADIOACTIVE LEUCINE INTO NEURONAL AND GOAL NUCLEAR PROTEINS OF RAT BRAIN

Purified neuronal and glial nuclei were separated from rat brain cells. The fraction rich in neuronal nuclei contained 68 ± 9 per cent neuronal nuclei and the fraction rich in glial nuclei contained 89 ± 6 per cent glial nuclei. The fraction rich in neuronal nuclei isolated from cells of adult rat brain incorporated l -[4,5-³H]leucine into TCA-insoluble material at a rate comparable to those of the microsomal and the soluble fractions of the brain, and at a much higher rate than the fraction rich in glial nuclei. The proteins soluble in buffered-saline, the acid-soluble deoxyribonucleoproteins, and the residual proteins of the neuronal nuclei are apparently the proteins which account for the higher specific activity of neuronal proteins compared with glial nuclear proteins. In liver and kidney, the incorporation of [³H]leucine into nuclear proteins was lower than into other subcellular fractions from the same organs.     

Concerning the subcellular distribution of 3beta-hydroxysteroid dehydrogenase/isomerase in the rat adrenal.

The distribution of 3beta-hydroxysteroid dehydrogenase/isomerase in the subcellular fractions of rat adrenal gland has been determined. Based on the total activity found, 55% was in the microsomal fraction, 10% in the heavy-mitochondrial fraction, 3% in the light-mitochondrial fraction and 26% in the fraction consisting of cell-debris plus nuclei. Ninety-five percent of the total activity was recovered in the fractions. Approximately half the activity in the heavy-mitochondrial fraction could be accounted for by microsomal contamination.     

Characterization of the phenol soluble and insoluble nonhistone proteins of in vivo (32)P-labelled rat liver nuclei by high-resolution gel electrophoresis

The (32)P-labelling patterns of phenol soluble and insoluble nonhistone proteins of in vivo labelled rat liver nuclei freed of the soluble nuclear proteins have been determined after separation by high-resolution gel electrophoresis. The bulk of the proteins of the nuclear residues was phenol soluble. Seven percent of the proteins of the nuclear residues was obtained with the aqueous phase. As shown in this paper both fractions contain (32)P-labelled proteins but they represent different types of nonhistone proteins.     

NONHISTONE NUCLEAR PROTEINS OF RAT BRAIN

Abstract— The rat brain was dissected into cerebral cortex, cerebellum and the remaining regions. From the nuclei, isolated from these three brain sections, were extracted two fractions of nuclear sap proteins (proteins soluble in 014 M NaCl and proteins soluble in 01 M Tris-HCl buffer pH 7-6) and two fractions of nonhistone chromosomal proteins (one soluble in 0-35 M NaCl and one which is not soluble at this salt concentration). Each of these four fractions of the nonhistone nuclear proteins was further separated by polyacrylamide gel electrophoresis. The electrophoretic patterns of the studied fractions of nuclear proteins are qualitatively identical regardless of the brain section from which the analysed protein fraction was isolated. In addition, there arc no qualitative differences in the electrophoretic patterns of nonhistone chromosomal proteins which are and which are not soluble in 0-35 M NaCl. In contrast to the qualitative similarity of the electrophoretic patterns of proteins from different sections of the brain, the amount of the nonhistone nuclear proteins is characteristic for each studied brain section. The ratio of the total nonhistone nuclear proteins to DNA is highest in the brain cortex and lowest in the cerebellum. The most expressed difference between the nuclei is in the ratio of the nonhistone chromosomal proteins soluble in 0-35 M NaCl to DNA. This ratio is 0-52 in the cortex. 0-38 in the mixture of noncortical and noncerebel-lar regions and only 0-18 in the cerebellum. The amount of the three fractions of nonhistone nuclear proteins in the nuclei of individual brain sections is proportional to the activity of the genome in these nuclei. The only exception are the nonhistone chromosomal proteins which are not soluble in 0-35 M NaCl. These proteins and the histones are present in the same amounts in nuclei isolated from all three studied sections of the brain. The results support a proposal that the nonhistone nuclear proteins are involved in the expression of the genetic activity of the cell, without the majority of the proteins in any of the four fractions being the specific regulatory molecules.     

Cyclic GMP and lymphocyte proliferation: effects on DNA-dependent RNA polymerase I and II activities.

Cyclic GMP (guanosine 3′:5′-cyclic monophosphate) accumulation and Ca²⁺ influx have been correlated with early nuclear events associated with increases in RNA polymerase I activity and decreases in RNA polymerase II activity in phytohemagglutinin-stimulated lymphocytes. In the present study we demonstrate that cyclic GMP in the presence of Ca²⁺ stimulates RNA polymerase I activity in lymphocyte nuclei isolated from both non-stimulated and phytohemagglutinin-stimulated lymphocytes. In addition, cyclic GMP in the presence of Ca²⁺ decreases RNA polymerase II activity in both non-stimulated and phytohemagglutinin-stimulated lymphocyte nuclei. These observations suggest that cyclic GMP and Ca²⁺ may represent components of a plasma membrane-to-nucleus “mitogen signal sequence”.     

