On the role of ATP in phosphodiester bond hydrolysis catalyzed by the recBC deoxyribonuclease of Escherichia coli.

The deoxyribonuclease specified by the recB and recC genes of Escherichia coli (recBC DNase; exonuclease V) has been purified to near homogeneity by a new procedure. Although hydrolysis of even a single nucleotide from a duplex DNA molecule by the pure enzyme is absolutely dependent upon ATP, the extent of phosphodiester hydrolysis is strongly inhibited by ATP concentrations of 0.2 mm or greater, and the initial rate is unaffected. Under these conditions, the extent of DNA hydrolysis is proportional to enzyme concentration. In contrast, neither the rate nor the extent of hydrolysis of single-stranded DNA nor ATP is affected by high concentrations of ATP. The amount of large single-stranded polynucleotide generated by the action of the recBC DNase increases as the ATP concentration increases and, at 0.5 mM ATP, becomes equivalent to the amount of acid-soluble nucleotide formed. These findings suggest that high intracellular concentrations of ATP affect the mechanism of the recBC DNase so as to limit the extent of hydrolysis of duplex DNA, while at the same time favoring the formation of single-stranded regions within the duplex. Such regions may be essential intermediates in the recombination process.     

Structural and enzymological characterization of a deoxyribonucleic acid dependent adenosine triphosphatase from KB cell nuclei

We have purified to near homogeneity the single DNA-dependent ATPase activity that we have identified in extracts of KB cell nuclei. The protein structure of the enzyme was defined by sodium dodecyl sulfate gel electrophoresis, which revealed a single protein band of 75000 daltons that was coincident with the profile of ATPase activity resolved by the final step of agarose-ATP chromatography or by isoelectric focusing. The enzyme has a pI of 8.5, a Stokes' radius by gel filtration of 3.8 nm, and a sedimentation coefficient in high salt of 5.3 S. At low ionic strength the enzyme activity sediments at 7.0 S, suggesting that it may dimerize under these conditions. The purified enzyme has a specific activity of 5.9 X 10(5) nmol of ATP hydrolyzed per h per mg of protein and is devoid of endonuclease, exonuclease, RNA or DNA polymerase, nicking-closing, and gyrase activities at exclusion limits of 10(-6)-10(-8) of the ATPase activity. The enzyme can hydrolyze only ATP or dATP, to generate ADP or dADP plus Pi, but the other NTPs and dNTPs are competitive inhibitors of the enzyme with respect to ATP. A divalent cation (Mg2+ greater than Mn2+ greater than Ca2+) as well as a nucleic acid cofactor is required for activity. Single-stranded DNA or deoxyhomopolymers are most effective, but blunt-ended linear and nicked circular duplex DNA molecules are also used at Vmax values approximately 20% of that obtained with single-stranded DNA. Intact duplex DNA and polyribonucleotides are unable to support ATP hydrolysis. Velocity gradient sedimentation studies corroborate the interpretations of the kinetic analyses and demonstrate enzyme binding to single-stranded DNA and nicked duplex DNA but not to intact duplex DNA. Although we have not succeeded directly in demonstrating DNA unwinding by this protein, preliminary results suggest that in the presence of ATP, the ATPase can stimulate the reactivity of homogeneous human DNA polymerases alpha and beta on nicked duplex DNA substrates.     

A nucleoside triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. Purification and characterization of the enzyme.

A deoxyribonuclease, which requires nucleoside triphosphate for reaction, has been purified about 150-fold from extracts of Bacillus laterosporus. Potassium phosphate and ethylene glycol stabilize the purified enzyme. The enzyme degrades double-stranded DNA about 100 times faster than heat-denatured DNA in the presence of nucleoside triphosphate. Double-stranded DNA is not degraded to any measurable extent in the absence of ATP, but the enzyme exhibits activity toward denatured DNA in the absence of nucleoside triphosphate, and this activity seems to be an intrinsic property of this enzyme protein. The optimum pH is 8.5 and the maximum activity is obtained in the copresence of Mg2+ (8.0 X 10(-3)M) and Mn2+ (7.0 X 10(-5)M). ATP and dATP are most effective and nucleoside di- or monophosphates are ineffective. ATP is converted to ADP and inorganic phosphate during the reaction and the ratio of the amount of ATP cleaved to that of hydrolyzed phosphodiester bonds of DNA is about 3:1. An inhibitor of the enzyme was observed in bacterial extracts prepared by sonic disruption; the inhibitory substance is produced in the bacteria in the later stages of cell growth. Preliminary results show that the inhibitor emerged near the void volume of a Sephadex G-200 column, and was relatively heat-stable, RNase-resistant, and DNase-sensitive.     

