Manganese as a mutagenic agent during in vitro DNA synthesis.

The effect of Mn²⁺, a known mutagen, on the fidelity of DNA synthesis $\text{in vitro}$ by avian myeloblastosis DNA polymerase has been determined. Substitution of Mn²⁺ for Mg²⁺ leads to an enhanced incorporation of noncomplementary deoxynucleotides as well as complementary ribonucleotides with either poly (A) or poly (C) as templates. Since this polymerase lacks any detectable deoxyribonuclease activity, the $\text{in vitro}$ mutagenic effect of Mn²⁺ in promoting errors in base-pairing does not result from any diminished proof-reading function.     

A temperature-sensitive mutant of Escherichia coli with an altered RNA polymerase beta' subunit.

$\text{Penicillium charlesii}$ extracts contain UDP-galactose:NAD⁺ 2-hexosyl oxidoreductase (1). ADP-ribose also serves as a substrate resulting in formation of NADH and an oxidized ADP-ribose derivative. Treatment of the oxidized product with NaBH₄ followed by hydrolysis at pH 2 and 100° releases xylose as well as ribose. We conclude that ADP-D-$\text{glycero}$-D-$\text{glycero}$-3-pentosulose (ADP-3-ketoribose) is the product derived from ADP-ribose.     

Effects of physical and chemical treatments on chloroplast manganese. NMR and ESR studies

Measurements were made of the water proton relaxation rate (T^?1₂ = R₂), electron spin resonance (ESR) six-line signal of ‘free’ Mn²⁺, and O₂-evolution activity in thylakoid membranes from pea leaves. The main results are: (1) Aging of thylakoids at 35°C causes a parallel decrease in O₂-evolution activity, in R₂ and in the content of bound Mn, suggesting that R₂ may be related to the loosely bound Mn involved in O₂ evolution. (2) Treatment of thylakoids with tetraphenylboron (TPB) at [TPB] > 2 mM produces a 2-fold increase in R₂, without release of Mn²⁺. The titration curve exhibits three sharp end points. The first end point occurs at a $\text{[}\text{TPB}\text{]}\text{[}\text{chlorophyll}\text{]}$ of 1.25, at which the O₂ evolution is completely inhibited. (3) Treatment of thylakoids with NH₂OH also increases R₂ by nearly 2-fold, either by the reduction of the higher oxidation states of Mn to Mn²⁺ and / or by exposing the Mn to solvent protons. Also, progressive release of bound Mn occurs at [NH₂OH] ≥ 1 mM as shown by an increase increase in the Mn²⁺ ESR signal and a decrease in R₂. (4) Addition of H₂O₂ (0.1–1.0%) to thylakoids causes an enhancement of R₂ similar to that by NH₂OH, but without the release of Mn²⁺. (5) Heat treatment of thylakoids at 40–50°C releases Mn²⁺ and increases R₂. Conversely, pH values of 7 to 4 release Mn²⁺ without changing R₂ while pH values of 7–9 increase R₂ without releasing Mn²⁺. Thus, both high and low pH values as well as the heat treatment cause structural changes enhancing the relaxivity of the bound Mn or of other paramagnetic species.     

Terminal deoxynucleotidyl transferase: characterization of extraction and assay conditions from human and calf tissue.

Methods of extraction and assay of terminal deoxynucleotidyl transferase (TdT) from human lymphoblasts and calf thymus were compared. A high salt concentration was mandatory for complete enzyme extraction, while dialysis of the crude extract resulted in a major loss of enzyme activity. In addition, TdT was partially purified from lymphoblasts of patients with acute lymphoblastic leukemia. The K_m for the monomer, deoxy-guanosine 5′-triphosphate (dGTP), is high (~0.1 mm) in the presence of either Mg²⁺ or Mn²⁺, whereas the K_m for the initiator, poly(deoxyadenylic acid $\text{[poly(d(pA)}\text{50}\text{)]}$, with an average chain length of 50 residues, is 2.5 μm in the presence of Mg²⁺ and 0.3 μm in the presence of Mn²⁺. The maximum velocity is higher for the calf thymus TdT in the presence of Mg²⁺ than in Mn²⁺. Human TdT catalyzes the polymerization of dGTP at a higher rate in the presence of Mn²⁺ than with Mg²⁺. These data illustrate that partially purified human TdT differs in catalytic properties from the purified calf thymus enzyme. Therefore, optimal conditions for assay of TdT in extracts from calf and human tissues differ.     

