Purification and properties of xanthine dehydrogenase from Pseudomonas acidovorans.

Xanthine dehydrogenase (EC 1.2.1.37) from Pseudomonas acidovorans has been purified to near homogeneity (approx. 65-fold). The enzyme has a molecular weight of about 275 000. Electrophoresis in gels containing sodium dodecyl sulphate showed the presence of two types of subunit with molecular weights of about 81 000 and 63 000. Thus the intact molecule probably contains two of each type of subunit. Xanthine and hypoxanthine are good substrates, and NAD+ is an effective electron acceptor. With xanthine and NAD+ as substrates the purified enzyme has a specific activity of about 20 mumol NADH formed/min per mg protein. Michaelis constants for xanthine and NAD+ are 0.07 and 0.12 mM, respectively, and for hypoxanthine and NAD+ 0.29 and 0.16 mM, respectively.     

Purification and characterization of Escherichia coli guanine-xanthine phosphoribosyltransferase produced by a high efficiency expression plasmid utilizing a lambda PL promoter and CI857 temperature-sensitive repressor

The gene for Escherichia coli guanine-xanthine phosphoribosyltransferase was placed after the high efficiency lambda phage leftward promoter in plasmid pHEGPT also containing the lambda CI857 temperature-sensitive repressor. Guanine-xanthine phosphoribosyltransferase increases 780-fold when cells containing pHEGPT are shifted from 30 to 42 degrees C. Guanine-xanthine phosphoribosyltransferase represents approximately 5% of the protein in a crude extract of induced cells. Guanine-xanthine phosphoribosyltransferase may be purified to apparent homogeneity by ammonium sulfate fractionation, Sephadex G-100, and DEAE-cellulose column chromatography. The enzyme has a subunit molecular weight of 18,600 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and behaves as a trimer during Sephadex G-100 column chromatography. Guanine-xanthine phosphoribosyltransferase is active from pH 7.5 to 10.5 with maximum activity at pH 9.5. The enzyme is protected from heat inactivation by phosphoribosylpyrophosphate (PRPP). At 65 degrees C, the enzyme has a half-life of 2 min in the absence of PRPP and 90 min in the presence of PRPP. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants for guanine, xanthine, hypoxanthine, and PRPP of 2.6, 39, 167, and 95 microM, respectively. The activity of the enzyme with guanine is 2-fold greater than that with xanthine and 3-fold greater than that with hypoxanthine.     

Tunicamycin Inhibits Biosynthesis of Acid Mucopolysaccharides in Cultures of Chick Embryo Fibroblasts

Enterobacter cloacae KY 3074 grown in a medium containing xanthine, hypoxanthine, guanine, or their nucleosides and nucleotides produced xanthine oxidase. The purified enzyme preparation showed a major protein band and a few minor bands in acrylamide gel electrophoresis. Molecular oxygen was the most effective electron acceptor. Ferricyanide and 2,6-dichlorophenolindophenol also served as electron acceptors, but NAD and NADP did not. Xanthine and hypoxanthine were good substrates, and guanine was also an effective substrate. The activity was inhibited by Ag²⁺, Cu²⁺, PCMB, and ascorbate. The spectrum of the Enterobacter enzyme resembled that of some known xanthine oxidizing enzymes, and this suggests a similarity in the prosthetic groups of these enzymes. The molecular weight of the native enzyme and subunit was 128,000 and 69,000, respectively.     

Purification and properties of an NAD(P)+-linked formaldehyde dehydrogenase from Methylococcus capsulatus (Bath).