Two pH optima of adenosine 5'-triphosphate dependent deoxyribonuclease from Bacillus laterosporus

The various catalytic activities of the ATP-dependent deoxyribonuclease (DNase) of Bacillus laterosporus have pH optima at 6.3 and 8.3. Although the pH profile of ATP-dependent DNase activity on duplex DNA is bell shaped with a maximum at about pH 8.3, ATP-dependent DNAse activity on single-stranded DNA has optima at pH 6.3 and 8.3. ATPase activities dependent on double-stranded and single-stranded DNA have a high bell-shaped peak with a maximum at pH 6.3 with a low and broad shoulder at about pH 8.3. ATP-independent DNase activity also has optima at pH 6.3 and 8.3. The ratio of the amount of ATP hydrolyzed per number of cleaved phosphodiester bonds in DNA increases with decrease in the pH value of the reaction. The ratios obtained at pH 8.3 and 6.3 were respectively about 3 and 22 with duplex DNA as substrate and 5 and 17 with single-stranded DNA as substrate. Formation of a single-stranded region of 15000-20000 nucleotides, which is linked to duplex DNA and about half of which has 3'-hydroxyl termini, was observed at about pH 6.3, but not at above pH 7.5. Furthermore, the optimum concentrations of divalent cations for the activity producing the single-stranded region and the activity hydrolyzing ATP were identical (3 mM Mn2+ or 5 mM Mg2+). Thus the two activities are closely related. These results indicate that the enzyme has two different modes of action on duplex DNA which are modulated by the pH.     

Purification and properties of a manganese-stimulated deoxyribonuclease produced during sporulation of Bacillus subtilis.

A DNAase (deoxyribonuclease) was isolated from culture supernatants of sporulating Bacillus subtilis 168. The purified enzyme migrated as a single band during polyacrylamide-gel electrophoresis. The enzyme differs from other DNAases of B. subtilis in molecular weight, metal-ion requirement and mode of action. The enzyme was inactive in the absence of metal ions, and exhibited optimum activity with 10 mM-Mn2+, although Mg2+, Cd2+ and Co2+ could also permit some activity. The pH optimum for the enzyme was pH 7.5, and it degraded linear-duplex DNA or closed-circular-duplex DNA to acid-soluble material. There was little or no activity on single-stranded DNA or rRNA. Sucrose-gradient analysis of the products of DNAase action on bacteriophage T7 DNA showed that endonucleolytic cleavage had occurred by the introduction of single-strand breaks in both strands of the duplex. The molecular weight of the enzyme was determined, by gel filtration on Sephadex G-75, to be 12000.     

Fe2+ and other divalent metal ions uncouple Ca2+ transport from (Ca2+-Mg2+)-ATPase in rat liver plasma membranes

The addition of nanomolar concentrations of free Fe2+, Mn2+, or Co2+ to rat liver plasma membranes resulted in an activation of ATP hydrolysis by these membranes which was not additive with the Ca2+-stimulated ATPase activity coupled to the Ca2+ pump. Detailed analysis showed that, if fact, (i) as for the stimulation of (Ca2+-Mg2+)-ATPase by Ca2+, activation of ATP hydrolysis by Fe2+, Mn3+, or Co2+ followed a cooperative mechanism involving two ions; (ii) two interacting sites for ATP were involved in the activation of both Fe2+- and Ca2+-stimulated ATPase activities; (iii) micromolar concentrations of magnesium caused the same dramatic inhibition of both activities; and (iv) the subcellular distribution of Fe2+-activated ATP hydrolysis activity corresponded to that of plasma membrane markers. This suggests that the (Ca2+-Mg2+)-ATPase might be stimulated not only by Ca2+, but also by Fe2+, Mn2+, or Co2+. However, interaction of (Ca2+-Mg2+)-ATPase with Fe2+, Mn2+, or Co2+ inhibited the Ca2+ pump activity. Furthermore, neither the formation of the phosphorylated intermediate of (Ca2+-Mg2+)-ATPase, nor ATP-dependent (59Fe) uptake could be detected in the presence of Fe2+ concentrations which stimulated ATP hydrolysis. We conclude that: (i) under the influence of certain metal ions, the Ca2+ pump in the liver plasma membrane may be switched to an uncoupled state which displays ATP hydrolysis activity, but does not insure ion transport; (ii) therefore the Ca2+ pump in liver plasma membranes specifically insures Ca2+ transport.     

