Studies on the Kinetic Mechanism and Salt Activation of Bovine Brain Choline Acetyltransferase

Abstract: :The kinetic mechanism of bovine brain choline acetyltransferase has been studied using acetylaminocholine as a dead-end inhibitor and di-methylaminoethanol as an alternate substrate. Acetylaminocholine inhibition is competitive with respect to acetylcholine and noncompetitive with respect to choline. Dimethylaminoethanol exhibits one-sixth the V_max obtained with choline. These results suggest that the reaction obeys a sequential random kinetic mechanism. Salt activation of the enzyme is nonspecific with respect to monovalent anions, and results in a parallel increase in the K^m for choline and the Kⁱ for acetylcholine. These results support the conclusion that salt activation of choline acetyltransferase is a nonspecific effect and that no specific chloride ion regulation of this enzyme occurs in vivo .     

Inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex by reduced nicotinamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition

Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex [Lawlis, V. B., & Roche, T. E. (1980) Mol. Cell. Biochem. 32, 147-152]. Product inhibition patterns from initial velocity studies conducted at less than 10(-9) M or at 1.5 X 10(-5) M Ca2+ with NAD+, CoA, or alpha-ketoglutarate as the variable substrate showed that NADH was a noncompetitive inhibitor with respect to each of these substrates, except at high NAD+ concentrations, where reciprocal plots were nonlinear and the inhibition pattern for NADH vs. NAD+ changed from a noncompetitive to a competitive pattern. From slope and intercept replots, 2-fold to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 X 10(-5) M Ca2+ than for inhibition at less than 10(-9) M Ca2+. These inhibition patterns and the lack of an effect of Ca2+ on the inhibition of the dihydrolipoyl dehydrogenase component suggested that Ca2+-modulated NADH inhibition occurs at an allosteric site with competitive binding at the site by high levels of NAD+. Decarboxylation of alpha-keto[1-14C]glutarate by the resolved alpha-ketoglutarate dehydrogenase component was investigated in the presence of 5.0 mM glyoxylate which served as an efficient acceptor. NADH (0.2 mM) or 1.0 mM ATP inhibited the partial reaction whereas 15 muM Ca2+, 1.0 mM ADP, or 10 mM NAD+ stimulated the partial reaction and reduced NADH inhibition of this reaction. Thus these effectors alter the activity of the alpha-ketoglutarate dehydrogenase complex by binding at allosteric sites on the alpha-ketoglutarate dehydrogenase component. Inhibition by NADH over a wide range of NADH/NAD+ ratios was measured under conditions in which the level of alpha-ketoglutarate was adjusted to give matching control activities at less than 10(-9) M Ca2+ or 1.5 X 10(-5) M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies establish that both Ca2+ and ADP decreased NADH inhibition under conditions compensating for the effects of Ca2+ and ADP on S0.5 for alpha-ketoglutarate. ADP was particularly effective in reducing NADH inhibition; further studies are required to determine whether this occurs through binding of NADH and ADP at the same, overlapping, or interacting sites.     

Ionic strength dependence of the inhibition of acetylcholinesterase activity by Al3+

Inhibition of acetylcholinesterase activity by Al3+ has been examined by initial velocity kinetics and by a first-order kinetic method. Both methods yield an inhibition constant of approx. 1.7 mM at 0.1 M ionic strength. The initial velocity study indicates a noncompetitive mechanism of inhibition by Al3+. Inhibition at 10 mM ionic strength shows a Ki of 0.03 mM. Evaluation of the ionic strength dependence concurs with the results of Nolte et al. (Biochemistry 19 (1980) 3705). An effective charge in the binding site of -9 predicts the ratio of inhibition constants at high and low ionic strength. Extrapolation to zero ionic strength gives a Ki0 = 0.34 microM.     

H4-isozyme of lactate dehydrogenase in the solution of sodium chloride--3. The enzymatic activity and the pyruvate inhibition

1. Low enzymatic activities in low pyruvate concentrations and high Km were observed in sodium chloride solutions. 2. The pyruvate inhibition shown by the % activity at 1 mM pyruvate was lower sodium chloride than in 0.1 M sodium phosphate. 3. At 40 degrees C, as compared with results at 20 degrees C, less pyruvate inhibition was observed in phosphate buffer and in sodium chloride solutions. 4. By using the equilibrium constants between dimer and tetramer, a theoretical explanation is proposed for the pyruvate inhibition. In this explanation, it is suggested that the quaternary complex which is composed of tetrameric enzyme, coenzyme and two kinds of pyruvates was the main cause of the pyruvate inhibition.     

