ATP sulfurylase from Penicillium chrysogenum. Molecular basis of the sigmoidal velocity curves induced by sulfhydryl group modification

ATP sulfurylase from Penicillium chrysogenum is a noncooperative homooligomer containing three free sulfhydryl groups per subunit. Under nondenaturing conditions, one SH group per subunit was modified by 5,5'-dithiobis-(2-nitrobenzoate), or N-ethylmaleimide. Modification had only a small effect on kcat, but markedly increased the [S]0.5 values for the substrates, MgATP and SO4(2-). MgATP and adenosine-5'-phosphosulfate protected against modification. The SH-modified enzyme displayed sigmoidal velocity curves for both substrates with Hill coefficients (nH) of 2. Fluorosulfonate (FSO3-) and other dead-end inhibitors competitive with SO4(2-) activated the SH-modified enzyme at low SO4(2-) concentration. In order to determine whether the sigmoidicity resulted from true cooperative binding (as opposed to a kinetically based mechanism), the shapes of the binding curves were established from the degree of protection provided by a ligand against phenylglyoxal-dependent irreversible inactivation under noncatalytic conditions. Under standard conditions (0.05 M Na-N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid buffer, pH 8, 30 degrees C, and 3mM phenylglyoxal) the native enzyme was inactivated with a k of 2.67 +/- 0.25 X 10-3 s-1, whereas k for the SH-modified enzyme was 5.44 +/- 0.27 X 10-3 s-1. The increased sensitivity of the modified enzyme resulted from increased reactivity of ligand-protectable groups. Both the native and the SH-modified enzyme displayed hyperbolic plots of delta k (i.e. protection) versus [MgATP], or [FSO3-], or [S2O3(2-]) in the absence of coligand (nH = 0.98 +/- 0.06). The plots of delta k versus [ligand] for the native enzyme were also hyperbolic in the presence of a fixed concentration of coligand. However, in the presence of a fixed [FSO3-] or [S2O3(2-]), the delta k versus [MgATP] plot for the SH-modified enzyme was sigmoidal, as was the plot of delta k versus [FSO3-] or [S2O3(2-]) in the presence of a fixed [MgATP]. The nH values were 1.92 +/- 0.09. The results indicate that substrates (or analogs) bind hyperbolically to unoccupied SH-modified subunits, but in a subunit-cooperative fashion to form a ternary complex.     

A novel method to differentiate between ping-pong and simultaneous exchange kinetics and its application to the anion exchanger of the HL60 cell.

We have developed a new test to differentiate between ping-pong and simultaneous mechanisms for tightly coupled anion exchange. This test requires the use of a dead-end reversible noncompetitive inhibitor. As an example, we have applied the test to the anion exchanger of the HL60 cell using the salicylic acid derivative 3,5-diiodosalicylic acid (DIS), which reversibly inhibits HL60 cell Cl/Cl exchange. The concentration of DIS that causes 50% inhibition (ID50) increased only slightly as either intra- or extracellular chloride was increased, indicating that DIS inhibits HL60 anion exchange in a noncompetitive manner. In agreement with this observation, plots of the slope of the Dixon plot as a function of 1/[Clo] or 1/[Cli] were fit with straight lines with nonzero intercepts, indicating that DIS does not compete with either of the substrates ([Clo] and [Cli]). The secondary Dixon slope test is based on the fact that, for a dead-end inhibitor such as DIS, the slope of the Dixon plot slope vs. 1/[Cli] (secondary Dixon slope or SDS) is independent of extracellular Cl when the exchange mechanism follows ping-pong kinetics. Similarly, the SDS calculated from a plot as a function of 1/[Clo] is also independent of intracellular Cl for a ping-pong exchanger. In contrast to this prediction, we found that for DIS inhibition of Cl/Cl exchange in HL60 cells the slope of the Dixon plot slope vs. 1/[Cli] decreased by a factor of 2.5-fold when [Clo] was increased from 1 to 11 mM (P < 0.0001). This change in the SDS rules out ping-pong kinetics, but is consistent with a simultaneous model of Cl/Cl exchange in which there are extra- and intracellular anion binding sites, both of which must be occupied by suitable anions in order to allow simultaneous exchange of the ions.     