Nucleolar and nuclear envelope proteins of the yeast Saccharomyces cerevisiae

We have developed a fast and reliable purification protocol to obtain yeast nuclei in intact and pure form and in a reasonable yield. The purified nuclei appear homogeneous at the light and electron microscopic level, are highly enriched in the nuclear marker histone H2B and devoid of mitochondrial, vacuolar and cytosolic marker proteins. On sodium dodecyl sulfate (SDS)-polyacrylamide gels, the nuclear fraction contains unique proteins which distinguishes them from the major yeast subcellular fractions. Yeast nuclei were separated by detergent/salt extraction into soluble, insoluble and membrane fractions. Antibodies raised against subnuclear fractions lead to the identification of an integral nuclear membrane protein and a high-abundance 38-kDa protein which is located in the yeast nucleolus.     

Lysosomal localisation of cobalamin during absorption by the ileum of the dog

The subcellular distribution of cobalamin during absorption in the dog ileum has been studied using analytical subcellular fractionation. Animals dosed orally with cyano[57Co]cobalamin were killed 2 h later, and postnuclear supernatant fractions prepared from homogenates of the ileal mucosa were subjected to isopycnic centrifugation on reorientating sucrose density gradients. Marker enzymes for the principal subcellular organelles and cyano[57Co]cobalamin were assayed in the gradient fractions. At 2 h, the distribution of cyano[57Co]cobalamin exhibited a major lysosomal localisation with only 30% of the counts being recovered in the soluble fractions. This observation was confirmed by preparing postnuclear supernatant fractions in digitonin, which selectively disrupted lysosomes and released their contents into the soluble fractions. Lysosomal localisation during passage through the ileal enterocyte strongly supports absorption of cobalamin by a process of receptor-mediated endocytosis in the dog.     

Distribution subcellulaire des protéine-kinases stimulables par l'AMP cyclique et des protéines réceptrices de l'AMP cyclique dans la thyroïde de porc

The distribution of cyclic AMP-dependent protein kinase activity in porcine thyroid glands has been studied. Enzyme activity catalyzing phosphorylation of exogenous substrate (protamine) from ATP, and cyclic AMP binding were determined in parallel in subcellular fractions purified by differential centrifugation and flotation on sucrose density layers. Both activities were found in all the studied fractions; they were quantitatively the highest in the cytosol but particles showed the highest specific activities.Latent protein-kinase activity was unmasked by action of detergents on microsomes (× 5–10 fold) and solubilized (85 to 99 p. cent of the initial total activity). Cyclic AMP binding capacity was also recovered in detergent-treated microsomal extracts in spite of reduced cyclic AMP binding in the presence of detergent.Protein kinase activity and cyclic AMP-binding proteins were less represented in purified nuclei than in microsomes. Again both activities were unmasked by detergent.Preparations highly enriched in Golgi membranes were compared to rough microsomal preparations. Higher protein kinase activity was detected in rough microsomes as compared to Golgi membranes, whereas the reverse was true for cyclic AMP binding. Both activities were equalized after detergent treatment. Since unmasking of protein kinase activity was the highest in Golgi membranes, this fraction contains more enzyme activity and cyclic AMP binding capacity than rough microsomes.The localization of endogeneous protein substrates of cyclic AMP-dependent protein kinases was investigated using purified soluble protein kinase subcellular fractions. The better endogeneous substrates seemed to be localized in the rough microsomal and in the nuclear fractions.     

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.     

Study on chemical species of iodine in human liver

The distribution and chemical species of iodine in various subcellular fractions of human liver were studied by using epithermal neutron activation analysis combined with chemical and biochemical separation techniques, such as gradient centrifugation and gel chromatography. It was found that the total iodine content orders in various subcellular fractions is as follows: nuclei > cytosol > mitochondria > lysosome > microsome. In the lysosomal fraction, iodine is mainly bound to macromolecules, whereas in the nuclei and mitochondrial fractions, mainly with lower-molecular-weight organic compounds. In the cytosol fraction, iodine is combined with three proteins, in which iodine is chiefly bound with mid- and high-molecular-weight proteins.     

Manganese, copper, zinc, and iron concentrations and subcellular distribution in two types of skeletal muscle

To clarify trace element distribution in red and white muscle, and to verify two populations of muscle mitochondria, the iron, zinc, copper, and manganese concentrations of whole muscle and their subcellular fractions were determined. The iron, zinc, copper, and manganese concentrations of red muscle were 1.83, 4.31, 2.05, and 1.67 times higher than those of white muscle, respectively. In skeletal muscle subcellular distribution or iron, zinc, and copper were entirely different and that of manganese was relatively similar as compared with those in liver reported previously. The pattern of mineral distribution in all fractions of red muscle was similar to that of white muscle, but their concentrations in some fractions were different between red and white muscle, e.g., iron, zinc, and manganese in supernatant fraction and copper in nuclear and microsomal fractions. The difference between subsarcolemmal and interfibrillar mitochondria were ascertained by the distribution of trace elements.     

