Kinetic analysis of oligodeoxyribonucleotide-directed triple-helix formation on DNA

Pyrimidine oligonucleotides recognize extended purine sequences in the major groove of double-helical DNA by triple-helix formation. The resulting local triple helices are relatively stable and can block DNA recognition by sequence-specific DNA binding proteins such as restriction endonucleases. Association and dissociation kinetics for the oligodeoxyribonucleotide 5'-CTCTTTCCTCTCTTTTTCCCC (bold C's indicate 5-methylcytosine residues) are now measured with a restriction endonuclease protection assay. When oligonucleotides are present in greater than 10-fold excess over the DNA target site, the binding reaction kinetics are pseudo first order in oligonucleotide concentration. Under our standard conditions (37 degrees C, 25 mM Tris-acetate, pH 6.8, 70 mM sodium chloride, 20 mM magnesium chloride, 0.4 mM spermine tetrahydrochloride, 10 mM beta-mercaptoethanol, 0.1 mg/mL bovine serum albumin) the value of the observed pseudo-first-order association rate constant, k2obs, is 1.8 x 10(3) +/- 1.9 x 10(2) L.(mol of oligomer-1.s-1. Measurement of the dissociation rate constant yields an equilibrium dissociation constant of approximately 10 nM. Increasing sodium ion concentration slightly decreased the association rate, substantially increased the dissociation rate, and thereby reduced the equilibrium binding constant. This effect was reversible by increasing multivalent cation concentration, confirming the significant role of multivalent cations in oligonucleotide-directed triple-helix formation under these conditions. Finally, a small reduction in association rate, a large increase in dissociation rate, and a resulting reduction in the equilibrium binding constant were observed upon increasing the pH between 6.8 and 7.2.     

Kinetic analysis of phasic inhibition of neuronal sodium currents by lidocaine and bupivacaine.

Phasic ("use-dependent") inhibition of sodium currents by the tertiary amine local anesthetics, lidocaine and bupivacaine, was observed in voltage-clamped node of Ranvier of the toad, Bufo marinus. Local anesthetics were assumed to inhibit sodium channels through occupation of a binding site with 1:1 stoichiometry. A three-parameter empirical model for state-dependent anesthetic binding to the Na channel is presented: this model includes two discrete parameters that represent the time integrals of binding and unbinding reactions during a depolarizing pulse, and one continuous parameter that represents the rate of unbinding of drug between pulses. The change in magnitude of peak sodium current during a train of depolarizing pulses to 0 mV was used as an assay of the extent of anesthetic binding at discrete intervals; estimates of model parameters were made by applying a nonlinear least-squares algorithm to the inhibition of currents obtained at two or more depolarizing pulse rates. Increasing the concentration of drug increased the rate of binding but had little or no effect on unbinding, as expected for a simple bimolecular reaction. The dependence of the model parameters on pulse duration was assessed for both drugs: as the duration of depolarizing pulses was increased, the fraction of channels binding drug during each pulse became significantly larger, whereas the fraction of occupied channels unbinding drug remained relatively constant. The rate of recovery from block between pulses was unaffected by pulse duration or magnitude. The separate contributions of open (O) and inactivated (I) channel binding of drug to the net increase in block per pulse were assessed at 0 mV: for lidocaine, the forward binding rate ko was 1.3 x 10(5) M-1 s-1, kl was 2.4 x 10(4) M-1 s-1; for bupivacaine, ko was 2.5 x 10(5) M-1 s-1, kl was 4.4 x 10(4) M-1 s-1. These binding rates were similar to those derived from time-dependent block of maintained Na currents in nodes where inactivation was incomplete due to treatment with chloramine-T. The dependence of model parameters on the potential between pulses (holding potential) was examined. All three parameters were found to be nearly independent of holding potential from -70 to -100 mV. These results are discussed with respect to established models of dynamic local anesthetic-Na channel interactions.     

Cyanide binding to canine myeloperoxidase

Equilibria and kinetics of cyanide binding to canine myeloperoxidase were studied. Spectral results support the presence of two heme binding sites; an isosbestic point at 444 nm and a linear Scatchard plot suggest that the binding affinity of cyanide to the two subunits of the enzyme is the same. The dissociation constant is 0.53 microM. The pH dependence of the apparent second order rate constant indicates the presence of an acid-base group on the enzyme with a pKa of 3.8 +/- 0.1. The protonated form of cyanide binds to the basic enzyme with a rate constant of (4.3 +/- 0.3) x 10(6) M-1 s-1.     