Crude soluble extracts of Methylococcus capsulatus strain Bath, grown on methane, were found to contain NAD(P)+-linked formaldehyde dehydrogenase activity. Activity in the extract was lost on dialysis against phosphate buffer, but could be restored by supplementing with inactive, heat-treated extract (70 degrees C for 12 min). The non-dialysable, heat-sensitive component was isolated and purified, and has a molecular weight of about 115000. Sodium dodecyl sulphate gel electrophoresis of the protein suggested there were two equal subunits with molecular weights of 57000. The heat-stable fraction, which was necessary for activity of the heat-sensitive protein, was trypsin-sensitive and presumed to be a low molecular weight protein or peptide. A number of thiol compounds and other common cofactors could not replace the component present in the heat-treated soluble extract. The purified formaldehyde dehydrogenase oxidized three other aldehydes with the following Km values: 0.68 mM (formaldehyde); 0.075 mM (glyoxal); 7.0 mM (glycolaldehyde); and 2.0 mM (DL-glyceraldehyde). NAD+ or NADP+ was required for activity, with Km values of 0.063 and 0.155 mM respectively, and could not be replaced by any of the artificial electron acceptors tested. The enzyme was heat-stable at 45 degrees C for at least 10 min and had temperature and pH optima of 45 degrees C and pH 7.2 respectively. A number of metal-binding agents and substrate analogues were not inhibitory. Thiol reagents gave varying degrees of inhibition, the most potent being p-hydroxymercuribenzoate which at 1 mM gave 100% inhibition. The importance of possessing an NAD(P)+-linked formaldehyde dehydrogenase, with respect to M. capsulatus, is discussed.     

Isolation and characterization of valine dehydrogenase from Streptomyces aureofaciens.

Valine dehydrogenase was purified to homogeneity from the crude extracts of Streptomyces aureofaciens. The molecular weight of the native enzyme was 116,000 by equilibrium ultracentrifugation and 118,000 by size exclusion high-performance liquid chromatography. The enzyme was composed of four subunits with molecular weights of 29,000. The isoelectric point was 5.1. The enzyme required NAD+ as a cofactor, which could not be replaced by NADP+. Sulfhydryl reagents inhibited the enzyme activity. The pH optimum was 10.7 for oxidative deamination of L-valine and 9.0 for reductive amination of alpha-ketoisovalerate. The Michaelis constants were 2.5 mM for L-valine and 0.10 mM for NAD+. For reductive amination the Km values were 1.25 mM for alpha-ketoisovalerate, 0.023 mM for NADH, and 18.2 mM for NH4Cl.     

Purification and Properties of Hypoxanthine Phosphoribosyltransferase from Streptomyces cyanogenus

Hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) of a strain of Streptomyces cyanogenus was purified 1,900-fold to an apparent homogenity from cell-free extracts. The enzyme had a molecular weight of 150,000 and consisted of eight identical subunits with a molecular weight of 18,000. The isoelectric point was at pH 4.4. The enzyme required Mg²⁺ or Ma²⁺ for activity and had a pH optimum at 8.5. Hypoxanthine and guanine were good substrates for the enzyme. Xanthine was a very poor substrate and adenine was not a substrate. Apparent Km values of the enzyme for hypoxanthine, guanine and 5-phosphoribose-1-pyro-phosphate were 1.6 × 10^?8, 2.7 × 10^?6 and 6.3 × 10^?5 m, respectively. All purine nucleotides tested inhibited the activity significantly, apparently by competing with 5-phosphoribose-1-pyrophosphate.     

Purification and characterization of Escherichia coli xanthine-guanine phosphoribosyltransferase produced by plasmid pSV2gpt

The enzyme xanthine-guanine phosphoribosyltransferase from Escherichia coli cells harboring the plasmid pSV2gpt has been purified 30-fold to near homogeneity by single-step GMP-agarose affinity chromatography. It has a Km value of 2.5, 42 and 182 microM for the substrates guanine, xanthine and hypoxanthine, respectively, with guanine being the most preferred substrate. The enzyme exhibits a Km value of 38.5 microM for PRib-PP with guanine as second substrate and of 100 microM when xanthine is used as the second substrate. It is markedly inhibited by 6-thioguanine, GMP and to a lesser extent by some other purine analogues. Thioguanine has been found to be the most potent inhibitor. The subunit molecular weight of xanthine-guanine phosphoribosyltransferase was determined to be 19 000. The in situ activity assay on a nondenaturing polyacrylamide gel electrophoresis gel has indicated that a second E. coli phosphoribosyltransferase preferentially uses hypoxanthine as opposed to guanine as a substrate, and it does not use xanthine.     

Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM 462. Purification, characterization and kinetic mechanism

Alanine dehydrogenase (L-alanine: NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 degrees C for at least 60 min and with incubation in pH 5.5-9.5 at 75 degrees C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (-20 degrees C, at pH 7.2) for over 5 months. The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2-oxobutyrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L-alanine then NAD+. A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction. A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM.     

Effect of NADH on hypoxanthine hydroxylation by native NAD+-dependent xanthine oxidoreductase of rat liver, and the possible biological role of this effect.