Analysis of the hexagonal II phase and its relations to lipidic particles and the lamellar phase A freeze-fracture study

Model systems of phosphatidylethanolamine (PE) and cardiolipin (DPG), as pure components and in binary mixtures with phosphatidylcholine (PC) have been morphologically analysed. The relation between the hexagonal_II (H_II) phase and lipidic particles as well as between the H_II phase and the lamellar phase has been studied. Moreover, the periodicity of the various H_II tubes was determined. (1) The periodicity of the H_II phase of cardiolipin is dependent on the cation involved. DPG-Ca exhibits the smallest tube to tube distance when compared to Mg²⁺ and Mn²⁺. Moreover, the DPG-Ca tubes are quite straight, in contrast to the Mg²⁺ and Mn²⁺ tubes, which appear to be frequently curved. (2) H_II tubes with two distinct diameters have been observed in H_II phase containing lipid mixtures. The thickness of the H_II tube is related to the composition of the tube. In the cardiolipin-lecithin system, structural separation of the pure cardiolipin H_II phase has been suggested with Mg²⁺ and Mn²⁺, but not with Ca²⁺. (3) Models for the H_II to lamellar phase transition and for the H_II phase to the lipidic particles are presented. (4) Lipidic particles are exclusively found in lipid model systems, which contain H_II phase favouring lipids. Morphological evidence is presented which suggests these lipidic particles represent inverted micelles. These observations include: (${}_{\mathrm{<mtext>i</mtext>}}$) there is a strong topological and quantitative relation between H_II tubes and lipidic particles, (${}_{\mathrm{<mtext>ii</mtext>}}$) lipidic particles occur densely packed in conglomerates without the presence of a smooth layer.     

Electron exchange coupling in a naturally occurring tetramangano cluster in the mineral helvite, (Mn4S)(SiBeO4)3

The mineral helvite, (Mn₄S)(BeSiO₄)₃, contains discrete tetrahedral Mn₄S⁺⁶ clusters in which the S^?2 is tetrahedrally coordinated and each Mn(II) is in a distorted tetrahedron of one S^?2 and three oxygens; the cluster is situated within an encompassing lattice of SiO₄^?4 and BeO₄^?6 tetrahedra. Mn₄S⁺⁶ centers provide an interesting model for comparison to the polynuclear manganese center that is associated with photosynthetic water oxidation. Magnetic susceptibility data between 77 and 298 K have been measured for a natural helvite sample containing principally Mn₄S⁺⁶ centers but with significant contamination from Mn₃FeS⁺⁶ and Mn₃CaS⁺⁶. The data exhibited Curie-Weiss behavior with μ_eff = 5.969 B.M. and θ = 178.3 K. An analysis of the magnetic susceptibility, based on Van Vleck's formalism, demonstrated the presence of antiferromagnetic coupling, with a coupling constant J = ?5.83 cm^?1. Mössbauer spectra of Mn₃FeS centers in helvite and of Fe₄S centers in the related mineral danalite have also been recorded. Isomer shifts show little temperature dependence and lie in the range 1.23–1.43 $\text{mm}\text{sec.}$. This range is typical of tetrahedrally coordinated Fe(II) in several ionic crystals but is significantly above that of Fe(II) in ferredoxins and below that in the [quinone-Fe(II)-quinone] complex of the photosynthetic bacterium,Rhodopseudomonas sphaeroides. Quadrupole splittings are highly temperature dependent, ranging from 2.4 $\text{mm}\text{sec}$ at 4.2 K to less than 0.5 $\text{mm}\text{sec}$ at 248 K.     

