Properties of purified calf thymus poly(adenosine diphosphate ribose) polymerase. Comparison of the DNA-independent and the DNA-dependent enzyme.

The physicochemical properties of the purified calf thymus poly(ADP-ribose) polymerase were investigated. The enzyme purified to homogeneity was shown to contain about 10% DNA on a weight basis and its activity to be DNA independent. After removing this fragment of DNA, called the sDNA fraction, the enzyme becomes DNA dependent. The activity of this enzyme preparation was entirely dependent on, and completely restored by, added calf thymus DNA or sDNA. However, the calf thymus DNA concentration needed was a hundred times higher than that of sDNA. The properties of the two enzyme preparations, DNA independent and DNA dependent, were essentially the same. They both reacted against the specific antibody obtained with the DNA-independent poly(ADP-ribose) polymerase. The pH optimum was around 8; the activity was stimulated by Mg2+, Mn2+ and Ca2+, and inhibited by high ionic strength, p-chloromercuribenzoate, ADP-ribose, AMP and polylysine. Nicotinamide, thymidine and NADP were shown to be competitive inhibitors. The enzymatic activity was stimulated by histone H1 when the ratio of DNA to histone H1 was 2. Histones H2A, H2B, H3 and H4 had little effect on the DNA-independent enzyme activity, but were strongly inhibitory for the DNA-dependent enzyme. This inhibitory effect could be reversed by allowing the DNA-dependent enzyme to react with the sDNA fraction before adding histone subfractions. The apparent Km for NAD of the DNA-dependent poly(ADP-ribose) polymerase was shown to vary with the DNA concentration. It was minimum when the amount of sDNA was 10% of that of the enzyme. The ratio of the apparent Km for sDNA to the enzyme concentration was constant at any enzyme concentration. The minimum estimation of the number of base pairs of sDNA required for maximal activation of one enzyme molecule was 16. For calf thymus DNA, this estimation was of 640. These results suggest that the activation of the enzyme needs the formation of some complex between the protein and a specific part of the DNA. This complex was preserved in the DNA-independent enzyme preparation.     

Immunohistochemical demonstration of poly(adenosine diphosphate-ribose) synthetase in bovine tissues

The inter- and intracellular localization of poly(adenosine diphosphate-ribose)(poly(ADP-ribose] synthetase was investigated using an indirect immunofluorescence technique and a specific antibody against the enzyme purified from calf thymus. In various bovine tissues, including liver, heart, pancrease, thyroid, spleen, adrenal, and skeletal muscle, the specific immunofluorescence of poly(ADP-ribose) synthetase was localized exclusively in the nucleus. Immunostaining was inhibited by preabsorption of the antibody with purified calf thymus poly(ADP-ribose) synthetase. Nuclear immunofluorescence appeared to be more prominent in the marginal area than in the central region in most nuclei. This staining pattern is similar to that of naturally occurring poly(ADP-ribose). In bovine peripheral blood the immunofluorescence of poly(ADP-ribose) synthetase was detected in nuclei of lymphocytes, but not in granulocytes, in agreement with the finding that the enzymatic activity of poly(ADP-ribose) synthetase was barely detectable in nuclei isolated from granulocytes.     

Identification of minimal size requirements of DNA for activation of poly(ADP-ribose) polymerase

Poly(ADP-ribose) polymerase requires DNA as an essential enzyme activator. Using enzyme purified from lamb thymus and double-stranded deoxynucleotide oligomers of defined length, we conducted studies to identify the smallest size DNA fragment capable of successfully activating poly(ADP-ribose) polymerase. These studies revealed that a double-stranded hexadeoxynucleotide activated the enzyme 30% as effectively as highly polymerized calf thymus DNA and a double-stranded octadeoxynucleotide activated the enzyme even more effectively than calf thymus DNA. When histone H1 was also included in the reaction system, the enzyme could be activated by even smaller DNA fragments. Thus, in the presence of histone H1, a double-stranded tetradeoxynucleotide activated the enzyme 25% as effectively as calf thymus DNA, and a double-stranded hexadeoxynucleotide was equally as effective as calf thymus DNA. The time courses for activation and the stabilities of the products were identical when the enzyme was activated by a double-stranded hexadeoxynucleotide or by calf thymus DNA. Double-stranded oligodeoxynucleotides containing dephosphorylated termini were more effective activators than those containing 3'-phosphorylated termini which in turn were more effective than those containing 5'-phosphorylated termini.     

