Purification and characterization of type II DNA topoisomerase from mouse FM3A cells: phosphorylation of topoisomerase II and modification of its activity

Type II topoisomerase has been purified from mouse FM3A cells by using P4 phage knotted DNA as a substrate. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 167 and 151 kDa. Partial digestion of the two bands with Staphylococcus aureus V8 protease indicated that the two polypeptides were structurally related. The enzyme required ATP and Mg2+ for activity. dATP could substitute for ATP, and ITP was slightly effective at 5-10 mM. The activity was sensitive to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), coumermycin, and ethidium bromide. A protein kinase activity was detected in the partially purified topoisomerase II fraction, and this protein kinase was further purified. The protein kinase phosphorylated the purified topoisomerase II, and the phosphorylation of topoisomerase II by the kinase increased the activity by 8.6-fold over that of the unmodified enzyme. The treatment of the purified topoisomerase II with alkaline phosphatase abolished the enzyme activity almost completely, and the treatment of the dephosphorylated topoisomerase II with the protein kinase restored the enzyme activity. The protein kinase activity was not stimulated by Ca2+ or cyclic nucleotides, and the aminoacyl residue phosphorylated by the kinase was serine. Enzymatic properties of the kinase were very similar to those of the kinase reported to be tightly associated with the Drosophila topoisomerase II [Sander, M., Nolan, J. M., & Hsieh, T.-S. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6938-6942]. The immunoprecipitation of nuclear extracts prepared from 32P-labeled cells with anti-mouse topoisomerase II antiserum indicated that DNA topoisomerase II existed in mouse cells as a phosphoprotein.     

Purification and characterization of DNA-dependent ATP phosphohydrolases from KB cells

DNA-dependent ATPases have been purified from logarithmically growing KB cells by chromatography on single-stranded DNA cellulose and phosphocellulose. Phosphocellulose resolved the DNA-dependent ATPases into three activities designated ATPase I, II and III, respectively. From gel filtration and sedimentation analysis ATPases II and III were found to be very similar, both with calculated molecular weights of 78,000. Due to the extreme lability these enzymes were not purified further. The molecular weight of ATPase I determined by gel filtration and sedimentation analysis was calculated to be 140,000. ATPase I was further purified by gradient elution on ATP-agarose, revealing two peaks of activity (IA and IB), and by sucrose gradient sedimentation. Analysis of the fractions from the sucrose gradient by sodium dodecylsulphate gel electrophoresis revealed only one broad polypeptide band co-sedimenting with both ATPase IA and ATPase IB. This band was composed of four closely spaced polypeptides with apparent molecular weights of 66,000, 68,000, 70,000 and 71,000. Comparison of the native molecule weight (140,000) with these results suggests that ATPase I is a dimer. ATPase IA and IB were indistinguishable in their structural and enzymatic properties and presumably represent the same enzyme. The purified enzyme has an apparent Km of 0.5 mM for ATP producing ADP + Pi. A maximum activity of 2,100 molecules of ATP hydrolyzed per enzyme molecular per minute was found. Hydrolysis of ATP requires the presence of divalent cations (Mg2+ greater than Ca2+ greater than Mn2+ greater than Co2+). A broad pH optimum (pH 6--8) was observed. The enzyme uses ATP or dATP preferentially as a substrate, while other deoxyribonucleoside or ribonucleoside triphosphates were inactive. ATPase I prefers denatured DNA as cofactor. The activity with native DNA is 40% of that with denatured DNA.     

Interaction of FliI, a component of the flagellar export apparatus, with flagellin and hook protein.

