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.     

Purification and properties of the major apurinic/apyrimidinic endodeoxyribonuclease of rat-liver chromatin

Two nucleases active on alkylated-depurinated DNA have been extracted from rat liver chromatin with 1 M KCl. The major enzyme was purified to near homogeneity; it has a molecular weight of 12 500 (although some dimerization might occur), needs Mg2+ or Mn2+ for activity. The endonuclease activity is specific for apurinic/apyrimidinic sites in DNA; the enzyme has no associated exonuclease activity.     

Purification and properties of an endodeoxyribonuclease from nuclei of bovine small intestinal mucosa

An endodeoxyribonuclease has been purified from nuclei of bovine small intestinal mucosa to a homogeneous state by a procedure involving affinity chromatography on heparin-agarose. The endonuclease, which was found to be bound to chromatin, has a pH optimum of 5.4. It requires Mn2+ or Co2+ for activity and its maximum activity with Mg2+ is about 80% of that with Mn2+. Its activity is strongly inhibited by sulfhydryl-blocking agents, and by ethidium bromide. The enzyme does not attack RNA and is inhibited by it. Its isoelectric point is 8.5 +/- 0.1, and its molecular weight is 49,000 +/- 3,000, determined by sucrose gradient sedimentation and gel filtration on Sephadex G-100. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two nonidentical subunits with molecular weights of 30,000 and 23,000. The enzyme catalyzes the endonucleolytic cleavage of circular duplex ColE1 DNA via single strand scissions from the initial stage of degradation. The average size of the limit products of native phage T7 or ColE1 DNA is about 2,000 to 1,500 base pairs, estimated by neutral sucrose gradient sedimentation or agarose gel electrophoresis. The enzyme degrades denatured DNA about 20 times faster than native DNA. The products contain 5'-phosphoryl and 3'-hydroxyl termini, and all four deoxymononucleotides are present in almost equal amounts at the 5'-termini.     

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.     

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.     

Purification and properties of a 3'-phosphoryl former endodeoxyribonuclease from eggs of Asterias forbesi.

A DNA endonuclease has been purified from eggs of Asterias forbesi by a simple four-step-purification procedure. The purified enzyme is at least 96% pure and is free of phosphatase, phosphodiesterase, and RNase. It has a pH optimum of 6.5 and does not require divalent cations. The enzyme produces 3'-phosphoryl and 5'-hydroxyl end groups. The products of exhaustive hydrolysis can be grouped in two fractions. The first fraction, 40%, contains a small amount of mononucleotides and di-, tri-, tetra-, penta-, and hexanucleo-tides. The second fraction, 60%, contains oligonucleotides larger than hexanucleotides.     

Purification and properties of an aminopeptidase from rat-liver cytosol.

An aminopeptidase was purified from the rat-liver cytosolic fraction to apparent electrophoretic homogeneity. The enzyme is a monomeric protein of 95 kDa, having an isoelectric point of 4.9. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cysteine. The enzyme hydrolyzed a broad spectrum of amino acid beta-naphthylamides at a neutral pH. The enzyme also hydrolyzed di-, tri-, and oligopeptides, including physiologically active peptides such as enkephalins and Met-Lys-bradykinin. The enzyme was inhibited by metal-chelating agents, sulfhydryl-reactive reagents, N-P-tosyl-L-phenylalaninechloromethyl ketone, N-P-tosyl-L-lysinechloromethyl ketone, and puromycin but not by protease inhibitors of microbial origin. The enzyme was activated by the addition of Co2+ and sulfhydryl compounds. The aminopeptidase enhanced proteolysis when the enzyme was added to the protease assay system with purified rat-liver cytosolic neutral protease, suggesting the cooperative action of aminopeptidase in the overall process of protein degradation.     

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 characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain

The magnesium-dependent, plasma membrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homologies of bovine N-SMase to any known protein. Peptide-specific antibodies recognized a single 97-kDa protein in Western blot analysis of cell lysates. The purified enzyme displayed a K(m) of 40 microM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg(2+), whereas Zn(2+) and Ca(2+) proved inhibitory. The highly purified bovine N-SMase was effectively blocked by glutathione and scyphostatin. Scyphostatin proved to be a potent inhibitor of N-SMase with 95% inhibition observed at 20 microM scyphostatin. The results of this study define a N-SMase that fulfills the biochemical and functional criteria characteristic of the tumor necrosis factor-responsive membrane-bound N-SMase.     