Bovine thymus poly(adenosine diphosphate ribose) polymerase.

About 1,300-fold purification of poly(adenosine diphosphate ribose) polymerase has been achieved from the extract of bovine thymus with a recovery of 10 to 20%. The final preparation has a purity of 99%, and the enzyme is composed of a single peptide with a molecular weight of 130,000. The purified enzyme required NAD+, Mg2+, a thiol compound, DNA, and histones for full activity. Whereas DNA is essential for activation of the enzyme, histones are not. The observed stimulation of the reaction by histones is shown to be due to masking of the inhibitory effect of contaminating denartured DNA in native DNA preparation. The concentration of DNA required for half-maximal enzyme activity (apparent Km for DNA) is proportional to the concentration of enzyme in the reaction mixture. The minimum estimation of the number of nucleotide pairs of DNA required for half-maximal activation of one enzyme molecule is 220 to 240 for bulk of calf thymus DNA, while the value is 10 for a calf thymus DNA fraction, "active DNA," which was separated from the enzyme fraction in a stage of the purification. These results suggest that the enzyme is activated by binding to a specific site on calf thymus DNA. The apparent Km for NAD+ and the maximum velocity of the enzyme are estimated to be 60 micrometer and 0.91 mumolper min per mg, respectively.     

Microcalorimetric Investigation of DNA,poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] Melting in the Presence of Water Soluble (Meso tetra (4 N oxyethylpyridyl) Porphyrin) and its Zn Complex

Abstract

Thermodynamic parameters of melting process (δH_m, T_m, δT_m) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C))·poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased T_m of satellite fraction by 24°C, but ZnTOEpyp(4), on the contrary, predominately bound with AT-rich sites and increased DNA main stage T_m by 18°C, and T_m of poly(dA)poly(dT) increased by 40 °C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes—strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode—strong binding—took place. The weak binding is characterized with shifting of T_m by some grades, and for the strong binding T_m shifts by ～ 30–40°C. Invariability of ΔH_m of DNA and poly(dA)poly(dT), and sharp increase of T_m in the range of r from 0.08 to 0.25 for thymus DNA and 0.01–0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H₂O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width ΔT_m caused by increase of added ZnTO- Epyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which ΔT～T_GC-T_AT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193–205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.     

Purification and characterization of poly (ADP-ribose) synthetase from human placenta

Poly(ADP-ribose) synthetase has been purified 2,000-fold to apparent homogeneity from human placenta. The purification procedure involves affinity chromatography with 3-aminobenzamide as the ligand. The purified enzyme absolutely requires DNA for the catalytic activity and catalyzes poly(ADP-ribosyl)ation of the synthetase itself (automodification) and histone H1. Mg2+ enhances both the automodification and poly(ADP-ribosyl)ation of histone H1. The enzyme is a monomeric protein with a pI of 10.0 and an apparent molecular weight of 116,000. The sedimentation coefficient and Strokes radius are 4.6 S and 5.9 nm, respectively. The frictional ratio is 1.82. Amino acid analysis and limited proteolysis with papain and alpha-chymotrypsin indicate that the human placental enzyme is very similar to the enzyme from calf thymus, although some differences are noted. Mouse antibody raised against the placental enzyme completely inhibits the activity of enzymes from human placenta and HeLa cells and cross-reacts with the enzymes from calf thymus and mouse testis. Immunoperoxidase staining with this antibody demonstrates the intranuclear localization of the enzyme in human leukemia cells. All these results indicate that molecular properties as well as antigenic determinants of poly(ADP-ribose) synthetase are highly conserved in various animal cells.     

