Activation of yeast enolase by Cd(II)

Activation of yeast enolase by Cd2+ exhibits properties similar to activation by the physiological cofactor Mg2+. The activity is weakly stimulated, then inhibited by increasing ionic strength. The activity increases, then falls with increasing Cd2+ concentration. The effect of pH on activity produced by Cd2+ is very similar to that produced by Mg2+, except that the Cd2+ profile is shifted one pH unit to more alkaline values, and the maximum activity of the Cd2+-enzyme is about 10% of that of the Mg2+-enzyme. The apparent kinetic parameters of Cd2+ activation show little effect of pH except for inhibition by high concentrations of Cd2+: the apparent Ki increases sharply with pH. This is interpreted as the result of Cd2+ being a less effective "catalytic" metal ion, and Cd2+ being more effective in stabilizing the enzyme at alkaline pH's. The similarity of effects of ionic strength, divalent cation, and pH may be due to interaction with the same six sites per mole of enzyme. We also characterized the dependence of what is believed to be the enzyme-catalyzed enolization of a substrate analog, D-tartronate semialdehyde-2-phosphate (TSP) on similar parameters of pH, ionic strength, etc. The putative enolization is dependent on catalytic metal ion, although the TSP binds to the conformational Cd2+-enzyme complex. The reaction is very slow and very pH dependent, increasing with pH with a midpoint of reaction velocity at pH 8.7. There is a strong qualitative correlation between pH dependencies of reaction velocity of substrate conversion and TSP enolization and absorbance of the enzyme-bound TSP enolate, whether with Mg2+ or Cd2+ as cofactor. The slowness of the Cd2+-TSP reaction is not limited by proton release or any reaction involving covalent bonds to hydrogen. The apparent reaction rate constant increases linearly with Cd2+ concentration. Addition of excess ethylenediaminetetraacetic acid reverses the TSP reaction, but again very slowly. The binding of Cd2+ to the catalytic sites is characterized by low association and dissociation rate constants.     

Ionic conditions for the cleavage of the tRNA-like structure of turnip yellow mosaic virus by the catalytic RNA of RNase P

The 3'-end of the RNA genome of turnip yellow mosaic virus can form a pseudoknotted tRNA-like structure that can be recognized by several tRNA-specific enzymes. We have found that the catalytic RNA component of Bacillus subtilis RNase P can cleave this structure in unusually low ionic strength buffers at a site analogous to the 5'-end of an aminoacyl stem of a tRNA. Most other precursors can only be processed under low ionic strength conditions if the RNase P holoenzyme is used; processing by the catalytic RNA component alone requires a higher ionic strength buffer. The cleavage of the turnip yellow mosaic virus tRNA-like structure demonstrates the importance of the substrate in determining the optimal buffer conditions for this reaction and also shows that high ionic strength buffers are not always necessary for cleavage by the catalytic RNA.     

Ionic control of enzymic degradation of double-stranded RNA

The pattern of the degradation of various double-stranded polyribonucleotides by several ribonucleases (bovine RNAase A and its cross-linked dimer, bovine seminal RNAase, and pike-whale pancreatic RNAase) has been studied as a function of ionic strength and pH. It appears that (1) there is no direct correlation between the secondary structure of double-stranded RNA and its resistance against enzymatic breakdown, i.e., the stability of the secondary structure of double-helical RNA is not the main variable in the process. (2) The acstivity responses of the enzymes examined to changes of ionic strength and pH suggest that enzymic degradation of double-stranded RNA is mainly controlled by ion concentration, and that the process may fall within the phenomena interpreted by the theory of the ionic control of biochemical reactions advanced by Douzou and Maurel (Douzou, P. and Maurel, P. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1013--1015). (3) The activity curves of the enzyme studied show, at a given pH, a shift toward higher ionic strengths as a function of the basicity of the enzyme protein. This finding explains the already observed correlation between number and/or density of positive charges of a ribonuclease molecule and its ability to attack double-stranded RNA in 0.15 M sodium chloride/0.015 M sodium citrate (SSC). (4) A careful analysis of the influence of ionic strength and pH on the reaction appears to be necessary in order to characterize a ribonuclease which shows activity towards double-stranded RNAs, and to allow a meaningful comparison between different enzymes capable of attacking these substrates.     