The course of the reaction sequence hypoxanthine leads to xanthine leads to uric acid, catalysed by the NAD+-dependent activity of xanthine oxidoreductase, was investigated under conditions either of immediate oxidation of the NADH formed or of NADH accumulation. The enzymic preparation was obtained from rat liver, and purified 75-fold (as compared with the 25000 g supernatant) on a 5'-AMP-Sepharose 4B column; in this preparation the NAD+-dependent activity accounted for 100% of total xanthine oxidoreductase activity. A spectrophotometric method was developed for continuous measurements of changes in the concentrations of the three purines involved. The time course as well as the effects of the concentrations of enzyme and of hypoxanthine were examined. NADH produced by the enzyme lowered its activity by 50%, resulting in xanthine accumulation and in decreases of uric acid formation and of hypoxanthine utilization. The inhibition of the Xanthine oxidoreductase NAD+-dependent activity by NADH is discussed as a possible factor in the regulation of IMP biosynthesis by the 'de novo' pathway or (from unchanged hypoxanthine) by ther salvage pathway.     

Purification and Properties of Uricase from Enterobacter cloacae

Uricase activity was found in Enterobacter cloacae KY3074 grown on guanine, hypoxanthine, uric acid, and xanthine media. The enzyme was purified from cells grown on uric acid as a source of nitrogen. The purification procedure included ammonium sulfate fractionation, gel filtration on Sephadex G-150, and column chromatography on DEAE-cellulose and DEAE-Sephadex. The enzyme had a molecular weight of about 105,000 and was specific for uric acid. The optimum pH was around 9.5, and the activity was inhibited by the presence of potassium cyanide, Ag⁺ or Cu²⁺. This uricase can be used for estimation of uric acid.     

Isolation and characterization of xanthine dehydrogenase from Chlamydomonas reinhardtii

Xanthine dehydrogenase (XDH, EC 1.2.1.37) of Chlamydomonas reinhardtii (Sager) 6145c wild strain has been isolated and characterized for the first time in a unicellular green alga. The enzyme has an M_r of 330 kDa, and FAD, molybdenum and iron are cofactors required for its activity as deduced from results obtained using specific inhibitors, ⁵⁹Fe-labelling experiments, activity protection by FAD, physiological responses in vivo to iron and molybdenum deficiencies in the culture medium and work with mutants lacking molybdenum cofactor. Xanthine dehydrogenase exhibited Mi-chaelian kinetics typical for a bisubstrate enzyme with apparent K_m values for NAD ⁺, hypoxanthine and xanthine of 35, 160 and 70 μ M , respectively. Under phototrophic conditions enzyme activity was repressed by ammonium, but xanthine was not required for the enzyme to be induced, since high levels of enzyme activity were found in cells grown on ammonium and transferred to either N-frec media or media containing either of the nitrogen sources adenine, urea, urate, xanthine, hypoxanthine and guanine.     

Purification and characterization of the NAD-dependent glutamate dehydrogenase in the ectomycorrhizal fungus laccaria bicolor (Maire) orton

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) from Laccaria bicolor was purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at -75 degrees C, 4 days at 4 degrees C, and 1 h at 50 degrees C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at -75 degrees C. NAD-GDH activity was stimulated by Ca2+ and Mg2+ but strongly inhibited by Cu2+ and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 &mgr;M, 89 &mgr;M, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and its Km value increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid. Copyright 1997 Academic Press. Copyright 1997 Academic Press     

Purification and characterization of hypoxanthine-guanine phosphoribosyltransferase from Schistosoma mansoni. A potential target for chemotherapy

Hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase activities are essential for the supply of guanine nucleotides in Schistosoma mansoni schistosomules. In crude extracts of adult S. mansoni, these two activities co-elute in size exclusion, ion exchange, and chromatofocusing chromatography and exhibit similar stabilities to heat treatment, suggesting that they are associated in one enzyme protein hypoxanthine-guanine phosphoribosyltransferase. This enzyme has been purified by a combination of heat treatment at 85 degrees C and chromatofocusing chromatography with elution at an apparent pI of 5.27 +/- 0.15. Pore gradient electrophoresis of the native enzyme followed by subsequent activity staining demonstrate an enzyme molecular weight of 105,000. The activity staining pattern remains the same whether hypoxanthine or guanine is used as the substrate, further supporting the existence of a single protein, hypoxanthine-guanine phosphoribosyltransferase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein results in a single protein band with a subunit molecular weight estimate of 64,000, suggesting that the native enzyme is a dimer. Preliminary kinetic studies showed that the purified hypoxanthine-guanine phosphoribosyltransferase reacted with guanine at a rate twice as fast as it did with hypoxanthine, but it did not act on xanthine at all. A full-length mouse neuroblastoma hypoxanthine-guanine phosphoribosyltransferase cDNA clone pHPT5 and a plasmid pSV2-gpt containing the xanthine-guanine phosphoribosyltransferase gene for Escherichia coli were utilized as probes on Southern blots of S. mansoni DNA digests, and no significant hybridization was found under relatively relaxed conditions. Polyclonal antibodies made against human erythrocyte hypoxanthine-guanine phosphoribosyltransferase and E. coli xanthine-guanine phosphoribosyltransferase were tested in enzyme-linked immunosorbent assays of S. mansoni protein extracts, and no detectable cross-reacting protein was found. S. mansoni hypoxanthine-guanine phosphoribosyltransferase thus may bear rather limited homology to mammalian hypoxanthine-guanine phosphoribosyltransferase or bacterial xanthine-guanine phosphoribosyltransferase and could be an attractive target for antischistosomal chemotherapeutic drug design.     

Characterization of recombinant Drosophila melanogaster myo-inositol-1-phosphate synthase expressed in Escherichia coli

Cloned myo-inositol-1-phpsphate synthase (INOS) of Drosophila melanogaster was expressed in Escherichia coli, and purified using a His-affinity column. The purified INOS required NAD+ for the conversion of glucose-6-phosphate to inositol-1-phosphate. The optimum pH for myo-inositol-1-phosphate synthase is 7.5, and the maximum activity was measured at 40 degrees C. The molecular weight of the native enzyme, as determined by gel filtration, was approximately Mr 271,000 +/- 15,000. A single subunit of approximately Mr 62,000 +/- 5,000 was detected upon SDS-polyacrylamide gel electrophoresis. The Michaelis (Km) and dissociation constants for glucose-6-phosphate were 3.5 and 3.7 mM, whereas for the cofactor NAD+ these were 0.42 and 0.4 mM, respectively.     

Comparison of xanthine:NAD+ oxidoreductase from liver of two uricotelic species: hen Gallus gallus and snake Natrix natrix

1. Kinetic properties of xanthine:NAD+ oxidoreductase from liver of two uricotelic species of vertebrates (hen Gallus gallus and snake Natrix natrix) are compared. 2. Hen enzyme is saturated by hypoxanthine and xanthine at higher concentrations than the snake enzyme. For both species the enzyme-saturating concentration and hydroxylation rate of hypoxanthine are higher than those of xanthine, and the rate of uric acid production in the hypoxanthine----xanthine----uric acid reaction sequence is independent of the initial hypoxanthine concentration. 3. Km's for xanthine are the same, but Km for NAD+ of the hen enzyme is approximately 5-fold lower. The enzyme from both species is inhibited by NADH only slightly and at high non-physiological concentrations.     

Soybean nodule xanthine dehydrogenase: a kinetic study

Xanthine dehydrogenase was purified from soybean nodules and the kinetic properties were studied at pH 7.5. Km values of 5.0 +/- 0.6 and 12.5 +/- 2.5 microM were obtained for xanthine and NAD+, respectively. The pattern of substrate dependence suggested a Ping-Pong mechanism. Reaction with hypoxanthine gave Km's of 52 +/- 3 and 20 +/- 2.5 microM for hypoxanthine and NAD+, respectively. The Vmax for this reaction was twice that for the xanthine-dependent reaction. The pH dependence of Vmax gave a pKa of 7.6 +/- 0.1 for either xanthine or hypoxanthine oxidation. In addition the Km for xanthine had a pKa of 7.5 consistent with the protonated form of xanthine being the true substrate. Km for hypoxanthine varied only 2.5-fold between pH 6 and 10.7. Product inhibition studies were carried out with urate and NADH. Both products gave mixed inhibition with respect to both substrates. Xanthine dehydrogenase was able to use APAD+ as an electron acceptor for xanthine oxidation, with a Km at pH 7.5 of 21.2 +/- 2.5 microM and Vmax the same as that obtained with NAD+. Reduction of APAD+ by NADH was also catalyzed by xanthine dehydrogenase with a Km of 102 +/- 15 microM; Vmax was approximately 2.5 times that for the xanthine-dependent reaction, and was independent of pH between 6 and 9. Reaction with group-specific reagents indicated the possibility of an essential histidyl group. A thiol-modifying reagent did not cause inactivation of the enzyme. A role for the histidyl side chain in catalysis is proposed.     

