Sub-nuclear fractionation. I. Procedure and characterization of fractions

A procedure for fractionation of nuclei from rat liver, Xenopus liver and Xenopus erythrocytes is described. It is based on mild sonication of isolated nuclei for 7–12 sec in a nearly isotonic medium, separation of nuclear sap and centrifugation on a discontinuous sucrose density gradient containing Na and K citrate. Nuclei are thus separated in a single operation into 8 fractions representing nucleoplasm, euchromatin, nucleoli, heterochromatin and nuclear membranes. The sub-nuclear fractions were characterized by chemical composition (DNA, protein, RNA and phospholipid), electron microscopy, thermal denaturation properties of chromatin, relative binding of ${}^3\text{H-actinomycin D}$, polyacrylamide gel electrophoresis of nuclear proteins and titration of membranes against Triton X-100. Approx. 10% of total DNA was recovered as heterochromatin associated with membranes but the bulk of nuclear membranes co-sedimented with the major euchromatin zones. Subnuclear fractions prepared in this way retain virtually all the RNA polymerase activity bound to chromatin [41].     

Nuclear membranes from mammalian liver. VI. Glucose-6-phosphatase in rat liver, a cytochemical and biochemical study

Activities of glucose-6-phosphatase (G-6-Pase) and other phosphatases were determined in nuclei, nuclear membrane and microsomal fractions and subfractions, and condensed chromatin isolated from the liver of adult, newly born and prenatal rats. The purity of the fractions was controlled by electron microscopic morphometry and by measurement of various marker enzymes. The specific G-6-Pase activity of the nuclear membranes was found to be about 60% that of the microsomes. However, when calculated on the basis of the phospholipid content, all fractions had similar activities. Determinations of G-6-Pase enrichments and recoveries were also made. The correspondence of the hydrolysing activities of glucose-6-phosphate, mannose-6-phosphate, and inorganic pyrophosphate, together with various phosphotransferases, showed the same association of the G-6-Pase with these enzymes in the nuclear envelope as in the microsomal membranes. G-6-Pase was also demonstrated in the fractions by cytochemistry, and the activity was localized alongside the cisternal surfaces of both, inner and outer, nuclear membrane. ‘Free’ inner nuclear membrane fragments contained also G-6-Pase. No activity was observed at the nuclear pore complexes. Both, nuclear and microsomal membranes revealed a parallel rapid perinatal increase of G-6-Pase activity climaxing at 23 to 28 h after birth. Triton-X-100 treatment of isolated nuclei, which was found not to selectively release outer nuclear membranes, resulted in a great decrease of G-6-Pase activity as well as in losses of membrane phospholipids. The results clarify the divergence of earlier reports concerning the presence of G-6-Pase in the perinuclear cisterna and add biochemical evidence to the morphologically derived view of the nuclear envelope as being a special form of the ER system.     

Cellular localization of the apurinic/apyrimidinic endodeoxyribonucleases in rat liver

A method has been developed to purify rat liver nuclei; the isolated nuclei keep both nuclear membranes and retain more than 90% of the cell apurinic/apyrimidinic (AP) endodeoxyribonuclease activity. The nuclear enzyme is located mostly in chromatin non-histones; there is also an important amount of activity in the nuclear sap and some in the nuclear membranes. The cytoplasmic AP endodeoxyribonuclease activity is shared between mitochondria, cytosol and membranes. Different cell compartments appear to contain different AP endodeoxyribonuclease species: the membrane enzyme is activated by Triton whereas the other enzymes are rather inhibited; the nuclear sap enzyme has a higher molecular weight and a higher thermal resistance than the chromatin enzyme. A hypothesis is formulated according to which: (1) the chromatin enzyme is the only species important for nuclear DNA repair; (2) the species present in the other cell compartments might be precursors of the chromatin AP endodeoxyribonuclease.     

Intranuclear distribution of mouse liver nuclear DNA polymerase

Adult mouse liver nuclei and their subfractions corresponding to heterochromatin, nucleoli, membranes, and euchromatin were studied for DNA-polymerase activity. The intact nuclei and the two heavy nuclear fractions contained rather low activity while the two light fractions (membranes and euchromatin) had no activity at all. In the two heavy fractions, the activity was stimulated by β-mercaptoethanol and depressed by p-hydroxymercuribenzoate and by omission of one or more nucleotides. A nuclease activity, detected in the intact nuclei, may also be present in the nuclear subfractions. DNA-polymerase activity in the heavy fractions of mouse liver nuclei is discussed in relation to other published results.     