Substrate and product inhibition of hydrogen production by the extreme thermophile,Caldicellulosiruptor saccharolyticus

Substrate and product inhibition of hydrogen production during sucrose fermentation by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was studied. The inhibition kinetics were analyzed with a noncompetitive, nonlinear inhibition model. Hydrogen was the most severe inhibitor when allowed to accumulate in the culture. Concentrations of 5-10 mM H(2) in the gas phase (identical with partial hydrogen pressure (pH(2)) of (1-2) x 10(4) Pa) initiated a metabolic shift to lactate formation. The extent of inhibition by hydrogen was dependent on the density of the culture. The highest tolerance for hydrogen was found at low volumetric hydrogen production rates, as occurred in cultures with low cell densities. Under those conditions the critical hydrogen concentration in the gas phase was 27.7 mM H(2) (identical with pH(2) of 5.6 x 10(4) Pa); above this value hydrogen production ceased completely. With an efficient removal of hydrogen sucrose fermentation was mainly inhibited by sodium acetate. The critical concentrations of sucrose and acetate, at which growth and hydrogen production was completely inhibited (at neutral pH and 70 degrees C), were 292 and 365 mM, respectively. Inorganic salts, such as sodium chloride, mimicked the effect of sodium acetate, implying that ionic strength was responsible for inhibition. Undissociated acetate did not contribute to inhibition of cultures at neutral or slightly acidic pH. Exposure of exponentially growing cultures to concentrations of sodium acetate or sodium chloride higher than ca. 175 mM caused cell lysis, probably due to activation of autolysins.     

THE LACK OF SPECIFICITY TOWARDS SALTS IN THE ACTIVATION OF CHOLINE ACETYLTRANSFERASE FROM HUMAN PLACENTA

Abstract— The effects of monovalent and divalent anions on the choline acetyltransferase reaction have been determined at high (5.0 mM) and low (0.58 mM) choline. At 0.58 mM-choline, both monovalent and divalent anions activate the enzyme ±9 fold; however, at 5.0mM-choline, monovalent anions activate the enzyme ±25 fold, while divalent anions activate ±9 fold. Both monovalent and divalent anions show uncompetitive activation with respect to choline. When either dimethylaminoethanol, N -(2-hydroxyethyl)- N -methyl piperidinium iodide, or N -(2-hydroxyethyl)- N -propyl pyrrolidinium iodide was substituted for choline, activation by monovalent or divalent anions was only 2.5-4 fold. With AcCoA as substrate the ChA reaction can be increased ±20 fold by increased salts; however, with acetyl dephosphoCoA as substrate, the reaction is insensitive to the salt concentration. Similar salt effects on the ChA reaction, as measured in the direction of acetylcholine synthesis, have been demonstrated in the reverse reaction. In addition, inhibition of the forward reaction by acetylcholine has been measured as a function of sodium chloride concentration. Although the K₁ for acetylcholine increases with increasing salt, this change in K ₁, parallels the increase in the K _m for choline. These results support the hypothesis that both monovalent and divalent anions activate choline acetyltransferase by the same singular mechanism; which is to increase the rate of dissociation of coenzyme A from the enzyme.     

THE REACTION OF CHOLINE AND 3,3-DIMETHYL-1-BUTANOL WITH THE ACETYLENZYME FROM ACETYLCHOLINESTERASE

—A comparison was made of the manner in which choline chloride and 3,3-dimethyl-1-butanol react with the acetylenzyme formed during the hydrolysis of esters of acetic acid by acetylcholinesterase. Acetylcholine and acetylthiocholine were the substrates. The ratio of the formation of alkyl acetate to that of acetic acid increased linearly with the concentration of dimethylbutanol, but approached a limiting value as the concentration of choline was increased. Total enzymic activity was inhibited by choline and activated slightly by dimethylbutanol. The effects of varying ionic strength, pH and substrate concentration were examined. The effects of tetraethyl- and tetramethylammonium ions on the reaction of dimethylbutanol with the acetylenzyme were also studied. The results suggest that dimethylbutanol and choline bind to different regions of the active site. A mechanism for the reaction of choline and substrate with acetylcholinesterase is suggested.     

