Polyamine-dependent deoxyribonuclease activity from rat-liver nuclei.

When nuclei isolated from rat liver in a low salt buffer were washed with 0.1 M NaCl solution, the supernatant showed a deoxyribonuclease (DNase) activity. The activity required Mg2+ and in addition spermine or spermidine, and its optimal pH was 7.2-7.4. The activity was higher on denatured (single stranded) DNA than on double-helical DNA. With both substrates the activity was highest at a polyamine concentration at which the DNA-polyamine complex began to precipitate. No Mg2++Ca2+ dependent DNase activity was detected in the preparation.     

Polynucleotide kinase from rat-liver nuclei. Purification and properties.

A polynucleotide kinase, which catalyzes the phosphorylation of 5'-hydroxyl ends of deoxyribonucleic acid in the presence of adenosine triphosphate, has been purified 260-fold with a yield of 14% from 0.15 M NaCl extracts of rat liver nuclei. The purified enzyme has a pH optimum of 5.5. The enzyme is reversible inhibited by p-chloromercuribenzoate. The S0.5 value (ligand concentration required for a half-maximal activity) for ATP is 2.5 muM. A bivalent cation is essential for the reaction and S0.5 values for Mg2+, Ca2+ and Mn2+ are 3.3 mM, 4 mM and 0.05 mM respectively. Pyrophosphate remarkable inhibits the activity with I0.5 value (ligand concentration required for a half-maximal inhibition) of 0.2 mM, and sulfate, with I0.5 of 0.5 mM, whereas phosphate weakly inhibits the activity with I0.5 of about 20 mM. An apparent molecular weight of the purified enzyme is estimated to be 8 X 10(4) by gel filtration on a column of Sephadex G-150, and the Stokes radius of the enzyme molecule is shown to be about 0.36 nm. Sucrose density gradient centrifugation reveals that the enzyme has a sedimentation coefficient of about 4.4 S.     

A nuclease from rat-liver nuclei with endo- and exonucleolytic activity.

An Mg2(+)-dependent endonuclease endogenous to rat-liver nuclei had an exonuclease activity for single-stranded DNA, but not for duplex DNA. The activity was about twice as high in the 3'----5' direction as in the 5'----3' direction. The products by 3'----5' activity were mononucleotides alone. The 5'----3' activity released mononucleotides as main products and small amounts of di-, tri-, tetra- and oligonucleotides. Another major endonuclease endogenous to the nuclei, a Ca2+/Mg2(+)-dependent endonuclease, did not have such exonuclease activities.     

Purification and properties of an endonuclease endogenous to rat-liver nuclei

An endonuclease endogenous to rat-liver nuclei has been purified by a series of chromatographic procedures and finally by isoelectric focusing (IEF) electrophoresis. The nuclease fraction prepared by the IEF electrophoresis (IEF fraction) showed a pI value of 5.7 and migrated as a single band to a molecular weight position of 46,000 on an SDS-polyacrylamide gel. The activity for single-stranded DNA was enhanced by 10 mM MgCl2 and/or by 5-15 mM MgCl2 in the presence of 2 mM CaCl2 (an optimum pH, 7.0), but was lowered by CaCl2 alone and inhibited strongly by ZnCl2 or MnCl2. The activity for duplex DNA was rather low, although an optimum condition was 10 mM MgCl2. In fact, even under this condition, the activity was about 40% lower than that for single-stranded DNA. Moreover, the IEF fraction formed single-strand nicks much more rapidly than double-strand cuts in pBR322 DNA, and preferentially produced deoxyadenosine 5'-monophosphate termini in the DNA. In addition, RNAase activity was also detected in this fraction.     

Characterization of the ribonucleic acid synthesized by isolated rat-liver nuclei

1. Isolated rat-liver nuclei incorporated [¹⁴C]UMP into RNA when incubated in the presence of Mg²⁺ and all four ribonucleoside triphosphates. The addition of bentonite to the system diminished the breakdown of the newly synthesized RNA. 2. AMP and CMP were incorporated in the absence of the other added triphosphates, and in the presence of deoxyribonuclease. 3. RNA synthesized in the presence of Mg²⁺ contained a high proportion of CMP and GMP, and sedimented in the regions of ribosomal RNA and of heavier molecules. About 1% of this RNA hybridized with homologous DNA, and hybrid formation was more effectively inhibited by nuclear RNA than by ribosomal RNA. 4. RNA synthesized in the presence of Mn²⁺ plus ammonium sulphate had a composition intermediate between that of ribosomal RNA and of DNA, and about 4% of this RNA formed hybrids with DNA. 5. Less than 2% of the newly synthesized RNA was capable of forming ribonuclease- and deoxyribonuclease-resistant complexes. 6. It was concluded that the newly synthesized RNA arose as a result of an asymmetric process and included both ribosomal and DNA-like species.     

