Absence Of MgCl2 Research Articles

Aggregation equilibria of Escherichia coli RNA polymerase: evidence for anion-linked conformational transitions in the protomers of core and holoenzyme.

The aggregation equilibria of Escherichia coli RNA polymerase core and holoenzyme have been studied by velocity sedimentation as a function of [NaCl] both in the presence and in the absence of MgCl2. Effects of other anions (F- and I-), pH, and temperature have also been examined. Diffusion coefficients obtained by quasi-elastic light scattering (QLS) at high and low salt concentrations were used in conjunction with sedimentation coefficients under these conditions to obtain molecular weights of the protomer and aggregates of the core enzyme. At low salt concentration, core aggregates to a tetramer in the absence of MgCl2 and to an octamer in the presence of MgCl2. Some ambiguity exists in the interpretation of the sedimentation and QLS data for holoenzyme. The sedimentation results are consistent with the formation of dimers at low salt, both in the presence and in the absence of MgCl2. In all cases, equilibrium constants were calculated assuming a simple monomer--j-mer stoichiometry. These equilibrium constants are extremely sensitive functions of the concentration and type of monovalent anion. In Cl-, aggregation of both core and holoenzyme begins abruptly when the salt concentration is reduced below approximately 0.2 M (at a protein concentration of approximately 0.30 mg/mL); for core, substitution of I- for Cl- suppresses aggregation while F- enhances aggregation at a fixed anion concentration. No specific effect of monovalent cations (Na+, NH4+) is observed; Mg2+ has no effect on holoenzyme dimerization and has little effect on the salt range of core aggregation, though the stoichiometries of the core aggregates in the presence and absence of Mg2+ differ. Anion effects on these equilibria were modeled by assuming that a class of anion-binding sites on the protomer is not present in the aggregate, so that anion release accompanies aggregation. Analytical expressions for several models of the effect of anions on the aggregation equilibria were derived by using the method of binding polynomials. The salt dependence of the aggregation equilibria in the absence of Mg2+ appears inconsistent with a model in which the anion-binding sites on the protomer are independent (noncooperative), but it is well described by a model in which anion binding to the protomers occurs in a completely cooperative manner. The molecular basis of this apparent cooperative effect of anions on the aggregation equilibria is proposed to be an allosteric effect of anions on conformational equilibria of the protomers of core polymerase and the holoenzyme. Implications of such a salt-dependent conformational transition for the DNA-binding interactions of the enzyme are considered.

31P nuclear magnetic resonance study of phosphoribosyldiphosphate and its interaction with magnesium ions.

31P NMR has been used to study phosphoribosyldiphosphate (P-Rib-PP) over a wide range of pH values, both in the absence and presence of MgCl2. In the absence of MgCl2, the chemical shift variations of the three 31P nuclei in the molecule, over the pH range 4 to 9, were found to be largest for the terminal 1-diphosphate (1P beta) oxyanion and the 5-phosphate (5P) moiety. Apparent pK alpha values of approximately 6.1 and 6.3 were estimated for protonation of the 1P beta and 5P groups, respectively. Variations in the apparent pK alpha values associated with 1P beta and 5P oxyanions in the presence of various concentrations of MgCl2 were consistent with P-Rib-PP having two independent metal ion binding sites with different affinities for Mg2+ ions. The binding of Mg2+ reduced the apparent pK alpha of the 1P beta moiety by approximately 1.6 units and the apparent pK alpha of the 5P group by approximately 0.7 unit. This behavior is analogous to the situation reported for the terminal phosphooxyanion of ADP and observed for the phosphate group of ribose 5-phosphate, respectively. In the presence of an equimolar concentration of added MgCl2, the 1P alpha and 1P beta resonances of P-Rib-PP were shifted downfield and the 31P-31P coupling constant was decreased. Changes in both these parameters were very similar to those reported for the MgADP- complex. The observed chemical shifts and spin-spin coupling constants suggest that the diphosphate and monophosphate moieties of P-Rib-PP act as independent binding sites for Mg2+ in a manner similar to the phosphooxyanion groups of ADP and ribose 5-phosphate, respectively.