Kinetics of phosphoenolpyruvate carboxylase from Zea mays leaves at high concentration of substrates

At low concentrations of phosphoenolpyruvate and magnesium, the substrate of phosphoenolpyruvate carboxylase (PEPC) from Zea mays leaves is the MgPEP complex and free phosphoenolpyruvate (fPEP) is an allosteric activator [A. Tovar-Méndez, R. Rodríguez-Sotres, D.M. López-Valentín, R.A. Mu?oz-Clares, Biochem. J. 332 (1998) 633-642]. To further the understanding of this photosynthetic enzyme, we have re-investigated its kinetics covering a 500-fold range in fPEP and free Mg(2+) (fMg(2+)) concentrations. Apparent V(max) values were dependent on the concentration of the fixed free species, suggesting that these species are substrates of the PEPC-catalyzed reaction. However, when substrate inhibition was taken into account, similar V(max) values were obtained in all saturation curves for a given varied free species, indicating that MgPEP is indeed the reaction substrate. As substrate inhibition may be the result of the rise in ionic strength of the assay medium, we studied its effects on the kinetics of the enzyme. Mixed inhibition against MgPEP was found, with apparent K(ic) and K(iu) values of 36 and 1370 mM, respectively. Initial velocity patterns determined at constant ionic strength, 600 mM, were consistent with MgPEP being the true PEPC substrate, fPEP an allosteric activator, and fMg(2+) a weak, non-competitive inhibitor, thus confirming the kinetic mechanism determined previously at low concentrations of PEP and Mg(2+), and indicating that apparent substrate inhibition by MgPEP in maize leaf PEPC is caused by inhibition by high magnesium and ionic strength.     

The use of gel filtration to follow conformational changes in proteins. Conformational flexibility of bovine myelin basic protein.

The hydrodynamic behavior of bovine myelin basic protein was studied by gel filtration through Sephadex G-100 under conditions which included variations in pH from 2 to 12, variations in ionic strength from 0.01 to 1.5 M at pH 2 and from 0.1 to 2 M at pH 7, and variations in guanidinium chloride concentration from 0 to 6 M. A number of well characterized compact globular proteins were subjected to the same conditions for comparison. Compact globular proteins showed major conformational transitions due to acid, alkali, and guanidinium chloride denaturation and, possibly, minor transitions as well. Myelin basic protein behaved like a flexible linear polyelectrolyte, expanding continuously between pH 11 and pH 2 to 3 at ionic strength 0.1 M and contracting continuously with increase in ionic strength at pH 2 and at pH 7 to the point of salting-out. Relatively low concentrations of guanidinium chloride (approximately 0.5 M) were sufficient to cause the basic protein to expand. With increasing concentration of the denaturant the molecule continued to expand, but in a noncooperative manner. These results demonstrated the lack of significant intramolecular stabilization in the protein.     

Kinetic mechanism of NAD:malic enzyme from Ascaris suum in the direction of reductive carboxylation

Initial velocity studies in the absence and presence of product and dead-end inhibitors suggest a steady-state random mechanism for malic enzyme in the direction of reductive carboxylation of pyruvate. For this quadreactant enzymatic reaction (Mn2+ is a pseudoreactant), initial velocity patterns were obtained under conditions in which two substrates were maintained at saturating concentrations while one reactant was varied at several fixed concentrations of the other. Data from the resulting reciprocal plots, analyzed in terms of a bireactant mechanism, are consistent with a sequential mechanism with an obligatory order of addition of metal prior to pyruvate. NAD is competitive against NADH whether pyruvate and CO2 are maintained at low or high concentrations, whereas it is noncompetitive against pyruvate and CO2. Thio-NADH, alpha-ketobutyrate, and nitrite were used as dead-end analogs of NADH, pyruvate, and CO2, respectively. Thio-NADH is competitive against NADH, whereas it is noncompetitive against pyruvate and CO2, in accordance with a random mechanism. alpha-Ketobutyrate and nitrite gave noncompetitive inhibition against all substrates. The noncompetitive patterns observed for alpha-ketobutyrate versus pyruvate and nitrite versus CO2 suggest binding of the inhibitor to both the E.Mn.NADH and E.Mn.NAD complexes. Primary deuterium isotope effects are equal on all kinetic parameters, in agreement with the random mechanism, and suggest equal off-rates for NAD from E.Mn.NAD as well as pyruvate and NADH from E.Mn.NADH.pyruvate. Data are consistent with an overall symmetry in the malic enzyme reaction in the two reaction directions with a requirement for metal bound prior to pyruvate and malate.     

