Purification and partial characterization of the DNA-dependent RNA polymerase from Rickettsia prowazekii

The DNA-dependent RNA polymerase was purified from Rickettsia prowazekii, an obligate intracellular bacterial parasite. Because of limitation of available rickettsiae, the classical methods for isolation of the enzyme from other procaryotes were modified to purify RNA polymerase from small quantities of cells (25 mg of protein). The subunit composition of the rickettsial RNA polymerase was typical of a eubacterial RNA polymerase. R. prowazekii had beta' (148,000 daltons), beta (142,000 daltons), sigma (85,000 daltons), and alpha (34,500 daltons) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The appropriate subunits of the rickettsial RNA polymerase bound to polyclonal antisera against Escherichia coli core polymerase and E. coli sigma 70 subunit in Western blots (immunoblots). The enzyme activity was dependent on all four ribonucleoside triphosphates, Mg2+, and a DNA template. Optimal activity occurred in the presence of 10 mM MgCl2 and 50 mM NaCl. Interestingly, in striking contrast to E. coli, approximately 74% of the rickettsial RNA polymerase activity was associated with the rickettsial cell membrane at a low salt concentration (50 mM NaCl) and dissociated from the membrane at a high salt concentration (600 mM NaCl).     

Interaction of sodium and potassium ions with Na+,K+-ATPase. II. General properties of ouabain-sensitive K+ binding

General properties of ouabain-sensitive K+ binding to purified Na+,K+-ATPase [EC 3.6.1.3] were studied by a centrifugation method with 42K+. 1) The affinity for K+ was constant at pH values higher than 6.4, and decreased at pH values lower than 6.4. 2) Mg2+ competitively inhibited the K+ binding. The dissociation constant (Kd) for Mg2+ of the enzyme was estimated to be about 1 mM, and the ratio of Kd for Mg2+ to Kd for K+ was 120 : 1. The order of inhibitory efficiency of divalent cations toward the K+ binding was Ba2+ congruent to Ca2+ greater than Zn2+ congruent to Mn2+ greater than Sr2+ greater than Co2+ greater than Ni2+ greater than Mg2+. 3) The order of displacement efficiency of monovalent cations toward the K+ binding in the presence or absence of Mg2+ was Tl+ greater than Rb+ greater than or equal to (K+) greater than NH4+ greater than or equal to Cs+ greater than Na+ greater than Li+. The inhibition patterns of Na+ and Li+ were different from those of other monovalent cations, which competitively inhibited the K+ binding. 4) The K+ binding was not influenced by different anions, such as Cl-, SO4(2-), NO3-, acetate, and glycylglycine, which were used for preparing imidazole buffers. 5) Gramicidin D and valinomycin did not affect the K+ binding, though the former (10 micrograms/ml) inhibited the Na+,K+-ATPase activity by about half. Among various inhibitors of the ATPase, 0.1 mM p-chloromercuribenzoate and 0.1 mM tri-n-butyltin chloride completely inhibited the K+ binding. Oligomycin (10 micrograms/ml) and 10 mM N-ethylmaleimide had no effect on the K+ binding. In the presence of Na+, however, oligomycin decreased the K+ binding by increasing the inhibitory effect of Na+, whether Mg2+ was present or not. 6) ATP, adenylylimido diphosphate and ADP each at 0.2 mM decreased the K+ binding to about one-fourth of the original level at 10 microM K+ without MgCl2 and at 60 microM K+ with 5 mM MgCl2. On the other hand, AMP, Pi, and p-nitrophenylphosphate each at 0.2 mM had little effect on the K+ binding.     

Purification and kinetic properties of polynucleotide kinase from rat testes

Polynucleotide kinase (EC 2.7.1.78) has been purified from rat testes, and an approximately 2000-fold purification was obtained. The purified enzyme had an Mr of 38000 +/- 3800. The enzyme phosphorylated micrococcal nuclease-treated calf thymus DNA and (dT)10 while 5'-HO-tRNA was a very poor substrate. A certain degree of specificity towards purine-containing 5'-HO-nucleotides was observed. The polynucleotide kinase had an absolute requirement for a divalent cation. Both Mg2+ and Mn2+ could be used, but 10 mM MgCl2 gave optimal activity. The monovalent cations Na+, K+ and NH4+ all stimulated enzyme activity, and the optimal concentration was 0.1 M. The enzyme was inhibited by inorganic phosphate, pyrophosphate and sulphate. A 50% inhibition was obtained with 20, 0.3 and 2 mM, respectively. At 2 mM MgCl2, 1 mM spermine enhanced the enzyme activity 3-times. The apparent KATP was estimated to be 36 microM and KHO-DNA was found to be 2 microM.     