Properties of the Avian Viral Protein p12

The avian RNA tumour virus structural protein p12 was purified from avian myeloblastosis virus (AMV) by nucleic acid affinity chromatography to apparent homogeneity as judged from SDS--polyacrylamide gel electrophoresis. A filter binding assay was used for the identification of p12. High concentrations of p12 precipitated nucleic acids out of solution in the absence of MgCl2. Binding of p12 to single-stranded nucleic acids protected them from digestion with nucleases and resulted in a hyperchromic effect. These phenomena were reversible in the presence of salt. The affinity of p12 to nucleic acids was determined by competing for the binding of p12 to denatured radioactive DNA by various other nuclei acids. It was found that p12 bound preferentially to single-stranded nucleic acids and showed a higher affinity to poly(rI) than to poly(rC) and poly(rA). Purified RNA-dependent DNA polymerase activity from AMV was stimulated up to sixfold by p12, depending on the template. Solubilization of RNA in RNA--DNA hybrids by RNase H was inhibited in the presence of p12.

Fluorescence titration and fluorescence stopped-flow studies on skeletal troponin C labeled with fluorescent maleimide reagent or dansylaziridine.

Skeletal muscle troponin C (TN-C) labeled with N-(p-(2-benzimidazolyl)phenyl)-maleimide (BIPM) shows about 5% fluorescence increase and 82% fluorescence increase upon Ca2+ binding and Mg2+ binding to the high affinity Ca2+-binding sites (sites III and IV) of TN-C, respectively. TN-C labeled with N-(1-anilinonaphthyl-4)maleimide (ANM) shows about 26% fluorescence decrease and 22% fluorescence increase upon Ca2+ binding and Mg2+ binding to the high affinity Ca2+-binding sites, respectively. These findings indicate that environmental change around Cys-98, where the maleimide reagent bind, induced by Ca2+ binding to the high affinity Ca2+-binding sites is very different from that induced by Mg2+ binding to the same sites. These dye-protein conjugates do not show fluorescence intensity change upon Ca2+ binding to the low affinity Ca2+-binding sites (sites I and II). Dansylaziridine (DANZ)-labeled TN-C shows more than 100% fluorescence increase upon Ca2+ binding to the low affinity Ca2+-binding sites of TN-C. Hence, we can observe the kinetic processes which TN-C undergoes upon Ca2+ binding to or removal from the high affinity Ca2+-binding sites by following the fluorescence intensity change of ANM-labeled TN-C, respectively. THe kinetic processes of the fluorescence intensity change associated with the Ca2+ binding and removal reactions with the high affinty Ca2+-binding sites (rate constants, 3.7-157 s-1) are slower than the kinetic processes associated with the low affinity Ca2+-binding sites (rate constants, equal to or higher than 230 s-1) in the absence of MgCl2. In the presence of 2 mM MgCl2, a new slow phase (rate constant, 10-16 s-1) appears in the kinetic processes associated with the low affinity Ca2+-binding sites, in addition to the rapid phase which is already observed in the absence of MgCl2. Kinetic properties associated with the high affinity Ca2+-binding sites are not essentially altered by addition of 2 mM MgCl2 to the system, but ANM-labeled TN-C shows lower rate constants (0.67-26 s-1).

?-1-3- and ?-1-4-glucan synthesis by membrane fractions from the fungus Saprolegnia

The β-glucan synthetase activity of the fungus Saprolegnia monoica was assayed by supplying UDP-glucose to membrane fractions of mycelial homogenate. The analysis of glucan products by hydrolysis with various β-glucanases and by chromatography show that both β-1-3- and β-1-4-linkages are formed at high substrate concentrations. In the absence of MgCl2, β-1-3-linked glucans are mainly produced. By increasing MgCl2 concentrations the total synthesis activity and β-1-3-linkages production are reduced. At low substrate concentrations in the presence of MgCl2, β-1-4-linked glucans are the only polysaccharide synthesized. Electron microscopy of radioactive products, synthesized by original membrane fractions or by membrane fractions isolated from continuous sucrose density gradients, shows microfibrils when the assays are conducted at high substrate concentrations in the absence of MgCl2.

