The binding of spermine to the ribosomes and ribosomal ribonucleic acid from Bacillus stearothermophilus

1. The total intracellular concentrations of Na(+), K(+), Mg(2+), spermine, spermidine and RNA were measured in Bacillus stearothermophilus. 2. The binding of spermine to ribosomes and to ribosomal RNA from B. stearothermophilus was studied under various conditions by using a gel-filtration technique. 3. The affinity of spermine for ribosomes and for ribosomal RNA decreased with increasing ionic strength of the medium in which they were suspended. 4. The extent of spermine binding did not change appreciably in the temperature range 4-60 degrees . 5. Optimum binding occurred at about pH7.0. 6. The number of binding sites for spermine on either ribosomes or ribosomal RNA was 0.10-0.13/RNA phosphate group. 7. A high proportion of the intracellular spermine is likely to be bound to the ribosomes in vivo; spermine competes with Mg(2+) on equal terms for sites on the ribosomes.     

Interactions of some naturally occurring cations with phenylalanine and initiator tRNA from yeast as reflected by their thermal stability

The thermal unfolding of phenylalanine and initiator tRNA from yeast was investigated over a broad range of solution conditions by differential ultraviolet absorption at 260 nm. Under most conditions, the initiator tRNA exhibits two clearly separated transitions in its differential melting curve which were assigned to unfolding of tertiary and secondary structure elements, respectively. The tertiary transition of this tRNA and the overall transition observed for tRNAPhe do not show a maximum in a curve of Tm values plotted as a function of [Na+]. Such a maximum is usually observed for other nucleic acids at about 1 M Na+. In the presence of 5 mM of the divalent cation Mg2+ (or Ca2+), an overall destabilization of the tRNAs is observed when increasing the sodium concentration. The largest fall in Tm (approximately 15 degrees C) is observed for the tertiary transition of the initiator tRNA. Among various cations tested the following efficiency in the overall stabilization of tRNAPhe is observed: spermine greater than spermidine greater than putrescine greater than Na+ (approximately NH4+). Mg2+ is most efficient at concentrations above 5 mM, but below this concentration spermine and spermidine appear to be more efficient. The same hierarchy in stabilizing power of the polyamines and Na+ is observed for both transitions of the initiator tRNA. However, when compared with Mg2+, the polyamines are far less capable of stabilizing the tertiary structure. In contrast, spermine and spermidine are slightly better than Mg2+ in stabilizing the secondary structure. At increasing concentrations of the polyvalent cations (at fixed [Na+] ) the Tm values of the tRNAs attain a constant value.     

Structural analysis of DNA interactions with biogenic polyamines and cobalt(III)hexamine studied by Fourier transform infrared and capillary electrophoresis

Biogenic polyamines, such as putrescine, spermidine, and spermine are small organic polycations involved in numerous diverse biological processes. These compounds play an important role in nucleic acid function due to their binding to DNA and RNA. It has been shown that biogenic polyamines cause DNA condensation and aggregation similar to that of inorganic cobalt(III)hexamine cation, which has the ability to induce DNA conformational changes. However, the nature of the polyamine.DNA binding at the molecular level is not clearly established and is the subject of much controversy. In the present study the effects of spermine, spermidine, putrescine, and cobalt(III)hexamine on the solution structure of calf-thymus DNA were investigated using affinity capillary electrophoresis, Fourier transform infrared, and circular dichroism spectroscopic methods. At low polycation concentrations, putrescine binds preferentially through the minor and major grooves of double strand DNA, whereas spermine, spermidine, and cobalt(III)hexamine bind to the major groove. At high polycation concentrations, putrescine interaction with the bases is weak, whereas strong base binding occurred for spermidine in the major and minor grooves of DNA duplex. However, major groove binding is preferred by spermine and cobalt(III)hexamine cations. Electrostatic attractions between polycation and the backbone phosphate group were also observed. No major alterations of B-DNA were observed for biogenic polyamines, whereas cobalt(III)hexamine induced a partial B --> A transition. DNA condensation was also observed for cobalt(III)hexamine cation, whereas organic polyamines induced duplex stabilization. The binding constants calculated for biogenic polyamines are K(Spm) = 2.3 x 10(5) M(-1), K(Spd) = 1.4 x 10(5) M(-1), and K(Put) = 1.02 x 10(5) M(-1). Two binding constants have been found for cobalt(III)hexamine with K(1) = 1.8 x 10(5) M(-1) and K(2) = 9.2 x 10(4) M(-1). The Hill coefficients indicate a positive cooperativity binding for biogenic polyamines and a negative cooperativity for cobalt(III)hexamine.     

