Purine ribonucleoside and deoxyribonucleoside kinase activities in thymocytes. Specificity and optimal assay conditions for phosphorylation

The optimal assay conditions and specificity for the principal reactions of purine nucleoside phosphorylation were studied in mouse thymocytes. The following relative activities were obtained for the nucleoside substrates: adenosine, 100; deoxyguanosine, 24; and deoxyadenosine, 14. The phosphorylation of adenosine, 45 microM, was optimal between pH 5.8 and 6.0 with a millimolar Mg:ATP ratio of 1:5. This activity was insensitive to inhibition by other nucleosides and dCTP. Optimal phosphorylation of deoxyguanosine, 350 microM, occurred at pH 8.4 with a millimolar Mg:ATP ratio of 10:3.5. Phosphorylation of 80 microM deoxyguanosine was inhibited approximately 90% by 10 microM deoxycytidine or dCTP and was inhibited 70% by 200 microM deoxyadenosine but unaffected by adenosine. Deoxyadenosine, 450 microM, phosphorylation was optimal between pH 6.5 and 8.5 with a millimolar Mg:ATP ratio of 5:1. Phosphorylation of deoxyadenosine, 100 microM, was partially inhibited by 200 microM adenosine, 34%; 200 microM deoxyguanosine, 10%; and 100 microM deoxycytidine or dCTP, 33%. Only deoxyadenosine phosphorylation was inhibited by 200 microM deoxyinosine, 10%. These results and those obtained from isokinetic sucrose density gradient analysis are consistent with there being a specific adenosine kinase, a faster sedimenting deoxycytidine kinase of broad specificity which also catalyzes the phosphorylation of deoxyguanosine and deoxyadenosine, and a specific deoxyguanosine kinase sedimenting more rapidly than either of the other activities.     

Deoxyguanosine kinase from human placenta

Deoxyguanosine kinase (ATP:deoxyguanosine 5'-phosphotransferase) has been purified up to a specific activity of 10.3 nmol/min per mg protein from human placenta. The enzyme appears to have a molecular weight of 58 000 from the results of Sephadex G-75 gel filtration. The enzyme catalyzed phosphorylation of deoxyguanosine and deoxyadenosine, but deoxycytidine was not phosphorylated. An apparent Km value for deoxyguanosine was 2.5 micro M. When ATP was used as a phosphate donor, the pH optimum was at pH 6.0, but the optimum was shifted to pH 6.8 by the addition of dTTP. At physiological pH, the activity was stimulated 3-4-fold by dTTP. dTTP was also an effective phosphate donor, but using dTTP as a phosphate donor, a broad pH optimum of 7.0 was observed. Two Km values of 0.13 and 2.2 mM were obtained for both MgATP2- and MgdTTP2-. The activity was strongly inhibited by dGTP and dGDP; 50% inhibition by 1.0 micro M dGTP and 2.1 micro M dGDP, respectively. The enzyme required the presence o Mg2+ or Mn2+.     

Identification of purine deoxyribonucleoside kinases from human leukemia cells: substrate activation by purine and pyrimidine deoxyribonucleosides

Cell extracts from human leukemic T lymphoblasts and myeloblasts were chromatographed on DEAE-cellulose columns to separate purine deoxyribonucleoside, deoxyadenosine (dAdo) and deoxyguanosine (dGuo), phosphorylating activities. Three distinct purine deoxyribonucleoside kinases, a deoxycytidine (dCyd) kinase, an adenosine (Ado) kinase, and a deoxyguanosine (dGuo) kinase (the latter appears to be localized in mitochondria), were resolved. dCyd kinase contained the major phosphorylating activity for dAdo, dGuo, and 9-beta-D-arabinofuranosyladenine (ara-A). Ado kinase represented a second kinase for dAdo and ara-A while a third kinase for dAdo was found in mitochondria. dCyd kinase was purified about 2000-fold with ion-exchange, affinity, and hydrophobic chromatographies. On gel electrophoresis, both dCyd and dAdo phosphorylating activities comigrated, indicating that the activities are associated with the same protein. The enzyme showed a broad pH optimum ranging from pH 6.5 to pH 9.5. Divalent cations Mg2+, Mn2+, and Ca2+ stimulated dCyd kinase activity; Mg2+ produced the maximal activity. dCyd kinase from either lymphoid or myeloid cells showed broad substrate specificity. The enzyme used several nucleoside triphosphates, but ATP, GTP, and dTTP were the best phosphate donors. dCyd was the best nucleoside substrate, since dCyd kinase had an apparent Km of 0.3, 85, 90, and 1400 microM for dCyd, dAdo, dGuo, and ara-A, respectively. The enzyme exhibited substrate activation with both pyrimidine and purine deoxyribonucleosides, suggesting that there is more than one substrate binding site on the kinase. These studies show that, in lymphoblasts and myeloblasts, purine deoxyribonucleosides and their analogues are phosphorylated by dCyd kinase, Ado kinase, and dGuo kinase.     