Purification and characterization of chromatin-bound DNA-dependent RNA polymerase I from parsley (Petroselinum crispum). Influence of nucleoside triphosphates.

The isolation and purification of DNA-dependent RNA polymerase I (EC 2.7.7.6) from parsley (Petroselinum crispum) callus cells grown in suspension culture is described. The enzyme was solubilized from isolated chromatin. Purification was achieved by using DEAE- and phospho-cellulose in batches, followed by column chromatography on DEAE- and phospho-cellulose (two columns) and density-gradient centrifugation. The highly purified enzyme was stable over several months. The properties of purified parsley RNA polymerase I were investigated. Optimum concentration for Mn2+ was 1 mM, and for Mg2+ 4-6 mM, Mn2+ was slightly more stimulatory than Mg2+. The enzyme was most active at low ionic strengths [10-20 mM-(NH4)SO4]. The influence of various phosphates was tested: pyrophosphate inhibited RNA polymerase at low concentrations, whereas orthophosphate had no effect on the enzyme activity. ADP was slightly inhibitory, and AMP had no effect on the enzyme reaction. Nucleoside triphosphates and bivalent cations in equimolar concentrations in the range 4-11 mM did not influence the RNA synthesis in vitro. Free nucleoside triphosphates in excess of this 1:1 ratio inhibited the enzyme activity, unlike free bivalent cations, which stimulated RNA polymerase I.     

Kinetic properties of the purified Ca2+-translocating ATPase from human erythrocyte plasma membrane

The basic kinetic properties of the solubilized and purified Ca2+-translocating ATPase from human erythrocyte membranes were studied. A complex interaction between the major ligands (i.e., Ca2+, Mg2+, H+, calmodulin and ATP) and the enzyme was found. The apparent affinity of the enzyme for Ca2+ was inversely proportional to the concentration of free Mg2+ and H+, both in the presence or absence of calmodulin. In addition, the apparent affinity of the enzyme for Ca2+ was significantly increased by the presence of calmodulin at high concentrations of MgCl2 (5 mM), while it was hardly affected at low concentrations of MgCl2 (2 mM or less). In addition, the ATPase activity was inhibited by free Mg2+ in the millimolar concentration range. Evidence for a high degree of positive cooperativity for Ca2+ activation of the enzyme (Hill coefficient near to 4) was found in the presence of calmodulin in the slightly alkaline pH range. The degree of cooperativity induced by Ca2+ in the presence of calmodulin was decreased strongly as the pH decreased to acid values (Hill coefficient below 2). In the absence of calmodulin, the Hill coefficient was 2 or slightly below over the whole pH range tested. Two binding affinities of the enzyme for ATP were found. The apparent affinity of the enzyme for calmodulin was around 6 nM and independent of the Mg2+ concentration. The degree of stimulation of the ATPase activity by calmodulin was dependent on the concentrations of both Ca2+ and Mg2+ in the assay system.     