Interrelationships among quinidine, amiloride, and lithium as inhibitors of the renal Na+-H+ exchanger

We examined the effects of quinidine, amiloride and Li+ on the kinetics of Na+-H+ exchange in microvillus membrane vesicles isolated from the rabbit renal cortex. Quinidine reversibly inhibited the initial rate of Na+-H+ exchange (I50 200 microM). The plot of 1/V versus [quinidine] was curvilinear, with Hill coefficient greater than 1.0, indicating that the drug interacts at two or more inhibitory sites or at a single site on at least two different conformations of the transporter. Quinidine decreased the Vmax for Na+-H+ exchange and increased the Km for Na+, indicating a mixed-type mechanism of inhibition. In contrast, plots of 1/V versus [amiloride] and 1/V versus [Li+] were linear, indicating single inhibitory sites; amiloride and Li+ each increased the Km for Na+ with no effect on Vmax, indicating a competitive mechanism of inhibition. Addition of Li+ increased the intercept with no change in slope of the 1/V versus [amiloride] plot, indicating that Li+ and amiloride are mutually exclusive inhibitors of Na+-H+ exchange. Addition of quinidine increased the slopes of the plots of 1/V versus [amiloride] and 1/V versus [Li+], indicating that the binding of quinidine is not mutually exclusive with the binding of amiloride and Li+. Results from this and previous studies are consistent with the concept that the inhibitor amiloride and the transportable substrates Na+, H+, Li+, and NH+4 all mutually compete for binding to a single site, the external transport site of the renal Na+-H+ exchanger. However, our findings indicate that quinidine interacts with the Na+-H+ exchanger on at least one additional site that is not shared by Na+, Li+, or amiloride.     

Changes in pH associated with clotting of fibrinogen. Kinetic studies of the pH shift and correlation of the pH change with the release of fibrinopeptides and the ensuing polymerization

The effect of the initial pH and the concentrations of thrombin, fibrinogen, and Ca2+ upon the rate of pH change associated with clotting of bovine fibrinogen by human thrombin was investigated at pH 6.80, 7.80, and 8.80, 0.3 ionic strength, 25 degrees C, and 19.5 mg/mL final fibrinogen concentration. At pH 6.80 and 7.80, the reaction was first order, with rate constant k1. At pH 8.80, a first-order reaction of the release of H+ (k1) was followed by a partial rebinding of these in a reaction consecutive to the first one (k2). At each of the above pH values, k1 was proportional to thrombin concentration in the 0.05-3.0 min-1 range investigated. The k1 constants were 0.111 +/- 0.001, 0.250 +/- 0.005, and 0.190 +/- 0.002 min-1 (NIH thrombin units)-1 mL-1 at pH 6.80, 7.80, and 8.80, respectively. Plots of log rate vs log thrombin concentration of these data were linear with slopes close to 1 at all three pH values. The rate of the second reaction (k2) was independent of both the thrombin and the initial fibrinogen concentration. The pH dependence of k1 exhibited a bell-shaped curve that could be resolved into the effect of one group with a pK of 7.27 that increased the rate and another with a pK of 9.22 that decreased the rate. With constant thrombin concentration but varying fibrinogen concentration, plots of 1/k1 vs [fibrinogen] were linear, but the lines did not pass through the origin. From the slope and intercept, kcat and KM of the Michaelis-Menten equation could be calculated. The same parameters were obtained also from initial velocity vs [fibrinogen] plots. Values of kcat were consistent and accurate; those of KM were more scattered. KM was (22.4-34.2) X 10(-6) M at pH 6.80 and approximately 7 X 10(-6) M in the pH 7.26-8.80 range. The latter value, pertaining to the release of H+ ions, is in agreement with values in the literature for KM of the release of fibrinopeptide A by thrombin in the 7.4-8.0 pH range. The value of kcat s-1 (unit of thrombin)-1 mL-1 increases from 1.2 X 10(-10) s-1 unit of thrombin-1 mL-1 at pH 6.80 to 2.46 X 10(-10) at pH 7.80 and then decreases to 2.01 X 10(-10) 10(-1) (units of thrombin)-1 mL-1 at pH 8.80. The kcat values are significantly lower than those in the literature for the release of fibrinopeptide A.(ABSTRACT TRUNCATED AT 400 WORDS)     