On the NADPH dependent reaction of cytoplasmic sn-glycerol-3-phosphate dehydrogenase

Cytoplasmic sn-glycerol-3-phosphate dehydrogenase (EC. 1.1.1.8.) can reduce dihydroxy acetone phosphate with NADPH as coenzyme under in vitro conditions, in solutions of low ionic strengths, at pH values lower than 7. The reaction is inhibited by phosphoenolpyruvate, NAD, ATP, ADP and Pi. In the cell this reaction can occur apparently only in case of specific metabolic conditions, i.e. when the local pH is low and the enzyme is protected from the inhibition by the above listed metabolites.     

Inhibition of Sucrose:Sucrose Fructosyl Transferase by Cations and Ionic Strength

Fructans are storage carbohydrates found in many temperate grasses. The first enzyme in the biosynthetic pathway of most fructans is sucrose:sucrose fructosyl transferase (SST). In this report, we demonstrate that K+ and ionic strength noncompetitively inhibit the activity of SST from wheat (Triticum aestivum L.) stems. The Ki for this inhibition is high, 122 mM, but in the range of concentrations of K+ found in the tissue (205-314 mM). Addition of KCl to the assay system had no effect on the pH optimum (5.5) or the Km for sucrose (266 mM) but reduced the Vmax. At equivalent ionic strengths, inhibition by choline chloride was about half that of KCl, indicating that inhibition by ionic strength might be responsible for approximately 50% of the KCl inhibition. Inhibition by LiCl and (NH4)2SO4 was similar to that by choline chloride. Soluble invertase activity found in the SST preparations was less sensitive to KCl and more sensitive to choline chloride than was SST. SST from barley (Hordeum vulgare L.) stems and leaves, as well as SST from leaves of orchardgrass (Dactylis glomerata), was also inhibited by KCl. SST from onion (Allium cepa L.) bulbs and asparagus (Asparagus officinalis L.) stems was not inhibited by KCl; thus, inhibition of activity by KCl is not a universal characteristic of SST from all sources.     

Mutagen Stability of Alkylation-Sensitive Mutants of Bacillus subtilis

The phosphotransacetylase of Veillonella alcalescens catalyzes a reversible reaction with Michaelis-Menten kinetics for all substrates. The rate of the reverse reaction (the synthesis of acetyl coenzyme A from acetyl phosphate) was 6.5 times greater than the rate of the forward reaction (the synthesis of acetyl phosphate from acetyl coenzyme A). The apparent K(m) values determined for the forward reaction were 8.6 x 10(-6)m for acetyl coenzyme A and 9.3 x 10(-3)m for phosphate. In the reverse reaction, the K(m) values were 3.3 x 10(-4)m for coenzyme A and 5.9 x 10(-4)m for acetyl phosphate. The results of an analysis of the inhibition by end products in the forward and reverse directions were compatible with a random bi- bi- mechanism. The enzyme was inhibited by adenosine triphosphate and adenosine diphosphate but was not affected by reduced nicotinamide adenine dinucleotide or pyruvate. The inhibition by adenosine triphosphate was noncompetitive with respect to acetyl phosphate and competitive with respect to coenzyme A. MgCl(2) reversed the inhibition by adenosine triphosphate or adenosine diphosphate. The role of Mg(2+) and adenylates in the regulation of phosphotranscetylase activity is discussed.     

Salt-dependent and protein-concentration-dependent changes in the solution structure of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis.

The solution structure of the histone-like DNA-binding protein, HBsu, from Bacillus subtilis in 2 mM sodium cacodylate, pH 7.5, is sensitive to the ionic strength of the buffer. This was shown by circular dichroism measurements at different concentrations of sodium chloride and potassium fluoride. The stability of HBsu is also influenced; at HBsu concentrations of about 0.1 mg.ml-1, melting temperatures of 32 degrees C and 55 degrees C were found in the absence of potassium fluoride and in the presence of 0.5 M potassium fluoride, respectively, exhibiting drastic ionic-strength-dependent differences in the temperature-induced unfolding of HBsu. Furthermore, at low ionic strength, circular dichroism spectra vary markedly depending on the HBsu concentration in the approximate range 0.2-3 mg.ml-1. Such protein-concentration-dependent differences in the spectra were not observed in the presence of 0.5 M potassium fluoride. Very similar circular dichroism spectra of HBsu and the histone-like DNA-binding protein of Bacillus stearothermophilus (HBst) at high ionic strength, indicate comparable structures of both proteins under these conditions. Estimation of the secondary structure content from the circular dichroism spectra yields data which are in satisfactory agreement with the values obtained from the crystal structure of HBst. Transition temperatures of 45 degrees C and 61 degrees C were found in differential scanning calorimetric measurements performed with HBsu in potassium-fluoride-free buffer and in the presence of 0.5 M potassium fluoride, respectively. The thermodynamic data point to the melting of native HBsu dimers into two denatured monomers.     