Ca(2+) efflux in mitochondria from the yeast Endomyces magnusii.

Calcium release pathways in Ca(2+)-preloaded mitochondria from the yeast Endomyces magnusii were studied. In the presence of phosphate as a permeant anion, Ca(2+) was released from respiring mitochondria only after massive cation loading at the onset of anaerobiosis. Ca(2+) release was not affected by cyclosporin A, an inhibitor of the mitochondrial permeability transition. Aeration of the mitochondrial suspension inhibited the efflux of Ca(2+) and induced its re-uptake. With acetate as the permeant anion, a spontaneous net Ca(2+) efflux set in after uptake of approximately 150 nmol of Ca(2+)/mg of protein. The rate of this efflux was proportional to the Ca(2+) load and insensitive to aeration, protonophorous uncouplers, and Na(+) ions. Ca(2+) efflux was inhibited by La(3+), Mn(2+), Mg(2+), tetraphenylphosphonium, inorganic phosphate, and nigericin and stimulated by hypotonicity, spermine, and valinomycin in the presence of 4 mm KCl. Atractyloside and t-butyl hydroperoxide were without effect. Ca(2+) efflux was associated with contraction, but not with mitochondrial swelling. We conclude that the permeability transition pore is not involved in Ca(2+) efflux in preloaded E. magnusii mitochondria. The efflux occurs via an Na(+)-independent pathway, in many ways similar to the one in mammalian mitochondria.     

The role of Mg2+ cofactor in the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family GTP-binding proteins

The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg(2+) cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA. Differing from Ras and Rab proteins, which require Mg(2+) for GDP and GTP binding, the Rho GTPases bind the nucleotides in the presence or absence of Mg(2+) similarly, with dissociation constants in the submicromolar concentration. The presence of Mg(2+), however, resulted in a marked decrease in the intrinsic dissociation rates of the nucleotides. The catalytic activity of the guanine nucleotide exchange factors (GEFs) appeared to be negatively regulated by free Mg(2+), and GEF binding to Rho GTPase resulted in a 10-fold decrease in affinity for Mg(2+), suggesting that one role of GEF is to displace bound Mg(2+) from the Rho proteins. The GDP dissociation rates of the GTPases could be further stimulated by GEF upon removal of bound Mg(2+), indicating that the GEF-catalyzed nucleotide exchange involves a Mg(2+)-independent as well as a Mg(2+)-dependent mechanism. Although Mg(2+) is not absolutely required for GTP hydrolysis by the Rho GTPases, the divalent ion apparently participates in the GTPase reaction, since the intrinsic GTP hydrolysis rates were enhanced 4-10-fold upon binding to Mg(2+), and k(cat) values of the Rho GTPase-activating protein (RhoGAP)-catalyzed reactions were significantly increased when Mg(2+) was present. Furthermore, the p50RhoGAP specificity for Cdc42 was lost in the absence of Mg(2+) cofactor. These studies directly demonstrate a role of Mg(2+) in regulating the kinetics of nucleotide binding and hydrolysis and in the GEF- and GAP-catalyzed reactions of Rho family GTPases. The results suggest that GEF facilitates nucleotide exchange by destabilizing both bound nucleotide and Mg(2+), whereas RhoGAP utilizes the Mg(2+) cofactor to achieve high catalytic efficiency and specificity.     