FliI is a key component of the flagellar export apparatus in Salmonella typhimurium. It catalyzes the hydrolysis of ATP which is necessary for flagellar assembly. Affinity blotting experiments showed that purified flagellin and hook protein, two flagellar axial proteins, interact specifically with FliI. The interaction of either of the two proteins with FliI, increases the intrinsic ATPase activity. The presence of either flagellin or hook protein stimulates ATPase activity in a specific and reversible manner. A Vmax of 0.12 nmol Pi min-1 microgram-1 and a Km for MgATP of 0.35 mM was determined for the unstimulated FliI; the presence of flagellin increased the Vmax to 0.35 nmol Pi min-1 microgram-1 and the Km for MgATP to 1.1 mM. The stimulation induced by the axial proteins was fully reversible suggesting a direct link between the catalytic activity of FliI and the export process.     

Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro

DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, [gamma-32P]ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. We conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, we speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.     

A homogeneous type II DNA topoisomerase from HeLa cell nuclei

Using kinetoplast DNA networks as a substrate in a decatenation assay, we have purified to apparent homogeneity a type II DNA topoisomerase from HeLa cell nuclei. The most pure preparations contain a single polypeptide of 172,000 daltons as determined by sodium dodecyl sulfate-gel electrophoresis. The molecular weight of the native protein, based on sedimentation and gel filtration analyses, is estimated to be 309,000. These results suggest that the enzyme is a dimer of 172,0090-dalton subunits. The enzyme is a type II topoisomerase as demonstrated by its ability to change the linking number of DNA circles in steps of two and to decatenate or unknot covalently closed DNA circles. No gyrase activity is detectable. ATP is required for the relaxation, decatenation, and unknotting of DNA, and a DNA-dependent ATPase activity is present in the most pure fractions. ATP is hydrolyzed to ADP in this properties to T4 DNA topoisomerase (Liu, L. F., Liu, C. C., and Alberts, B. M. (1979) Nature 281, 456-461).     

Protease Ti, a new ATP-dependent protease in Escherichia coli, contains protein-activated ATPase and proteolytic functions in distinct subunits

In addition to protease La (the lon gene product), Escherichia coli contains another ATP-dependent protease, Ti. This enzyme (approximately 340 kDa) is composed of two components, both of which are required for proteolysis. Both have been purified to homogeneity by conventional procedures using [3H]casein as the substrate. The ATP-stabilized component, A, has a subunit molecular weight of 80,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate, but it behaves as a dimer (140 kDa) upon gel filtration. Component P, which is relatively heat stable, is inactivated by diisopropyl fluorophosphate and can be labeled with [3H] diisopropyl fluorophosphate. It has a subunit size of 23 kDa, but the isolated component behaves as a complex (260 kDa) of 10-12 subunits. The isoelectric point of component A is 7.0 and that of P is 8.2, and their amino acid compositions differ considerably. The purified enzyme has an ATPase activity that is stimulated 2-4-fold by casein and other protein substrates but not by nonhydrolyzed proteins. Component A also shows ATPase activity which can be stimulated by casein. Addition of component P (which lacks ATPase activity) inhibits basal ATP hydrolysis by A and makes this ATPase more responsive to casein. Although component P contains the serine active site for proteolysis, it shows no proteolytic activity in the absence of component A, Mg2+, and ATP or dATP. Other nucleoside triphosphates are not hydrolyzed and do not support proteolysis. Protease Ti has a Km for ATP of 210 microM for hydrolysis of both casein and ATP. Casein increases the Vmax for ATP without affecting the Km. A Mg2+ concentration of 5 mM is necessary for half-maximal rates of ATP and casein hydrolysis. Ca2+ and Mn2+ partially support these activities. Thus, protease Ti shares many unusual properties with protease La (e.g. coupled ATP and protein hydrolysis and protein-activated ATPase), but these functions in protease Ti are associated with distinct subunits that modify each other's activities.     

Identification of a cysteine residue important for the ATPase activity of C. elegans fidgetin homologue

Based on the amino acid alignment, Caenorhabditis elegans F32D1.1 was identified to be a homologue of the mammalian fidgetin. We produced and purified the F32D1.1 protein by using a baculovirus-expression system. F32D1.1 has an ATPase activity, which is sensitive to N-ethylmaleimide. Km and Vmax for the ATPase activity of F32D1.1 were estimated to be 0.44 mM and 225 nmol/mg/min, respectively. When the cysteine at the position of 368 was mutated to alanine, the ATPase activity was greatly decreased; Vmax was decreased to one-sixth, while Km remained similar. These results suggest that the unique position of cysteine 368, located immediately downstream of the Walker A motif, plays an important role in the ATP hydrolysis process of C. elegans F32D1.1 protein.     