A rate determining step change in the pre-steady state of acetylcholinesterase inhibitions by 1,n-alkane-di-N-butylcarbamates

Alkane-1-N-butylcarbamate-n-ols (1-7) and 1,n-alkane-di-N-butylcarbamates (8-14) are potent pseudo-substrate inhibitors of acetylcholinesterase. For inhibitors 1-7, the pre-steady state -logK(s) values and steady state -logK(i), values are linearly correlated with the tether length (N). However, for inhibitors 8-14, correlation of the -logK(s) or -logK(i) values against N deviates from linearity. A discontinuity of the -logK(s) versus N plot, concave downwards, is indicative of a rate determining step change in the pre-steady state of acetylcholinesterase inhibitions by inhibitors 8-14.     

Kinetic studies of four enzyme forms of beta-N-acetylhexosaminidase from embryonic chicken brain

The separation of four different hexosaminidase forms from embryonic chicken brain (16-day-old) has been performed by ion-exchange chromatography. Two different DEAE-cellulose columns have been used: a first one at pH 7.2 and a second one at pH 6.0. Km and Vmax values were estimated from the Lineweaver-Burk or Dixon plots and ki from the Dixon plots, using N-acetyl-D-glucosamine or N-acetyl-D-galactosamine as inhibitors. In both cases we found a kind of competitive inhibition in which Lineweaver-Burk and Dixon plots curve downwards.     

Irreversibility of the interaction of human growth hormone with its receptor and analysis of irreversible reactions in radioreceptor assays—Theoretical considerations

Kinetic studies of the binding and dissociation of [¹²⁵I]-human growth hormone to rabbit liver and mammary gland membrane receptors have showed that the binding of [¹²⁵I]-human growth hormone was largely irreversible to liver membrane receptors and completely to the solubilised mammary gland receptor. As Scatchard analysis assumes complete reversibility of the hormone-receptor interaction the validity of estimates of affinity and capacity of receptors derived by this analysis may be questionable. Theoretical considerations show that in unimolecular irreversible interactions of hormone and receptor, a nonlinear (concave) or a linear Scatchard plot can be obtained. In linear Scatchard plots the capacity of the receptor obtained by extrapolation represents an overestimation of true capacity. This overestimation correlates with the value of the intercept in the Scatchard plot.     

Adenine nucleotides regulate the functional transition in mitochondrial H+-ATPase and the kinetic behaviour of its ATP-synthetase form

The kinetics of the SMP-catalyzed Pi-ATP exchange and oxidative phosphorylation was studied at variable [MgATP] + + [MgADP] and [MgATP]/[MgADP]. The existence on F1 of a center with a low affinity was demonstrated (KM = 0.4-2.7 mM). Saturation of this center with the Mg2+-complex of one of the nucleotides is obligatory for H+-ATPase to exhibit its ATP synthetase activity. It was found that with a decrease of [MgATP]/[MgADP] the lag periods, tau, of the reactions and KM(Pi) also show a decrease. Besides, in the Pi-ATP exchange reactions delta microH+ (steady-state) diminishes and SMP coupling is enhanced (the Vhydr/Vsynth ratio is decreased). Preincubation of SMP with MgADP eliminates the lags but does not affect the course of the steady-state reaction. It is concluded that F1 when bound to MgATP or MgADP changes to a "more" or "less coupled" conformational state, thus determining the rate of conversion to the ATP-synthetase functional state (ko = tau-1), the threshold potential of this conversion and the kinetic behaviour of ATP-synthetase (KM for Pi).     

Importance of coulombic end effects on cation accumulation near oligoelectrolyte B-DNA: a demonstration using 23Na NMR.