Monitoring the structure of Escherichia coli RNase P RNA in the presence of various divalent metal ions

Lead(II)-induced cleavage can be used as a tool to probe conformational changes in RNA. In this report, we have investigated the conformation of M1 RNA, the catalytic subunit of Escherichia coli RNase P, by studying the lead(II)-induced cleavage pattern in the presence of various divalent metal ions. Our data suggest that the overall conformation of M1 RNA is very similar in the presence of Mg(2+), Mn(2+), Ca(2+), Sr(2+) and Ba(2+), while it is changed compared to the Mg(2+)-induced conformation in the presence of other divalent metal ions, Cd(2+) for example. We also observed that correct folding of some M1 RNA domains is promoted by Pb(2+), while folding of other domain(s) requires the additional presence of other divalent metal ions, cobalt(III) hexamine or spermidine. Based on the suppression of Pb(2+) cleavage at increasing concentrations of various divalent metal ions, our findings suggest that different divalent metal ions bind with different affinities to M1 RNA as well as to an RNase P hairpin-loop substrate and yeast tRNA(Phe). We suggest that this approach can be used to obtain information about the relative binding strength for different divalent metal ions to RNA in general, as well as to specific RNA divalent metal ion binding sites. Of those studied in this report, Mn(2+) is generally among the strongest RNA binders.     

Divalent cations and polyamines bind to loop 8 of 14-3-3 proteins, modulating their interaction with phosphorylated nitrate reductase

Binding of 14-3-3 proteins to nitrate reductase phosphorylated on Ser543 (phospho-NR) inhibits activity and is responsible for the inactivation of nitrate reduction that occurs in darkened leaves. The 14-3-3-dependent inactivation of phospho-NR is known to require millimolar concentrations of a divalent cation such as Mg2+ at pH 7.5. We now report that micromolar concentrations of the polyamines, spermidine(4+) and spermine(3+), can substitute for divalent cations in modulating 14-3-3 action. Effectiveness of the polyamines decreased with a decrease of polycation charge: spermine(4+) > spermidine(3+) > cadavarine(2+) approximately putrescine(2+) approximately agmatine(2+) approximately N1-acetylspermidine(2+), indicating that two primary and at least one secondary amine group were required. C-terminal truncations of GF14 omega, which encodes the Arabidopsis 14-3-3 isoform omega, indicated that loop 8 (residues 208-219) is the likely cation-binding site. Directed mutagenesis of loop 8, which contains the EF hand-like region identified in earlier studies, was performed to test the role of specific amino acid residues in cation binding. The E208A mutant resulted in a largely divalent cation-independent inhibition of phospho-NR activity, whereas the D219A mutant was fully Mg(2+)-dependent but had decreased affinity for the cation. Mutations and C-terminal truncations that affected the Mg(2+) dependence of phospho-NR inactivation had similar effects on polyamine dependence. The results implicate loop 8 as the site of divalent cation and polyamine binding, and suggest that activation of 14-3-3s occurs, at least in part, by neutralization of negative charges associated with acidic residues in the loop. We propose that binding of polyamines to 14-3-3s could be involved in their regulation of plant growth and development.     

Altering the intermediate in the equilibrium folding of unmodified yeast tRNAPhe with monovalent and divalent cations