Equilibrium constants under physiological conditions for the reactions of choline kinase and the hydrolysis of phosphorylcholine to choline and inorganic phosphate.

The observed equilibrium constants (Kobs) of the P-choline hydrolysis reaction have been determined under physiological conditions of temperature (38 degrees) and ionic strength (0.25 M) and physiological ranges of pH and free [Mg2+]. Using sigma and square brackets to indicate total concentrations: (see article.) The value of Kobs has been found to be relatively insensitive to variations in pH and free [Mg2+]. At pH 7.0 and taking the standard state of liquid water to have unit activity ([H2O] = 1), Kobs = 26.6 M at free [Mg2+] = 0 [epsilon G0obs = -2.03 kcal/mol(-8.48 kJ/mol)], 26.8 M at free [Mg2+] = 10(-3) M, and 28.4 M at free [Mg2+] = 10(-2) M. At pH 8.0, Kobs = 18.8 M at free [Mg2+] = 0, 19.2 M at free [Mg2+] = 10(-3), and 22.2 M at free [Mg2+] = 10(-2) M. These values apply only to situations where choline and Pi concentrations are both relatively low (such as the conditions found in most tissues). At higher concentrations of phosphate and choline, the value of Kobs becomes significantly increased since HPO42- complexes choline weakly (association constant = 3.3 M-1). The value of K at 38 degrees and I = 0.25 M is calculated to be 16.4 +/- 0.3 M [epsilonG0 = 1.73 kcal/mol (-7.23 kJ/mol)]. The K for the P-choline hydrolysis reaction has been combined with the K for the ATP hydrolysis reaction determined previously under physiological conditions to calculate a value of 4.95 X 10(-3 M [deltaG0 j.28 kcal/mol (13.7 kJ/mol] for the K of the choline kinase reaction (EC 2.7.1.32), an important step in phospholipid metabolism: (see article.) Likewise, values for Kobs for the choline kinase reaction at 38 degrees, pH 7.0, and I = 0.25 M have been calculated to be 5.76 X 10(4) [deltaG0OBS = -6.77 KCAL/MOL (-28.3 KJ/mol)] at [Mg2+] = 0; 1.24 X 10(4) [deltaG0obs = -5.82 kcal/mol (-24.4 kJ/mol)] at [Mg2+] = 10(-3) M and 8.05 X 10(3) [delta G0obs = -5.56 kcal/mol (-23.3 kJ/mol)] at [Mg2+ = 10(-2) M. Attempts to determine the Kobs of the choline kinase reaction directly were unsuccessful because of the high value of the constant. The results indicate that in contrast to the high deltaG0obs for the hydrolysis of the ester bond of acetylcholine, the deltaG0obs for the hydrolysis of the ester bond of P-choline is quite low, among the lowest known for phosphate ester bonds of biological interest.     

Heparin and ionic strength-dependent conversion of antithrombin III from an inhibitor to a substrate of alpha-thrombin

The stoichiometry of antithrombin III (AT) inhibition of alpha-thrombin (T) has been investigated in the presence and absence of heparin as a function of ionic strength by quantitative titration of enzyme active sites. In contrast to the ionic strength-independent stoichiometry of 1.0 mol of AT/mol of T observed in the absence of heparin, the presence of high-affinity heparin (HAH) resulted in an ionic strength-dependent increase in the apparent stoichiometry of inhibition from a molar ratio of 1.1 AT/T at an ionic strength of 0.3 to 9.8 mol of AT/T when the ionic strength was lowered to 0.01. Reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the reaction products revealed that the increased AT/T stoichiometry was due to preferential formation of a specific proteolytically cleaved form of AT that was indistinguishable from the previously characterized reactive site-cleaved AT (ATM). Using high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to quantitate ATM, the cleaved inhibitor was shown to be formed rapidly and concomitant with the stable thrombin-antithrombin complex (TAT) and quantitatively accounted for the apparent increase in reaction stoichiometry at low ionic strength in the presence of HAH. The levels of HAH required to produce maximum ATM were catalytic at mu greater than or equal to 0.15, but became stoichiometric as the ionic strength decreased below 0.1. Substantially less ATM was produced in the presence of low-affinity heparin, while a low molecular weight HAH, virtually inactive in accelerating T inhibition by AT, was unable to promote significant ATM formation. These results indicate competition between substrate and inhibition reactions of AT with T which are affected by an ionic strength-dependent heparin interaction. A reaction mechanism accounting for these observations is proposed.     