The local cation concentration at the surface of oligomeric or polymeric B-DNA is expected, on the basis of MC simulations (Olmsted, M. C., C. F. Anderson, and M. T. Record, Jr. 1989. Proc. Natl. Acad. Sci. USA. 86:7766-7770), to decrease sharply as either end of the molecule is approached. In this paper we report 23Na NMR measurements indicating the importance of this "coulombic" end effect on the average extent of association of Na+ with oligomeric duplex DNA. In solutions containing either 20-bp synthetic DNA or 160-bp mononucleosomal calf thymus DNA at phosphate monomer concentrations [P] of 4-10 mM, measurements were made over the range of ratios 1 < or = [Na]/[LP] < or = 20, corresponding to Na+ concentrations of 4-200 nM. The longitudinal 23Na NMR relaxation rates measured in these NaDNA solutions, Robs, are interpreted as population-weighted averages of contributions from "bound" (RB) and "free" (RF) 23Na relaxation rates. The observed enhancements of Robs indicate that RB significantly exceeds RF, which is approximately equal to the 23Na relaxation rate in an aqueous solution containing only NaCl. Under salt-fre-tconditions ([Na]/[P] = 1), where the enhancement in Robs is maximal, we find that Robs--RF in the solution containing 160-bp DNA is approximately 1.8 times that observed for the 20-bp DNA. For the 160-bp oligomer (which theoretical calculations predict to be effectively polyion-like), we find that a plot of Robs v. [P]/[Na] is linear, as observed previously for sonicated (approximately 700 bp) DNA samples. For the 20-bp oligonucleotide this plot exhibits a marked departure from linearity that can be fitted to a quadratic function of [P]/[Na]. Monte Carlo simulations based on a simplified model are capable of reproducing the qualitative trends in the 23Na NMR measurements analyzed here. In particular, the dependences of Robs--RF on DNA charge magnitude of Z(320 vs. 38 phosphates) and (for the 20-bp oligomer) on [Na]/[P] are well correlated with the calculated average surface concentration of Na+. Thus, effects of sodium concentration on RB appear to be of secondary importance. We conclude that 23Na NMR relaxation measurements are a sensitive probe of the effects of oligomer charge on the extent of ion accumulation near B-DNA oligonucleotides, as a function of [Na] and [P].     

Kinetics of suicide substrates. Practical procedures for determining parameters.

Many clinically important or mechanistically interesting inhibitors react with enzymes by a branched pathway in which inactivation of the enzyme and formation of product are competing reactions. The steady-state kinetics for this pathway [Waley (1980) Biochem. J. 185, 771-773] gave equations for progress curves that were cumbersome. A convenient linear plot is now described. The time (t1/2) for 50% inactivation of the enzyme (this is also the time for 50% formation of product), or for 50% loss of substrate, is measured in a series of experiments in which the concentration of inhibitor, [I]0, is varied; in these experiments the ratio of the concentration of enzyme to the concentration of inhibitor is kept fixed. Then a plot of [I]0 X t1/2 against [I]0 is linear, and the kinetic parameters can be found from the slope and intercept. Furthermore, simplifications of the equations for progress curves are described that are valid when the concentration of inhibitors is high, or is low, or when the extent of reaction is low. The use of simulated data has shown that the recommended methods are not unduly sensitive to experimental error.     

Determination of the dissociation constants of pea diamine oxidase.

The activity of diamine oxidase [EC 1.4.3.6] (DAO) isolated from pea cotyledons was measured in Britton-Robinson buffers at pH range 5.0-9.6 by spectrophotometric method with E-1,4-diamino-2-butene as substrate. The enzyme has the highest activity at pH = 7.7 and in pH greater than 8.0 it is irreversible denaturated with time. The dissociation constants of the enzyme and enzyme-substrate complex were calculated by Dixon's method from plots of log Vmax, log KM and log Vmax/KM against pH. The pKEA = 6.5 suggests that histidine is in active site of DAO.     

The kinetics of intact microsome glucose-6-phosphatase are sigmoid at physiologic glucose 6-phosphate concentrations