Magnetic resonance studies of the mitochondrial divalent cation carrier

Measurements of water proton spin relaxation enhancements (ε) can be used to discriminate high-affinity binding of Mn²⁺ or Gd³⁺ to biological membranes, from low-affinity binding. In rat liver mitochondria, ε_b values of approx. 11 are observed upon binding of Mn²⁺ to the inner membrane, while internal or low-affinity binding remains invisible to this technique. Energy-driven Mn²⁺ uptake by liver mitochondria results in the subsequent decay of ε¹.Comparison of ε¹ with the initial velocity of Mn²⁺ uptake in rat liver mitochondria reveals a linear correlation, which holds at all temperatures between 0 °C and 40 °C, regardless of the mitochondrial protein concentration. Consequently, enhancement appears to reflect the binding of Mn²⁺ to the divalent cation pump.Binding of Mn²⁺ to blowfly flight muscle also results in substantial ε¹, which is associated with the glycerol-1-phosphate dehydrogenase instead of divalent cation transport. Consequently, no decay in ε¹ due to uptake occurs after Mn²⁺ is bound.Lanthanide ions are also bound and transported by mitochondria. Addition of Gd³⁺ to pigeon heart or rat liver mitochondria results in ε_b ≈ 5–6, which decays with similar kinetics in both systems. The uptake velocity of Gd³⁺ in rat liver mitochondria is about $\text{1}\text{6}$ the rate with which Mn²⁺ is transported. Lanthanides also diminish ε¹ due to the addition of Mn²⁺, and greatly retard the Mn²⁺ uptake kinetics. The presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone depresses ε¹ upon addition of Mn²⁺ or Gd³⁺ and also uncouples energy-driven uptake. On the other hand, prolonged anaerobic incubation in the presence of antimycin and rotenone exhausts the mitochondria of their energy stores, blocks the uptake of Mn²⁺, but does not affect ε¹ significantly. Evidently, the uncoupler-induced disappearance of divalent cation binding sites is not the result of “de-energization”.Measurements of ε¹ at several NMR frequencies indicate a correlation time (τ_b) for carrier-bound Mn²⁺ in rat liver mitochondria between 20 ns and 4 ns as one varies the temperature between 10 °C and 30 °C. The 13 Kcal/mole activation energy for τ_b suggests that the 11 ns time constant at room temperature represents the movement of the Mn^II-carrier complex. On the other hand, τ_b is probably approx. 100 times too short to represent the rotational motion of a carrier protein. Apparently, Mn²⁺ binds to a small arm of the carrier which moves independently of the main body of any protein.In addition to Mn(H₂O)₆²⁺, other complexes of Mn²⁺ may also be bound and transported by rat liver mitochondria. Only a small increase in ε¹ occurs upon addition of MnHPO₄, yet this species is accumulated by the mitochondria. Consequently, the carrier does not recognize divalent metal ions on the basis of charge.     

Stimulation by manganese and other divalent cations of the electron donation reactions of Photosystem II

Using inside-out thylakoid membranes, it has been shown that the oxidation of water and associated reduction of dichlorophenol indophenol is partially inhibited by low concentrations of cation chelators. This inhibition correlates with a removal of two manganese ions per Photosystem II reaction centre. The chelator-induced inhibition was completely reversed by the addition of low levels of Mn²⁺ ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 20 μ}\text{M}$) and higher levels of Mg²⁺ and Ca²⁺ ($\text{C}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 1}\text{mM}$). Other cations were not effective, indicating that the ability to overcome the inhibition did not involve a general electrostatic screening process. The degree of inhibition by chelators was greater at lower light intensities and after treatment with glutaraldehyde. In the presence of glutaraldehyde the stimulatory effect of Mn²⁺ was lost, while pretreatment with Mn²⁺ prevented the glutaraldehyde effect. These results are discussed in terms of conformational changes of the electron donation chains involving cation- (preferentially Mn-) dependent coupling between the oxygen evolving and reaction-centre complexes of Photosystem II.     