Regulation of deoxyadenosine and nucleoside analog phosphorylation by human placental adenosine kinase

The enzymes responsible for the phosphorylation of deoxyadenosine and nucleoside analogs are important in the pathogenesis of adenosine deaminase deficiency and in the activation of specific anticancer and antiviral drugs. We examined the role of adenosine kinase in catalyzing these reactions using an enzyme purified 4000-fold (2.1 mumol/min/mg) from human placenta. The Km values of deoxyadenosine and ATP are 135 and 4 microM, respectively. Potassium and magnesium are absolute requirements for deoxyadenosine phosphorylation, and 150 mM potassium and 5 mM MgCl2 are critical for linear kinetics. With only 0.4 mM MgCl2 in excess of ATP levels, the Km for deoxyadenosine is increased 10-fold. ADP is a competitive inhibitor with a Ki of 13 microM with variable MgATP2-, while it is a mixed inhibitor with a Ki and Ki' of 600 and 92 microM, respectively, when deoxyadenosine is variable. AMP is a mixed inhibitor with Ki and Ki' of 177 and 15 microM, respectively, with variable deoxyadenosine; it is a non-competitive inhibitor with a Ki of 17 microM and Ki' of 27 microM with variable ATP. Adenosine kinase phosphorylates adenine arabinoside with an apparent Km of 1 mM using deoxyadenosine kinase assay conditions. The Km values for 6-methylmercaptopurine riboside and 5-iodotubercidin, substrates for adenosine kinase, are estimated to be 4.5 microM and 2.6 nM, respectively. Other nucleoside analogs are potent inhibitors of deoxyadenosine phosphorylation, but their status as substrates remains unknown. These data indicate that deoxyadenosine phosphorylation by adenosine kinase is primarily regulated by its Km and the concentrations of Mg2+, ADP, and AMP. The high Km values for phosphorylation of deoxyadenosine and adenine arabinoside suggest that adenosine kinase may be less likely to phosphorylate these nucleosides in vivo than other enzymes with lower Km values. Adenosine kinase appears to be important for adenosine analog phosphorylation where the Michaelis constant is in the low micromolar range.     

Purification and properties of a kinase from Escherichia coli K-12 that phosphorylates two periplasmic transport proteins

The phosphorylation in vivo and in vitro of the arginine-ornithine and the lysine-arginine-ornithine (LAO) periplasmic transport proteins of Escherichia coli K-12 was previously reported (Celis, R. T. F. (1984) Eur. J. Biochem. 145, 403-411). The phosphorylative reaction required ATP (as a direct energy donor), Mg2+, and a kinase that can be released by osmotic shock treatment of the cells. The enzyme was purified to electrophoretic homogeneity. The enzyme exhibited an ATPase activity and a kinase activity. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gave an apparent molecular weight of 43,000 for the enzyme. The native protein showed the same molecular weight, suggesting that the protein is a monomer. The protein showed an apparent isoelectric point of 4.8 on isoelectric focusing. The two enzymatic reactions required a divalent cation and the apparent Km value for Mg2+ for the kinase activity was 0.5 mM. Mn2+ and Co2+ served as well as Mg2+, whereas Zn2+ and Ca2+ did not support activity. The ATPase activity of the enzyme yielded an apparent Km value for ATP of 50 microM. A similar value, Km of 100 microM, was calculated for the kinase activity with different concentrations of ATP. The enzyme showed a pH optimum of 7.3.     

Measurement and kinetic study of the formation of adrenaline sulphate in vitro.