Transferrins, the mechanism of iron release by ovotransferrin.

Iron release from ovotransferrin in acidic media (3 < pH < 6) occurs in at least six kinetic steps. The first is a very fast (相似文献   

Effects of MgATP and MgADP on the cross-bridge kinetics of rabbit soleus slow-twitch muscle fibers.

The elementary steps surrounding the nucleotide binding step in the cross-bridge cycle were investigated with sinusoidal analysis in rabbit soleus slow-twitch muscle fibers. The single-fiber preparations were activated at pCa 4.40, ionic strength 180 mM, 20 degrees C, and the effects of MgATP (S) and MgADP (D) concentrations on three exponential processes B, C, and D were studied. Our results demonstrate that all apparent (measured) rate constants increased and saturated hyperbolically as the MgATP concentration was increased. These results are consistent with the following cross-bridge scheme: [cross-bridge scheme: see text] where A = actin, M = myosin, S = MgATP, and D = MgADP. AM+S is a collision complex, and AM*S is its isomerized form. From our studies, we obtained K0 = 18 +/- 4 mM-1 (MgADP association constant, N = 7, average +/- sem), K1a = 1.2 +/- 0.3 mM-1 (MgATP association constant, N = 8 hereafter), k1b = 90 +/- 20 s-1 (rate constant of ATP isomerization), k-1b = 100 +/- 9 s-1 (rate constant of reverse isomerization), K1b = 1.0 +/- 0.2 (equilibrium constant of isomerization), k2 = 21 +/- 3 s-1 (rate constant of cross-bridge detachment), k-2 = 14.1 +/- 1.0 s-1 (rate constant of reversal of detachment), and K2 = 1.6 +/- 0.3 (equilibrium constant of detachment). K0 is 8 times and K1a is 2.2 times those in rabbit psoas, indicating that nucleotides bind to cross-bridges more tightly in soleus slow-twitch muscle fibers than in psoas fast-twitch muscle fibers. These results indicate that cross-bridges of slow-twitch fibers are more resistant to ATP depletion than those of fast-twitch fibers. The rate constants of ATP isomerization and cross-bridge detachment steps are, in general, one-tenth to one-thirtieth of those in psoas.     

Dependency of the force-velocity relationships on Mg ATP in different types of muscle fibers from Xenopus laevis.

MgATP binding to the actomyosin complex is followed by the dissociation of actin and myosin. The rate of this dissociation process was determined from the relationship between the maximum velocity of shortening and the MgATP concentration. It is shown here that the overall dissociation rate is rather similar in different types of muscle fibers. The relation between MgATP concentration and the maximum shortening velocity was investigated in fast and slow fibers and bundles of myofibrils of the iliofibularis muscle of Xenopus laevis at 4 degrees C from which the sarcolemma was either removed mechanically or made permeable by means of a detergent. A small segment of each fiber was used for a histochemical determination of fiber type. At 5 mM MgATP, the fast fibers had a maximum shortening velocity (Vmax) of 1.74 +/- 0.12 Lo/s (mean +/- SEM) (Lo: segment length at a sarcomere length of 2.2 microns). For the slow fibers Vmax was 0.41 +/- 0.15 Lo/s. In both cases, the relationship between Vmax and the ATP concentration followed the hyperbolic Michaelis-Menten relation. A Km of 0.56 +/- 0.06 mM (mean +/- SD) was found for the fast fibers and of 0.16 +/- 0.03 mM for the slow fibers. Assuming that Vmax is mainly determined by the crossbridge detachment rate, the apparent second order dissociation rate for the actomyosin complex in vivo would be 3.8.10(5) M-1s-1 for the fast fibers and 2.9.10(5) M-1 s-1 for the slow fibers. Maximum power output as a function of the MgATP concentration was derived from the force-velocity relationships.(ABSTRACT TRUNCATED AT 250 WORDS)     

High acetylcholine concentrations cause rapid inactivation before fast desensitization in nicotinic acetylcholine receptors from Torpedo.