The structure of the fluorophosphate-pyruvate kinase complex investigated by 31P relaxation rate studies

The interaction of fluorophosphate with muscle pyruvate kinase was investigated by ³¹P nuclear relaxation rate measurements. The fluorophosphate samples were highly purified and were first monitored by ¹⁹F and ³¹P relaxation rate measurements in the formation of the binary FPO₃-Mn complex. The results of the binary complex demonstrated that FPO₃^2? binds in the first coordination sphere of Mn²⁺ via the oxygen atoms but not via the fluorine. The enzyme experiments were designed under conditions where a significant fraction of the ligand is in the ternary enzyme-Mn-FPO₃ complex. These studies demonstrate that the ³¹P relaxation rate of bound FPO₃ ($\text{1}\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>m</mtext>}}\text{= 1.58 ± 0.05 × 10}{}^5\text{s}{}^{\mathrm{?1}}$) is consistent with the binding of this ligand in the first coordination sphere of enzyme-bound Mn²⁺ with an elongated Mn-O-P distance ($\text{r}{}_{\mathrm{<mtext>Mn-P</mtext>}}\text{= 3.3 ± 0.2}\text{A}\text{?}$). Such a structure is demonstrated in the ternary enzyme-Mn-FPO₃ complex, in the complex containing HCO₃^?, and in the complex also containing HCO₃^? and ADP. The data further substantiate the binding of phosphoenolpyruvate analogs in the first coordination sphere of pyruvate kinase-bound Mn²⁺.     

Interaction of manganese with fragments, complementary fragment recombinations, and whole molecules of yeast phenylalanine specific transfer RNA

Binding of Mn²⁺ to the whole molecule, fragments and complementary fragment recombinations of yeast tRNA^Phe, and to synthetic polynucleotides was studied by equilibrium dialysis. The comparison of the binding patterns of the fragments, fragment recombinations and synthetic polynucleotides with that of intact tRNA^Phe permits reasonable conclusions concerning the nature and location of the various classes of sites on tRNA^Phe. Binding of Mn²⁺ to intact tRNA^Phe consists of a co-operative and a non-co-operative phase. There are about 17 “strong” sites and several “weak” ones. Five of the 17 strong sites are associated with the co-operative phase. This phase is completely lacking in the binding of Mn²⁺ to tRNA^Phe fragments (5′-$\text{1}\text{2}$, 3′-$\text{1}\text{2}$, 5′-$\text{3}\text{5}$, 3′-$\text{2}\text{5}$), poly-(A):poly(U) and poly(I):poly(C) helices, and single stranded poly(A) and poly(U). This argues that the co-operative sites arise from the tRNA tertiary structure. This conclusion is further strengthened by the observation that cooperativity is present in a tRNA^Phe molecule which has been split in the anticodon loop, but it is absent in one which has been split in the extra loop. It is in the vicinity of the latter loop, but not the former, that tertiary interactions are seen in the crystal structure. The remaining 12 strong sites are “independent” and appear to be associated with cloverleaf helical sections.     

Spatial relationships among the active and allosteric sites of carbamoyl-phosphate synthetase