Tartrate-resistant acid phosphatase in free-living Amoeba proteus

Tartrate-resistant acid phosphatase (TRAP) of Amoeba proteus (strain B) was represented by 3 of 6 bands (= electromorphs) revealed after disc-electrophoresis in polyacrylamide gels with the use of 2-naphthyl phosphate as a substrate at pH 4.0. The presence of MgCl2, CaCl2 or ZnCl2 (50 mM) in the incubation mixture used for gel staining stimulated activities of all 3 TRAP electromorphs or of two of them (in the case of ZnCl2). When gels were treated with MgCl2, CaCl2 or ZnCl2 (10 and 100 mM, 30 min) before their staining activity of TRAP electromorphs also increased. But unlike 1 M MgCl2 or 1 M CaCl2, 1 M ZnCl2 partly inactivated two of the three TRAP electromorphs. EDTA and EGTA (5 mM), and H2O2 (10 mM) completely inhibited TRAP electromorphs after gel treatment for 10, 20 and 30 min, resp. Of 5 tested ions (Mg2+, Ca2+, Fe2+, Fe3+ and Zn2+), only the latter reactivated the TRAP electromorphs previously inactivated by EDTA or EGTA treatment. In addition, after EDTA inactivation, TRAP electromorphs were reactivated better than after EGTA. The resistance of TRAP electromorphs to okadaic acid and phosphatase inhibitor cocktail 1 used in different concentrations is indicative of the absence of PP1 and PP2A among these electromorphs. Mg2+, Ca2+ and Zn2+ dependence of TRAP activity, and the resistance of its electromorphs to vanadate and phosphatase inhibitor cocktail 2 prevents these electromorphs from being classified as PTP. It is suggested that the active center of A. proteus TRAP contains zinc ion, which is essential for catalytic activity of the enzyme. Thus, TRAP of these amoebae is metallophosphatase showing phosphomonoesterase activity in acidic medium. This metalloenzyme differs from both mammalian tartrate-resistant PAPs and tartrate-resistant metallophosphatase of Rana esculenta.     

Kinetic studies on the (Na+ + K+)-dependent ATPase evidence for coexisting sites for Na+, K+ and Mg2+

Na+-ATPase activity of a dog kidney (Na+ + K+)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 microM ATP and 50 microM MgCl2, but stimulated by 100 mM NaCl in the presence of 30 microM ATP and 3 mM MgCl2. The K0.5 for the effect of MgCl2 was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na+ -ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 microM MgCl2. Similar thimerosal treatment reduced (Na+ + K+)-ATPase activity by half but did not appreciably affect the K0.5 for activation by either Na+ or K+, although it reduced inhibition by high Na+ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na+: Na+-discharge sites at which Na+-binding can drive E2-P back to E1-P, thereby inhibiting Na+-ATPase activity, and sites activating E2-P hydrolysis and thereby stimulating Na+-ATPase activity, corresponding to the K+-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na+-discharge and K+-acceptance sites. Mg2+ may stimulate Na+-ATPase activity by favoring E2-P over E1-P, through occupying intracellular sites distinct from the phosphorylation site or Na+-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na+-ATPase reaction and lessen Na+-inhibition of the (Na+ + K+)-ATPase reaction by increasing the efficacy of Na+ in activating E2-P hydrolysis.     

Ca2+-dependent inhibition of smooth muscle adenylate cyclase activity

We have examined the inhibitory regulation by Ca2+ of the adenylate cyclase activity associated with microsomes isolated from bovine aorta smooth muscle. In the presence of 2 mM MgCl2, Ca2+ (0.8-100 microM) inhibited in a noncompetitive manner activation of the enzyme by GTP, Gpp[NH]p, or forskolin. In all instances the value for half-maximal inhibition was between 2 and 3 microM. In contrast, Ca2+ inhibited the activation by MgCl2 (2-50 mM), alone or in the presence of GTP, in a competitive manner. The inhibition of adenylate cyclase by 10 microM Ca2+ was reversed in the presence of either 5 or 25 microM calmodulin or troponin C. These data show that (i) Ca2+, at concentrations similar to those which activate smooth muscle contraction, inhibits the stimulation of adenylate cyclase by several activators; (ii) Ca2+ and Mg2+ compete for a common site on the smooth muscle adenylate cyclase complex; and (iii) the reversal of Ca2+-dependent inhibition by Ca2+-binding proteins may be produced by chelation of the metal by these proteins.     

Polyadenylate polymerase from cytoplasm and nuclei of N.I.H.-Swiss mouse embryos.

Poly (A) polymerase activity from cytoplasm and nuclei of 12-16-day-old mouse embryos has been partially purified by (NH4)2SO4 fractionation, DEAE-cellulose, phosphocellulose and tRNA-Sepharose affinity chromatography, and their properties have been compared. The nuclear and cytoplasmic enzymes exhibit similar chromatographic elution profiles, and similar biochemical and physical properties. Poly(A) polymerase has an absolute requirement for a divalent cation, ATP and an oligo- or polyribonucleotide primer. With tRNA, the divalent salt concentrations for optimum enzyme activity are 1 mM MnCl2 or 10 mM MgCl2. The enzyme activity with MnCl2 is 10-15-fold higher than that with MgCl2. The molecular weight of the native enzyme is about 65 000 and its sedimentation coefficient is around 4.5 S. The average chain length synthesized by the enzyme is between 10 and 13 nucleotides. The inhibitors of RNA polymerase do not affect poly (A) polymerase activity; however, some synthetic rifamycin SV derivatives are potent inhibitors of this enzyme.     