Purification and properties of a novel azide-sensitive ATPase of Exiguobacterium aurantiacum

Exiguobacterium aurantiacum BL77/1 possesses at least two distinct membrane-bound ATPases. One of them was solubilized with decanoyl N-methylglucamide, a non-ionic detergent, and purified by successive chromatography on DEAE-Sepharose and hydroxyapatite. The purified ATPase appears to consist of a single polypeptide component with an apparent molecular mass of 54 kDa. Among the triphosphates of various nucleosides tested, ATP was the best substrate. The enzyme exhibited a Km of 0.5 mM for ATP and a Vmax of 109 micromol ATP (mg protein)(-1) min(-1); the optimum pH for activity was near 6.5. The enzyme was sensitive to azide and inactivated by N,N'-dicyclohexylcarbodiimide. Analysis of the inhibition kinetics by N,N'-dicyclohexylcarbodiimide suggested that binding of the drug to a single carboxyl group per ATPase molecule is sufficient for inactivation.     

Properties of a DNA-dependent ATPase from rat mitochondria.

A DNA-dependent ATPase has been highly purified from rat liver mitochondria and characterized. The enzyme catalyzes the hydrolysis of ATP or dATP in the presence of single-stranded DNA cofactor and a divalent cation. The Km values for ATP and dATP are 0.15 mM and 0.35 mM, respectively. The enzyme activity is highly sensitive to N-ethylmaleimide. The sedimentation coefficient of the enzyme is 8.3 S in a glycerol gradient. From this and data on Sephadex G-200 gel filtration, the molecular weight of the native enzyme was calculated to be about 190,000. All the natural single-stranded DNAs tested were equally effective for the ATPase activity, but synthetic deoxyhomopolymer poly(dC) was found to be more effective than natural single-stranded DNAs. Synthetic and natural RNAs had no effect on the activity.     

ATP-dependent reactions catalyzed by inner membrane vesicles of rat liver mitochondria. Kinetics, substrate specificity, and bicarbonate sensitivity.

Three ATP-dependent reactions catalyzed by the inner membrane of rat liver mitochondria and the ATPase reaction catalyzed by purified mitochondrial ATPase (F1), were studied with respect to kinetic properties, substrates specificity, and sensitivity to bicarbonate. The ATP-dependent transhydrogenase reaction (reduction of NADP+ by NADH) catalyzed by inner membrane vesicles displays typical Michaelis-Menten kinetics in both Tris-Cl and Tris-bicarbonate buffers, with Km (ATP) values of 0.035 mM and 0.054 mM respectively. The Vmax of transhydrogenase activity (25 nmol min-1 mg-1) is the same in Tris-bicarbonate or Tris-Cl buffer. ITP and GTP readily substitute for ATP in the transhydrogenase reaction. The ATP-P1 exchange reaction catalyzed by inner membrane vesicles displays typical Michaelis-Menten kinetics in both Tris-Cl and Tris-bicarbonate buffers with Km (ATP) values of 1.0 mM and 1.4 mM respectively. The Vmax of exchange (200 nmol min-1 mg-1) is the same in either buffer. ITP and GTP do not effectively replace ATP in the exchange reaction.     

Presence of a high-affinity Ca2+- and Mg2+-dependent ATPase in rat peritoneal mast-cell membranes.