A distinct vesicle population targets membranes and pore complexes to the nuclear envelope in Xenopus eggs

Extracts from Xenopus eggs capable of nuclear envelope assembly in vitro were fractionated by differential and density gradient centrifugation. Nuclear envelope assembly was found to require soluble components in the cytosol and two distinct particulate fractions, which we have called nuclear envelope precursor fractions A and B (NEP-A and NEP-B). Both NEP-A and NEP-B are sensitive to treatments with trypsin, sodium carbonate, and detergents, but can be distinguished from each other by their sensitivities to high salt and N-ethylmaleimide and by their levels of alpha-glucosidase activity. Vesicles in NEP-B bind to chromatin, whereas those in NEP-A do not. NEP-B may therefore be involved in the targeting of membranes to the surface of the chromatin, whereas NEP-A may provide a pool of vesicles that contributes many of the nuclear envelope membranes. NEP-B may also play a role in the assembly of nuclear pore complexes because the density of nuclear pores in the resulting envelope is dependent on the ratio of NEP-B to NEP-A in the reconstituted extract.     

Phospholipid composition of nuclear membranes and cell nuclei of rat liver and hepatoma 27]

Phospholipid composition of nuclei and nuclear membranes from rat liver and hepatoma-27 were investigated. Hepatoma nuclei and nuclear membranes were found to contain cardiolipin which was absent in the same fractions of rat liver. In the nuclei and nuclear membranes of hepatoma the content of sphingomyelin was higher and that of lecithin is lower than in the corresponding subcellular fractions of rat liver. The content of acid phospholipids was much higher in hepatoma nuclear membranes than those of the normal liver. The data obtained show that the general trend of lipid dedifferentiation which earlier was demonstrated for various membranes of different tumor cells is observed also in the case of nuclear membranes.     

The possible role of endogenous nucleases in the structural organization of chromatin]

Two fractions of rat liver nuclei with different buoyant density have been obtained. The electrophoretic analysis of the oligonucleosome patterns of DNA out of nuclei of these two fractions revealed different levels of activity in endonucleases. In case of inhibition during the extraction of activity in Ca, Mg-dependent endonucleases, the average size of high polymeric DNA is larger for nuclei with bigger buoyant density (fraction I) than for nuclei with smaller ones (fraction II). This finding is evidence of in situ existence of two pools of liver nuclei with different endogenic nuclease activities. In nuclear chromatin fraction I DNA is torsionally stressed; in fraction II it is relaxed that correlates with larger activity of endonucleases and smaller buoyant density of this fraction. A hypothesis on a possible role of endonucleases in chromatin structure organization has been put forward. According to this hypothesis a modulation of activity in nuclear endonucleases can determine different packaging and activity of chromatin from different pools of cellular nuclei.     

Intracellular distribution of 5' nucleotidase in rat liver

The intracellular distribution of 5' nucleotidase was investigated in rat liver by biochemical analysis of cell fractions obtained by differential centrifugation. The enzymatic activity was measured by determination of the inorganic phosphorus liberated from 5' nucleotides. The 5' nucleotidase activity was mainly found in the nuclear and microsomal fractions. An attempt to extract the enzyme from these fractions with Mg(++) ion solutions was unsuccessful. It is concluded that 5' nucleotidase is actually present in the nuclear and microsomal fractions of rat liver cells.     

Intracellular Distribution of 5' Nucleotidase in Rat Liver

The intracellular distribution of 5' nucleotidase was investigated in rat liver by biochemical analysis of cell fractions obtained by differential centrifugation. The enzymatic activity was measured by determination of the inorganic phosphorus liberated from 5' nucleotides. The 5' nucleotidase activity was mainly found in the nuclear and microsomal fractions. An attempt to extract the enzyme from these fractions with Mg⁺⁺ ion solutions was unsuccessful. It is concluded that 5' nucleotidase is actually present in the nuclear and microsomal fractions of rat liver cells.     

Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs

We have studied the pathway of nuclear assembly from demembranated sperm chromatin by fractionating a cell-free system from Xenopus eggs (Lohka, M. J., and Y. Masui. 1983. Science (Wash. DC). 220:719-721). Both the soluble fraction and a washed vesicular fraction are required for formation of normal nuclei that initiate replication in vitro. The soluble fraction alone decondenses chromatin and the vesicular fraction alone surrounds chromatin with membranes. Both fractions are required for formation of nuclear pore complexes. Recombining these two fractions recovers approximately 100% of the nuclear assembly and DNA replication activities. Restricting the proportion of the vesicular fraction slows acquisition of the nuclear membrane and allows observation of immature nuclear pores ("prepores"). These form as arrays around and within the chromatin mass before membranes form. Subsequently membrane vesicles bind to these prepores, linking them by a single membrane throughout the chromatin mass. At the periphery this single membrane is surrounded by an outer membrane. In mature nuclei all membranes are at the periphery, the two membranes are linked by pores, and no prepores are seen. Nuclear assembly and replication are inhibited by preincubating the chromatin with the vesicular fraction. However nuclear assembly is accelerated by preincubating the condensed chromatin with the soluble fraction. This also decreases the lag before DNA replication. Initiation of DNA replication is only observed after normal nuclei have fully reassembled, increasing the evidence that replication depends on nuclear structure. The pathway of nuclear assembly and its relationship to DNA replication are discussed.     

An increase of the regulating factor for chromatin-dependent RNA polymerase II reactions in the tightly-bound chromatin fraction by glucocorticoid injection

A sugar-containing factor for chromatin-dependent RNA polymerase II reactions exists in rat liver nuclei (1). In this communication the localization of this factor in cell nuclei was investigated. The major activity of the factor was observed in the nuclear soluble fraction whereas a minor activity was detected in the tightly-bound chromatin fraction, but not in the loosely bound chromatin fraction. The factor in the tightly-bound chromatin fraction was considerably increased by glucocorticoid injection, but not in other fractions.     

Cellular distribution of the hamster liver specific nucleolar antigens

The immunochemical localization of hamster liver nucleolar antigens in subcellular fractions (nuclei, 10,000 x g pellet, 100,000 x g pellet and supernatant), nuclear substructures (chromatin, nuclear matrix, nuclear envelope, nucleoli, RNP particles and nucleosomes), and three classes of nonhistone chromosomal proteins with different affinities to DNA (NHCP1, NHCP2 and NHCP3) from nuclease-sensitive and nuclease-resistant chromatin fractions of hamster liver were studied. Six main nucleolar antigens with mol. wts 27,000; 29,000; 30,000; 36,000; 45,000; and 46,000 were found in subcellular fractions, nuclear substructures and classes of non-histone proteins of hamster liver. The antigens with mol.wts of approx. 27,000; 29,000; and 36,000 which were absent in hamster pancreas, spleen and Kirkman--Robbins hepatoma nuclei, seem specific for liver tissue.     

Lamin activity is essential for nuclear envelope assembly in a Drosophila embryo cell-free extract

The role of the Drosophila lamin protein in nuclear envelope assembly was studied using a Drosophila in vitro assembly system that reconstitutes nuclei from added sperm chromatin or naked DNA. Upon incubation of the embryonic assembly extract with anti-Drosophila lamin antibodies, the attachment of nuclear membrane vesicles to chromatin surface and nuclear envelope formation did not occur. Lamina assembly and nuclear membrane vesicles attachment to the chromatin were inhibited only when the activity of the 75-kD lamin isoform was inhibited in both soluble and membrane-vesicles fractions. Incubation of decondensed sperm chromatin with an extract that was depleted of nuclear membranes revealed the presence of lamin molecules on the chromatin periphery. In addition, high concentrations of bacterially expressed lamin molecules added to the extract, were able to associate with the chromatin periphery, and did not inhibit nuclear envelope assembly. After nuclear reconstitution, a fraction of the lamin pool was converted into the typical 74- and 76-kD isoforms. Together, these data strongly support an essential role of the lamina in nuclear envelope assembly.     

Assembly/disassembly of the nuclear envelope membrane: cell cycle-dependent binding of nuclear membrane vesicles to chromatin in vitro.

Dissociation and association of membranes with chromatin at the beginning and end of mitosis are critical in controlling nuclear dynamics during these stages of the cell cycle. Employing purified membrane and cytosolic fractions from Xenopus eggs, a simple assay was developed for the reversible binding of nuclear membrane vesicles to chromatin. We have shown, using phosphatase and kinase inhibitors, that membrane-chromatin association is regulated by a phosphatase/kinase system. In interphase, the balance in this system favors dephosphorylation, possibly of a membrane receptor, which then mediates chromatin binding. At mitosis the membrane receptor is phosphorylated, causing release of chromatin-bound membrane. Purified MPF kinase does not directly cause membranes to dissociate from chromatin. Rather, binding of membranes to chromatin at mitosis appears to be regulated indirectly by MPF through its action on a phosphatase/kinase system that directly modulates the phosphorylation state of a nuclear membrane component.     