The kinetics of rat liver glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) were studied with intact and detergent-disrupted microsomes from normal and diabetic rats. Glucose-6-P concentrations employed (12 microM to 1.0 mM) spanned the physiologic range. With the enzyme of intact microsomes from both groups, plots of v versus [glucose-6-P] were sigmoid. Hanes plots (i.e. [glucose-6-P]/v versus [glucose-6-P]) were biphasic (concave upwards). A Hill coefficient of 1.45 was determined with substrate concentrations between 12 and 133 microM. Disruption of microsomal integrity abolished these departures from classic kinetic behavior, indicating that sigmoidicity may result from cooperative interaction of glucose-6-P with the glucose-6-phosphatase system at the substrate translocase specific for glucose-6-P. With the enzyme from normal rats the [glucose-6-P] at which the enzyme was maximally sensitive to variations in [glucose-6-P] (which we term "Smax"), determined from plots of dv/d [glucose-6-P] versus [glucose-6-P], was in the physiologic range. The Smax of 0.13 mM corresponded well with the normal steady-state hepatic [glucose-6-P] of 0.16 mM, consistent with glucose-6-phosphatase's function as a regulatory enzyme. With the diabetic enzyme, in contrast, values were 0.30 and 0.07 mM for the Smax and steady-state level, respectively. We suggest that the decreasing sensitivity of glucose-6-phosphatase activity to progressively diminishing glucose-6-P concentration, inherent in its sigmoid kinetics, constitutes a mechanism for the preservation of a residual pool of glucose-6-P for other hepatic metabolic functions in the presence of elevated concentrations of glucose-6-phosphatase such as in diabetes.     

Sodium fluxes in human fibroblasts: kinetics of serum-dependent and serum-independent pathways

Sodium influx in serum-deprived human fibroblasts is by way of a pathway which shows saturation kinetics. A plot of initial Na influx versus [Na]0 ([Na]i approximately equal to 10 mM) gives a simple Michaelis-Menten type of curve with a K1/2 = 70.0 +/- 8.1 mM and a Vmax = 14.5 +/- 1.9 mumol/g prot/min. A similar plot of initial Na influx versus [Na]0 in the presence of 10% fetal bovine serum (FBS) gives a nonsaturating curvilinear response which appears to be biphasic. A plot of the serum-dependent Na influx versus [Na]0 (obtained by subtracting the curve in the absence of FBS from the curve in the presence of 10% FBS) shows that there is a linear relationship between serum-induced Na influx and external [Na]. At physiological Na concentrations, in the presence of FBS, the serum-induced Na influx is equal to the amiloride-sensitive Na flux, whereas in the absence of serum amiloride inhibits less than 10% of the Na influx. The effect of intracellular Na on Na flux was tested by preloading cells with Na in a digitoxin-containing medium prior to measurement of Na flux. A plot of steady-state Na exchange flux versus [Na]0 ([Na]i approximately equal to [Na]0) in the absence of serum gives a curve that appears to saturate at approximately 100 mM Na (flux = 100 mumol/g prot/min) and then declines with increasing [Na] (flux = 40 mumol/g prot/min at 150 mM). In contrast to Na influx in control serum-deprived cells, Na flux in Na-loaded cells in dramatically inhibited by the presence of amiloride. Since the peak Na exchange flux of 100 mumol/g prot/min is greatly in excess of the Vmax for Na influx in control serum-deprived cells and the enhanced Na flux is amiloride-sensitive, elevating intracellular Na must somehow activate the amiloride-sensitive Na transport system, which is normally only minimally active in the absence of serum.     

Initial rates. A new plot.

Excellent estimations of initial rates can be obtained from plots of delta P/t versus product formed (where P is the instantaneous concentration of the product). delta P/t is the chord from P0,t0 to P,t on an ordinary P-versus-t plot. When the chord is plotted as a function of product, the intercept at P0 of the resulting curve is necessarily dP/dt0. This curve approximates to a straight line extremely closely in all cases tested thus far. If delta P/t versus product is calculated from the integrated rate equation for a first-order reaction, and if a straight line is fitted through points representing the first 50% of the reaction, the discrepancy between the true initial rate and dP/dt0 estimated from the plot is 0.68%. For the most common form of the integrated rate equation for catalysed reactions the discrepancy varies between 0 and 0.90%. Because of the complexities of the integrated rate equations, catalysed second-order reactions have not been evaluated directly; uncatalysed reactions have been done instead. For a reaction with one reactant and two products, the discrepancy varies from 0.68 to 2.02%. For two reactants and one product, it varies from 0 to 0.68%; for two and two, 0 to 2.02%. The larger discrepancies occur only when unfavourable equilibrium constants are being overcome by the initial conditions.     