Purified perigranular and plasma membranes isolated from rat peritoneal mast cells were examined for Ca2+- and Mg2+-dependent ATPase activity. Isolated perigranular membranes contained only a low-affinity Ca2+- or Mg2+-dependent ATPase (Km greater than 0.5 mM). The plasma membranes contained both a low-affinity Ca2+- or Mg2+-dependent ATPase (Km = 0.4 mM, Vmax. = 20 nmol of Pi/min per mg), as well as a high-affinity Ca2+- and Mg2+-dependent ATPase (Km = 0.2 microM, Vmax. = 6 nmol of Pi/min per mg).     

Mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus: purification, characterization, and kinetic studies.

Mitochondrial F1-ATPase was purified from the mycelium of Phycomyces blakesleeanus NRRL 1555(-) and its kinetic characteristics were studied. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme reveals five bands (alpha, beta, gamma, delta, and epsilon) characteristic of the F1 portion with apparent molecular weights of 60,000, 53,000, 31,000, 25,000, and 21,000, respectively. The molecular weight of the native F1-ATPase from Phycomyces blakesleeanus was in agreement with the stoichiometry alpha 3 beta 3 gamma delta epsilon. The MgATP complex is the true substrate for ATPase activity which has a Km value of 0.15 mM. High concentrations of free ATP or free Mg2+ ions inhibit the ATPase activity. ADP appears to act as a negative allosteric effector with regard to MgATP hydrolysis, with the apparent Vmax remaining unchanged.     

Properties of a purified nuclear topoisomerase from L1210 cells

A nuclear type I topoisomerase from mouse leukemia L1210 cells has been partially purified and characterized. The sedimentation coefficient of the enzyme by velocity sedimentation is 4.3 S, consistent with a globular protein of 68 kDa. Enzyme activity is stimulated 20-fold in the presence of magnesium over that achieved in KCl alone. The enzyme is completely inhibited in the presence of the berenil congeners HOE 13548 and 15030 while berenil itself caused only partial inhibition at concentrations below 200 micrograms/ml. An acid soluble protein of 30 kDa (by SDS-polyacrylamide gel electrophoresis) co-purified with the topoisomerase but could be separated by precipitation in a low salt buffer. This protein, as well as a protein of similar characteristics, histone H1, stimulated topoisomerase activity over a narrow concentration range. The role of topoisomerase in the DNA strand scission observed in L1210 cells following exposure to intercalating agents remains conjectural as the purified enzyme did not produce nicks in plasmid DNA in the presence of adriamycin.     

Purification and characterization of ferulate and p-coumarate decarboxylase from Bacillus pumilus.

Bacillus pumilus PS213 isolated from bovine ruminal fluid was able to transform ferulic acid and p-coumaric acid to 4-vinylguaiacol and 4-vinylphenol, respectively, by nonoxidative decarboxylation. The enzyme responsible for this activity has been purified and characterized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extract from a culture induced by ferulic acid or p-coumaric acid shows three bands that are not present in the crude extract of an uninduced culture, while the purified enzyme shows a single band of 23 kDa; the molecular mass calculated by size exclusion chromatography is 45 kDa. Enzyme activity is optimal at 37 degrees C and pH 5.5 and is not enhanced by any cation. Kinetic studies indicated a Km of 1.03 mM and a Vmax of 0.19 mmol.min-1/mg.liter-1 for ferulic acid and a Km of 1.38 mM and a Vmax of 0.22 mmol.min-1/mg.liter-1 for p-coumaric acid.     

Adenosine triphosphatases associated with bovine brain microtubules. II. Properties of ATPases I and II