Similarity between alkaline phosphatase and nucleotide pyrophosphatase from skipjack liver.

Skipjack liver alkaline phosphatase (EC 3.1.3.1) and nucleotide pyrophosphatase (EC 3.6.1.9) were compared in respect to some physico-chemical and enzymatic properties. Neuraminidase storgly inactivated both enzymes in a similar fashion, whereas muramidase inactivated alkaline phosphatase more slowly than the other enzyme. Nucleotide pyrophosphatase was also inactivated by several proteolytic enzymes more easily than alkaline phosphatase. Sodium lauryl sulfate, urea, and guanidine hydrochloride inactivated both enzymes in a similar manner, and the protective effect of MgCl2 was also similar. In the presence of MgCl2, alkaline phosphatase showed the optimum temperature at 35°C and nucleotide pyrophosphatase at 45°C. In the absence of MgCl2, however, both enzymes showed the identical optimal temperature at 45°C. All the substrates common to both enzymes inhibited them competitively. Nucleotide pyrophosphatase was inhibited by all the substrates specific to alkaline phosphatase but the inhibition types were different for both enzymes with some substrates. All the substrates for nucleotide pyrophosphatase also inhibited alkaline phosphatase by the competitive or the mixed type manner. These results suggested that the two skipjack enzymes are closely similar to each other in their primary or higherdimentional structure.

Binding to the high-affinity substrate site of the (Na+ + K+)-dependent ATPase.

The (Na+ + K+)-dependent ATPase exhibits substrate sites with both high affinity (Km near 1 microM) and low affinity (Km near 0.1 mM) for ATP. To permit the study of nucleotide binding to the high-affinity substrate sites of a canine kidney enzyme preparation in the presence as well as absence of MgCl2, the nonhydrolyzable beta-gamma imido analog of ATP, AMP-PNP, was used in experiments performed at 0-4 degrees C by a centrifugation technique. By this method the KD for AMP-PNP was 4.2 microM in the absence of MgCl2. Adding 50 microM MgCl2, however, decreased the KD to 2.2 microM; by contrast, higher concentrations of MgCl2 increased the KD until, with 2 mM MgCl2, the KD was 6 microM. The half-maximal effect of MgCl2 on increasing the KD occurred at approximately 1 mM. This biphasic effect of MgCl2 is interpreted as Mg2+ in low concentrations favoring AMP-PNP binding through formation at the high-affinity substrate sites of a ternary enzyme-AMP-PNP-Mg complex; inhibition of nucleotide binding at higher MgCl2 concentrations would represent Mg2+ acting through the low-affinity substrate sites. NaCl in the absence of MgCl2 increased AMP-PNP binding, with a half-maximal effect near 0.3 mM; in the presence of MgCl2, however, NaCl increased the KD for AMP-PNP. KCl decreased AMP-PNP binding in the presence or absence of MgCl2, but the simultaneous presence of a molar excess of NaCl abolished (or masked) the effect of KCl. ADP and ATP acted as competitors to the binding of AMP-PNP, although a substrate for the K+-dependent phosphatase reaction also catalyzed by this enzyme, p-nitrophenyl phosphate, did not. This lack of competition is consistent with formulations in which the phosphatase reaction is catalyzed at the low-affinity substrate sites.

The chemical reactivity of fully maleylated cytochrome c.