Purification and some properties of a deoxyribonucleic acid endonuclease endogenous to rat liver chromatin

A deoxyribonucleic acid (DNA) endonucleolytic activity has been purified from a 0.3 M KCl extract of rat liver chromatin by a combination of selective precipitation and ion-exchange and gel filtration chromatography. The purified protein has a molecular weight of 35 000 as determined by Sephadex G-200 gel filtration and sodium dodecyl sulfate-acrylamide gel electrophoresis. The nuclease activity is stimulated by the addition of Mg2+ and thus may represent the Mg2+-activated DNase endogenous to chromatin. The purified enzyme has the ability to make both single-strand nicks and double-strand cuts in DNA.     

Purification and properties of a thiol protease from rat liver nuclei

A thiol protease was purified about 800-fold from the chromatin fraction of rat liver by employing Sepharose 6B gel filtration, chromatofocusing and Sephadex G-100 gel filtration. It was nearly homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and its molecular weight was about 29000. The isoelectric point of the enzyme was 7.1. The pH optimum for degradation of 3H-labelled ribosomal proteins was 4.5. It is noticeable that the maximal activity was shifted to pH 5.5 by DNA, and that 30-40% of the maximal activity was observed at neutral pH in the presence of DNA. The activity was increased about twice by 2-4 mM dithiothreitol. The protease may be specific for the nuclei because it is different from all lysosomal thiol proteases ever known.     

Purification and some properties of a nuclease from rye germ nuclei

1. An endonuclease has been isolated from the nuclei of rye (Secale cereale L) germ and partially purified. The enzyme shows optimum activity over the pH range 5.4-7.4 towards both DNA and RNA, and has no phosphomonoesterase or phosphodiesterase activity. 2. DNA is degraded by the rye germ nuclease to oligonucleotides of similar size, and RNA to oligonucleotides and mononucleotides containing a C-terminal 5'-phosphate group. 3. The rate of hydrolysis of nuclear acids by the enzyme decreases in the following order: native DNA greater than denatured DNA greater than RNA. Synthetic polynucleotides are hydrolysed at a rate decreasing in the order: poly(A) greater than poly(U) greater than poly(C) greater than poly(G).     

Intracellular localization of rat-liver uracil-DNA glycosylase. Purification and properties of the chromatin enzyme

Most of the uracil-DNA glycosylase of the rat liver cell is located in chromatin; there is, however, some activity in the nuclear sap and in the cytoplasm. The chromatin uracil-DNA glycosylase has been purified; the preparation is devoid of endonuclease and exonuclease activities; the enzyme does not need divalent cations, has a broad optimum pH around 8, is strongly inhibited by increasing ionic strength and free uracil. The apparent Km is independent of the strandedness of the DNA substrate containing uracil, but V is slightly higher with the single-stranded substrate. The frequency of uracil substitution in the double-stranded DNA influences the kinetic parameters: a higher frequency increases both Km and V. The inhibitory effects of NaCl and free uracil are greater when the substrate is double-stranded rather than single-stranded. It is speculated that, acting either on the DNA or on the enzyme, both oppose the opening of the double helix necessary for the formation of the enzyme-substrate complex. The increased reaction rate with a higher frequency of uracil residues in double-stranded DNA is interpreted as a tendency for the repair enzyme to work in a processive way. It is supposed that processivity also occurs with single-stranded DNA and that it is opposed by both NaCl and free uracil, explaining a greater inhibition when the single-stranded substrate has a higher uracil content.     

Localization of the phosphoester bond hydrolyzed by the major apurinic/apyrmidinic endodeoxyribonuclease from rat-liver chromatin

The major apurinic/apyrimidinic (AP) endodeoxyribonuclease from rat liver chromatin, an enzyme specific for AP sites in DNA, cleaves the phosphodiester bridge which is the immediate neighbour of the AP site on its 5' side leaving 3'-hydroxyl and 5'-phosphate ends. In contrast with Escherichia coli endonuclease VI, this chromatin enzyme is inactive on reduced AP sites.     

Purification and properties of a cyclic AMP-independent protein kinase from calf thymus nuclei

A phosphoprotein kinase (ATP : protein phosphotransferase, EC 2.7.1.37) from calf thymus nuclei was purified by DEAE-cellulose chromatography, hydroxyapatite, and Sepharose 6B gel filtration. The enzyme is a cyclic AMP-independent protein kinase by the following criteria: (a) the protein kinase did not bind cyclic AMP; (b) no inhibition of activity was obtained with the heat-stable protein kinase inhibitor from rabbit skeletal muscle; (c) the regulatory subunit of cyclic AMP-dependent protein kinase had no effect on activity; and (d) no inhibition was obtained with antibody to cyclic AMP-dependent protein kinase. The nuclear cyclic AMP-independent protein kinase readily phosphorylated protamine on serine and to a lesser extent on threonine. Homologous nucleoplasmic RNA polymerase (EC 2.7.7.6) is a better substrate than arginine-rich histone, phosvitin or casein. Physical characteristics of the enzyme are described.