Microsomal activation of thioacetamide-S-oxide to a metabolite(s) that covalently binds to calf thymus DNA and other polynucleotides

In the presence of NADPH liver microsomes isolated from phenobarbital-pretreated rats catalyze the conversion of [³H]thioacetamide-S-oxide to a reactive intermediate(s) which covalently binds to calf thymus DNA, calf liver RNA, polyguanylic acid (poly(G)) and polyadenylic acid (poly(A)). The highest level of binding of radioactivity was obtained with poly(G), followed by poly(A), RNA and DNA. The incorporation of radioactivity into DNA was linear for 30 min and there was a requirement for NADPH for time-dependent covalent binding to occur. Performing the microsomal incubations in an atmosphere of 80% CO/20% O₂ or adding partially purified anti cytochrome P-450 immune serum to the microsomal incubations inhibited the total metabolism of thioacetamide-S-oxide and had a small, but insignificant, inhibitory effect on binding of radioactivity to calf thymus DNA. Using a reconstituted monooxygenase system containing cytochrome P-450 purified from phenobarbital-treated rats we were unable to detect any metabolism of thioacetamide-S-oxide. Only background levels of radioactivity were incorporated into calf thymus DNA when microsomes isolated from phenobarbital-treated rats were incubated with [³H]thioacetamide in the presence of NADPH. These results suggest that thioacetamide-S-oxide is an obligatory intermediate in the metabolic activation of thioacetamide to a reactive metabolite(s) which binds to calf thumus DNA.     

Isolation and purification of poly(ADP-ribose) glycohydrolase from pig thymus

Poly(ADP-ribose) glycohydrolase has been purified about 12 300-fold from pig thymus with a recovery of 8.5%. The specific activity of the purified enzyme is 13.8 mumol min -1 mg protein -1. The molecular weight was estimated to be 59 000 by gel filtration through Sephadex G-100 in a non-denaturing solvent. Analysis of the final preparation by sodium dodecyl sulphate gel electrophoresis reveals two protein bands of molecular weight, 61 500 and 67 500. The Km value for poly(ADP-ribose) is estimated to be 1.8 microM monomer units. The enzyme preparation is free from phosphodiesterase, NADase and ADP-ribosyltransferase activities. The purified enzyme is inhibited by cyclic AMP, ADP-ribose, naphthylamine, histones H1, H2A, H2B, H3, polylysine, polyarginine, polyornithine and protamine. The inhibition by histone is relieved by an equal mass of DNA. Single-stranded DNA, poly(A), poly(I) and polyvinyl sulphate were inhibitory, but double-stranded DNA was not inhibitory.     

Purification and properties of poly(ADP-ribose) synthetase from mouse testicle

Poly(ADP-ribose) synthetase has been purified to apparent homogeneity from mouse testicle by a rapid and simple procedure using column chromatography on DNA-agarose and on Cibacron blue F₃G-A-Sephadex G-150. The purified enzyme absolutely requires DNA for activity, and half-maximal activation occurs at a DNA concentration of 25 μg/ml. The K_m for NAD and V at pH 8.0 and 25 °C are 47 μm and 1400 nmol/min/ mg, respectively. The molecular weight is 116,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicates that the mouse testicle enzyme is very similar to calf thymus enzyme, but there is a difference in the contents of several amino acid residues between the two enzymes. This difference appears to reflect species or tissue specificity of poly(ADP-ribose) synthetase.     

Changes in poly(adenosine diphosphate-ribose) and poly(adenosine diphosphate-ribose) polymerase in synchronous HeLa cells.

An antibody has been prepared which is highly specific for poly(adenosine diphosphate-ribose). Neither poly(A), DNA, nor a variety of adenine-containing nucleosides or nucleotides were effective in competing with poly(ADP-ribose) for binding to the antibody. Of all compounds tested, only adenosine diphosphate-ribose competed for binding to the antibody. Unlabeled poly(adenosine diphosphate-ribose) was about 10 000 times more effective in competing with labeled polymer for antibody binding than was adenosine diphosphate-ribose. Using the antibody, the amount of poly(adenosine diphosphate-ribose) was found to increase from early S phase to a peak at mid S with a second, even larger increase seen at the S-G2 transition point in synchronously dividing HeLa cells. Pulse labeling of the polymer with [2-3H]adenosine was also maximal at the same time points. Changes in the levels of poly(adenosine diphosphate-ribose) polymerase activity measured in isolated nuclei coincided with the changes in amounts of polymer present in intact cells during progression from S phase into G2.     

Synthetic RNA-dependent DNA polymerase from calf thymus

A RNA dependent-DNA polymerase was purified about 450-fold from the soluble fraction of calf thymus. This enzyme was able to copy the polyribonucleic acid strand of synthetic ribonucleic acid primed with complementary oligodeoxynucleotides, i.e., poly(rA)·(dT)₁₀. This enzyme activity was separated from the DNA-dependent DNA polymerases by both DEAE-cellulose columm chromatography and glycerol gradient centrifugation. Some properties of this enzyme were described.     