High activity of NADP-dependent malic enzyme in mitochondria from abdomen muscle of the crayfish Orconectes limosus.

1. Mitochondria isolated from abdomen muscle of crayfish Orconectes limosus exhibit malic enzyme activity in the presence of L-malate, NADP and Mn2+ ions after addition of Triton X-100. Under optimal conditions about 230 nmole of reduced NADP and an equivalent amount of pyruvate are produced per min per mg of mitochondrial protein. 2. The pH optimum for decarboxylation of L-malate is about 7.5. 3. The apparent Km for L-malate, NADP and Mn2+ ions was found to be 0.66, 0.012, and 0.0025 mM, respectively. 4. The requirement for Mn2+ can be replaced by Mg2+, Co2+ and Ni2+ ions; however, higher concentrations of these ions than Mn2+ are required for a full stimulation of malic enzyme activity. 5. Oxaloacetate and pyruvate inhibited the enzyme activity in a competitive manner with apparent Ki values of 0.05 mM and 5.4 mM, respectively.     

Outer membrane-associated deoxyribonuclease activity of Porphyromonas gingivalis

Extracellular outer membrane vesicles which are produced by Gram-negative bacteria may enclose deoxyribonucleic acid (DNA). While characterizing vesicles of Porphyromonas gingivalis, it was found that they do not contain detectable amount of DNA. It was also shown that the presence of deoxyribonuclease activity on whole cells and vesicles can degrade plasmidic and linear DNA. Deoxyribonuclease activity was also demonstrated in several other Gram-negative oral bacterial species. The nuclease activity of P. gingivalis was further characterized. When deoxyribonuclease activity was analyzed by zymography, only one active band was detected under the conditions tested. This nuclease enzyme showed a molecular weight of approximately 50 kDa. The activity was inhibited by 5 mM ZnCl2 or 100 mM EDTA whereas Mg2+ or Ca2+ ions were not required for activity. Activity was totally destroyed by treatment at 70 degrees C for 15 min. Although the enzyme may participate in virulence or provide nucleic acid precursors for bacterial growth, its exact role is still unknown.     

Effect of Mg2+ and Mn2+ on hydrolysis of calf thymus DNA by pancreatic deoxyribonuclease I

Divalent cations are activators for DNA hydrolysis by pancreatic deoxyribonuclease I. Apparent Vm and Km changes have been studied in presence of Mn2+ or Mg2+. The activation process modifies both Vm and Km, their relationship with Mg2+ or Mn2+ being a linear one. Deoxyribonucleotides inhibit the DNA hydrolysis, whether Mg2+ or Mn2+ is the activator; the purine deoxyribonucleotides are more effective as inhibitors than the pyrimidine ones. The effect of some derivatives of adenine has been studied: the inhibition is maximum with 5'-dAMP and minimum with adenine or adenosine. A kinetic mechanism of enzymatic activation by Mn2+ or Mg2+ is discussed.     

Characterization of an adenylyl cyclase activity in particulate preparations from epimastigote forms of Trypanosoma cruzi.