The isothermal equilibrium folding of the unmodified yeast tRNA(Phe) is studied as a function of Na(+), Mg(2+), and urea concentration with hydroxyl radical protection, circular dichroism, and diethyl pyrocarbonate (DEPC) modification. These assays indicate that this tRNA folds in Na(+) alone. Similar to folding in Mg(2+), folding in Na(+) can be described by two transitions, unfolded-to-intermediate-to-native. The I-to-N transition has a Na(+) midpoint of approximately 0.5 M and a Hill constant of approximately 4. Unexpectedly, the urea m-value, the dependence of free energy on urea concentration, for the I-to-N transition is significantly smaller in Na(+) than in Mg(2+), 0.4 versus 1.7 kcal mol(-1) M(-1), indicating that more structure is formed in the Mg(2+)-induced transition. DEPC modification indicates that the I state in Na(+)-induced folding contains all four helices of tRNA and the I-to-N transition primarily corresponds to the formation of the tertiary structure. In contrast, the intermediate in Mg(2+)-induced folding contains only three helices, and the I-to-N transition corresponds to the formation of the acceptor stem plus tertiary structure. The cation dependence of the intermediates arises from the differences in the stability of the acceptor stem and the tertiary structure. The acceptor stem is stable at a lower Na(+) concentration than required for the tertiary structure formation. The relative stability is reversed in Mg(2+) so that the acceptor stem and the tertiary structure form simultaneously in the I-to-N transition. These results demonstrate that formation of the RNA secondary structure can be independent or coupled to the formation of the tertiary structure depending on their relative stability in monovalent and divalent ions.     

Distinct reaction pathway promoted by non-divalent-metal cations in a tertiary stabilized hammerhead ribozyme

Divalent ion sensitivity of hammerhead ribozymes is significantly reduced when the RNA structure includes appropriate tertiary stabilization. Therefore, we investigated the activity of the tertiary stabilized "RzB" hammerhead ribozyme in several nondivalent ions. Ribozyme RzB is active in spermidine and Na(+) alone, although the cleavage rates are reduced by more than 1,000-fold relative to the rates observed in Mg(2+) and in transition metal ions. The trivalent cobalt hexammine (CoHex) ion is often used as an exchange-inert analog of hydrated magnesium ion. Trans-cleavage rates exceeded 8 min(-1) in 20 mM CoHex, which promoted cleavage through outersphere interactions. The stimulation of catalysis afforded by the tertiary structural interactions within RzB does not require Mg(2+), unlike other extended hammerhead ribozymes. Site-specific interaction with at least one Mg(2+) ion is suggested by CoHex competition experiments. In the presence of a constant, low concentration of Mg(2+), low concentrations of CoHex decreased the rate by two to three orders of magnitude relative to the rate in Mg(2+) alone. Cleavage rates increased as CoHex concentrations were raised further, but the final fraction cleaved was lower than what was observed in CoHex or Mg(2+) alone. These observations suggest that Mg(2+) and CoHex compete for binding and that they cause misfolded structures when they are together. The results of this study support the existence of an alternate catalytic mechanism used by nondivalent ions (especially CoHex) that is distinct from the one promoted by divalent metal ions, and they imply that divalent metals influence catalysis through a specific nonstructural role.     

Characterization of lanthanides as competitors of Na+ and K+ in occlusion sites of renal (Na+,K+)-ATPase.

Lanthanides are useful probes in Ca2+ binding proteins, including sarcoplasmic reticulum (Ca2+,Mg2+)-ATPase. Here, we report that lanthanides compete with Rb+ and Na+ for occlusion in renal (Na+,K+)-ATPase. The lanthanides appear to bind at a single site and act as competitive antagonists, without themselves becoming occluded. All lanthanides tested are effective with the order of potencies Pr greater than Nd greater than La greater than Eu greater than Tb greater than Ho greater than Er, but differences are small. The presence of Mg2+ ions does not affect competition of La3+ with Na+ or K+ suggesting that the effects are not exerted via divalent metal sites. Lanthanides compete with Rb+ and Na+ in membranes digested with trypsin so as to produce 19-kDa and smaller fragments of the alpha-chain (Karlish, S.J.D., Goldshleger, R., and Stein, W. D. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4566-4570), also suggestive of a direct interaction of lanthanides with Na+ and K+ sites. Effects of lanthanides on conformational changes of fluorescein-labeled (Na+,K+)-ATPase are Na(+)-like. They stabilize the E1 state and compete with K+ ions. The Ki for La3+ is 0.445 microM. The apparent affinity in fluorescence assays is proportional to enzyme concentration (Ki = 32.4*[protein] + 0.445 microM La3+), suggesting that lanthanides are also bound nonspecifically (possibly to phospholipids). Direct assays confirm that Tb3+ binding is nonspecific. Measurements of the rate of various conformational transitions show that the rate of E2(K+)----E1(X) (X = Na+ or La3+) is significantly inhibited by La3+ compared to Na+. La3+ ions also slightly accelerate the rate of the E1----E2(K+) conformational transition. The dissociation rate of La3+ has been measured by monitoring the rate of E1(La3+)----E2(K+). It is 1.741 s-1 at 25 degrees C. Based on this value, it is unlikely that La3+ ions are stably occluded, consistent with the conclusion from occlusion experiments. In the future, lanthanides bound to monovalent cation sites with high affinity may become useful probes for location and characterization of sites, although it will be necessary to take into account the large amount of nonspecific binding.     