Presence of a thiol protease in regenerating rat-liver nuclei. Partial purification and some properties

1. Nuclei of regenerating rat liver washed with Triton X-100 were found to contain a new protease. Since the enzymatic activity for degrading ribosomal proteins was inhibited in vivo by administration of E-64, a thiol protease inhibitor, the enzyme may participate in the degradation of newly synthesized ribosomal proteins and histones in regenerating rat liver nuclei as reported previously by us [Biochem. Biophys. Res. Commun. 75, 525-531 (1077)]. The optimum pH was 5.5. 2. The enzyme was extracted from washed nuclei and partially purified by gel filtration through Sepharose 6B. Its molecular weight was about 40 000. A maximal activity of partially purified enzyme was observed in the presence of 1 mM EDTA and 2 mM dithiothreitol at pH 5.5 It was inhibited by thio reagents, E-64, leupeptin and hevy metal ions. The enzyme degraded ribosomal proteins endoproteolytically and degraded most proteins tested as substrates, although liver cell sap proteins and serum albumin were less degraded than ribosomal proteins and histones, alpha-N-Benzoylarginine-beta-naphthylamide and benzoylarginine amide were not hydrolyzed.     

Purification and properties of a magnesium-dependent endodeoxyribonuclease endogenous to rat-liver nuclei

An Mg2+-dependent endonuclease has been purified from a 0.6 M NaCl extract of rat-liver nuclei by a series of chromatographic procedures and finally by isoelectric focusing (IEF) electrophoresis. The nuclease fraction prepared by the IEF electrophoresis (IEF fraction) was shown to have a pI value of 7.1 and to migrate as a single band to a molecular-weight position of 36,500 on SDS-polyacrylamide gel. The IEF fraction was subjected to a sedimentation analysis. In a hypotonic buffer (10 mM Tris), the nuclease activity sedimented to have an S value of 4.1 S. However, in an isotonic buffer (0.15 M NaCl), this fraction exhibited two activity peaks of 2.8 and 4.3 S. In a hypertonic buffer (0.3 M NaCl), almost all of the nuclease activity sedimented at 2.7-2.8 S. In this connection, values of 2.8 and 4.3 S were determined to correspond to molecular weights of about 36,000 and 70,000, respectively. Thus, an Mg2+-dependent endonuclease, endogenous to rat-liver nuclei, has been inferred to exist in the reversible form of a monomer/homodimer as its native state. Moreover, the IEF fraction formed single-strand nicks more rapidly than double-strand cuts in pBR322 DNA, and preferentially produced deoxyguanosine 5'-monophosphate termini in the DNA at an early incubation time. In addition, RNAase activity was not detected in this fraction.     

Effect of DNA on thyroid-hormone binding by specific receptor proteins from rat-liver nuclei

Influence of double-stranded native DNA on the binding of thyroid hormone, 3,5,3'-triiodo-L-thyronine, by the isolated nuclear receptors was studied and the following results were obtained. (1) The receptor-triiodothyronine complexes bound to DNA with moderate affinities. (2) DNA enhanced the hormone binding of the receptors. (3) The stimulatory DNA effect on triiodothyronine binding of the receptors was dependent on DNA concentration, showing its maximum at 30 microgram/ml. (4) The increase in triiodothyronine binding was observed not only in the initial velocity but also in the plateau level which was attained after sufficient incubation time. (5) There were two types of specific receptors in the rat liver nuclear extract. The dissociation constants and the maximal binding capacities for triiodothyronine, which were determined by Scatchard plot analysis in the presence and absence of DNA, suggested that DNA exerted its effect through increasing binding capacity on one class of the receptors and through enhancing affinity for the hormone on the other class of the receptors. (6) Among various polynucleotides examined, the double-stranded eukaryotic DNA was most effective in enhancing the hormone binding by the receptors. These results indicate that the nuclear thyroid hormone receptors interact with double-stranded DNA in a specific manner and are induced to bind more thyroid hormone. We interpret these results as suggesting that a ternary complex of triiodothyronine, the receptor and DNA is formed in the cell nucleus in vivo, probably representing an intrinsic step in the hormone action. Possible physiological significance of this effect of DNA on the receptors is discussed.     