The sulpho-conjugation of [14C]adrenaline form inorganic sulphate and ATP or preformed adenosine 3'-phosphate 5'-sulphatophosphate was demonstrated in the high-speed supernatant prepared from the liver and small intestine of various animals. Hydrolysis with sulphatase indicated the sulphate nature of the conjugate. The overall sulphation reaction has a pH optimum of 9.0. Maximal activity was obtained with a ratio of ATP/Mg2+ of 1 at 4--6mM. Above their optimal concentrations, ATP and Mg2+, separately or in combination, were inhibitory. Dithiothreitol at 3 mM stimulated the reaction by about 30%. The Km for adrenaline, determined by the sulphotransferase reaction and by the three-step (sulphate-activating and sulphotransferase) reactions was 125 micrometer. The rate of synthesis of [14C]-adrenaline sulphate, expressed in pmol/min per mg of protein for the livers of dog, monkey, rat, guinea pig and rabbit were, respectively, 144, 77, 47, 11 and 6. The corresponding values for the small intestines of dog and monkey were 60 and 62. Brain and heart tissues showed no measurable activity.     

Nucleoside-dependent protein kinase from Trypanosoma gambiense.

A nucleoside-dependent protein kinase (EC 2.7.1.37) was partially purified from Trypanosoma gambiense, the pathogenic agent of sleeping sickness. This enzyme catalyzes the phosphorylation of histone and protamine. Various nucleosides at the concentration of 10(-4) M stimulated the histone kinase activity about two-fold, whereas cyclic AMP and cyclic GMP were without effect. The pH-optimum for histone phosphorylation was at about pH 7.0. The enzyme activity absolutely depends on Mg2+, Mn2+ or Co2+. The apparent Km-value for histone was 0.3 mg/ml and those for ATP were 2 - 10(-4) M and 6 - 10(-5) M in the absence or presence of 10(-4) M adenosine respectively. IDP and ADP complete with ATP. The inhibition constants were calculated to be 2 - 10(-4) M and 2.5 - 10(-4) M, respectively. The molecular weight of the histone kinase was found to be 95 000 by gel filtration and 88 000 by sedimentation in a sucrose gradient.     

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.     

Demonstration of protein kinase activities in the coronary artery of cattle]

Protein kinase activities were identified in a soluble and a particulate fraction from the A. coronaria of cattle. For both protein kinase activities Mg++ is essential. Protamine was used as a substrate of the protein kinase activity of the soluble fraction. The pH optimum of the protein kinase activity of the soluble fraction is around 6.5. The Km-value of the protein kinase for ATP is 1.9 +/- 0.4 - 10(-5) M. cAMP stimulates the protein kinase activity more effectively than cGMP. Ca++ cannot replace Mg++; monovalent cations (Na+ and K+) show no influence. The protein kinase activity of the fraction was determined via endogenous phosphorylation. By means of the cAMP-dependent particulate protein kinase 72 to 80 percent of the serine residues are phosphorylated. The pH optimum of the protein kinase activity of the particulate fraction lies around 7.0. The Km-value of the enzyme for ATP is 6.6 +/- 0.8 - 10(-5) M. cGMP stimulates the protein kinase of the particulate fraction better than cAMP. For the protein kinase activity of this fraction Ca++ replaces Mg++ in the endogenous phosphorylation but not in the exogenous phosphorylation (protamine). In the presence of Mg++ and in the additional presence of Na+ or K+, the protein kinase activity is suppressed in the endogenous phosphorylation whereas it is stimulated in the exogenous phosphorylation.     