Cloning of beta-isopropylmalate dehydrogenase gene from Bacillus coagulans in Escherichia coli and purification and properties of the enzyme

The beta-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate: NAD+ oxidoreductase, EC 1.1.1.85) gene from Baccilus coagulans was cloned and expressed in Escherichia coli C600, using pBR322 as a vector plasmid. The B. coagulans enzyme was purified to a homogeneous state from the E. coli carrying a pBR322 - the B. coaglulans enzyme gene hybrid plasmid. The enzyme consists of two subunits of equal molecular weight (4.4 X 10(4) ). The enzyme activity was stimulated by 0.5 mM Mn2+, Mg2+ and Co2+. The enzyme was strongly inhibited by 0.2 mM p-chloromercuribenzoate and the inhibition was completely recovered by 1 mM dithiothreitol. The B. coagulans enzyme was thermostabilized by 1.5 M NaCl. The B. coagulans enzyme is a composite of alpha-helix, beta-sheet and remainder. The secondary structure of the enzyme was appreciably altered by 0.5 mM MgCl2 and 1.5 M NaCl.     

Two distinct poly(A) polymerases isolated from the cytoplasm of Ehrlich ascites tumour cells

The poly(A) polymerases from the cytosol and ribosomal fractions of Ehrlich ascites tumour cells are isolated and partially purified by DEAE-cellulose and phosphocellulose column chromatography. Two distinct enzymes are identified: (a) a cytosol Mn2+-dependent poly(A) polymerase (ATP:RNA adenylyltransferase) and (b) a ribosome-associated enzyme defined tentatively as ATP(UTP): RNA nucleotidyltransferase. The cytosol poly(A) polymerase is strictly Mn2+-dependent (optimum at 1 mM Mn2+) and uses only ATP as substrate, poly(A) is a better primer than ribosomal RNA. The purified enzyme is free of poly(A) hydrolase activity, but degradation of [3H]poly(A) takes place in the presence of inorganic pyrophosphate. Most likely this enzyme is of nuclear origin. The ribosomal enzyme is associated with the ribosomes but it is found also in free state in the cytosol. The purified enzyme uses both ATP and UTP as substrates. The substrate specificity varies depending on ionic conditions: the optimal enzyme activity with ATP as substrate is at 1 mM Mn2+, while that with UTP as substrate is at 10--20 mM Mg2+. The enzymes uses both ribosomal RNA and poly(A) [but not poly(U)] as primers. The purified enzyme is free of poly(A) hydrolase activity.     

The interdependence of magnesium with spermidine and phosphoenolpyruvate in an enzyme-synthesizing system in vitro.

The DNA-dependent syntheses of different enzymes of the bacteriophages T3 and T7 were studied in an Escherichia coli system in vitro with respect to the optimal Mg2+ concentration and its interdependence with substituting (e.g. spermidine) and complexing agents (e.g. phosphoenolpyruvate). The following results were obtained. 1. The optimal conditions for the syntheses of the different enzymes were not identical. The optima for RNA polymerase synthesis were 8 mM Mg2+, 10 mM P-pyruvate and 3 mM spermidine; for S-adenosyl-L-methionine cleaving enzyme synthesis, 6 mM Mg2+, 6 mM P-pyruvate and 3 mM spermidine; and for lysozyme synthesis, 13-18 mM Mg2+, 28 mM P-pyruvate and 3-0 mM spermidine. 2. The optimal conditions for the synthesis of analog enzymes (RNA polymerases and lysozymes) from the two templates were identical with experimental error. 3. Mg2+ and spermidine substituted for each other in relation to the number of their charges. 4. The apparent complexing of one Mg2+ molecule required the addition of 3-5 P pyruvate molecules. 5. Under the optimal conditions the enzyme-synthesizing activity was higher by more than a factor of 10 compared to previously described systems.     