The catalytic properties of two ATPases which had been purified from bovine brain microtubules (Tominaga, S. & Kaziro, Y. (1983) J. Biochem. 93, 1085-1092) were studied. ATPase I, which had a molecular weight of 33,000, required the presence of 1.0 microM tubulin, 0.2 mM Mg2+, and 10 mM Ca2+ for maximal activity. The activation of ATPase I by tubulin was specific to the native form of tubulin, which could not be replaced by F-actin or tubulin denatured either by heat or more mildly by dialysis in the absence of glycerol. ATPase I was not specific to ATP, and GTP, and to a lesser extent, UTP and CTP were also hydrolyzed. Km for ATP of ATPase I was about 0.04 mM. ATPase I was inhibited by 5 mM Mg2+, 0.04 M K+, 10(-3) M vanadate, 10 mM N-ethylmaleimide, or 20% (v/v) glycerol. ATPase II, which was associated with membrane vesicles, required the presence of 0.2-2.0 mM Mg2+ and 20 mM KCl for activity. Tubulin stimulated the reaction of ATPase II only partially, and the addition of Ca2+ was rather inhibitory. ATPase II was specific to ATP with a Km value of 0.14 mM. It was inhibited by 1.6 mM N-ethylmaleimide and 20% (v/v) glycerol, but was not very sensitive to vanadate. Instead, ATPase II was inhibited by trifluoperazine, chlorpromazine, and nicardipin at 10(-3) M.     

Characterization of human erythrocyte cytoskeletal ATPase

Human erythrocyte cytoskeletal ATPase was extracted with 0.2 mM ATP (pH 8.0) from Triton X-100 treated ghosts. The ATPase fraction contained mainly spectrin, actin, and band 4.1. When the ATPase fraction was applied to a Sepharose 4B column, 90% of the ATPase activity was recovered in a spectrin, actin, and band 4.1 complex fraction and none was detected in the spectrin fraction. A specific activity of the complex ATPase was 60-120 nmol/(mg protein X h). No ATPase activity was detected in the presence of EDTA. The presence of magnesium in the incubation medium was essential for the ATPase activity. The activity was activated by KCl and was almost completely inhibited by 10(-5) M free calcium in the presence of 0.2 mM MgCl2. The Ki for Ca2+ was 7 X 10(-7) M. Phalloidin and DNase 1, which affect actin, inhibited this K,Mg-ATPase activity by 95%, but cytochalasin B did not inhibit it. N-Ethylmaleimide activated the ATPase 1.6-fold. The order of affinity for nucleotides was ATP greater than ITP greater than CTP, ADP, AMP-PNP, GTP. A specific ATPase activity of purified actin was 50 nmol/(mg X h). These results suggest that the cytoskeletal ATPase is actin ATPase and the actin ATPase is activated by spectrin and band 4.1.     

Purification and characterization of the proton translocating plasma membrane ATPase of red beet storage tissue

Plasma membranes were prepared from red beet (Beta vulgaris L.) storage tissue by partition in an aqueous two-phase system. A highly active proton-translocating ATPase was purified from these membranes by lysophosphatidylcholine extraction and glycerol density gradient centrifugation. The ATPase activity was inhibited by vanadate or dicyclohexyl carbodiimide, but was insensitive to azide, nitrate and molybdate at concentrations which inhibit the F1ATPase, the tonoplast ATPase, and acid phosphatase. Inhibition by vanadate was consistent with a non-competitive mechanism, with Ki = 10 microM. The Km for Mg-ATP was about 1 mM, magnesium ions were required, and the activity was stimulated by KCl and by lysophosphatidylcholine. The optimal pH was 6.5. The molecular mass by gel filtration in the presence of 2 g/liter octyl glucoside was 600 kDa, while dodecyl sulfate gel electrophoresis gave a polypeptide molecular mass of 100 kDa. After blotting onto nitrocellulose, the purified enzyme did not bind concanavalin A, although a concanavalin A-binding peptide of the plasma membrane runs to nearly the same position on the gel and showed some tendency to co-purify with the ATPase. Phospholipid vesicles into which the purified ATPase had been incorporated by the freeze-thaw technique showed vanadate-sensitive, ATP-dependent proton uptake. When the ATPase was reconstituted into lipid membranes at high protein to lipid ratios and incubated with ATP, two-dimensionally crystalline arrays of protein molecules were formed.     