Human T-lymphoblast deoxycytidine kinase: purification and properties

Previous observations present tremendous variations in the properties of deoxycytidine kinase. To clarify the properties and physiologic role of deoxycytidine kinase, we have undertaken its purification. Deoxycytidine kinase was purified from cultured human T-lymphoblasts (MOLT-4) to 90% purity with an estimated specific activity of 8 mumol min-1 (mg of protein)-1. The purification procedure included ammonium sulfate precipitation, Superose-12 HPLC gel filtration chromatography, DE-52 ion-exchange chromatography, AMP-Sepharose 4B affinity chromatography, and dCTP-Sepharose-4B affinity chromatography. Deoxyguanosine, deoxyadenosine, and cytidine phosphorylating activities copurified with deoxycytidine kinase to final specific activities of 7.2, 13.5, and 4 mumol min-1 (mg of protein)-1, respectively. The enzyme is very unstable at low protein concentration and is stabilized by storage at -85 degrees C with 1 mg/mL bovine serum albumin, 20% glycerol (v/v), 200 mM potassium chloride, and 25 mM dithiothreitol. The molecular weight was 60,000, and the Stokes radius was 32 A by gel filtration chromatography. The subunit molecular weight was 30,500. This enzyme had apparent Km values of 1.5, 430, 500, 450, and 40 microM for deoxycytidine, deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside, respectively. The pH optimum ranged from 6.5 to 9.0. Mg2+ and Mn2+ were the preferred divalent cations. ATP, GTP, dGTP, ITP, dITP, TTP, and XTP were substrates for the enzymes. Our study indicates that deoxycytidine kinase is a dimer with two subunits and has phosphorylating activity for deoxyguanosine, deoxyadenosine, cytidine, and cytosine arabinoside. This highly purified enzyme will facilitate the study of its regulation and phosphorylation of anticancer or antiviral nucleoside analogues.     

Isolation and characterization of adenosine kinase from Leishmania donovani

Adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) has been purified 3250-fold from Leishmania donovani promastigotes using ion-exchange, gel filtration, and affinity chromatography techniques. Both native and sodium dodecyl sulfate-gel electrophoresis of the enzyme revealed a single polypeptide of around 38,000 molecular weight. Biophysical and biochemical analyses of the enzyme reveal unique characteristics different from those of adenosine kinases from other eukaryotic sources. The isoelectric pH of the enzyme is 8.8. In native acrylamide gels the enzyme moves with an RF of about 0.62. The enzyme displays a maximum activity at pH between 7.5 and 8.5 and is dependent upon an optimum ATP/Mg2+ ratio. ATP at high concentration inhibits the reaction. Adenosine and Mg2+ are not inhibitory. EDTA completely knocks off the activity. Enzyme activity is dependent upon the presence of active thiol group(s) at or near the active center. Under a defined set of conditions the enzyme exhibited an apparent Km for adenosine and ATP of 33 and 50 microM, respectively. Of the nucleoside triphosphates tested ATP and GTP were the most effective phosphate donors. Marginal inhibition of activity was detected with other nucleosides as competitors. However, adenosine analogs, such as 7-deaza-adenosine (tubercidin) and 6-methylmercaptopurine riboside at very low concentrations, were found to be excellent inhibitors and substrates as well. S-Adenosylhomocysteine does not inhibit the reaction even at very high concentration.     

Purification and properties of the adenosine deaminase from the midgut gland of a marine bivalved mollusc, Atrina spp.

1. The adenosine deaminase has an approximate molecular weight of 130,000-140,000 and the composition of two polypeptide units (mol. wt about 68,000) is suggested, by means of SDS disc electrophoresis. 2. Both the alpha (Vm/Km) and beta (Vm) parameters were varied with pH and temperature. RSS (relative substrate specificity) adenosine and deoxyadenosine values for alpha and beta were 1.2 and 1.1, respectively. 3. Adenine, 2'-, 3', 5'-AMP, 5'-deoxyAMP, ADP and ATP were not deaminated by the enzyme. 4. Inhibition by Mg2+ was found in reaction with adenosine at pH 8 but not with deoxyadenosine at the same pH. Mn2+, which did not affect the reaction rate at pH 4 and 5, showed competitive inhibitory effects at pH 6, 7 and 8.     

Purification and characterization of a myosin heavy chain kinase from Dictyostelium discoideum

A Dictyostelium discoideum myosin heavy chain kinase has been purified 14,000-fold to near homogeneity. The enzyme has a Mr = 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and greater than 700,000 as determined by gel filtration on Bio-Gel A-1.5m. The enzyme has a specific activity of 1 mumol/min X mg when assayed at a Dictyostelium myosin concentration of 0.3 mg/ml. A maximum of 2 mol of phosphate/mol of myosin is incorporated by the kinase, and the phosphorylated amino acid is threonine. Phosphate is incorporated only into the myosin heavy chains, not into the light chains. The actin-activated Mg2+-ATPase of Dictyostelium myosin is inhibited 70-80% following maximal phosphorylation with the kinase. The myosin heavy chain kinase requires 1-2 mM Mg2+ for activity and is most active at pH 7.0-7.5. The activity of the enzyme is not significantly altered by the presence of Ca2+, Ca2+ and calmodulin, EGTA, cAMP, or cGMP. When incubated with Mg2+ and ATP, phosphate is incorporated into the myosin heavy chain kinase, perhaps by autophosphorylation.     