The chemical reactivity of fully maleylated horse heart cytochrome c with oxidants, reductants, and iron ligands was studied in the presence and absence of MgCl2. In the absence of salt, the partly unfolded protein reacts rapidly with cyanide in the ferric state (k = 235 M-1s-1); the reaction is exothermic (delta H = -9.4 kcal/mol) and entropically unfavored (delta S = -7.0 e.u.). In 0.01 M MgCl2, the refoleded protein recovers the behavior towards cyanide of native cytochrome c. The oxidation-reduction potential of the refolded species is 190 mV. In the course of reduction of the unfolded form with dithionite, a reduced intermediate species (t 1/2 = 5.9 s) is observed; this species binds carbon monoxide rapidly, but the ligand dissociates thereafter. This shows that full maleylation does not disrupt the conformational stability of reduced cytochrome c.

The effects of cytochalasins on actin polymerization and actin ATPase provide insights into the mechanism of polymerization.

Substoichiometric concentrations of cytochalasin D inhibited the rate of polymerization of actin in 0.5 mM MgCl2, increased its critical concentration and lowered its steady state viscosity. Stoichiometric concentrations of cytochalasin D in 0.5 mM MgCl2 and even substoichiometric concentrations of cytochalasin D in 30 mM KCl, however, accelerated the rate of actin polymerization, although still lowering the final steady state viscosity. Cytochalasin B, at all concentrations in 0.5 mM MgCl2 or in 30 mM KCl, accelerated the rate of polymerization and lowered the final steady state viscosity. In 0.5 mM MgCl2, cytochalasin D uncoupled the actin ATPase activity from actin polymerization, increasing the ATPase rate by at least 20 times while inhibiting polymerization. Cytochalasin B had a very much lower stimulating effect. Neither cytochalasin D nor B affected the actin ATPase activity in 30 mM KCl. The properties of cytochalasin E were intermediate between those of cytochalasin D and B. Cytochalasin D also stimulated the ATPase activity of monomeric actin in the absence of MgCl2 and KCl and, to a much greater extent, stimulated the ATPase activity of monomeric actin below its critical concentration in 0.5 mM MgCl2. Both above and below its critical concentration and in the presence and absence of cytochalasin D, the initial rate of actin ATPase activity, when little or no polymerization had occurred, was directly proportional to the actin concentration and, therefore, apparently was independent of actin-actin interactions. To rationalize all these data, a working model has been proposed in which the first step of actin polymerization is the conversion of monomeric actin-bound ATP, A . ATP, to monomeric actin-bound ADP and Pi, A* . ADP . Pi, which, like the preferred growing end of an actin filament, can bind cytochalasins.

Phosphorylation and dephosphorylation kinetics of potassium-stimulated ATP phosphohydrolase from hog gastric mucosa.

Partial reactions of potassium-stimulated ATP phosphohydrolase from hog gastric mucosa were studied by means of a rapid-mixing apparatus. At 21 degrees C, in the presence of 2 mM MgCl2 and 5 microM [gamma-32P]ATP there was a rapid phosphorylation of the enzyme with a pseudofirst order rate constant of 1400 min-1. Addition of the ATP about 120 ms before the MgCl2 increased this rate constant to 4400 min-1. In the absence of MgCl2 there was no phosphorylation. Addition of 4 or 10 mM KCl to the phosphoenzyme which had been formed in the absence of KCl produced a rapid initial rate of dephosphorylation (k = 2600 and 3200 min-1 respectively). An additional slow component of dephosphorylation was observed when unlabeled ATP was added together with the KCl (k = 700 to 900 min-1). At a 4 mM concentration, KCl stimulated the ATPase activity about 9-fold. At higher concentrations, the activity was reduced in parallel with a reduction of the steady state level of phosphoenzyme. Addition of KCl to the enzyme before the addition of ATP plus MgCl2 resulted in a low rate and extent of phosphorylation. KCl appeared to inhibit the phosphorylation at a level preceeding the E.ATP complex.

Transformation and transfection of Pseudomonas aeruginosa: effects of metal ions.