Particulate preparations from epimastigote forms of Trypanosoma cruzi contain an adenylyl cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) which could be stored at --20 degree C and resisted 5 cycles of freezing and thawing over 10 days without significant loss of activity. The enzyme reaction strictly required Mn2+, had a pH optimum of 7.7 and was not inhibited or stimulated by NaF. Particles prepared in the presence of 10 mM Mn2+ or Mg2+ were 3--4 times more active than particles prepared in the absence of these cations. However, Mg2+ could not substitute for Mn2+ during enzyme assay nor did it enhance activity in the presence of saturating concentrations of Mn2+. The binary complex Mn - ATP2- was shown to be the true substrate for the adenylyl cyclase and free ATP was highly inhibitory. Plots of enzyme activity against equimolar concentrations of ATP - Mn gave sigmoid curves with n values in Hill plots ranging between 1.5 and 2.0. Excess Mn2+ activated the cyclase catalyzed reaction at low but not at high concentrations of ATP - Mn. In the presence of an excess of 1 mM Mn2+, which transforms 97% of the added ATP to productive Mn - ATP2- complex, the substrate saturation curve assumed a Michaelian pattern with an apparent Km =0.2 mM.     

ATP hydrolysis and synthesis by the membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum.

The membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum showed maximum activity for ATP hydrolysis at pH 8, at temperatures between 65 and 70 degrees C, and at an ATP-Mg2+ ratio of 0.5. Anaerobic conditions were not prerequisite for enzyme activity. The enzyme showed a Km value for ATP of 2 mM, and activity was Mg2+ dependent; Mn2+, Co2+, Ca2+, and Zn2+ could replace Mg2+ to some extent. Other nucleoside triphosphates could be hydrolyzed. N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. A proton-motive force, artificially imposed by a pH shift or valinomycin, resulted in ATP synthesis in whole cells. The ATP synthetase complex of the thermophilic methanogenic bacterium is similar to those described in aerobic and anaerobic microorganisms.     

Effect of ATP on the Friend Murine leukemia virus-associated endonuclease activity and the endonuclease activity of the avian myeloblastosis virus RNA-directed DNA polymerase

The Mn2+-dependent endonuclease activity associated with the avian myeloblastosis virus RNA-directed DNA polymerase has been shown to be activated by ATP in the presence of Mg2+. In the presence of Mn2+ the endonucleolytic activity was stimulated about 3-fold by the addition of ATP. The earlier identified Mr = 40,000 Friend murine leukemia virus (F-MuLV)-associated endonuclease which functions in the presence of both Mg2+ and Mn2+ has also been shown to be similarly stimulated by ATP. For both endonuclease activities stimulation was only observed at ATP concentrations above 0.5 mM, and it did not increase upon elevating the ATP concentration above 2.5 mM. ADP and dATP also stimulated both activities, although not to the same extent as ATP. GTP had no apparent effect and AMP seemed to inhibit both activities. The effect ATP analogs had on the F-MuLV associated endonuclease activity could suggest that the endonuclease reaction in the presence of ATP might involve the cleavage of beta-gamma phosphate bonds in ATP. Neither adenyl-5'-yl imidodiphosphate nor (beta, gamma-methylene)adenosine 5'-triphosphate stimulated the activity, whereas significant stimulation was observed in the presence of (alpha, beta-methylene)adenosine 5'-triphosphate. Although no ATPase activity could be detected in the purified F-MuLV endonuclease preparation, the data do not exclude the possibility that ATP may be cleaved in amounts which are equivalent to the number of nicks introduced into DNA by the virus-associated endonuclease. In the presence of ATP and Mg2+ the F-MuLV-associated endonuclease nicked both supercoiled and linear DNA duplexes extensively, although the former was nicked more readily than the latter. Single-stranded DNA functioned poorly as a substrate. The nicks introduced by the enzyme contained a 5'-phosphoryl terminus and a 3'-hydroxyl group.     

Kinetic characteristics of ATP hydrolysis by a detergent-solubilized alkaline phosphatase from rat osseous plate