Pronounced and differential effects of ionic strength and pH on testosterone oxidation by membrane-bound and purified forms of rat liver microsomal cytochrome P-450

The aim of this study was to determine the effects of ionic strength and pH on the different pathways of testosterone oxidation catalyzed by rat liver microsomes. The catalytic activity of cytochromes P-450a (IIA1), P-450b (IIB1), P-450h (IIC11) and P-450p (IIIA1) was measured in liver microsomes from mature male rats and phenobarbital-treated rats as testosterone 7 alpha-, 16 beta-, 2 alpha- and 6 beta-hydroxylase activity, respectively. An increase in the concentration of potassium phosphate (from 25 to 250 mM) caused a marked decrease in the catalytic activity of cytochromes P-450a (to 8%), P-450b (to 22%) and P-450h (to 23%), but caused a pronounced increase in the catalytic activity of cytochrome P-450p (up to 4.2-fold). These effects were attributed to changes in ionic strength, because similar but less pronounced effects were observed with Tris-HCl (which has approximately 1/3 the ionic strength of phosphate buffer at pH 7.4). Testosterone oxidation by microsomal cytochromes P-450a, P-450b, P-450h and P-450p was also differentially affected by pH (over the range 6.8-8.0). The pH optima ranged from 7.1 (for P-450a and P-450h) to 8.0 (for P-450p), with an intermediate value of 7.4 for cytochrome P-450b. Increasing the pH from 6.8 to 8.0 unexpectedly altered the relative amounts of the 3 major metabolites produced by cytochrome P-450h. The decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h that accompanied an increase in ionic strength or pH could be duplicated in reconstitution systems containing purified P-450a, P-450b or P-450h, equimolar amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. This result indicated that the decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h was a direct effect of ionic strength and pH on these enzymes, rather than a secondary effect related to the increase in testosterone oxidation by cytochrome P-450p. Similar studies with purified cytochrome P-450p were complicated by the atypical conditions needed to reconstitute this enzyme. However, studies on the conversion of digitoxin to digitoxigenin bisdigitoxoside by liver microsomes, which is catalyzed specifically by cytochrome P-450p, provided indirect evidence that the increase in catalytic activity of cytochrome P-450p was also a direct effect of ionic strength and pH on this enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Do ribosomal RNAs act merely as scaffold for ribosomal proteins?

Investigations that are being carried out in various laboratories including ours clearly provide the answer which is in the negative. Only the direct evidences obtained in this laboratory will be presented and discussed. It has been unequivocally shown that the interaction between 16S and 23S RNAs plays the primary role in the association of ribosomal subunits. Further, 23S RNA is responsible for the Binding of 5S RNA to 16S.23S RNA complex with the help of three ribosomal proteins, L5, L18, L15/L25. The 16S.23S RNA complex is also capable of carrying out the following ribosomal functions, although to small but significant extents, with the help of a very limited number of ribosomal proteins and the factors involved in protein synthesis: (a) poly U-Binding, (B) poly U-dependent Binding of phenylalanyl tRNA, (c) EF-G-dependent GTPase activity, (d) initiation complex formation, (e) peptidyl transferase activity (puromycin reaction) and (f) polyphenylalanine synthesis. These results clearly indicate the direct involvement of rRNAs in the various steps of protein synthesis. Very recently it has Been demonstrated that the conformational change of 23S RNA is responsible for the translocation of peptidyl tRNA from the aminoacyl (A) site to the peptidyl (P) site. A model has Been proposed for translocation on the Basis of direct experimental evidences. The new concept that ribosomal RNAs are the functional components in ribosomes and proteins act as control switches may eventually turn out to Be noncontroversial.     