By using both a 3 to 4 ms quenched-86Rb+ flux assay and native acetylcholine receptor (AChR) rich electroplaque vesicles on which 50-60% of acetylcholine activation sites were blocked with alpha-BTX, we determined apparent rates of agonist-induced inactivation in AChR from Torpedo under conditions where measured flux response was directly proportional to initial 86Rb+ influx rate. Inactivation kinetics with acetylcholine in both the activating range (10 microM-10 mM) and the self-inhibiting range (15-100 mM) were measured at 4 degrees C. In the presence of 10 microM-1 mM acetylcholine, inactivation is characterized by a single exponential rate constant, kd (fast desensitization). Plots of kd vs. acetylcholine concentration display maximum kds [kd(max)] of 6.6-8.0 s-1, half-maximal kd at 102 +/- 16 microM, and a Hill coefficient of 1.6 +/- 0.3, closely paralleling the initial ion flux response of AChR. Thus, fast desensitization probably occurs from a doubly-liganded preopen state or the open channel state. In the self-inhibiting acetylcholine concentration range, inactivation is biphasic. A "rapid inactivation" phase is complete within 30 ms, followed by fast desensitization at a rate close to kd(max). Both the rate and extent of rapid inactivation increase with acetylcholine concentration, indicating that acetylcholine binds to its self-inhibition site with apparent kon approximately equal to 10(3) M-1s-1 and koff approximately equal to 40 s-1. This slow kon suggests either hindered access to the inhibitory allosteric site or that a fast binding step is followed by a slower conformational change leading to channel inhibition. Overall, our data suggest that acetylcholine binds preferentially to its inhibitory site when the receptor is in the open-channel conformation and that fast desensitization can occur from all multiple-liganded states.     

Rate of phosphate release after photoliberation of adenosine 5'-triphosphate in slow and fast skeletal muscle fibers.

Inorganic phosphate (Pi) release was determined by means of a fluorescent Pi-probe in single permeabilized rabbit soleus and psoas muscle fibers. Measurements of Pi release followed photoliberation of approximately 1.5 mM ATP by flash photolysis of NPE-caged ATP in the absence and presence of Ca2+ at 15 degrees C. In the absence of Ca2+, Pi release occurred with a slow rate of 11 +/- 3 microM . s-1 (n = 3) in soleus fibers and 23 +/- 1 microM . s-1 (n = 10) in psoas fibers. At saturating Ca2+ concentrations (pCa 4.5), photoliberation of ATP was followed by rapid force development. The initial rate of Pi release was 0.57 +/- 0.05 mM . s-1 in soleus (n = 13) and 4.7 +/- 0.2 mM . s-1 in psoas (n = 23), corresponding to a rate of Pi release per myosin head of 3.8 s-1 in soleus and 31.5 s-1 in psoas. Pi release declined at a rate of 0.48 s-1 in soleus and of 5.2 s-1 in psoas. Pi release in soleus was slightly faster in the presence of an ATP regenerating system but slower when 0.5 mM ADP was added. The reduction in the rate of Pi release results from an initial redistribution of cross-bridges over different states and a subsequent ADP-sensitive slowing of cross-bridge detachment.     

Solubilization of an alpha-bungarotoxin-binding component from rat brain.

Binding of [125I]-alpha-bungarotoxin to rat brain was investigated. Picomole quantities of specific toxin binding sites per gram of fresh tissue were found in particulate preparations as well as detergent extracts of whole brain. The toxin-binding macromolecules can be solubilized in low concentrations of Triton X-100. Specific binding occurs to a single class of sites with a dissociation constant of 5.6 X 10(-11) M. The association rate constant in 10 mM sodium phosphate, pH 7.4, was determined to be 6.8 X 10(5) M-1 s-1; the half-life of the complex was found to be 5.1 h, corresponding to a dissociation rate constant of 3.8 X 10(-5) s-1. The binding macromolecules resemble peripheral nicotinic acetylcholine receptors in toxin binding kinetics, solubility, isoelectric point, and hydrodynamic properties.     