The association of a magnesium-dependent endonuclease activity with a nucleosome fraction from rat-liver nuclei

The nucleosomes released by the incubation (autodigestion) of rat-liver nuclei were fractionated by sucrose-density gradient centrifugation, and subjected to nuclease assay with heat-denatured 3H-DNA from Escherichia coli as an exogenous substrate. With increasing incubation time, the nuclease activity was enhanced and localized in the mono/tetra-, hexa/hepta-, and long-chain oligonucleosome fractions. In contrast, independent of the nucleosome size, the activities of 0.35 M NaCl-soluble fractions from them were found to be almost equal in terms of specific activity (dpm/nucleosomal DNA). Such nuclease activity was not detected in the sucrose gradient (top region) lacking nucleosomes and/or chromatin. When the chromatin was dialyzed against a 0.35 M NaCl buffer and then fractionated in a sucrose gradient containing 0.35 M NaCl, most of the nuclease activity was solubilized into the above top region. On gel filtration of the mononucleosome fraction in the 0.35 M NaCl buffer, the nuclease activity was eluted at the position of 36,000 daltons. This nuclease cleaved heat-denatured DNA more rapidly than the native DNA in the presence of Mg2+, and had the ability to make both single-strand nicks and double-strand cuts in pBR322 DNA; in other words, it had an endonucleolytic activity. Moreover, four different classes of mononucleosomes were fractionated by electrophoresis of the nucleosomes released by autodigestion of the nuclei. These mononucleosomes also showed nuclease activity with the heat-denatured DNA. Thus, the present studies suggest that an Mg2+-dependent endonuclease of about 36,000 daltons is associated with the nucleosome particle(s) in rat-liver nuclei.     

Compartmentation of acetyl-coA in rat-liver mitochondria.

The ratio of the specific radioactivities of 3-hydroxybutyrate: citrate was determined in rat liver mitochondria which were incubated in the presence of [1-14C]palmitate, pyruvate, bicarbonate, ATP, phosphate and malonate. Without compartmentation this ratio would maximally be 2, however, under our conditions values of 2.5-3.7 were observed. In further experiments with mitochondria, the sensitivity of pyruvate carboxylase for acetyl-CoA produced from various precursors was tested. It was found that acetyl-CoA produced from L-acetylcarnitine or by oxidation from either pyruvate, octanoate or palmitylcarnitine but not from leucine led to a stimulation of pyruvate carboxylation. These results demonstrate a compartmentation of acetyl-CoA in liver mitochondria. The further finding that different mitochondrial fractions showed varying ratios of specific radioactivities of 3-hydroxybutyrate:citrate indicates that the observed compartmentation may be explained by the existence of different types of mitochondria with varying enzyme patterns and acetyl-CoA pools.     

Pyrimidine-rich oligonucleotides from rat-liver ribosome surface.

The distribution of oligonucleotides which are released from rat liver ribosomes by treatment with pancreatic ribonuclease has been studied. Rat liver monoribosomes lost from 15 to 17% of their nucleotides by treatment with pancreatic ribonuclease. This quantity was highly reproducible and did not depend significantly on the temperature (0-20 degrees C) and time (10-120 min) of incubation or on the concentration of enzyme (1:5000-1:50). Whereas the amounts of oligonucleotides liberated was 16%, it was shown by column chromatography that they consisted of 71% mononucleotides, 16% dinucleotides, 6% trinucleotides, 4% tetranucleotides and 2% pentanucleotides and that these oligonucleotides were enriched in uridine, containing approximately half of the uridine residues present in the high-molecular-weitht ribosomal RNA. The high molecular weight of the RNA from ribonuclease-treated ribosomes was preserved until it was heated; after heating, RNA fragments having sedimentation coefficients of 5 S and less were present. It is inferred that the olignucleotides are derived from pyrimidine-rich clusters located in single-stranded "hairpin" loops on the outside surface of the ribosome.     

Control of choline oxidation by rat-liver mitochondria.

The steady-state concentrations of choline and its reaction products in intact rat-liver mitochondria were determined under different conditions. From these measurements, it is concluded that in a sucrose medium choline dehydrogenation and betaine aldehyde dehydrogenation are the rate-limiting steps in overall choline oxidation under "State-3" or uncoupled conditions, respectively. Ageing of the mitochondria leads to changes in the mitochondrial membrane, resulting in a markedly different pattern of oxidation products. This finding explains why rotenone inhibits oxygen uptake with choline as substrate in fresh but not in aged mitochondria.