Distribution of elongation factors EF-1 and EF-2 among components of rabbit reticulocyte lysate

The distribution of activity of the elongation factors EF-1 and EF-2 among the components of rabbit reticulocyte lysate separated by sucrose density gradient centrifugation was studied. At low ionic strength (0.01 M KCl) about 30% of the EF-1 activity was found in polyribosomes. At moderate ionic strength (0.1 M KCl) the EF-1 activity was absent in the polyribosomes. An addition of RNA excess to the lysate prior to centrifugation at low ionic strength resulted in elimination of the EF-1 activity from the polyribosomes. This indicates that EF-1 is reversibly bound to the polyribosomes and that EF-1 may be retained on them due to interaction with RNA of polysomes mediated by its RNA-binding site. After dissociation of polyribosomes containing EF-1 in the presence of EDTA and subsequent fractionation of the dissociation products at low ionic strength (0.01 M KCl) the EF-1 activity was revealed in the ribosomal subparticles (predominantly in 60S). At 0.1 M KCl EF-1 mainly sedimented in the zone of distribution of polyribosomal informosomes. The elongation factor EF-2 was not revealed in polyribosomes during lysate centrifugation even at low ionic strength which corresponds to its lower affinity for RNA.     

Activation of rabbit muscle phosphofructokinase by F-actin and reconstituted thin filaments

Striking effects of F-actin and the reconstituted thin filament of muscle on the catalytic activity of rabbit muscle phosphofructokinase are demonstrated through direct measurements of enzymatic activity by using the pH stat. The addition of F-actin to solutions of phosphofructokinase at low ionic strength (10 mM KCl and 5 mM MgCl2) partially reverses the inhibition of the enzyme seen at high ATP concentrations and increases the apparent affinity of the enzyme for fructose 6-phosphate with slight effect on Vmax. F-Actin augments the activation of the enzyme obtained with AMP and partially counters the inhibition obtained with citrate. The maximum effect in the reversal of ATP inhibition is about the same for combinations of either F-actin or the thin filament with AMP as it is for AMP alone. In general, the effect of F-actin on the catalytic activity of phosphofructokinase is larger than that of the thin filament. The activation of phosphofructokinase by F-actin persists at physiological ionic strength.     

Studies on the secondary structure of nuclear ribonucleic acids

Rat liver nuclei were separated into two fractions, the nucleolus and the nucleoplasm, by a combined salt-enzymic extraction. RNA was purified from both sources and analysed on sucrose density gradients. In both fractions a prominent heterogeneous RNA class with mean sedimentation coefficient of 18S was found. This material was analysed by measuring the rate of reaction with formaldehyde, the ultraviolet absorbance-temperature profile, the spectrophotometric observation of conformational changes as a function of pH, the spectrophotometric titration of uracil and guanine residues and the effect of both temperature and ionic strength on the spectrophotometric titration of cytosine residues. Nucleoplasmic 18S RNA fraction exhibited, on heating and also by adjustment of the pH to 2.5, a hyperchromicity of about 16%, close to that observed, in control experiments, for ribosomal RNA (22%). Titration of cytosine residues in solutions containing 1mm-NaCl and 0.1m-NaCl yielded pK values equal to 4.41 and 3.84 respectively. These results suggest that this RNA fraction is composed of structurally complex polymers. The hypochromicity of the nucleolar 18S RNA fraction determined by heating or adjusting the pH to 2.5, was not greater than 6% of the initial value. The rate of reaction with formaldehyde was 88% of that observed for the hydrolysed 18S fraction which suggested only 12% hydrogen bonding. pK values for uracil and guanine residues were 10.1 and 10.05 respectively. Titration of cytosine residues yielded a pK of 4.10, which was found to be independent of temperature and ionic strength variations.     

Direct calorimetric analysis of the enzymatic activity of yeast cytochrome c oxidase.