Binding of ellipticine base and ellipticinium cation to calf-thymus DNA. A thermodynamic and kinetic study

The acid-basic properties of ellipticine have been re-estimated. The apparent pK of protonation at 3 microM drug concentration is 7.4 +/- 0.1. The ellipticine free base (at pH 9, I = 25 mM) intercalates into calf-thymus DNA with an affinity constant of 3.3 +/- 0.2 X 10(5) M-1, and a number of binding sites per phosphate of 0.23. The ellipticinium cation (pH 5, I = 25 mM) binds also to DNA with a constant of 8.3 +/- 0.2 x 10(5) M-1 and at a number of binding sites (n = 0.19). It is postulated that the binding of the drug to DNA at pH 9 is driven by hydrophobic and/or dipolar effects. Even at pH 5, where ellipticine exists as a cation, it is thought that the hydrophobic interaction is the main contribution to binding. The neutral and cationic forms share common binding within DNA sites but yield to structurally different complexes. The free base has 0.04 additional specific binding sites per phosphate. As determined from temperature-jump experiments, the second-order rate constant of the binding of the free base (pH 9) is 3.4 x 10(7) M-1 s-1 and the residence time of the base within the DNA is 8 ms. The rate constant for the binding of the ellipticinium cation is 9.8 x 10(7) M-1 s-1 when it is assumed that drug attachment occurs via a pathway in which the formation of an intermediate ionic complex is not involved (competitive pathway).     

The dependence of isometric tension, isometric ATPase activity, and shortening velocity of limulus muscle on the MgATP concentration.

The dependence of the isometric tension, the velocity of unloaded shortening, and the steady-state rate of MgATP hydrolysis on the MgATP concentration (range 0.01-5 mM MgATP) was studied in Ca-activated skinned Limulus muscle fibers. With increasing MgATP concentration the isometric tension increased to a peak at approximately 0.1 mM, and slightly decreased in the range up to 5 mM MgATP. The velocity of unloaded shortening depended on the MgATP concentration roughly according to the Michaelis-Menten law of saturation kinetics with a Michaelis-Menten constant Kv = 95 microM and a maximum shortening velocity of 0.07 muscle lengths s-1; the detachment rate of the cross-bridges during unloaded shortening was 24 s-1. The rate of MgATP splitting also depended hyperbolically on the MgATP concentration with a Michaelis-Menten constant Ka = 129 microM and a maximum turnover frequency of 0.5-1 s-1. The results are discussed in terms of a cross-bridge model based on a biochemical scheme of ATP hydrolysis by actin and myosin in solution.     

Interaction of local anesthetics with Torpedo californica membrane-bound acetylcholine receptor

The effects of local anesthetics on the rate of the agonist-induced increase in ligand affinity of membrane-bound acetylcholine receptor from Torpedo californica were examined. The rate of the transition in receptor affinity was determined by following the time-dependent increase in inhibition of iodinated alpha-bungarotoxin binding caused by 1 microM carbamylcholine. At concentrations below those that directly inhibited the binding of iodinated alpha-bungarotoxin, dibucaine increased the rate of the transition to a high-affinity state and tetracaine decreased this rate. The measured rate constants were 0.026 +/- 0.008 s-1 in the presence and 0.010 +/- 0.002 s-1 in the absence of dibucaine while tetracaine decreased the rate to 0.006 +/- 0.002 s-1 as compared to a control value of 0.012 +/- 0.003 s-1. A parallel was observed between the effectiveness of a compound in increasing or decreasing the rate of the agonist-induced transition in affinity and the change in its apparent inhibition constant in the presence of carbamylcholine (increase or decrease) measured by the displacement of tritiated perhydrohistrionicotoxin. This parallel could be explained by assuming (a) that local anesthetics bound directly to the specific histrionicotoxin binding site or (b) that they bound to a different site and the observed effects were caused by conformational changes.     