Polidocanol-solubilized alkaline phosphatase was purified to homogeneity with a specific activity of 822.3 U/mg. In the absence of Mg2+ and Ca2+ ions and at pH 9.4, the enzyme hydrolyzed ATP in a manner that could be represented by biphasic curves with V = 94.3 U/mg, K0.5 = 17.2 microM, and n = 1.8 and V = 430.3 U/mg, K0.5 = 3.2 mM, and n = 3.2 for high- and low-affinity sites, respectively. In the presence of saturating concentrations of Mg2+ or Ca2+ ions, the hydrolysis of ATP also followed biphasic curves. However, the specific activity increased to as much as 1,000 U/mg, whereas the K0.5 and n values remained almost unchanged. In the presence of nonsaturating concentrations of metal ions, the hydrolysis of ATP was similar to that observed in the absence of these ions, but with a marked decrease in K0.5 values. At pH 7.5, the enzyme also hydrolyzed ATP with K0.5 = 8.1 microM and V = 719.8 U/mg. Apparently, alkaline phosphatase was able to hydrolyze ATP in vivo, either at pH 7.5 or pH 9.4. These data contribute to the knowledge of the biological properties of skeletal alkaline phosphatase and suggest that this enzyme may have a high-affinity binding site for ATP at alkaline pH.     

Purification of a eukaryotic site-specific endonuclease, Endo.Sce I, from Saccharomyces cerevisiae and effectors on its specificity and activity

A site-specific endonuclease (Endo.Sce I) which caused double-strand scission of DNA was highly purified from a eukaryote, Saccharomyces cerevisiae IAM4274. The molecular weight of the active form of Endo.Sce I was estimated to be 120,000 and 110,000 by sedimentation analysis on a glycerol density gradient and gel filtration on Ultrogel AcA34, respectively. Analysis of the fractions from the last column chromatography by polyacrylamide gel-electrophoresis in the presence of sodium dodecyl sulfate and by an assay of the endonucleolytic activities suggested that Endo.Sce I consists of two non-identical subunits with molecular weights of 75,000 and 50,000. Unlike restriction endonucleases, Endo.Sce I was active on chromosomal DNA of the cells which produced Endo.Sce I. Single-stranded DNA was not cleaved by Endo.Sce I, but inhibited the endonucleolytic activity of the enzyme on double-stranded DNA. The endonucleolytic activity of Endo.Sce I required the magnesium ions (Mg2+) as a sole cofactor; Mg2+ could not be replaced by Ca2+ or Zn2+. When Mg2+ was replaced by manganese ions (Mn2+), extensively purified Endo.Sce I cleaved double-stranded DNA at many other sites in addition to the sites at which DNA was cleaved in the presence of Mg2+. Experiments indicated that this is not the activation of contaminating endonuclease in the preparation of Endo.Sce I, but the result of relaxation in the site-specificity of cleavage.     

The effect of divalent cations on the mode of action of DNase I. The initial reaction products produced from covalently closed circular DNA

The effect of different divalent metal ions on the hydrolysis of DNA by DNase I was studied with an assay which distinguishes between cleavage of one or both strands of the DNA substrate during initial encounters between enzyme and DNA. Using covalently closed superhelical SV40(I) DNA as substrate, initial reaction products consisting of relaxed circles or unit-length linears are resolved by electrophoresis of radioactively labeled DNA in agarose gels. Only in the presence of a transition metal ion, such as Mn2+ or Co2+, and only under certain reaction conditions, is DNase I able to cut both DNA strands at or near the same point, generating unit-length linears. This ability to cut both DNA strands is inhibited by such factors as temperature decrease, the addition of a monovalent ion or another divalent cation which is not a transition metal ion, or a reduction in the number of superhelical turns in the DNA substrate. All of these factors lead to a winding of the duplex helix and antagonize the unwinding of the duplex promoted by transition metal ion binding. Transition metal ions may thus convert the DNA substrate locally to a form in which DNase I can introduce breaks into both strands. In the presence of Mg2+, DNase I introduces single strand nicks into SV40(I), generating exclusively the covalently open, relaxed circular SV40(II) as the initial product of the reaction. In the presence of Mn2+, DNase I generates as initial products a mixture of SV40(II) and unit-length SV40 linear DNA molecules, formed by two nicks in opposite strands at or near the same point in the duplex. These circular SV40(II) molecules consist of two types. A minority class is indistinguishable from the nicked SV40(II) produced by DNase I in the presence of Mg2+. The majority class consists of molecules containing a gap in one of the two strands, the mean length of the gap being 11 nucleotides. The SV40(L) molecules produced in the presence of Mn2+ appear to have single strand extensions at one or both ends.     