The kinetic and thermodynamic parameters associated with the enzymatic reaction of yeast cytochrome c oxidase with its biological substrate, ferrocytochrome c, have been measured by using a titration microcalorimeter to monitor directly the rate of heat production or absorption as a function of time. This technique has allowed determination of both the energetics and the kinetics of the reaction under a variety of conditions within a single experiment. Experiments performed in buffer systems of varying ionization enthalpies allow determination of the net number of protons absorbed or released during the course of the reaction. For cytochrome c oxidase the intrinsic enthalpy of reaction was determined to be -16.5 kcal/mol with one (0.96) proton consumed for each ferrocytochrome c molecule oxidized. Activity measurements at salt concentrations ranging from 0 to 200 mM KCl in the presence of 10 mM potassium phosphate, pH 7.40, and 0.5 mM EDTA display a biphasic dependence of the electron transferase activity upon ionic strength with a peak activity observed near 50 mM KCl. The ionic strength dependence was similar for both detergent-solubilized and membrane-reconstituted cytochrome c oxidase. Despite the large ionic strength dependence of the kinetic parameters, the enthalpy measured for the reaction was found to be independent of ionic strength. Additional experiments involving direct transfer of the enzyme from low to high salt conditions produced negligible enthalpy changes that remained constant within experimental error throughout the salt concentrations studied (0-200 mM KCl). These results indicate that the salt effect on the enzyme activity is of entropic origin and further suggest the absence of a major conformational change in the enzyme due to changes in ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)     

各种因子对固定化烟草核酮糖1,5-二磷酸羧化酶/加氧酶解离作用的影响

pH,温度、离子强度及效应剂等对固定化烟草RuBP羧化酶在2.5mol/L尿素处理下的解离作用有各种不同的影响。在pH6.0时,仅小亚基从大亚基核(L_8)解离,当pH为中性偏碱时,大亚基核也解离。低温和低离子强度均促进酶的解离,而温度和离子强度对大亚基之间的解离的影响显著大于对大、小亚基之间的影响。这表明酶的亚基之间存在着不同的极性和疏水作用,而大亚基之间的疏水作用比大、小亚基之间的强。6-PG对大、小亚基之间解离的抑制作用表明大亚基上的催化位置与小亚基之间有一定的密切关系。     

Changes in the quaternary structure of phosphoenolpyruvate carboxylase induced by ionic strength affect its catalytic activity

Phosphoenolpyruvate carboxylase from maize leaves dissociated into dimers and/or monomers when exposed to increasing ionic strength (e.g. 200-400 mM NaCl) as indicated by gel filtration experiments. Changes in the oligomerization state were dependent on pH, time of preincubation with salt and protein concentration. A dissociation into dimers and monomers was observed at pH 8, while at pH 7 dissociation into the dimeric form only was observed. Exposure of the enzyme to higher ionic strength decreased the activity in a time-dependent manner. Turnover conditions and glucose 6-phosphate protected the carboxylase from the decay in activity, which was faster at pH 7 than at pH 8. The results suggest that changes in activity of the enzyme, following exposure to high ionic strength, are the consequence of dissociation. Tetrameric and dimeric forms of the phosphoenolpyruvate carboxylase seemingly reveal different catalytic properties. We suggest that the distinct catalytic properties of the different oligomeric species of phosphoenolpyruvate carboxylase and changes in the equilibrium between them could be the molecular basis for an effective regulation of metabolite levels by this key enzyme of C4 plants.     

Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome

The catalytic mechanism of peptide bond formation on the ribosome is not known. The crystal structure of 50S ribosomal subunits shows that the catalytic center consists of RNA only and suggests potential catalytic residues. Here we report rapid kinetics of the peptidyl transferase reaction with puromycin at rates up to 50 s(-1). The rate-pH profile of the reaction reveals that protonation of a single ribosomal residue (pK(a) = 7.5), in addition to protonation of the nucleophilic amino group, strongly inhibits the reaction (>100-fold). The A2451U mutation within the peptidyl transferase center has about the same inhibitory effect. These results suggest a contribution to overall catalysis of general acid-base and/or conformational catalysis involving an ionizing group at the active site.     