Specific effects of spermine on Na+,K+-adenosine triphosphatase

Specific effects of spermine on Na+,K+-ATPase were observed using an enzyme partially purified from rabbit kidney microsomes by extraction with deoxycholate. 1. Spermine competed with K+ for K+-dependent, ouabain-sensitive nitrophenylphosphatase. The K1 for spermine was 0.075 mm in the presence of 1 mM Mg2+ and 5 mM p-nitrophenylphosphate at pH 7.5. 2. spermine activated Na+,K+-ATPase over limited concentration ranges of K+ and Na+ in the presence of 0.05 mM ATP. The spermine concentration required for half maximal activation was 0.055 mM in the presence of 1 mM K+, 10 mM Na+, 1 mM Mg2+, and 0.05 mM ATP. 3. The activation of Na+,K4-ATPase was not due to substitution of spermine for K+, Na+, or Mg2+. 4. When the concentration of K+ or Na+ was extremely low, or in excess, spermine did not activate Na+,K+-ATPase, but inhibited it slightly. 5. Plots of 1/v vs. 1/[ATP] at various concentrations of spermine showed that spermine decreased the Km for ATP without changing the Vmax. 6. Plots of 1/v vs. 1/[ATP] at concentrations of K+ from 0.05 mM to 0.5 mM showed that K+ increased the Km for ATP with increase in the Vmax in the presence of 0.2 mM spermine similarly to that in the absence of spermine. The contradictory effects of spermine on this enzyme system suggest that the K+-dependent monophosphatase activity does not reflect the second half (the dephosphorylation step) of the Na+,K+-ATPase catalytic cycle.     

Kinetic properties of a magnesium ion- and calcium ion-stimulated adenosine triphosphatase from the outer-membrane fraction of rat spleen mitochondria

1. Isolated outer membranes from rat spleen mitochondria can be stored in liquid N(2) for several weeks without significant loss of ATPase (adenosine triphosphatase) activity. 2. The ATPase reaction has a broad pH optimum centering on neutral pH, with little significant activity above pH9.0 or below pH5.5. 3. A sigmoidal response of the ATPase activity to temperature is observed between 0 and 55 degrees C, with complete inactivation at 60 degrees C. The Arrhenius plot shows that the activation energy above the transition temperature (22 degrees C) (E(a)=144kJ/mol) is one-third of that calculated for below the transition temperature (E'(a)=408kJ/mol). 4. The outer-membrane ATPase (K(m) for MgATP=50mum) is inactive unless Mg(2+) is added, whereas the inner-membrane ATPase (K(m) for ATP=11mum) is active without added Mg(2+) unless the mitochondria have been depleted of all endogenous Mg(2+) (by using ionophore A23187). 5. The substrate for the outer-membrane ATPase is a bivalent metal ion-nucleoside triphosphate complex in which Mg(2+) (K(m)=50mum) can be replaced effectively by Ca(2+) (K(m)=6.7mum) or Mn(2+), and ATP by ITP. Cu(2+), Co(2+), Sr(2+), Ba(2+), Ni(2+), Cd(2+) and Zn(2+) support very little ATP hydrolysis. 6. Univalent metal ions (Na(+), K(+), Rb(+), Cs(+) and NH(4) (+), but not Li(+)) stimulate the MgATPase activity (<10%) at low concentrations (50mm), but, except for K(+), are slightly inhibitory (20-30%) at higher concentrations (500mm). 7. The Mg(2+)-stimulated ATPase activity is significantly inhibited by Cu(2+) (K(i)=90mum), Ni(2+) (K(i)=510mum), Zn(2+) (K(i)=680mum) and Co(2+) (K(i)=1020mum), but not by Mg(2+), Ca(2+), Ba(2+) or Sr(2+). 8. The outer-membrane ATPase is insensitive to the inhibitors oligomycin, NN'-dicyclohexylcarbodiimide, NaN(3), ouabain and thiol-specific reagents. A significant inhibition is observed at high concentrations of AgNO(3) (0.5mm) and NaF (10mm). 9. The activity towards MgATP is competitively inhibited by the product MgADP (K(i)=0.7mm) but not by the second product P(i) or by 5'-AMP.     