Binding of manganese ions to the Na+/K+-ATPase during phosphorylation by ATP

The aim of the present work was to study the Mg2+-Na+/K+-ATPase interaction that was proposed to lead to the formation of a stable Mg-enzyme complex during phosphorylation from ATP. Instead of Mg we used Mn, which can replace Mg as essential activator of Na+/K+-ATPase activity. The amounts of steady-state Mn bound to the enzyme were estimated at 0 degree C on the basis of the 54Mn remaining in the effluent after passing the reaction mixture through a cation exchange resin column. As a function of the MnCl2 concentration, the amount of Mn retained by the enzyme in the absence and presence of ATP showed a saturable and a linear component; the slope of the linear component was the same in both instances (0.016 nmol/mg per microM). The ATP-dependent Mn binding could be adjusted to a hyperbolic function with a Km of 0.76 microM. The ratio [ATP-dependent E-Mn]/[E-P] measured at 5 microM MnCl2 and 5 microM ATP was not different from 1.0, both in native (Mn-E2-P) as well as in a chymotrypsin treated enzyme (Mn-E1-P). When the Mn.E-P complex was allowed to react with KCl (E2-P form) or ADP (E1-P form), the enzyme was dephosphorylated and simultaneously lost the strongly bound Mn in such a way that the ratio [ATP-dependent E-Mn]/[E-P] remained 1:1. These results show the existence of strongly bound Mn ions to Na+/K+-ATPase during phosphorylation by ATP. That binding is (i) of high affinity for Mn, (ii) probably on a single site, and (iii) with a stoichiometry Mn-Pi of 1:1.     

Deoxyribonucleic acid polymerases of BHK-21/C13 cells. Partial purification and characterization of the enzymes.

DNA polymerase from BHK-21/C13 cells were separated into two species, DNA polymerase I corresponding to the heterogeneous enzyme with sedimentation coefficient of 6-8S, and DNA polymerase II, corresponding to the enzyme with sedimentation coefficient of 3.3S. DNA polymerase I was purified 114-fold and DNA polymerase II 154-fold by a simple extraction procedure followed by column chromatography on phosphocellulose and gel filtration through Sephadex G-100. The purified enzymes differed markedly in respect of pH optimum, stimulation and inhibition by K+, Km for the deoxyribonucleoside 5'-triphosphates, stability to heating at 45 degrees C, and inhibition by N-ethylmaleimide. The preferred primer-template for both enzymes was "activated" DNA (DNA submitted to limited degradation by pancreatic deoxyribonuclease); native or thermally denatured DNA templates were relatively very poorly copied. When certain synthetic templates were tested, substantial differences were revealed between the two enzymes. Poly[d(A-T)] was poorly used by polymerase I but was superior to "activated" DNA for polymerase II. Poly[d(A)]-oligo[d(pT)10] was used efficiently by polymerase I but not by polymerase II. Poly(A)-oligo[d(pT)10] was not an effective primer-template although polymerase I could use it to a limited extent when Mn2+ replaced Mg2+ in the polymerase reaction and when the temperature of incubation was lowered from 37 degrees to 30 degrees C. When only one or two or three triphosphates were supplied in the reaction mixture, the activity of polymerase I was more severly diminished than that of polymerase II.     

Metal requirements and phosphodiesterase activity of tRNase Z enzymes

The endonuclease tRNase Z from A. thaliana (AthTRZ1) was originally isolated for its tRNA 3' processing activity. Here we show that AthTRZ1 also hydrolyzes the phosphodiester bond in bis(p-nitrophenyl) phosphate (bpNPP) with a kcat of 7.4 s-1 and a KM of 8.5 mM. We analyzed 22 variants of AthTRZ1 with respect to their ability to hydrolyze bpNPP. This mutational mapping identified fourteen variants that lost the ability to hydrolyze bpNPP and seven variants with reduced activity. Surprisingly, a single amino acid change (R252G) resulted in a ten times higher activity compared to the wild type enzyme. tRNase Z enzymes exist in long and short forms. We show here that in contrast to the short tRNase Z enzyme AthTRZ1, the long tRNase Z enzymes do not have bpNPP hydrolysis activity pointing to fundamental differences in substrate cleavage between the two enzyme forms. Furthermore, we determined the metal content of AthTRZ1 and analyzed the metal requirement for bpNPP hydrolysis. AthTRZ1 shows a high affinity for Zn2+ ions; even upon incubation with metal chelators, 0.76 Zn2+ ions are retained per dimer. In contrast to bpNPP hydrolysis, pre-tRNA processing requires additional metal ions, Mn2+ or Mg2+, as Zn2+ ions alone are insufficient.     