Thiostrepton inhibition of tRNA delivery to the ribosome

Ribosome-stimulated hydrolysis of guanosine-5'-triphosphate (GTP) by guanosine triphosphatase (GTPase) translation factors drives protein synthesis by the ribosome. Allosteric coupling of GTP hydrolysis by elongation factor Tu (EF-Tu) at the ribosomal GTPase center to messenger RNA (mRNA) codon:aminoacyl-transfer RNA (aa-tRNA) anticodon recognition at the ribosomal decoding site is essential for accurate and rapid aa-tRNA selection. Here we use single-molecule methods to investigate the mechanism of action of the antibiotic thiostrepton and show that the GTPase center of the ribosome has at least two discrete functions during aa-tRNA selection: binding of EF-Tu(GTP) and stimulation of GTP hydrolysis by the factor. We separate these two functions of the GTPase center and assign each to distinct, conserved structural regions of the ribosome. The data provide a specific model for the coupling between the decoding site and the GTPase center during aa-tRNA selection as well as a general mechanistic model for ribosome-stimulated GTP hydrolysis by GTPase translation factors.     

Some properties and the possible role of intrinsic ATPase of rat liver 80S ribosomes in peptide bond elongation

The properties and role in peptide elongation of ATPase intrinsic to rat liver ribosomes were investigated. (i) Rat liver 80S ribosomes showed high ATPase and GTPase activities, whereas the GTPase activity of EF-1alpha and EF-2 was very low. mRNA, aminoacyl-tRNA, and elongation factors alone enhanced ribosomal ATPase activity and in combination stimulated it additively or synergistically. The results suggest that these translational components induce positive conformational changes of 80S ribosomes by binding to different regions of ribosomes. Translation inhibitors, tetracyclin and fusidic acid, inhibited ribosomal ATPase with or without elongational components. (ii) Two ATPase inhibitors, AMP-P(NH)P and vanadate, did not inhibit GTPase activities of EF-1alpha and EF-2 assayed as uncoupled GTPase, but they did inhibit poly(U)-dependent polyphe synthesis of 80S ribosomes. (iii) Effects of AMP-P(NH)P and ATP on poly(U)-dependent polyphe synthesis at various concentrations of GTP were examined. ATP enhanced the activity of polyphe synthesis even at high concentrations of GTP, suggesting a specific role of ATP. At low concentrations of GTP, the extent of inhibition by AMP-P(NH)P was very low, probably owing to the prevention of the reduction of the GTP concentration. (iv) Vanadate inhibited the translocation reaction by high KCl-washed polysomes. These findings together indicate that ribosomal ATPase participates in peptide translation by inducing positive conformational changes of mammalian ribosomes, in addition to its role of chasing tRNA from the E site.     

Preparation of immobilized NAD glycohydrolase from Neurospora crassa conidia by hydrophobic interaction--characteristics of the enzyme derivative

NAD glycohydrolase from Neurospora crassa conidia has been immobilized by hydrophobic interaction on Sepharose 4B beads coated with propyl residues through CNBr activation. The bond resulted stable under a wide range of conditions (ionic strength, temperature, pH). As a result of immobilization the pH optimum for catalytic activity shifted by about 0.2 pH unit in the acidic direction, to lie between 7.5 and 7.3. The stability of the enzymatic activity was largely enhanced by effect of immobilization but the Km value towards NAD+ was increased compared with that of the free enzyme (1 X 10(-3) and 2 X 10(-4) M respectively).     

Catalysis of superoxide dismutation by manganese aminopolycarboxylate complexes

Complexes of manganese, copper, cobalt, and iron with a variety of aminopolycarboxylates at concentrations from 2 X 10(-7) to 3 X 10(-6) M were tested for superoxide dismutase activity with horse ferricytochrome c as the competing reagent for superoxide. In the presence of excess ligand only manganous nitrilotriacetate and manganous ethylenediaminediacetate showed activity with catalytic rate constants of 2.2 X 10(7) and 1.8 X 10(7) M-1 S-1, respectively, at pH 6, 22 +/- 1 degree C, and 10 mM ionic strength. These rate constants decrease considerably at higher pH. Manganous N-hydroxyethylethylenediaminetriacetate is oxidized by superoxide, but does not appear to have catalytic activity. From the experimental conditions under which the two complexes mentioned above exhibit catalysis, and the inactivity of other metal chelates, it is concluded that an open coordination site is essential but not sufficient to catalyze the dismutation reaction.     

Direct covalent mercuration of nucleotides and polynucleotides.