Natural occurence of a biopolymer, poly (adenosine diphosphate ribose).

Evidence for the natural occurrence of poly(adenosine diphosphate ribose) in vivo was obtained using a sensitive radioimmunoassay and poly(adenosine diphosphate ribose) glycohydrolase, which specifically hydrolyzes poly(adenosine diphosphate ribose). Calf thymus, liver, kidney, brain, pancreas and spleen contained poly(adenosine diphosphate ribose). Naturally occurring poly(adenosine diphosphate ribose) in calf thymus is composed of molecules of various chain lengths, like that synthesized by an in vitro system. Calf thymus was estimated to contain about 0.02 microgram/mg DNA of poly(adenosine diphosphate ribose).     

Isolation and Characterization of a Relatively Guanine-specific Endoribonuclease in Rice Bran

A relatively guanine-specific endoribonuclease (RB-1) was isolated from rice bran. The pH optimum was 8.5 using yeast RNA as a substrate. The enzyme activity was inhibited by Cu²⁺, Zn²⁺, DTT and SDS, while EDTA, PCMB, IAA and heparin had no effect on the activity. The enzymic activity of RB-1 was inhibited by 3′-GMP as an end-product inhibitor. The enzyme protein was highly heat-stable. RB-1 did not hydrolyze native calf thymus DNA, heat-denatured DNA, poly A, poly U and poly C. Among synthetic substrates, only poly I was depolymerized. Only 2′,3′-cyclic GMP was identified in the hydrolysate of yeast RNA after 6hr hydrolysis.     

Characterization of DNA kinase from calf thymus

DNA kinase has been purified to homogeneity from calf thymus. The purified enzyme, with a specific activity of 16.7 units/mg protein at 25 degrees C, exhibited a sharp pH/activity curve with a pH optimum at 5.5 and low activity at alkaline pH. The molecular weight of the enzyme was estimated by dodecylsulfate/polyacrylamide gel electrophoresis to be 5.4 X 10(4). The enzyme has a sedimentation coefficient of 4.0 S. An apparent molecular weight of 5.6 X 10(4) and a Stokes' radius of 3.3 nm were estimated by gel-filtration on Sephadex G-100. The enzyme phosphorylates neither yeast RNA nor poly(A) instead of DNA. Compared with rat liver DNA kinase, calf thymus DNA kinase is relatively resistant to the inhibition by sulfate (Ki = 7 mM) and pyrophosphate (Ki = 5 mM). The enzyme activity is markedly stimulated by polyamines at the sub-optimal concentration of Mg2+ but not by monovalent cations.     

Antigenic determinant and interspecies cross-reactivity of a monoclonal antibody to poly(ADP-ribose) synthetase

A monoclonal antibody (1F4) was prepared against calf thymus poly(ADP-ribose) synthetase. It was classified as IgG1/kappa and its antigenic determinant was localized on the 46 kDa portion of the enzyme molecule which contains the site for the binding of DNA. When calf thymus DNA-binding proteins were subjected to immunostaining after electrophoresis and transblotting to a nitrocellulose filter, the native enzyme (120 kDa) and its endogenous degradation products (80, 64 and 32 kDa) were detected. When the interspecies cross-reactivity was examined using DNA-binding proteins from 6 different sources, 1F4 reacted with the 120- and 32-kDa protein bands in HeLa cells, mouse testis and chicken liver as in the case of calf thymus. These results indicate that the antigenic structures of poly(ADP-ribose) synthetase and its degradation products are highly conserved in various animal cells.     

Microcalorimetric investigation of DNA, poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] melting in the presence of water soluble (meso tetra (4 N oxyethylpyridyl) porphyrin) and its Zn complex

Thermodynamic parameters of melting process (DeltaHm, Tm, DeltaTm) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C)).poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased Tm of satellite fraction by 24 degrees C, but ZnTOEpyp(4), on the contrary, predominantly bound with AT-rich sites and increased DNA main stage Tm by 18 degrees C, and Tm of poly(dA)poly(dT) increased by 40 degrees C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes--strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode--strong binding--took place. The weak binding is characterized with shifting of Tm by some grades, and for the strong binding Tm shifts by approximately 30-40 degrees C. Invariability of DeltaHm of DNA and poly(dA)poly(dT), and sharp increase of Tm in the range of r from 0.08 to 0.25 for thymus DNA and 0.01-0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H2O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width DeltaTm caused by increase of added ZnTOEpyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which DeltaT approximately TGC-TAT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193-205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.     