The ability of different metal ions to promote transformation of Pseudomonas aeruginosa by deoxyribonucleic acid of the plasmid RP1 was examined. CaCl2, MgCl2, and MnCl2 were found to promote such transformation, although at different frequencies and with the optimum response at different concentrations. Only MgCl2 promoted transfection of P. aeruginosa by the linear deoxyribonucleic acid of phage F116. CaCl2 was demonstrated to allow adsorption and entry into the cell of F116 deoxyribonucleic acid such that it became resistant to exogenous deoxyribonuclease, but phage production occurred only when MgCl2 was provided. Inactivation of linear phage deoxyribonucleic acid taken up in the absence of MgCl2 was observed. The transfection frequencies at various concentrations of MgCl2 were compared, and the optimum response occurred at the concentration which promoted the highest frequency of transformation by RP1 deoxyribonucleic acid.

Novel properties of bacterial elongation factor Tu.

We have characterized novel properties of the bacterial protein synthesis elongation factor Tu which indicate that it may function as a structural protein. Under appropriate conditions, elongation factor Tu polymerizes to form filaments and, more often, bundles of filaments. It is also the predominant component of a complex of proteins from Escherichia coli that undergoes reversible polymerization in the presence of KCl and MgCl2. In addition, purified elongation factor Tu binds tightly to DNase I in the presence of 10 mM MgCl2. In crude extracts the factor shows no binding in the presence or absence of MgCl2. These properties suggest that elongation factor Tu may have certain actin-like properties and that it has cellular functions other than its role in protein synthesis.

The Effect of Dimethyladipimidate on the Stability of Cowpea Chlorotic Mottle and Brome Mosaic Viruses

SUMMARY Cross-linking lysyl residues of brome mosaic and cowpea chlorotic mottle viruses with dimethyladipimidate at pH 8.5 in the presence of MgCl2 resulted in particles that were stable under conditions in which the viruses normally came apart. Virus substituted in the absence of MgCl2 was not stabilized nor would it reassociate into normal particles. Substitution of isolated coat protein subunits did not, however, interfere with the assembly of capsids.

The mechanism of action of methionyl-tRNA synthetase from Escherichia coli. Equilibrium-dialysis studies on the binding of methionine, ATP and ATP-Mg2+ by the native and trypsin-modified enzymes.

A comparative study of the interactions of native and trypsin-modified methionyl-tRNA synthetases with methionine and ATP was undertaken. In imidazole buffer in the absence of MgCl2, the trypsin-modified enzyme possesses one binding site for methionine and one for ATP (KA= 20 mM−1 and 5.5 mM−1, respectively). Binding of each substrate is abolished in the presence of methioninyl adenylate, thereby implicating these sites in adenylate formation. Binding of ATP, but not of methionine, is strongly inhibited by phosphate ions. Furthermore, the unique ATP site on the trypsin-modified enzyme strongly discriminates against ATP-Mg2+ (KA≤ 0.7 mM−1). However, in the presence of structural analogues of methionine (methioninol and 3-methylthiopropylamine), the enzyme acquires the ability to interact efficiently with ATP-Mg2+ (KA= 10 mM−1). Native methionyl-tRNA synthetase possesses two sites for methionine and four for ATP. Occupation of the methionine sites and of two out of the four ATP sites by their respective ligands, is abolished in the presence of methioninyl adenylate, indicating that these sites are involved in adenylate formation. The two non-equivalent catalytic ATP sites (A sites) are strongly inhibited by phosphate ions and display a lower affinity for ATP-Mg2+ than for ATP. The ability of these sites to interact with ATP-Mg2+ is restored in the presence of analogues of methionine, as in the case of the trypsin-modified enzyme. Conversely, the two equivalent non-catalytic ATP sites (B sites) are unaffected by the presence of phosphate or magnesium ions. The implications of these findings on the mechanism of action of methionyl-tRNA synthetase are discussed.

Histone phosphatase and cyclic nucleotide-stimulated protein kinase from human lymphocytes.