An adenosine triphosphate-dependent deoxyribonuclease from Bacillus laterosporus. Improved purification, subunit structure and substrate specificity

The ATP-dependent deoxyribonuclease from Bacillus laterosporus has been purified to near homogeneity by a procedure involving ammonium sulfate fractionation, DEAE-cellulose chromatography, Sephadex G-150 gel filtration, DEAE-Sephadex A-25 chromatography and DNA-cellulose affinity chromatography. The purified enzyme has a molecular weight of 210,000 +/- 8,000 as determined by sucrose gradient sedimentation. It is composed of two nonidentical polypeptide chains with close molecular weights of around 110,000. The substrate preference of the pure enzyme is essentially identical with the previous result obtained with the partially purified enzyme preparation (Anai, M., Mihara, T., Yamanaka, M., Shibata, T., & Takagi, Y. (1975) J. Biochem. 78, 105-114). Thus, the enzyme degrades double-stranded DNA about 100 times faster than heat-denatured DNA in the presence of ATP. Double-stranded DNA is not degraded to any measurable extent in the absence of ATP, but the enzyme exhibits activity toward denatured DNA in the absence of ATP. Furthermore, no endonuclease activity is observed on covalently closed circular duplex DNA and open circular duplex DNA.     

Comparison between calcium transport and adenosine triphosphatase activity in membrane vesicles derived from rabbit kidney proximal tubules

Characteristics of Ca2+ uptake were studied in a vesicular preparation of proximal tubule plasma membranes from rabbit kidney and compared with the properties of both membrane-bound and solubilized Ca2+-ATPase activities. Calcium uptake required both ATP and MgCl2 and revealed two kinetic components with respect to Ca2+ concentration requirements, one with a high affinity for Ca2+ (1.8 microM), operative in the range of cytosolic Ca2+ activity, and one with a low affinity for Ca2+ (250 microM) which may become active only at abnormally high cytosolic Ca2+ concentrations. The high- and low-affinity components were stimulated to similar extents by phosphate, and required similar concentrations of ATP (0.6 mM) for half-maximal activity. The amount of membrane-bound phosphoenzyme formed from ATP in the presence of Ca2+ was the same regardless of whether only one or both sites were saturated, suggesting that occupancy of the second Ca2+ binding site accelerates the enzyme turnover. Inhibition of Ca2+ transport by Na+ was reversed by the addition of ouabain or an ATP-regenerating system, indicating that this inhibitory effect of Na+ on Ca2+ uptake may be due to the accumulation of ADP in the medium as a result of Na+ pump activity. Low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and valinomycin (2.5 and 1 microM, respectively) were without effect on Ca2+ uptake in the presence of phosphate, whereas higher concentrations of the ionophores (200 and 100 microM, respectively) reduced uptake by 60% or more. The calmodulin antagonist 48/80 also reduced Ca2+ uptake with half-maximal effectiveness at 100 micrograms/ml. None of these drugs affected either ATPase activity or the EGTA-induced Ca2+ efflux from preloaded vesicles. The Ca2+ dependence of ATP hydrolysis by the membrane-bound enzyme preparation was similar to that observed for Ca2+ uptake by the vesicles. However, with solubilized enzyme, concentrations of Ca2+ similar to that found in the plasma reduced Ca2+-stimulated ATP hydrolysis to one-half of its maximal rate. This indicates that peritubular Ca2+ may play a role in the regulation of Ca2+ transport across the tubular epithelium. ATP could not be replaced by ITP as a substrate for Ca2+ uptake, and the (Ca2+ + Mg2+)ITPase activity of soluble enzyme was 25-fold lower than in the presence of ATP. This is an indication that the active Ca2+ pumping mechanism in proximal tubules is critically dependent on the nucleoside moiety of the substrate.