NMR experiments were conducted to map distances among various loci on Escherichia coli carbamoyl-phosphate synthetase. Three paramagnetic probes, viz., Mn²⁺, Cr³⁺-ATP, and nitroxide spin-labels were used in experiments designed to measure the $\text{1}\text{T}{}_1$ (longitudinal relaxation rate) of various nuclei in enzyme complexes with these paramagnetic species. The distance between the monovalent cation activator site and enzyme-bound Cr³⁺-ATP was determined using three different monovalent cations, ¹³³Cs⁺, ¹⁵NH₄⁺, and Li⁺ (⁶Li and ⁷Li). Substantial paramagnetic effects were observed on the $\text{1}\text{T}{}_1$ values for all four nuclei and the M⁺ to Cr³⁺ distance was ～4 Å. Additional NMR data with ¹³³Cs⁺ and Mn²⁺ were used to obtain the distance between the two cation activator sites, monovalent and divalent, and a Mn²⁺ to Cs⁺ distance of 8.0 Å was calculated, corroborating earlier work [F. M. Raushel, P. M. Anderson, and J. J. Villafranca (1983)Biochemistry22, 1872–1876]. Three separate sulfhydryl sites on carbamoyl-phosphate synthetase were spin-labeled with 3-maleimido-2,2,5,5-tetramethylpyrrolidinyl-1-oxy. Each of these enzyme-nitroxide complexes was used to examine the paramagnetic influence on the ¹H of l-glutamate and l-ornithine and also the ¹H and ³¹P of IMP and UMP. Small paramagnetic effects were observed on these nuclei and only lower limits on the distance from each nitroxide could be obtained. Thus both l-ornithine and l-glutamate are >11 Å from each sulfhydryl site while IMP and UMP are >15 Å from these sites. A topographical map is presented based on these data and data from our previous NMR studies that show the spatial relationship among the active-site components of carbamoyl-phosphate synthetase.     

Influence of magnesium in the homogenization medium on the state of a divalent cation-stimulated,diethystilbestrol-sensitive adenylnucleotidyl phosphatase activity from pea stem microsomal preparations

The amount of divalent cation-activated, diethylstilbestrol-sensitive adenylnucleotidyl phosphatase activity recovered in the ‘microsomes’ ($\text{13 000–80 000 x g}\text{sediment}$) from pea stem tissue is strongly influenced by the concentration of Mg²⁺ in the homogenization medium. The absence of Mg²⁺ during homogenization results in a marked decrease of the activity found in the microsomal fraction, compensated by its increase in the soluble fraction. Part of the solubilized activity becomes sedimentable at $\text{80 000 × g}$ upon addition of 5–10 mM Mg²⁺ (or Mn²⁺, Ca²⁺, Zn²⁺) to the supernatant. This sediment shows a very high specific activity, and can be re-solubilized by treatment with either EDTA or 0.3 M monovalent salts, or deoxycholate. When the supernatant containing the solubilized activity is incubated together with low-adenylnucleotidyl phosphatase microsomes and with 10 mM MgCl₂ the activity recovered in the sediment is much larger than the sum of the activity of the microsomes plus that of the sediment obtained by incubating the same supernatant with Mg²⁺. Microsomes prepared with Mg²⁺ in the homogenization medium do not show this effect. The supernatant/microsomes saturation curves as well as a change of the temperature coefficient of the activity following combination of the soluble preparation with the microsomal particles suggest an at least partial reconstitution of the original enzyme-membrane structure.     

Multiple ion-stimulated adenosine triphosphatase activities associated with membranes of the diatom Nitzschia alba.

At least ten distinct ATP-hydrolyzing activities are associated with mitochondria, endoplasmic reticulum-, Golgi-, and plasma membrane-enriched fractions from the marine diatom, Nitzschia alba. These activities are divided into four groups: Ca²⁺-dependent, Mg²⁺-dependent monovalent cation-stimulated, Mg²⁺-anion-stimulated ATPases, and Mg²⁺-dependent nucleotidases.The Mg²⁺-dependent activities hydrolyze nucleoside triphosphates and, in some membranes, nucleoside diphosphates. Molar ratios of 1:2 $\text{ATP}\text{Mg}{}^{\mathrm{2+}}$ are preferred. However, their divalent cation requirements are not specific, and they can effectively utilize Ca²⁺, Mn²⁺, Mg²⁺, or Zn²⁺. The most effective inhibitors of the Mg²⁺-dependent activities are oligomycin, NaN₃, and NaF.Optimal activity of the Mg²⁺-dependent monovalent cation-stimulated ATPase is obtained at Na⁺, or Na⁺ plus K⁺ concentrations of 100–300 mm. Under these high salt conditions, ATP is hydrolyzed almost exclusively, and Mg²⁺ is specifically required for activation. Preference is for a molar ratio of $\text{ATP}\text{Mg}{}^{\mathrm{2+}}\text{≧ 2}$, and the sulfhydryl-blocking agents, p-chloromecuribenzoate, N-ethylmaleimide, and iodoacetamide strongly or completely inhibit ATP hyrolysis.     