Kinetics of a nucleotide pyrophosphatase in the plasma membrane of the fat cell

Fat cells from rat and rabbit hydrolyzed externally applied adenosine triphosphate at a rate of about 1.8 nmol times mg(-1) cells times min(-1) corresponding to about 0.3 mumol times mg(-1) protein tinus min(-1). Similar activities were found in cell homogenates. In purified adipocyte plasma membranes the rate of hydrolysis was about 1.8 mumol times mg(-1) protein times min(-1). The hydrolytic activity was dependent on divalent metal ions. Mg(2+), Mn(2+) and Ca(2+) gave highest activities. The activity was maximal at about equimolar concentrations of M(2+) and ATP. Km for MgATP was about 0.23 mM and for CaATP about 0.36 mM. Combinations of Mg(2+) and Ca(2+), or of Mg(2+), Na(+) and K(+) gave similar activities as did Mg(2+) only. At concentrations of 1 mM the following nucleotides were hydrolyzed with a decreasing rate: ATP > ITP > GTP > UTP = CTP. In isolated fat cells the beta-adrenergic drug isoproterenol and insulin slightly increased the rate of hydrolysis of external ATP, while the alpha-effector clonidine was inhibitory. The results suggest that a major portion of the ATP hydrolytic activity of the fat cell plasma membrane represents a nucleotide pyrophosphatase activity with access to externally applied ATP.     

D443 of the N domain of Na+,K+-ATPase interacts with the ATP-Mg2+ complex, possibly via a second Mg2+ ion

This paper provides evidence for an interaction of D443 in the N domain of Na(+),K(+)-ATPase with a Mg(2+) ion. Wild-type, D443N/A/C and S445A mutants of porcine Na(+),K(+)-ATPase (alpha1beta1) have been expressed in Pichia pastoris. By comparison with wild-type, D443N reduces the turn-over rate by about 40%. Binding affinity of ATP, measured directly, was not affected by D443N, D443A, or D443C mutations. AMP-PNP-Fe(2+)-catalyzed oxidative cleavage of Na(+),K(+)-ATPase produces two characteristic fragments, at (708)VNDS (P domain) and near (440)VAGDA (N domain), respectively. In the D443N and D443A mutants, both cleavages are suppressed, indicating an interaction between the residues with AMP-PNP-Fe(2+) bound. Previous work suggested that with ATP-Fe(2+) bound the N and P domains come into proximity, both D710 and D443 making contact with a single Fe(2+) (or Mg(2+)) ion. However, the crystal structure of Ca(2+)-ATPase with bound AMP-PCP and Mg(2+) confirm the involvement of D703 (D710) but show that E439 (D443) is too far to make contact with the Mg(2+). By contrast, in the crystal structure with bound ADP, AlF(4), and Mg(2+), representing the E(1)-P conformation, two Mg(2+) ions were observed. Significantly, ADP-Fe(2+)-mediated oxidative cleavage of renal Na,K-ATPase produces the fragment near (440)VAGDA (N domain), while the cleavage at (708)VNDS (P domain) is almost completely absent. The results are explained economically by the hypothesis that ATP is bound with two Mg(2+) (Fe(2+)) ions, a "catalytic" Mg(2+) interacting with D710 via the gamma phosphate and a "structural" Mg(2+) interacting with D443 via the alpha and beta phosphates and a water molecule, respectively.     