Selective localization of neptunium-237 in nuclei of mammalian cells]

After injection in the rat of soluble neptunium salt, the distribution of this element was studied at the subcellular level by electron microscopy and electron probe microanalysis. Abnormal structures have been observed by electron microscopy in the nuclei of hepatocytes, and the same structures have also been observed in the nuclei of the proximal tubules cells of the kidney. These structures are formed of clusters of very small and dense particles, several nanometers in diameter. The clusters are localized in the central part of the nuclei and they are separate from nucleoli and heterochromatin. Electron probe X-ray analysis of this cluster have shown that they contain neptunium associated with phosphorus. In the cell containing neptunium inclusions, other non specific lesions are also observed (nuclear pycnosis, mitochondrial depletion).     

Effect of iron transferrin on nucleic acid synthesis in phytohemagglutinin-stimulated human lymphocytes

In serum-free cultures of phytohemagglutinin-stimulated human lymphocytes, iron transferrin causes enhanced uptake of both tritiated thymidine and tritiated uridine over that seen with only phytohemagglutinin. This effect is specific for the iron transferrin complex, no enhancement produced by either free iron(III) or apotransferrin. Iron bound to transferrin is quantitatively taken up by stimulated lymphocyte cultures, while under similar conditions only 10% of transferrin-bound zinc is incorporated. The relative specificity of action of iron and zinc on nucleic acid synthesis is discussed.     

Nuclear location of the putative transforming protein of avian myelocytomatosis virus

The putative transforming protein of avian myelocytomatosis virus MC29 is a 110,000 dalton (P110gag-myc) polyprotein comprised of sequences derived from both the gag region and the MC29-specific myc region. Two approaches have been taken to determine the location of the MC29 gag-related proteins in transformed cells: subcellular fractionation and immunofluorescence. Analysis of subcellular fractions of MC29-transformed cells by immunoprecipitation indicates that the majority of the gag-myc polyprotein is found in the nuclear fractions of Q8 cells (a nonproducer line of MC29-transformed quail embryo fibroblasts) and nonproducer cells derived from a liver tumor of MC20-infected quail. This is in contrast to the distribution of gag-related helper virus proteins lacking myc, which are found only in nonnuclear fractions of superinfected Q8 cells. The purity of unlabeled nuclei was assessed by electron microscopy and enzyme assays, revealing little contaminating material from other subcellular fractions. Immunofluorescence experiments using monospecific anti-gag serum showed specific, intense immunofluorescence in the nuclei of fixed Q8 cells. In contrast, the majority of P75gag-erb, a candidate transforming protein produced by avian erythroblastosis virus (AEV), is absent from the nuclei of nonproducer AEV-transformed chick embryo fibroblasts. The nuclear association of the MC29 transforming protein may be related to some of the unique properties of MC29-transformed cells.     

Subcellular distribution of the protein kinase activity in the normal and neoplastic thyroid tissue of the rat]

Thyroid tissue has been fractionnated by centrifugation (105 000 q) of its homogenate. Protein-kinase activity in presence of histone is distributed in nuclei (11.5%) mitochondira (22.8%), microsomes (9.8%) and soluble fraction (56%); it is activated by cyclic AMP and GMP, mostly in soluble and nuclear fractions. Protein-kinase activity of total homogenate of neoplasic thyroid (strain 1-5G Wollman) in presence of histone is 3 times higher than in normal tissue and more activated by cAMP. In absence of histone, protein-kinase activity is the more important in mitochondrial and microsomal fractions of normal thyroid and in soluble and nuclear fraction of neoplastic tissue.     

Partitioning of bile acids into subcellular organelles and the in vivo distribution of bile acids in rat liver.

1. The subcellular distribution of conjugates of cholic acid and chenodeoxycholic acid between cytosol, nuclei, mitochondria and microsomes in rat liver has been determined. 2. The partition coefficients for the distribution of these bile acids between subcellular fractions and buffer have been measured and used to construct a compartmental model of the amounts of conjugated bile acids present in the different subcellular organelles in vivo. 3. This model indicates that a large percentage of the bile acid in the rat liver is found in the nuclear fraction; 42% of the cholic acid conjugates and 27% of the chenodeoxycholic acid conjugates. Substantial amounts of bile acid are also present in microsomes and mitochondria suggesting that published estimates of the amounts of bile acids in these fractions are underestimates. 4. The model also allows the amount of bile acid which is in free solution in cytosol to be determined; 10.9% of the cholic acid conjugates and 4.1% of the chenodeoxycholic acid conjugates in rat liver were present in this fraction. Knowlege of the amount of free bile acid allows possible roles of the cytosolic bile binding proteins to be assessed.