Ca 2+ -dependent allosteric regulation of nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa

Nicotinamide nucleotide transhydrogenase from $\text{Pseudomonas}\text{aeruginosa}$ exhibits allosteric properties and has been shown to be regulated by the prevailing $\text{[NADPH]}\text{[NADP}{}^{\mathrm{+}}\text{]}$ ratio or by 2′-AMP. The present data obtained with membrane fragments from $\text{P.}\text{aerug}$. show that Ca²⁺ strongly influences the concentration of 2′-AMP or NADPH required for half-maximal stimulation. Saturating concentrations of Ca²⁺ cause full activation of the enzyme; Mn²⁺, Mg²⁺ and K⁺ are considerably less efficient and antagonistic to Ca²⁺. Some implications of these findings for the regulatory mechanism and possible physiological function of the enzyme are considered.     

Lithium-7 NMR as a probe of monovalent cation sites at the active site of (Na+ + K+)-ATPase from kidney.

Lithium-7 nuclear magnetic resonance studies are used to characterize the binding of monovalent cations and substrate analogs to the (Na⁺ + K⁺)-ATPase. Li⁺ substitutes for K⁺ in the activation of the ATPase, while the longitudinal relaxation rate, $\text{1}\text{T}{}_1$, of ⁷Li⁺ is increased upon binding of either Mn²⁺ or CrATP to the enzyme. The effects of Mn²⁺ are consistent with the existence of a Li⁺ binding site 7.2A from the single catalytically active Mn²⁺ site on the ATPase. Temperature effects on the observed relaxation rates indicate that exchange of Li⁺ at the observed site is rapid, while the effects of added Na⁺ and K⁺ suggest that the observed site is a K⁺-type site not previously observed by other methods. These experiments also demonstrate that Li⁺ should be superior to other nuclei as NMR probes of the (Na⁺ + K⁺)-ATPase.     

Aminopeptidase Co,a new yeast peptidase

A new aminopeptidase — aminopeptidase Co — has been detected in the yeast $\text{Saccharomyces}\text{cerevisiae}$. The enzyme is only active in the presence of Co²⁺ions. Zn^2+- and Mn²⁺ions are inhibitory. The enzyme activity is also inhibited by chelating agents. Of the p-nitroanilide derivatives tested only those containing basic amino acids are cleaved.     

Electron spin resonance and photoreaction of Mn(II) in lettuce chloroplasts.