精胺和Mg~（2＋）对tRNA~（Ile）圆二向色谱的影响

由于精胺（ｓｐｅｒｍｉｎｅ）能特异地刺激哺乳动物ｔＲＮＡ~（Ｉｌｅ）的氨基酰化,本文用纯化的牛肝ｔＲＮＡ~（Ｉｌｅ）观察了精胺和Ｍｇ（２＋）对ｔＲＮＡ~（Ｉｌｅ）ＣＤ光谱的影响。结果显示：Ｍｇ（２＋）可使牛肝ｔＲＮＡ~（Ｉｌｅ）ＣＤ光谱峰向短波方向偏移２ｎｍ,波峰为２６３ｎｍ,峰值被增大约１０％,ΔθＭｇ（２＋）＝２．３×１０３ｄｅｇ·ｃｍ２／ｄｍｏｌ;而精胺使牛肝ｔＲＮＡ~（Ｉｌｅ）ＣＤ光谱峰减少４０％,Δθｓｐｅｒｍｉｎｅ＝１×１０（－４）ｄｅｇ·ｃｍ２／ｄｍｏｌ;精胺和Ｍｇ（２＋）对肝ｔＲＮＡ~（Ｉｌｅ）－ＩｌｅＲＳ复合物或ＩｌｅＲＳ的ＣＤ光谱基本无影响。表明Ｍｇ（２＋）和精胺可影响牛肝ｔＲＮＡ~（Ｉｌｅ）的构象。实验同时以酵母ｔＲＮＡ（Ｐｈｅ）和Ｅ·ｃｏｌｉｔＲＮＡ~（Ｉｌｅ）作为对照。     

Tryptophan fluorescence of (Na+ + K+)-ATPase as a tool for study of the enzyme mechanism.

1. The protein fluorescence intensity of (Na+ + K+)-ATPase is enhanced following binding of K+ at low concentrations. The properties of the response suggest that one or a few tryptophan residues are affected by a conformational transition between the K-bound form E2 . (K) and a Na-bound form E1 . Na. 2. The rate of the conformational transition E2 . (K) leads to E . Na has been measured with a stopped-flow fluorimeter by exploiting the difference in fluorescence of the two states. In the absence of ATP the rate is very slow, but it is greatly accelerated by binding of ATP to a low affinity site. 3. Transient changes in tryptophan fluorescence accompany hydrolysis of ATP at low concentrations, in media containing Mg2+, Na+ and K+. The fluorescence response reflects interconversion between the initial enzyme conformation, E1 . Na and the steady-state turnover intermediate E2 . (K). 4. The phosphorylated intermediate, E2P can be detected by a fluorescence increase accompanying hydrolysis of ATP in media containing Mg2+ and Na+ but no K+. 5. The conformational states and reaction mechanism of the (Na+ + K+)-ATPase are discussed in the light of this work. The results permit a comparison of the behaviour of the enzyme at both low and high nucleotide concentrations.     

Properties of the sarcolemmal calcium ion-stimulated adenosine triphosphatase of hamster skeletal muscle

1. A sarcolemmal fraction was isolated from hamster hind-leg skeletal muscles by successive treatment with lithium bromide and potassium chloride. The membranous fraction was observed to contain a highly active Ca(2+)-stimulated ATPase (adenosine triphosphatase), a Mg(2+)-stimulated ATPase, and an Na(+)+K(+)-stimulated Mg(2+)-dependent ouabain-sensitive ATPase. 2. The Ca(2+)-stimulated ATPase activity was pH-dependent, the optimum being pH7.6. 3. Optimum activation of this enzyme was obtained with 3-4mm-Ca(2+) when 4mm-ATP was present as a substrate, and was not influenced by Na(+), K(+) or ouabain, whereas 2,4-dinitrophenol, sodium azide, oligomycin, sodium fluoride and ethanedioxybis(ethylamine)tetra-acetate were inhibitory. 4. The Ca(2+)-stimulated ATPase was markedly inhibited by thiol-blocking reagents, and cysteine was able to reverse this inhibition. 5. Various bivalent cations stimulated ATP hydrolysis by the sarcolemmal fraction in the following decreasing order of potency: Mg(2+), Ca(2+), Mn(2+), Co(2+), Sr(2+), Ba(2+), Zn(2+), Cu(2+).     