Purification and characterization of phosphatidylinositol kinase from porcine liver microsomes

Phosphatidylinositol kinase was solubilized and purified from porcine liver microsomes to apparent homogeneity. The purification procedure includes: solubilization of microsomes by 2% Triton X-100, ammonium sulfate precipitation (20-35% saturation), Reactive blue agarose chromatography, DEAE-Sephacel chromatography and two consecutive hydroxyapatite chromatographies. A total of 4900-fold purification with 8% recovery of enzyme activity was achieved. The molecular weight of the enzyme as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 55000. The enzyme is stimulated in a decreasing order by Mg2+, Fe2+, Mn2+, Fe3+ and Co2+. Ca2+ inhibited Mg2+-stimulated activity with an I50 of 0.4 mM. Apparent Km values for phosphatidylinositol and ATP are 120 and 60 microM, respectively. The enzyme is inhibited by adenosine (I50 = 70 microM), ADP (I50 = 120 microM) and quercetin (I50 = 100 microM). The enzyme is also sensitive to sulfhydryl inhibitors. Using the purified enzyme as an immunogen, we have successfully prepared antibodies for phosphatidylinositol kinase in rabbits. The antibodies appear to recognize an antigen of Mr 55000 on SDS-polyacrylamide gel electrophoresis from various porcine tissues in Western blot analysis.     

Properties of rat heart adenosine kinase.

Adenosine kinase was purified 870-fold from rat heart by a combination of gel filtration and affinity chromatography. The preparation was free of purine-metabolizing enzymes that could interfere in the assay of the kinase. A study of the properties of the purified enzyme showed that it is activated by Na+ and K+, it possesses a broad pH optimum between 6 and 8, MgATP is the nucleotide substrate, free Mg2+ is an inhibitor with respect to both MgATP and adenosine, and the enzyme is subject to substrate inhibition by adenosine. The severity of this inhibition increases as the concentration of free Mg2+ increase. The Km for MgATP was calculated to be 0.8 mM and that for adenosine, at likely physiological concentrations of MgATP and free MgCl2, was about 0.2 microM. In vivo the enzyme is likely to be saturated with both MgATP and adenosine. Indeed, the adenosine concentration in rat heart in vivo is probably sufficient to cause substrate inhibition, and this would be increased by an increase in free Mg2+ concentration. Changes in the concentrations of adenosine and free Mg2+ may play a role in modifying the activity of the enzyme in vivo.     

Affinity chromatographic purification and properties of flavokinase (ATP:riboflavin 5'-phosphotransferase) from rat liver

Flavokinase (ATP:riboflavin 5'-phosphotransferase, EC 2.7.1.26) has been purified to apparent homogeneity from rat liver by affinity chromatography using flavinyl agarose beads (agarose-OCH2CONH(CH2)2NHCO(CH2)/N10-7,8-dimethylisoalloxazine). The specific activity of the pure enzyme is 9,900 units (nmol of FMN formed/h at 37 degrees C)/mg of protein, and reflects a one-step, 7000-fold purification. Flavokinase thus obtained, unlike previous preparations from mammalian sources, is free from contaminating phosphatase and FAD synthase. The purified enzyme rapidly loses activity upon storage but is stabilized by riboflavin and thiol-protecting reagents. The apparent molecular weight, estimated by gel filtration on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is 28,000 +/- 1,000. Flavokinase phosphorylates and/or is inhibited by a large number of riboflavin analogs; however, the physiologically important 8 alpha-(amino acid)riboflavins are poorly accommodated. The strongly preferred phosphate donors are ATP and dATP. Both Zn2+ and Mg2+, as well as several other divalent cations, activate flavokinase, but Zn2+ yields greatest activity (1.8 times that with Mg2+). The pH optimum for activity with either Zn2+ or Mg2+ is approximately 9.3; at pH 7.0, the activity is 40% of that at the pH optimum.     