Purification and properties of milk xanthine dehydrogenase.

Milk xanthine oxidase (XO) has been prepared in a dehydrogenase form (XDH) by purifying the enzyme in the presence of 2.5 mM dithiothreitol. Unlike XO, which reacts rapidly only with oxygen and not with NAD, the XDH form of the enzyme reacts rapidly with NAD. XDH has a turnover number for the NAD-dependent conversion of xanthine to urate of 380 mol/min/mol at pH 7.5, 25 degrees C, with a Km = < or = 1 microM for xanthine and a Km = 7 microM for NAD, but has very little O2-dependent activity. There is evidence that the two forms of the enzyme have different flavin environments: XDH stabilizes the neutral form of the flavin semiquinone and XO does not. Further, XDH binds the artificial flavin 8-mercapto-FAD in its neutral form, shifting the pK of this flavin by 5 pH units, while XO binds 8-mercapto-FAD in its benzoquinoid anionic form. XDH can be converted back to the XO form by the addition of three to four equivalents of the disulfide-forming reagent 4,4'-dithiodipyridine, suggesting that, in the XDH form of the enzyme, disulfide bonds are broken; this may cause a conformational change which creates a binding site for NAD and changes the protein structure near the flavin.     

Characterization of guanine and hypoxanthine phosphoribosyltransferases in Methanococcus voltae.

Phosphoribosyltransferase (PRTase) and nucleoside phosphorylase (NPase) activities were detected by radiometric methods in extracts of Methanococcus voltae. Guanine PRTase activity was present at 2.7 nmol min(-1) mg of protein(-1) and had an apparent Km for guanine of 0.2 mM and a pH optimum of 9. The activity was inhibited 50% by 0.3 mM GMP. IMP and AMP were not inhibitory at concentrations up to 0.6 mM. Hypoxanthine inhibited by 50% at 0.16 mM, and adenine and xanthine were not inhibitory at concentrations up to 0.5 mM. Guanosine NPase activity was present at 0.01 nmol min(-1) mg of protein(-1). Hypoxanthine PRTase activity was present at 0.85 nmol min(-1) mg of protein(-1) with an apparent Km for hypoxanthine of 0.015 mM and a pH optimum of 9. Activity was stimulated at least twofold by 0.05 mM GMP and 0.2 mM IMP but was unaffected by AMP. Guanine inhibited by 50% at 0.06 mM, but adenine and xanthine were not inhibitory. Inosine NPase activity was present at 0.04 nmol min(-1) mg of protein(-1). PRTase activities were not sensitive to any base analogs examined, with the exception of 8-azaguanine, 8-azahypoxanthine, and 2-thioxanthine. Fractionation of cell extracts by ion-exchange chromatography resolved three peaks of activity, each of which contained both guanine and hypoxanthine PRTase activities. The specific activities of the PRTases were not affected by growth in medium containing the nucleobases. Mutants of M. voltae resistant to base analogs lacked PRTase activity. Two mutants resistant to both 8-azaguanine and 8-azahypoxanthine lacked activity for both guanine and hypoxanthine PRTase. These results suggest that analog resistance was acquired by the loss of PRTase activity.     

Purine degradation in Pseudomonas aeruginosa and Pseudomonas testosteroni.

1. Adenine, hypoxanthine, xanthine and guanine are broken down in Pseudomonas aeruginosa and Pseudomonas testosteroni to allantoin by the concerted action of the enzymes adenine deaminase, guanine deaminase, NAD+-dependent xanthine dehydrogenase and uricase. 2. Uric acid is broken down by an unstable, membrane-bound uricase with an unusually low pH optimum. 3. In both strains adenine inhibits growth and xanthine dehydrogenase. A second type of inhibition is manifest only in Ps. testosteroni and concerns the regulation of the biosynthesis of amino acids of the aspartate family. Enzymic studies showed that in this strain aspartate kinase is inhibited by AMP.     

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.