High-yield purification of a pp60c-src related protein-tyrosine kinase from human platelets

A protein-tyrosine kinase (PTK, EC 2.7.1.112) from human platelets was purified with high yield. Purification of the enzyme involved sequential chromatography on casein-agarose, tyrosine-agarose, heparin-Sepharose and hydroxylapatite. The procedure resulted in substantially enriched 54/52 kDa polypeptides on SDS-polyacrylamide gel electrophoresis and a yield of about 25% in PTK activity. About 250 micrograms of purified protein could be obtained from 1 g of cell protein. The purification factor varied between 1000 and 1500. Determination of the molecular mass of the purified PTK under nondenaturating conditions by molecular sieve chromatography revealed that the enzyme is a monomer of about 50 kDa. Among various protein substrates tested, casein was most prominently phosphorylated. All substrates were exclusively phosphorylated at tyrosine residues. Autophosphorylation at tyrosine residues of the 54/52 kDa proteins was observed in the presence of Mg2+ or Mn2+. At each purification step, the 54/52 kDa proteins were precipitated by sera from tumor-bearing rabbits immunoprecipitating pp60src, but not by control sera. The amount of the immunoprecipitated purified 54/52 kDa phosphoproteins was directly proportional to the amount of antiserum used. Partial peptide mapping by V8 proteinase showed a 26 kDa tyrosine-phosphorylated fragment for the 54 and the 52 kDa proteins as well as for the pp60c-src molecules of intact platelets. All these data indicated that purified PTK is closely related to pp60c-src of human platelets. Using casein as a substrate for the purified enzyme, the Km for ATP was 4 microM and the Vmax for the reaction was 2.0 nmol/min per mg.     

Purification and properties of the 5 alpha-dihydrotestosterone 3 alpha(beta)-hydroxysteroid dehydrogenase from human prostatic cytosol.

5 alpha-Dihydrotestosterone 3 alpha(beta)-hydroxysteroid dehydrogenase [3 alpha(beta)-HSDH] [EC 1.1.1.50/EC 1.1.1.51] which catalyses the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) to both 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol was purified to an apparent homogeneous state using cytosol of three human hyperplastic prostates by a 4-step purification procedure. After each purification step 3 alpha-HSDH activity was coincident with 3 beta-HSDH activity. On average, specific 3 alpha-HSDH activity was enriched 856-fold, specific 3 beta-HSDH activity 749-fold compared to human prostatic cytosol using anion exchange, hydrophobic interaction, gel filtration and affinity chromatography. Examination of the purified enzyme by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) revealed a single protein band with silver staining. The molecular weight of the enzyme was estimated as 33 kDa by SDS-polyacrylamide gel electrophoresis and as 28 kDa by Sephacryl S-200 gel filtration indicating that the native 3 alpha(beta)-HSDH is a monomer. In the presence of the preferred co-factor, NADPH, the purified enzyme had a mean apparent Km for 5 alpha-DHT of 3.9 microM and a Vmax of 93.3 nmol (mg protein)-1 h-1 with regard to 3 alpha-HSDH activity, and a Km of 6.3 microM and a Vmax of 20.6 nmol (mg protein)-1 h-1 with regard to 3 beta-HSDH activity.     

A DNA helicase from Xenopus laevis ovaries

A DNA helicase was extensively purified from Xenopus laevis ovaries. The most purified fraction was free of DNA topoisomerase, DNA polymerase, and nuclease activities. The enzyme had a Stokes radius of 54 A and a sedimentation coefficient of 6-7.3 S, from which a native molecular weight of 140,000-170,000 was calculated. DNA helicase activity required Mg2+ or Mn2+ and was dependent on hydrolysis of ATP or dATP. Monovalent cations, K+ and Na+, stimulated DNA unwinding with an optimum at 130 mM. DNA-dependent ATPase activity copurified with the X. laevis DNA helicase. Double-stranded and single-stranded DNA were both cofactors for the ATPase activity, but single-stranded DNA was more efficient. The molecular weight, monovalent cation dependence, cofactor requirements, and elution from single-stranded DNA-cellulose suggest that the X. laevis DNA helicase is different from previously described eukaryotic DNA helicases.