Binding of S-adenosylhomocysteine to various domains of the plasma membrane and to the endoplasmic reticulum from rat liver: Relation between binding and phospholipid methyltransferase activity

S-Adenosylhomocysteine (AdoHcy) binding to various membrane fractions of rat liver was determined at pH 7.4, using an oil centrifugation technique. The highest binding activity was found in the heavy microsomal (M-H) fraction enriched in endoplasmic reticulum, but high binding activity was also observed in the light microsomal fractions enriched in blood sinusoidal membranes (M-L fraction), and the heavy nuclear fraction (N-H fraction) containing the contiguous area. A substantial portion of AdoHcy binding activity in the M-L fraction may be ascribed to contamination of this fraction with endoplasmic reticulum, as indicated by the distribution of NADPH cytochrome c reductase activity. Binding activity was low in the light nuclear (N-L) fraction corresponding to the bile canaliculi. Phospholipid methyltransferase activity was determined in the same membrane fractions under similar conditions (pH 7.4), and in the absence and presence of added phospholipids. The distribution of the enzyme activity was dependent on the presence of exogenous phospholipids, and grossly similar to AdoHcy binding, the highest activities being observed in the M-H and the M-L fractions. The N-H fraction, rich in AdoHcy-binding activity, demonstrated, however, a very low phospholipid methyltransferase activity. It is concluded that AdoHcy-binding activity is not confined to the plasma membranes, and a major fraction of the binding activity resides on membranes derived from the endoplasmic reticulum. Also, the present results add to previous data suggesting that phospholipid methyltransferase does not totally account for the AdoHcy-binding sites on rat liver membranes.     

Localization of particulate guanylate cyclase in plasma membranes and microsomes of rat liver.

The subcellular localization of guanylate cyclase was examined in rat liver. About 80% of the enzyme activity of homogenates was found in the soluble fraction. Particulate guanylate cyclase was localized in plasma membranes and microsomes. Crude nuclear and microsomal fractions were applied to discontinuous sucrose gradients, and the resulting fractions were examined for guanylate cyclase, various enzyme markers of cell components, and electron microscopy. Purified plasma membrane fractions obtained from either preparation had the highest specific activity of guanylate cyclase, 30 to 80 pmol/min/mg of protein, and the recovery and relative specific activity of guanylate cyclase paralleled that of 5'-nucleotidase and adenylate cyclase in these fractions. Significant amounts of guanylate cyclase, adenylate cyclase, 5'-nucleotidase, and glucose-6-phosphatase were recovered in purified preparation of microsomes. We cannot exclude the presence of guanylate cyclase in other cell components such as Golgi. The electron microscopic studies of fractions supported the biochemical studies with enzyme markers. Soluble guanylate cyclase had typical Michaelis-Menten kinetics with respect to GTP and had an apparent Km for GTP of 35 muM. Ca-2+ stimulated the soluble activity in the presence of low concentrations of Mn-2+. The properties of guanylate cyclase in plasma membranes and microsomes were similar except that Ca-2+ inhibited the activity associated with plasma membranes and had no effect on that of microsomes. Both particulate enzymes were allosteric in nature; double reciprocal plots of velocity versus GTP were not linear, and Hill coefficients for preparations of plasma membranes and microsomes were calculated to be 1.60 and 1.58, respectively. The soluble and particulate enzymes were inhibited by ATP, and inhibition of the soluble enzyme was slightly greater. While Mg-2+ was less effective than Mn-2+ as a sole cation, all enzyme fractions were markedly stimulated with Mg-2+ in the presence of a low concentration of Mn-2+. Triton X-100 increased the activity of particulate fractions about 3- to 10-fold and increased the soluble activity 50 to 100%.     

Partial purification of prolactin-like substance from snake (Ptyas mucosa) pituitaries

Pituitary extract of the common rat snake (Ptyas mucosa) was found to be capable of displacing the binding of 125I-labelled ovine prolactin to female rat liver membranes, suggesting the presence of prolactin-like substance in snake pituitary. The snake prolactin-like substance was unadsorbed on Concanavalin A-Sepharose, but adsorbed on DEAE-cellulose. The partially purified snake prolactin-like substance was also capable of displacing the binding of 125I-labelled ovine prolactin to snake kidney and large intestine membranes. Chromatographic fractions derived from snake pituitary and which possessed potent growth hormone receptor binding activity were devoid of prolactin receptor binding activity, suggesting the existence of distinct prolactin-like and growth hormone-like substances in snake pituitary.