1. Extracts of human peripheral blood lymphocytes contained a histone phosphatase that catalysed the release of P(i) from phosphorylated whole thymus histone. 2. Stimulation of the phosphatase was obtained by concentrations of KCl and NaCl of up to 75mm, and by MgCl(2); CaCl(2) inhibited the enzymic activity. 3. In the absence of MgCl(2), phosphoenol-pyruvate inhibited histone phosphatase activity; this inhibition could be partially reversed by adding MgCl(2) to assays. 4. Lymphocyte extracts contained a protein kinase activity which was maximally stimulated by 1mum-cyclic AMP (adenosine 3':5'-cyclic monophosphate) or by 0.1mm-cyclic GMP (guanosine 3':5'-cyclic monophosphate). 5. Incubation of the enzyme with histone in the absence of ATP or MgCl(2) resulted in the dissociation of the enzyme into a lower-molecular-weight species that was not stimulated by cyclic AMP. This effect could be prevented if ATP and MgCl(2) were present in reaction mixtures before histone and enzyme were allowed to interact. 6. Cyclic AMP also dissociated the kinase into a lower-molecular-weight species. 7. In the presence of 1mum-AMP, half-maximal activities were obtained with 0.92mm-MgCl(2), 6.0mum-ATP and 0.23mg of whole thymus histone/ml.

The effect of ATP upon the oxygen sensitivity of nitrogenase from Azotobacter chroococcum.

Crude preparations of nitrogenase (AS1) from Azotobacter chroococcum which were normally oxygen‐tolerant, became oxygen‐sensitive in the presence of either ATP, GTP, ITP, UTP, CTP or sodium pyrophosphate. MgCl2 antagonised this effect of ATP. ATP‐induced oxygen damage was faster at 4°C than at 30°C. ATP‐induced oxygen damage to prepn AS1 affected fraction 2 (Ac2) of the nitrogenase; fraction 1 (Ac1) was unaffected. Purified fraction Ac2 became hypersensitive to oxygen in the presence of ATP. Pre‐treatment of prepn AS1 with “Chelex 100” or EDTA under anaerobic conditions made the nitrogenase oxygen‐sensitive. Oxygen‐tolerance was partly restored by metal ions. Similar anaerobic pre‐treatment of prepn AS1 with ATP followed by removing the ATP by dialysis did not induce oxygen‐sensitivity. Pure fraction Ac2 bound significantly more [14C]ATP in the presence of MgCl2 than did EDTA‐treated fraction Ac2 in the absence of MgCl2. Hill plots of the ATP requirement for acetylene reduction, for ATP‐induced oxygen damage, for the MgCl2, requirement for acetylene reduction and for the MgCl2, requirement for protection against oxygen damage to EDTA‐treated prepn AS1 all yielded nvalues between 1 and 2. These results are discussed in terms of a role for ATP in nitrogen fixation.

ENZYMATIC ENHANCEMENT OF INFECTIVITY OF REOVIRUS.

Spendlove, R. S. (California State Department of Public Health, Berkeley), and F. L. Schaffer. Enzymatic enhancement of infectivity of reovirus. J. Bacteriol. 89:597-602. 1965.-Enhancement of infectivity by chymotrypsin treatment has been demonstrated with all three types of reovirus, although not in all viral preparations. Enzyme treatment did not produce a simultaneous increase in the hemagglutinating activity of reovirus type 1 (the only type tested). The infectivity of reovirus type 1 (Lang strain) was increased by treatment with chymotrypsin, trypsin, papain, and a filtrate from a culture of a Pseudomonas sp. but not by treatment with pepsin. Sedimentation experiments showed that the property of enhanceability was closely associated with the virus particles themselves. Results of studies involving various sequential treatments with chymotrypsin, and with heat in the presence or absence of 2 m MgCl(2), were compatible with the interpretation that inhibited virus is resistant to exposure to a temperature of 56 C in the absence of MgCl(2), whereas activated virus is thermolabile. Activation of reovirus infectivity by heat in the presence of MgCl(2) and by chymotrypsin was not additive.