Observation of a chloride-dependent intermediate during catalysis by angiotensin converting enzyme using radiationless energy transfer

Stopped-flow radiationless energy-transfer kinetics have been used to examine the effects of chloride on the hydrolysis of Dns-Lys-Phe-Ala-Arg by angiotensin converting enzyme. The kinetic constants for hydrolysis at pH 7.5 and 22 degrees C in the presence of 300 mM sodium chloride were KM = 28 microM and kcat = 110 s-1, and in its absence, KM = 240 microM and kcat = 68 s-1. The apparent binding constant for chloride was 4 mM, and the extent of chloride activation in terms of kcat/KM was 14-fold. The effects of chloride on the pre-steady-state were examined at 2 degrees C. In the presence of chloride, two distinct enzyme-substrate complexes were observed, suggesting multiple steps in substrate binding. The initial complex was formed during the mixing period (kobsd greater than 200 s-1) while the second complex was formed much more slowly (kobsd = 40 s-1 when [S] = 5 microM and [NaCl] = 150 mM). Strikingly, in the absence of chloride, only a single, rapidly formed enzyme-substrate complex was observed. These results are consistent with a nonessential activator kinetic mechanism in which the slow step reflects conversion of an initially formed complex, (E X Cl- X S)1, to a more tightly bound complex, (E X Cl- X S)2.     

Reversible DIDS binding to band 3 protein in human erythrocyte membranes

Reversible binding of DIDS [4,4'-diisothiocyanato-2,2'-stilbenedisulphonate] to Band 3 protein, the anion exchanger located in erythrocyte plasma membrane, was studied in human erythrocytes. For this purpose, the tritiated form of DIDS ([3H]DIDS) has been synthesized and the filtering technique has been used to follow the kinetics of DIDS binding to the sites on Band 3 protein. The obtained results showed monophasic kinetics both for dissociation and association of the 'DIDS--Band 3' complex at 0 degree C in the presence of 165 mM KCl outside the cell (pH 7.3). A pseudo-first order association rate constant k+1 was determined to be (3.72 +/- 0.42) x 10(5) M-1 s-1, while the dissociation rate constant K-1 was determined to be (9.40 +/- 0.68) x 10(-3) s-1. The dissociation constant KD, calculated from the measured values of k-1 and k+1, was found to be 2.53 x 10(-8) M. The standard thermodynamics parameters characterizing reversible DIDS binding to Band 3 protein at 0 degree C were calculated. The mean values of the activation energies for the association and dissociation steps in the DIDS binding mechanism were determined to be (34 +/- 9) kJ mole-1 and (152 +/- 21) kJ mole-1, respectively. The results provide, for the first time, evidence for the reversibility of DIDS binding to Band 3 protein at 0 degree C. The existence of a stimulatory site is suggested, nearby the transport site on the Band 3 protein. The binding of an anion to this site can facilitate (through electrostatic repulsion interaction between two anions) the transmembrane movement of another anion from the transport site.     

Transport of glucose and fructose in rat hepatocytes at 37 degrees C

The kinetic parameters for transport of the nonmetabolizable glucose analogue 3-O-methyl-D-glucose and the relationship between transport and metabolism of D-glucose and D-fructose were determined in isolated rat hepatocytes at 37 degrees C and pH 7.4. 3-O-Methylglucose at a very low concentration (0.1 mM) equilibrated with the intracellular water with a rate constant of 0.41 s-1. Km for equilibrium exchange entry was 5.5 mM and Vmax was 2.2 mM X s-1 and similar results were obtained when using the zero-trans entry protocol. The rate constant for entry of tracer D-glucose was 0.15 s-1 and Km for glucose was about 20 mM. The phosphorylation rate for D-glucose was much slower than the transport rate. The rate constant for D-fructose entry was about 0.04 s-1, the apparent Km was about 100 mM and Vmax about 5 mM X s-1. The concentration dependence of 3-O-methylglucose inhibition of labelled fructose transport revealed biphasic kinetics indicating that fructose was transferred by both the glucose transporter and a fructose transporter. At concentrations lower than 1 mM, fructose metabolism appeared to be limited by the transport step.     

Adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum. Determining ligand dissociation constants of binary and ternary complexes from the kinetics of enzyme inactivation

Adenosine 5-phosphosulfate (APS) kinase from Penicillium chrysogenum is irreversibly inactivated by trinitrobenzene sulfonate in a pseudo-first order process. Under standard assay conditions kapp was 1.9 X 10(-3) s-1. Saturating MgATP or MgADP decreased Kapp to a limit of 4.1 X 10(-4) s-1. There are several explanations for the partial protection, including the presence of two essential lysyl side chains, only one of which is at the active site. Analysis of the inactivation kinetics by means of linear plots derived for partial protection yielded dissociation constants for E X MgATP (Kia) and E X MgADP (Kiq) of 2.9 mM and 1.8 mM, respectively. Low concentrations of APS alone provided no protection against trinitrobenzene sulfonate inactivation, but in the presence of 1 mM MgADP, as little as 2 microM APS provided additional protection while 100 microM APS reduced kapp to the limit of 4.1 X 10(-4) s-1. The results confirm the formation of a dead end E X MgADP X APS proposed earlier as the cause of the potent substrate inhibition by APS. Linear plots of 1/delta k versus 1/[MgADP] at different fixed [APS] and of 1/delta k versus 1/[APS] at different fixed [MgADP] were characteristic of the ordered binding of MgADP before APS (or the highly synergistic random binding of the two ligands). The true APS dissociation constant of the dead end E X MgADP X APS complex (K'ib) was determined to be 1.9 microM. From the value of K'ib and the previously reported value of KIB (apparent inhibition constant of APS as a substrate inhibitor of the catalytic reaction at saturating MgATP), the ratio of the MgADP and PAPS release rate constants (k4/k3) was calculated to be 11. Inactivation kinetics was used to study the effects of Mg2+ and high salt on ADP and APS binding. The results indicated that free ADP binds to the enzyme more tightly than does MgADP at low ionic strength. High salt decreased free ADP binding, but had little effect on MgADP binding. APS binds more tightly to E X MgADP in the absence or presence of salt than to E X ADP.     

Transient kinetic studies of substrate inhibition in the horse liver alcohol dehydrogenase reaction

The rate-limiting step of ethanol oxidation by alcohol dehydrogenase (E) at substrate inhibitory conditions (greater than 500 mM ethanol) is shown to be the dissociation rate of NADH from the abortive E-ethanol-NADH complex. The dissociation rate constant of NADH decreased hyperbolically from 5.2 to 1.4 s-1 in the presence of ethanol causing a decrease in the Kd of NADH binding from 0.3 microM for the binary complex to 0.1 microM for the abortive complex. Correspondingly, ethanol binding to E-NADH (Kd = 37 mM) was tighter than to enzyme (Kd = 109 mM). The binding rate of NAD+ (7 X 10(5) M-1s-1) to enzyme was not affected by the presence of ethanol, further substantiating that substrate inhibition is totally due to a decrease in the dissociation rate constant of NADH from the abortive complex. Substrate inhibition was also observed with the coenzyme analog, APAD+, but a single transient was not found to be rate limiting. Nevertheless, the presence of substrate inhibition with APAD+ is ascribed to a decrease in the dissociation rate of APADH from 120 to 22 s-1 for the abortive complex. Studies to discern the additional limiting transient(s) in turnover with APAD+ and NAD+ were unsuccessful but showed that any isomerization of the enzyme-reduced coenzyme-aldehyde complex is not rate limiting. Chloride increases the rate of ethanol oxidation by hyperbolically increasing the dissociation rate constant of NADH from enzyme and the abortive complex to 12 and 2.8 s-1, respectively. The chloride effect is attributed to the binding of chloride to these complexes, destabilizing the binding of NADH while not affecting the binding of ethanol.     

Pre-steady-state and steady-state kinetic analysis of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.