Nucleotides of cytosine and uracil are readily mercurated by heating at 37-50 degrees in buffered aqueous solutions (pH 5.0-8.0) containing mercuric acetate. Proton magnetic resonance, elemental, electrophoretic, and chromatographic analyses have shown the products to be 5-mercuricytosine and 5-mercuriuracil derivatives, where the mercury atom is covalently bonded. Polynucleotides can be mercurated under similar conditions. Cytosine and uracil bases are modified in RNA while only cytosine residues in DNA are substituted. There is little, if any, reaction with adenine, thymine, or guanine bases. The rate of polymer mercuration is, unlike that of mononucleotides, markedly influenced by the ionic strength of the reaction mixture: the lower the ionic strength the faster the reaction rate. Pyrimidine residues in single- and double-stranded polymers react at essentially the same rate. Although most polynucleotides can be extensively mercurated at pH 7.0 in sodium or Trisacetate buffers, tRNA undergoes only limited substitution in Tris buffers. The mild reaction conditions give minimal single-strand breakage and, unlike direct iodination procedures, do not produce pyrimidine hydrates. Mercurated polynucleotides can be exploited in a variety of ways, particularly by crystallographic and electron microscopic techniques, as tools for studying polynucleotide structure.     

Metal-mediated reduction of cytochrome c by thioglycolic acid

The reduction of cytochrome c by thioglycolic acid was found to be extremely sensitive to metal catalysis. The rate of the uncatalyzed reaction was negligible in comparison with rates obtained from reactions supplemented with catalytic amounts of copper or iron. Both the catalyzed and uncatalyzed reactions were independent of pH (near neutrality) but when o-phenanthroline was included in the reaction mixture, a pH dependence was induced. This pH dependence is the result of an interference of oxygen with the metal complexes. A comparison of the rate constants at zero ionic strenght, which were obtained from the application of the Debye-Hückel theory for the ionic strength dependence, demonstrated that copper complexes are superior catalysts as compared with iron complexes. Our results suggest that in the copper-mediated reaction, the catalyst is a cupric thioglycolate complex with a net charge of ?2. The addition of o-phenanthroline to the reaction mixture results in a tenfold decrease in the catalytic activity and in a change in the net charge of the catalyst to ?1. At pH 8 the iron-mediated reduction is catalyzed by a ferric thioglycolate complex, whereas at pH 7 a ferrothioglycolate complex provides the catalytic activity. Both complexes have a net negative charge of ?2. At both pH's the catalytic activity is completely abolished by the addition of o-phenanthroline. The results demonstrate the effectiveness by which metal-sulfur complexes can facilitate one-electron transfer reactions and could there-fore serve as a model in the study of various biological oxidations.     

Phosphoryl Group Exchange between ATP and ADP Catalyzed by H+-ATPase from Oat Roots

ATP-ADP exchange was estimated in the presence of plasma membrane H+-ATPase of oat (Avena sativa) roots partially purified with Triton X-100 by measuring [14C]ATP formation from [14C]ADP. Most studies were done at 0[deg]C. At pH 6.0 the exchange showed: (a) Mg2+ requirement with a biphasic response giving maximal activity at 152 [mu]M and (b) insensitivity to ionic strength, [Na+], and [K+]. ATP and ADP dependence were analyzed with a model in which nucleotide-enzyme interactions are at rapid-random equilibrium, whereas E1ATP [left right arrow] E1P-ADP transitions occur in steady state. The results indicated competition between ADP and ATP for the catalytic site, whereas ATP interaction with the ADP site was extremely weak. At 0[deg]C the exchange showed a 3-fold pH increase, from pH 5.5 to 9.0. At an alkaline pH the reaction was not affected by sodium azide and carbonyl cyanide p-trifluometoxyphenyl-hydrazone, had a biphasic response to Mg2+ (maximal at 513 [mu]m), and was insensitive to ionic strength. At 20[deg]C ATP-ADP exchange was pH insensitive. At both temperatures ATP hydrolysis displayed a bell-shaped response, with a maximum around pH 6.0 to 6.5. Because no adenylate kinase activity was detected under any condition, these results demonstrate the existence of an ATP-ADP exchange reaction catalyzed by the plant H+-ATPase.