Clotting of bovine fibrinogen. Kinetic analysis of the release of fibrinopeptides by thrombin and of the calcium uptake upon clotting at high fibrinogen concentrations

Kinetic data on the release of fibrinopeptides A and B from bovine fibrinogen by human thrombin were obtained at high fibrinogen concentrations, within the 0.8-8.8% range (0.227 X 10(-4) to 2.60 X 10(-4) M), at 25 degrees C, pH 7.26, 0.30 ionic strength, and 10(-4) M free Ca2+ concentration. Release of fibrinopeptide A followed strictly first-order kinetics at all concentrations, in spite of the fact that the highest concentration was 26 times larger than the value of KM found in the literature. This behavior can be explained by inhibition of thrombin by the reaction products, with KI = KM. The equation describing the course of the reaction under these conditions can be rearranged into a linear relationship between 1/kobsd and substrate concentration. The slope of the line is equal to 1/kcat and the intercept to KM/kcat. The data points fell accurately on a straight line, and with the parameters of the latter, kcat and KM were calculated as (6.3 +/- 0.11) X 10(-10) M s-1 (unit of thrombin)-1 L-1 and (11.0 +/- 3.0) X 10(-6) M, respectively. These values agree well with those found in the literature. Release of fibrinopeptide B follows complex kinetics. Higgins et al. [Higgins, D. L., Lewis, S.D., & Shafer, J.A. (1983) J. Biol. Chem. 258, 9276-9282] suggested that it can be described as the result of two consecutive reactions, the first one being the release of fibrinopeptide A and the second one of fibrinopeptide B from those molecules that have already lost fibrinopeptide A in the previous step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of KCNQ2 subunits in [3H]dopamine release triggered by depolarization and pre-synaptic muscarinic receptor activation from rat striatal synaptosomes

KCNQ2 and KCNQ3 subunits encode for the muscarinic-regulated current (I(KM)), a sub-threshold voltage-dependent K+ current regulating neuronal excitability. In this study, we have investigated the involvement of I(KM) in dopamine (DA) release from rat striatal synaptosomes evoked by elevated extracellular K+ concentrations ([K+]e) and by muscarinic receptor activation. [3H]dopamine ([3H]DA) release triggered by 9 mmol/L [K+]e was inhibited by the I(KM) activator retigabine (0.01-30 micromol/L; Emax = 54.80 +/- 3.85%; IC50 = 0.50 +/- 0.36 micromol/L). The I(KM) blockers tetraethylammonium (0.1-3 mmol/L) and XE-991 (0.1-30 micromol/L) enhanced K+-evoked [3H]DA release and prevented retigabine-induced inhibition of depolarization-evoked [3H]DA release. Retigabine-induced inhibition of K+-evoked [3H]DA release was also abolished by synaptosomal entrapment of blocking anti-KCNQ2 polyclonal antibodies, an effect prevented by antibody pre-absorption with the KCNQ2 immunizing peptide. Furthermore, the cholinergic agonist oxotremorine (OXO) (1-300 micromol/L) potentiated 9 mmol/L [K+]e-evoked [3H]DA release (Emax = 155 +/- 9.50%; EC50 = 25 +/- 1.80 micromol/L). OXO (100 micromol/L)-induced [3H]DA release enhancement was competitively inhibited by pirenzepine (1-10 nmol/L) and abolished by the M3-preferring antagonist 4-diphenylacetoxy N-methylpiperidine methiodide (1 micromol/L), but was unaffected by the M1-selective antagonist MT-7 (10-100 nmol/L) or by Pertussis toxin (1.5-3 microg/mL), which uncouples M2- and M4-mediated responses. Finally, OXO-induced potentiation of depolarization-induced [3H]DA release was not additive to that produced by XE-991 (10 micromol/L), was unaffected by retigabine (10 micromol/L), and was abolished by synaptosomal entrapment of anti-KCNQ2 antibodies. Collectively, these findings indicate that, in rat striatal nerve endings, I(KM) channels containing KCNQ2 subunits regulate depolarization-induced DA release and that I(KM) suppression is involved in the reinforcement of depolarization-induced DA release triggered by the activation of pre-synaptic muscarinic heteroreceptors.     

Bacterial DNA supercoiling and [ATP]/[ADP] ratio: changes associated with salt shock.