The complete time course of the hydrolysis of p-nitrophenyl phosphate catalyzed by the low molecular weight (acid) phosphotyrosyl protein phosphatase from bovine heart was elucidated and analyzed in detail. Burst titration kinetics were demonstrated for the first time with this class of enzyme. At pH 7.0, 4.5 degrees C, a transient pre-steady-state "burst" of p-nitrophenol was formed with a rate constant of 48 s-1. The burst was effectively stoichiometric and corresponded to a single enzyme active site/molecule. The burst was followed by a slow steady-state turnover of the phosphoenzyme intermediate with a rate constant of 1.2 s-1. Product inhibition studies indicated an ordered uni-bi kinetic scheme for the hydrolysis. Partition experiments conducted for several substrates revealed a constant product ratio. Vmax was constant for these substrates, and the overall rate of hydrolysis was increased greatly in the presence of alcohol acceptors. An enzyme-catalyzed 18O exchange between inorganic phosphate and water was detected and occurred with kcat = 4.47 x 10(-3) s-1 at pH 5.0, 37 degrees C. These results were all consistent with the existence of a phosphoenzyme intermediate in the catalytic pathway and with the breakdown of the intermediate being the rate-limiting step. The true Michaelis binding constant Ks = 6.0 mM, the apparent Km = 0.38 mM, and the rate constants for phosphorylation (k2 = 540 s-1) and dephosphorylation (k3 = 36.5 s-1) were determined under steady-state conditions with p-nitrophenyl phosphate at pH 5.0 and 37 degrees C in the presence of phosphate acceptors. The energies of activation for the enzyme-catalyzed hydrolysis at pH 5.0 and 7.0 were 13.6 and 14.1 kcal/mol, respectively. The activation energy for the enzyme-catalyzed medium 18O exchange between phosphate and water was 20.2 kcal/mol. Using the available equilibrium and rate constants, an energetic diagram was constructed for the enzyme-catalyzed reaction.     

Mode of action of iberiotoxin, a potent blocker of the large conductance Ca(2+)-activated K+ channel.

Iberiotoxin, a toxin purified from the scorpion Buthus tamulus is a 37 amino acid peptide having 68% homology with charybdotoxin. Charybdotoxin blocks large conductance Ca(2+)-activated K+ channels at nanomolar concentrations from the external side only (Miller, C., E. Moczydlowski, R. Latorre, and M. Phillips. 1985. Nature (Lond.). 313:316-318). Like charybdotoxin, iberiotoxin is only able to block the skeletal muscle membrane Ca(2+)-activated K+ channel incorporated into neutral-planar bilayers when applied to the external side. In the presence of iberiotoxin, channel activity is interrupted by quiescent periods that can last for several minutes. From single-channel records it was possible to determine that iberiotoxin binds to Ca(2+)-activate K+ channel in a bimolecular reaction. When the solution bathing the membrane are 300 mM K+ internal and 300 mM Na+ external the toxin second order association rate constant is 3.3 x 10(6) s-1 M-1 and the first order dissociation rate constant is 3.8 x 10(-3) s-1, yielding an apparent equilibrium dissociation constant of 1.16 nM. This constant is 10-fold lower than that of charybdotoxin, and the values for the rate constants showed above indicate that this is mainly due to the very low dissociation rate constant; mean blocked time approximately 5 min. The fact that tetraethylammonium competitively inhibits the iberiotoxin binding to the channel is a strong suggestion that this toxin binds to the channel external vestibule. Increasing the external K+ concentration makes the association rate constant to decrease with no effect on the dissociation reaction indicating that the surface charges located in the external channel vestibule play an important role in modulating toxin binding.     

Studies on rat liver plasma membrane. Altered protein and phospholipid metabolism after injection of D-galactosamine.

The displacement of NADH from cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by using NAD+, 1,10-phenanthroline, ADP-ribose, deamino-NAD+ and pyridine-3-aldehyde-adenine dinucleotide as displacing agents, by following the decrease in fluorescence as a function of time. The data obtained could be fitted by assuming two first-order processes were occurring, a faster process with an apparent rate constant of 0.85 +/- 0.20 s-1 and a relative amplitude of 60 +/- 10% and a slower process with an apparent rate constant of 0.20 +/- 0.05 s-1 and a relative amplitude of 40 +/- 10% (except for pyridine-3-aldehyde-adenine dinucleotide, where the apparent rate constant for the slow process was 0.05 s-1). The displacement rates did not change significantly when the pH was varied from 6.0 to 9.0. Kinetic data are also reported for the dependence of the rate of binding of NADH to the enzyme on the total concentration of NADH. Detailed arguments are presented based on the isolation and purification procedures, the equilibrium coenzyme-binding studies and the kinetic data, which lead to the following model for the release of NADH from the enzyme: (formula: see article). The parameters that best fit the data are: k + 1 = 0.2 s-1; k - 1 = 0.05 s-1; k + 2 = 0.8 s-1 and k - 2 = 5 X 10(5)litre-mol-1-s-1. The slow phase of the NADH release is similar to the steady-state turnover number for substrates such as acetaldehyde and propionaldehyde and appears to contribute significantly to the limitation of the steady-state rate.