Enzymic characteristics of ecto-adenosine triphosphatase in rat epididymal intact spermatozoa.

Ecto-ATPase in rat cauda-epididymal intact spermatozoa has a high degree of substrate specificity for the hydrolysis of ATP and dATP rather than of ADP, AMP, GTP, dGTP, CTP, dCTP, TTP and UTP. The enzyme is activated by bivalent metal ions in the order Mg2+ greater than Mn2+ greater than Co2+ greater than Ca2+. The apparent Km values of the enzyme for Mg2+, Mn2+, Co2+ and Ca2+ are approx. 80, 100, 100 and 150 microM respectively. Addition of Ca2+ (0.1 or 1 mM) gives no further stimulation of the Mg2+-activated ecto-ATPase activity. The apparent Km value of the enzyme for ATP is 95 microM. Pi (16 mM) inhibits the enzymic activity (by 25%), whereas Na+ (50 mM) or K+ (10 mM) alone or in combination, polyamines (spermine and spermidine; 1--12.5mM) and nucleic acids (yeast RNA and calf thymus DNA; 0.12 or 0.62 mg/ml) had no significant effect on the activity of the enzyme. Orthovanadate at a relatively low concentration (20 microM) strongly inhibits (approx. 50%) the ecto-ATPase activity. Vanadate inhibition can be reversed by noradrenaline (2.5 mM). The vanadate-sensitivity of the enzyme increases markedly during spermatozoal maturation in the epididymis. However, the activity of the spermatozoal ecto-ATPase decreases progressively during the epididymal transit of the testicular spermatozoa.     

Purification and characterization of a thermostable α-galactosidase from Thielavia terrestris NRRL 8126 in solid state fermentation

Several seeds and husks of some plants belonging to leguminosae, Graminae, Compositae and Palmae were evaluated as carbon substrates to produce α-galactosidase (α-Gal) by the thermophilic fungus, Thielavia terrestris NRRL 8126 in solid substrate fermentation. The results showed that Cicer arietinum (chick pea seed) was the best substrate for α-Gal production. The crude enzyme was precipitated by ammonium sulphate (60%) and purified by gel filtration on sephadex G-100 followed by ion exchange chromatography on DEAE-Cellulose. The final purification fold of the enzyme was 30.42. The temperature and pH optima of purified α-Gal from Thielavia terrestris were 70 °C and 6.5, respectively. The enzyme showed high thermal stability at 70 °C and 75 °C and the half-life of the α-Gal at 90 °C was 45 min. Km of the purified enzyme was 1.31 mM. The purified enzyme was inhibited by Ag2+, Hg2+, Zn2+, Ba2+, Mg2+, Mn2+ and Fe2+ at 5 mM and 10 mM. Also, EDTA, sodium arsenate, L-cysteine and iodoacetate inhibited the enzyme activity. On the other hand, Ca2+, Cu2+, K+ and Na+ slightly enhanced the enzyme activity at 5 mM while at 10 mM they caused inhibition. The molecular weight of the α-Gal was estimated to be 82 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme displays a number of biochemical properties that make it a potentially strong candidate for biotechnological and medicinal applications.     

Effects of polyamines and methylglyoxal bis(guanylhydrazone) on Escherichia coli ribonucleic acid polymerase and the template activity of hepatic cell nuclei in vitro

Escherichia coli RNA polymerase was assayed with 4 mM Mg2+ and 1 mM Mn2+ using native DNA, heat-denatured DNA, histone-nucleate and isolated rat liver nuclei as the template source. With purified DNA and either or both divalent metal ions, 0.1--5 mM amine stimulated enzyme activity. Spermidine resulted in the greatest stimulation (1.7-fold at 5 mM); whereas, spermine or methylglyoxal bis(guanylhydrazone) first stimulated, then above 3 mM inhibited, the reaction. The addition of unfractionated histone to purified DNA inhibited the reaction by 90%. The subsequent addition of amines resulted in a slight stimulation in incorporation (1.5-fold) in the range of 1--3 mM amine. Alternatively, when enzyme was combined with DNA before histone, only a 20% inhibition was observed and this could be completely prevented by 3 mM spermidine. The addition of amines to isolated nuclei resulted in marked alterations in ultrastructure and Mg2+ content; however, relatively small effects on RNA polymerase activity were observed. With the E. coli enzyme, 0.1--1.0 mM amine stimulated RNA synthesis (1.5-fold) whereas, none of the amines stimulated endogeneous activity in the absence or presence of 300 mM (NH4)2SO4.     