Isolation and characterization of tropomyosin kinase from chicken embryo

Tropomyosin kinase is partially purified from 14-day-old chicken embryos using DEAE-cellulose, cellulose phosphate and gel filtration chromatography. The purest enzyme preparation consists of two major bands of Mr = 76,000 and 43,000 on SDS-polyacrylamide gel electrophoresis. The molecular weight of the enzyme is 250,000 determined by gel filtration chromatography. It phosphorylates casein and skeletal tropomyosin equally well but histone and phosvitin at a much slower rate. Smooth muscle myosin light chain, tropomyosin from platelet, erythrocyte and smooth muscle are not phosphorylated. The apparent Km for skeletal alpha-tropomyosin and ATP is 50 microM and 200 microM, respectively. Vmax varies between 100-300 nmol/min per mg depending on the purity of the preparation. Mg2+ and dithiothreitol are essential for activity but Ca+, calmodulin and cAMP are not required. The optimum temperature is 37 degrees C and optimum pH is about 7.5. Heparin, a potent inhibitor of casein kinase II, has no inhibitory effect on the enzyme. Similar tropomyosin kinase activity is not detected in skeletal muscle in adult rabbit and chicken. The tropomyosin kinase described here represents a hitherto uncharacterized kinase responsible for phosphorylation of tropomyosin in the chicken embryo.     

Purification and properties of mitochondrial adenosine triphosphatase of hamster brown adipose tissue.

1. Oligomycin-insensitive ATPase (ATP phosphohydrolase, EC 3.6.1.3) was purified from brown adipose tissue mitochondria. It had a specific activity of 50 units/mg which could be increased up to 85 units/mg by KHCO3. The isolated enzyme represented less than 0.5% of the initial membrane proteins.2. The enzyme had a molecular weight equal to beef heart ATPase and was composed of five subunits with molecular weights of 56 200, 54 300, 33 500, 13 400 and 9500 respectively. 3. Isolated ATPase was labile while cold and was activated by the divalent cations Mn2+, Mg2+, Co2+ and Cd2+. The optimum ATP/Mg2+ ratio found was 1.58 and the enzyme had a maximum activity at pH 8.5; the Km was 220 micrometer. 4. The ATPase activity was 55% inhibited by aurovertin. The isolated enzyme enhanced the fluorescence of aurovertin, quenched by ATP and Mg2+ and enhanced by ADP. 5. Oligomycin sensitivity and cold stability of isolated ATPase was restored by its reconstitution with both brown adipose tissue and beef heart particles depleted of ATPase. 6. The results presented demonstrate that the low ATPase activity of brown adipose tissue mitochondria is due to a reduced content of ATPase.     

Purification and various properties of pantothenate kinase from the rat liver

Homogeneous (according to disc gel electrophoresis data) ATP: D-pantothenate-4'-phosphotransferase (pantothenate kinase, EC 2.7.1.33) was obtained from rat liver cytosol of heterogeneous stock rats. The enzyme was purified 199-fold with a 9.3% yield. The enzyme was relatively unstable but retained its activity in the presence of 10% glycerol containing 5.10(-4) M ATP over 10 days at 4 degrees C. The pH optimum was 6.5; the apparent Km values were equal to 1.2 X 10(-5) M and 1.4 X 10(-3) M for pantothenate and ATP, respectively, at the ATP/Mg2+ ratio of 1. Pantetheine produced a competitive inhibition of pantothenate kinase. Pantethine or pantetheine disulfide did not inhibit the enzyme.     

A deoxyribonucleic acid kinase from nuclei of rat liver. Purification and properties.

A DNA kinase has been partially purified from rat liver nuclei by a procedure which also yields DNA ligase. The kinase uses ATP to phosphorylate specifically the 5'-hydroxyl termini of oligodeoxynucleotides and of single- or double-stranded DNA, yielding 5'-phosphate termini and ADP. The kinase is inactive on RNA, or on oligodeoxynucleotides of chain length less than approximately 10 to 12 residues. The kinase requires a divalent cation (Mg2+, Mn2+, Co2+, Zn2+, Ni2+, or Ca2+) for activity and has an acidic pH optimum. It is inhibited by a variety of nucleotides as well as by very low levels of inorganic and organic sulfate compounds and sulfate analogues. The molecular weight of the kinase is estimated to be 8 times 10(4) from gel filtration.