When Escherichia coli K-12 was shifted from a medium lacking salt to one containing 0.5 M NaCl, both the [ATP]/[ADP] ratio and negative supercoiling of plasmid DNA increased within a few minutes. After about 10 min both declined, eventually reaching a level slightly above that observed with cells growing exponentially in the absence of salt. Since in vitro the [ATP]/[ADP] ratio influences the level of supercoiling generated by gyrase (H. Westerhoff, M. O'Dea, A. Maxwell, and M. Gellert, Cell Biophys. 12:157-181, 1988), the physiological response of supercoiling to salt shock is most easily explained by the sensitivity of gyrase to changes in the intracellular [ATP]/[ADP] ratio. This raises the possibility that the [ATP]/[ADP] ratio is an important factor in the control of supercoiling.     

Interaction of chlorpromazine with hemoglobin

Binding of chlorpromazine (CPZ), a widely used antidepressant tranquilizer, with hemoglobin has been studied by equilibrium dialysis method. r/Cf versus r plot was typically concave downwards revealing the positive cooperative nature of binding. Binding parameters, namely the affinity constant (K) and the degree of cooperativity (nH) were determined from the Hill plot. Oxygen was found to be released gradually from hemoglobin with gradual addition of CPZ, the extent of oxygen release depending on the stoichiometric ratio of CPZ: hemoglobin (D/P).     

Kinetic study of soybean beta-amylase. The effect of pH.

1) Beta-Amylase [EC 3.2.1.2] was prepared from defatted hawk eye soybean flour. The enzyme concentration dependence of the initial velocity for the hydrolytic reaction was investigated at pH 5.4 in the range of the enzyme concentration from 6.6 x 10(-10) M to 5.0 x 10(-6) M. It was found that the initial velocity was proportional to the enzyme concentration in this range. 2) The hydrolyses of maltodextrin (DPn = 74.4) and soluble starch catalyzed by soybean beta-amylase were investigated in the pH range from 3.0 to 9.1 at 25 degrees C, and the Michaelis constant, Km, and the maximum velocity, V, for each substrate were determined at each pH. The pH-rate profile showed a bell-shaped curve, and the pH "optimum" was at 5.85. From Dixon plots of V and V/Km, the pK values were found to be 3.5 and 8.2 for the free enzyme, and 3.5 and 8.5 for the enzyme-substrate complex. The pH-rate profile in the presence of 25% methanol (v/v) was also obtained at alkaline pH. The pKe values were the same as those in the absence of methanol. Based on these results, it was estimated that the ionizable acidic group was an amino group and the basic group was a carboxyl one.     

Characteristics of the Inhibition of Rat Brain Monoamine Oxidase In Vitro by MD780515

Abstract: The inhibition of type A and B MAO in rat forebrain crude membrane preparation by MD780515. (3-{4-[(3-cyanophenyl)methoxy]phenyl)-5-(methoxymethyl)-2-oxazolidinone Centre de Recherche Delalande, France) has been investigated in vitro with 5-hydroxytryptamine and β-phenylethyl-amine as substrates. The inhibition of the high-affinity binding of [³H]harmaline, a specific marker of type A MAO, was also studied. In the experimental conditions used, MD780515 appeared to be a pure mixed MAO inhibitor (MAOI) of 5-HT deamination, both K_m , and V_max being altered [K₁ (Dixon) = K_i , (slope) = 2 nM; K_i (intercept) = 12 nM]. Phenylethylamine oxidation could be considered to be noncompetitively inhibited by MD780515 (K_i (slope) = 78 nM; K_i , (intercept) = 103 nM). Dixon and intercept replots were hyperbolic, suggesting that, at high concentrations, PEA could be deaminated by both forms of MAO. This hypothesis was confirmed by biphasic inhibition curves of 80 μM-PEA obtained when MD7805 15 , clorgyline, harmaline and deprenyl were used as MAOIs. MD780515 was a potent inhibitor (IC₅₀= 1–2 nM) of [³H]harmaline binding. Comparatively, clorgyline, 'cold' harmaline and Lilly 51641 inhibited ³H ligand binding, with IC₅₀ of 5, 7 and 40 nM respectively. In conclusion, MD780515 is a reversible, specific and potent type A MAOI.