How much is secondary structure responsible for resistance of double-stranded RNA to pancreatic ribonuclease A?

1. Double-stranded f2 sus11 or Qbeta RNAs, resistant to bovine pancreatic RNAase A in 0.15 M NaCl/0.015 M sodium citrate (SSC), are quickly and completely degraded at 10-fold lower ionic strength (0.1 X SSC) under otherwise similar conditions. At this ionic strength the secondary structure of double-stranded RNA is maintained, as judged by the following: (a) the unchanged resistance of double-stranded RNA and DNA, under similar low ionic strength conditions, to nuclease S1 from Aspergillus oryzae, in contrast with the sensitivity of the corresponding denatured nucleic acids to this enzyme, specific for single-stranded RNA and DNA; (b) the co-operative pattern of the thermal-transition profile of double-stranded RNA (with a Tm of 89 degrees C) in 0.1 X SSC. 2. Whereas in SSC bovine seminal RNAase (RNAase BS-1) and whale pancreatic RNAase show an activity on double-stranded RNA significantly higher than that of RNAase A, in 0.1 X SSC the activity of the latter enzyme on this substrate becomes distinctly higher than that of RNAase BS-1, and similar to that of whale RNAase. 3. From these results it is deduced that the secondary structure is probably not the only nor the most important variable in determining the susceptibility double-stranded RNA to ribonuclease. Other factors, such as the effect of ionic strength on the enzyme and/or the binding of enzyme to nucleic acids, may play an important role in the process of double-stranded RNA degradation by ribonucleases specific for single-stranded 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.     

Role of ions in the regulation of porcine lactate dehydrogenase.

Different ions affect the H4 and M4 isoenzymes of porcine lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) in the same way, inhibiting the enzyme at low pyruvate concentrations, whereas at high pyruvate concentrations, the activities were enhanced. The inhibition was competitive with regard to pyruvate and NADH. The enhancement of the enzyme activity at high pyruvate concentration is due to the increase in the Km value for pyruvate, implying that higher substrate concentrations are needed to obtain substrate inhibition. Sulphate behaved differently from the other ions. It inhibited in a noncompetitive manner with regard to pyruvate and did not activate the enzyme at high pryvuate concentration. The effect of ions increased with the size of the anion. The ionic strength was of less importance.     

Effect of humic acids on the inhibition of pea choline esterase and choline acyltransferase with malathion

Humic acids in a concentration of up to 100 μg ml^-1 affect the activity of choline esterase of pea only weakly during sinapin (choline ester of sinapic acid) hydrolysis. The inhibition is stronger at higher concentrations. A similar course of inhibition with humic acids was also observed during the synthesis of sinapin catalysed with choline acyltransferase present in the same enzyme extract. The organophosphate malathion is a strong inhibitor of both the hydrolysis and the synthesis of sinapin. Thus, for example, at a 3.5 × 10^-5 M concentration it causes 85% inhibition of pea choline esterase. However, in combination with 100 μg of humic acids this inhibition decreases to about 8%. It was observed that humic acids prevent the inhibition of both the hydrolysis and the synthesis of sinapin with malathion even at low concentrations. The interaction of the organophosphate malathion with humic acids seems specific. Humic acids do not affect the inhibition of choline esterase with physostigmine or the inhibition of the hydrolysis and the synthesis of sinapin with chlorocholine chloride, alar and hexamine either. This is also true of the activation of the processes with the K⁺ ions. Humic acids represent competitive inhibitors of choline esterase. Malathion shows an acompetitive — noncompetitive character of the inhibition. The interaction of humic acids with malathion in the roots of the plants may also be assumed in the soil when malathion is applied as an insecticide.