Electron spin resonance (esr) of lettuce chloroplasts yields three types of signals: (i) a broad (~900 G) signal around g = 2.22 (apparently due to Cu²⁺ complexes); (ii) an Mn²⁺ spectrum around g = 2.003 consisting of six hyperfine lines (A = 94.5 G) of ~30 G width; and (iii) a sharp signal at g = 2.00 due to photosignals I and II. The present work is concerned with the Mn²⁺ signal and its relation to the photosynthetic process. Intensity measurements were performed by comparing the intensities of the Mn²⁺ signals of two identical chloroplast preparations, one of which was slightly acidified. The integrated intensity of the signal in the normal preparation was approximately one-fourth of that in the acidified sample, suggesting that only the?$\text{1}\text{2}\text{?}\text{1}\text{2}$ fine structure band is observed in untreated chloroplasts. This indicates that the manganese in the chloroplasts is bound in an asymmetric environment, apparently in protein complexes. The Mn²⁺ signal is light sensitive, decreasing on illumination and reappearing in the dark. Typical values for the half-lives of the light and dark processes in normal chloroplasts are 0.25 and 2.1s, respectively. The effect is interpreted in terms of the photooxidation of Mn²⁺ to higher oxidation states which are invisible to esr spectroscopy. In order to determine whether this process is related to photosynthesis the effect of certain reagents and treatments that are known to affect the photosynthetic system was studied. It was found that the oxygen evolution inhibitors 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU) and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) as well as the electron donors, phenylenediamine and sodium ascorbate, reduce or completely eliminate the light effect on the Mn²⁺ signal. Heat treatment and Tris washing caused deceleration of both the light and dark reactions. These effects indicate that the photooxidation of the Mn²⁺ is related to the photosynthetic cycle, the most probable site being the water splitting apparatus of photosystem II.     

Monovalent concanavalin A

The preparation of monovalent concanavalin A was achieved by incubating metal-free concanavalin A with trypsin at room temperature for 50 hrs. The digest was subjected to affinity chromatography on a column of ovomucoid-agarose to remove the trypsin and subsequent chromatography on Bio-Gel P-150 to resolve the monovalent fragment. Monovalent concanavalin A bound Mn²⁺, Ca²⁺ and p-nitrophenyl-α-D-mannopyranoside as determined by ultraviolet difference spectroscopy. The modified protein would not precipitate glycogen or agglutinate $\text{Bacillus subtilis}$ cells. The fragment did, however, prevent the agglutination of $\text{B. subtilis}$ by native concanavalin A. Preparation of monovalent concanavalin A could not be achieved unless metals were first removed from the native protein.     

Ruthenium red as a carrier of electrons between external NADH and cytochrome c in rat liver mitochondria.

We have determined that Co²⁺, Ni²⁺ or Zn²⁺ may substitute for Mg²⁺ during DNA synthesis with $\text{E.}\text{coli}$ DNA polymerase I, sea urchin nuclear DNA polymerase and the DNA polymerase from avian myeloblastosis virus (AMV). In addition, the frequency of non-complementary nucleotide incorporation using AMV DNA polymerase was increased using Co²⁺ or Mn²⁺ as the metal activator. These results suggest that the fidelity of DNA synthesis may be influenced by the metal activator used during catalysis.     

Kinetic studies of 18O exchange from inorganic phosphate catalyzed by Mg2+-activated unadenylylated glutamine synthetase (E. coli w).

Oxygen-18 exchange out of [¹⁸O]Pi catalyzed by Mg²⁺-activated unadenylated glutamine synthetase from $\text{E.}$$\text{coli}$ was followed by ³¹P-NMR in the presence of the other substrates, ADP and L-glutamine. The pattern of the ${}^{16}\text{O}{}^{18}\text{O}$ in the species P¹⁸O₄, P¹⁸O₃¹⁶O₁, P¹⁸O₂¹⁶O₂, P¹⁸O₁¹⁶O₃, P¹⁶O₄ during the exchange followed a binomial distribution consistent with indiscriminate removal of any of the four oxygens of P_i. The rate constant for ${}^{16}\text{O}{}^{18}\text{O}$ exchange was 410±40 min^?1 while the rate constant for net reaction (ATP formation) was 62±4 min^?1. Thus exchange proceeds ～7 times faster than net reaction, a finding in accord with that of Stokes and Boyer ($\text{J.}$$\text{Biol.}$$\text{Chem.}$ (1976) $\text{251}$, 5558) for the Mn²⁺-activated adenylylated glutamine synthetase. A model for the overall catalytic events first derived from rapid kinetic fluorescence experiments (Rhee and Chock, Proc. Natl. Acad. Sci. USA, (1976) $\text{73}$, 476) was successfully used to fit the oxygen exchange data in this paper.