Effects of some salts and sodicity on the growth of a Rhizobium leguminosarum bv. viceae strain isolated from a salt-affected soil.

The effects of sodium (Na+), calcium (Ca2+), magnesium (Mg2+), and boron (B) concentrations and sodicity, as measured by the sodium adsorption ratio (SAR), on the growth of a Rhizobium leguminosarum bv. viceae strain isolated from a salt-affected soil were studied. The rate of growth was measured in a yeast extract-mannitol broth, amended with salts having electrical conductivity (EC) of 4, 8, and 16 dS x m(-1). Each salinity level was prepared to achieve SAR values of 10, 20, and 30 with or without graded B concentrations of 0.5, 1, 3, and 5 mg x L(-1). We found that salinity levels equal to or more than 8 dS x m(-1) had negative effects on Rhizobium growth during the first days of incubation, but the effects became less pronounced after 1 week. Na+ concentrations of more than 1.1 g x L(-1) retarded growth, especially at high SAR values (i.e., at low Ca2+ concentrations). The retardation of growth increased with increases in EC up to 16 dS x m(-1), at all sodicity levels. Mg2+ added together with Na+ or with Ca2+ + Na+ affected growth more negatively than Ca2+ + Na+ alone. The effect of Mg2+ became more pronounced with increased salinities and sodicities. It was concluded that EC of more than 4 dS x m(-1) retarded growth of Rhizobium, but only at high sodicity levels. The relative specific ion effect on growth was in the order Na+ < Ca2+ < Mg2+. The harmful effect of Mg2+ on this strain was accentuated by adding Ca2+ to the cultural medium. When SAR increased from 10 to 30, Na+ had no clear effect on growth, irrespective of the accompanied cations, i.e, Ca2+, Mg2+, or Ca2+ + Mg2+. Growth was reduced by B concentrations as low as 0.5 mg x L(-1), and the B effect was enhanced by increased salinity.     

Inhibition of Na+/K(+)-ATPase and Mg(2+)-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na(+)/K(+)-ATPase and Mg(2+)-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu(2+), Zn(2+), Fe(2+) and Co(2+)) and heavy metals (Hg(2+) and Pb(2+)) ions were studied. All investigated metals produced a larger maximum inhibition of Na(+)/K(+)-ATPase than Mg(2+)-ATPase activity. The free concentrations of the key species (inhibitor, MgATP(2-), MeATP(2-)) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu(2+)/Fe(2+) or Hg(2+)/Pb(2+) caused additive inhibition, while Cu(2+)/Zn(2+) or Fe(2+)/Zn(2+) inhibited Na(+)/K(+)-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu(2+)/Fe(2+) or Cu(2+)/Zn(2+) inhibited Mg(2+)-ATPase activity synergistically, while Hg(2+)/Pb(2+) or Fe(2+)/Zn(2+) induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na(+)/K(+)-ATPase activity by reducing the maximum velocities (V(max)) rather than the apparent affinity (Km) for substrate MgATP(2-), implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg(2+)-ATPase activity by Zn(2+), Fe(2+) and Co(2+) as well as kinetic analysis indicated two distinct Mg(2+)-ATPase subtypes activated in the presence of low and high MgATP(2-) concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na(+)/K(+)-ATPase with various potencies. Furthermore, these ligands also reversed Na(+)/K(+)-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg(2+)-induced inhibition was not obtained.