Histone Kinase from Soybean Hypocotyls: PURIFICATION, PROPERTIES, AND SUBSTRATE SPECIFICITIES

A histone-type protein kinase (EC 2.7.1.37) has been partially purified (320-fold) from the crude extracts of soybean hypocotyls by means of a combination of gel filtration and anion exchange procedures. The purified enzyme fraction is devoid of the activities of phosphoprotein phosphatase (EC 3.1.3.16), histone protease, and casein (or phosvitin)-type kinase. The soybean histone kinase uses ATP to phosphorylate specifically lysine-rich histone H1 from either pea seedlings or calf thymus.     

In vitro acylation of the ϵ-amino group of l-lysine in calf thymus histones by the carcinogen, β-propiolactone

We had previously reported that the carcinogen, β-propiolactone (BPL) reacted in vitro with histones in whole mouse skin chromatin and that among the histone classes BPL was preferentially bound to the lysine-rich histones H1 and H1°. In order to determine if in vitro reaction of BPL with calf thymus histones resulted in binding of BPL to l-lysine, we synthesized the model compounds ?-N-(3-hydroxypropionyl)lysine (HPL) and ?-N-(2-carboxyethyl)lysine (CEL) from BPL and l-lysine. The α-amino group of l-lysine was protected from reaction with BPL by the formation of a copper chelate.Structures were assigned on the basis of infrared spectra, pKa values and chemical analyses. BPL was reacted in vitro with calf thymus histones and the BPL-reacted calf thymus histones and control calf thymus histones were digested with trypsin followed by pronase. The respective digests were each chromatographed on a column of AA-15 cation-exchange resin. The elution profiles of the two digests were very similar except for the appearance of a new ninhydrin-positive peak (NNPP) in the eluate of the trypsin-pronase digest of BPL-reacted calf thymus histones. When compounds HPL and CEL were added to the trypsin-pronase digest of control calf thymus histones and the mixture chromatographed on AA-15, both compounds were resolved from the other peptide (or amino acid) peaks. HPL was eluted in the same fractions as NNPP, HPL and NNPP exhibited identical R_F values on silica gel TLC with acidic, alkaline and neutral solvents. CEL was not identified as a product of the reaction between BPL and calf thymus histones.     

Inhibition of ribonucleases by ribonucleotides and transition state analogs in cell-free extracts from Ehrlich ascites tumor cells.

We investigated the ribonucleolytic breakdown of poly(U), poly(A), RNA trascribed from calf thymus DNA with E. coli RNA polymerase, ribosomal RNA, tRNA and mengovirus RNA by an enzyme fraction obrained from a postribosomal supernatant of Ehrlich ascites tumor cells. The single-stranded homopolyribonucleotides are preferentially degraded by the enzyme fraction with the production of ribonucleoside 5'-monophosphates. The RNase activity is completely dependent on the presence of Mg2+ ions and is highest at Mg2+ and K+ concentrations optimal for cell-free protein synthesis. Ribonucleoside 5'-monophosphates, ribonucleoside 2'(3')-monophosphates, ribonucleoside 2'(3'),5'-bisphosphates and transition state analogs consisting of vanadyl sulfate and either ribonucleosides or ribonucleoside 5'-monophosphates in a molar ratio 1:1 inhibit the ribonucleolytic activity of the enzyme fraction. The ribonucleoside 2'(3'),5'-bisphosphates and the transition state analogs are the most effective inhibitors. However, only in the presence of ribonucleoside 2'(3'),5'-bisphosphates a concomitant stimulation by 50 to 60% of poly(U)-directed polyphenylalanine synthesis is observed; all the other RNase inhibitors tested also inhibit polypeptide synthesis. The results of preliminary experiments show that poly(U) and ribonucleoside 2'(3'),5'-bisphosphates are well suited as ligands for affinity chromatography of ribonucleases from Ehrlich ascites tumor cells.