On the role of sulfhydryl groups in the structure and function of the Azotobacter vinelandii RNA polymerase.

Exposure of sulfhydryl groups as indicated by titration kinetics is decreased under conditions where RNA polymerase exists as a dimer or higher aggregate (low salt), in the presence of Mn2+, or when bound to d(A-T). Incubation of phenylmercurisulfonate with RNA polymerase above pH 9.0 results in loss of d(A-T) binding ability. Poly(U) binding is more sensitive to sulfhydryl modification and is lost as pH's above 8.0. The presence of 4 mM Mn2+ has an obvious effect in stabilizing the polymerase-poly(U) complex when incubated with 10 muM phenylmercurisulfonate + 1 M urea. Incubation of the enzyme with the mercurial and urea results in disaggregation to subprotomeric forms and release of the alpha subunit. Similar treatment in the presence of 4 mM MnSO4 stabilizes the protomeric structure of the enzyme. During chain elongation the enzyme exists as a ternary d(A-T)n-enzyme-r(U-A)n complex in which the bound d(A-T)n is refractory to the destabilizing effect of the mercurial; however, further phosphodiester bond formation is inhibited. The results are defined in terms of a role which reflects the involvement of polymerase sulfhydryl groups in the various conformations necessary for subunit-subunit interaction, tight template binding and catalytic activity.     

Phosphatase activity and potassium transport in liposomes with Na+,K(+)-ATPase incorporated.

We have used liposomes with incorporated pig kidney Na+,K(+)-ATPase to study vanadate sensitive K(+)-K+ exchange and net K+ uptake under conditions of acetyl- and p-nitrophenyl phosphatase activities. The experiments were performed at 20 degrees C. Cytoplasmic phosphate contamination was minimized with a phosphate trapping system based on glycogen, phosphorylase a and glucose-6-phosphate dehydrogenase. In the absence of Mg2+ (no phosphatase activity) 5-10 mM p-nitrophenyl phosphate slightly stimulated K(+)-K+ exchange whereas 5-10 mM acetyl phosphate did not. In the presence of 3 mM MgCl2 (high rate of phosphatase activity) acetyl phosphate did not affect K(+)-K+ exchange whereas p-nitrophenyl phosphate induced a greater stimulation than in the absence of Mg2+; a further addition of 1 mM ADP resulted in a 35-65% inhibition of phosphatase activity with an increase in K(+)-K+ exchange, which sometimes reached the levels seen with 5 mM phosphate and 1 mM ADP. The net K+ uptake in the presence of 3 mM MgCl2 was not affected by acetyl phosphate or p-nitrophenyl phosphate, whereas it was inhibited by 5 mM phosphate (with and without 1 mM ADP). The results of this work suggest that the phosphatase reaction is not by itself associated to K+ translocation. The ADP-dependent stimulation of K(+)-K+ exchange in the presence of phosphatase activity could be explained by the overlapping of one or more step/s of the reversible phosphorylation from phosphate with the phosphatase cycle.     

Purification and characterization of RNase P from Clostridium sporogenes

RNase P is a multi-subunit enzyme responsible for the accurate processing of the 5' terminus of all tRNAs. The RNA subunit from Clostridium sporogenes has been partially purified and characterized. The RNA is approximately 400 nucleotides long and makes a precise endonucleolytic cleavage at the mature 5' terminus of tRNA. The RNA requires moderate concentrations of Mg2+ (20 mM) and relatively high concentrations of NH4Cl (800 mM) for optimal activity. Mn2+ effectively substitutes for Mg2+ at 2 mM. Zn2+, Ni2+, Ca2+, and Co2+ are ineffective at stimulating activity. Monovalent ions are, in general, more effective the greater the ionic radius (NH+4 greater than Cs greater than Rb greater than K greater than Na). In contrast to the activity of Bacillus subtilis, C. sporogenes RNase P RNA is significant more active in (NH4)2SO4 than in NH4Cl.