Preparation and characterization of nucleotide-free and metal ion-free p21 "apoprotein"

p21 isolated under nondenaturing conditions is obtained as a complex with guanosine nucleotides and magnesium ions. We have developed a high performance liquid chromatography method which removes greater than 95% of bound nucleotide and the metal ion very rapidly under mild conditions. At the same time, p21 is purified from minor protein impurities. The protein thus prepared is thermally much less stable than the complexed p21, but can be used for studying its interaction with nucleotides and metal ions at low temperatures. The association rate constant for p21 and GDP is 1.47 X 10(6) M-1 s-1 and for GTP is 2.9 X 10(6) M-1 s-1 at 0 degree C. By using appropriately determined dissociation rate constants we have determined the binding constant for p21.GDP and p21.GTP in the presence of excess Mg2+ to be 5.7 X 10(10) M-1 and 6.0 X 10(10) M-1, respectively, at 0 degree C.     

Kinetic analysis of the hydrolysis of GTP by p21N-ras. The basal GTPase mechanism

The rate constants have been determined for elementary steps in the basal GTPase mechanism of normal p21N-ras (Gly-12) and an oncogenic mutant (Asp-12): namely GTP binding, hydrolysis, phosphate release, and GDP release. By extrapolation from data at lower temperatures, the GTP association rate constant at 37 degrees C is 1.4 x 10(8) M-1 s-1 for the normal protein and 4.8 x 10(8) M-1 s-1 for the mutant. Other rate constants were measured directly at 37 degrees C, and three processes have similar slow values. GTP dissociation is at 1.0 x 10(-4) s-1 (normal) and 5.0 x 10(-4) s-1 (mutant). The hydrolysis step is at 3.4 x 10(-4) s-1 (normal) and 1.5 x 10(-4) s-1 (mutant). GDP dissociates at 4.2 x 10(-4) s-1 (normal) and 2.0 x 10(-4) s-1 (mutant). GDP association rate constants are similar to those for GTP, 0.5 x 10(8) M-1 s-1 for normal and 0.7 x 10(8) M-1 s-1 for mutant. Both hydrolysis and GDP release therefore contribute to rate limitation of the basal GTPase activity. There are distinct differences (up to 5-fold) between rate constants for the normal and mutant proteins at a number of steps. The values are consistent with the reduced GTPase activity for this mutant and suggest little difference between normal and mutant proteins in the relative steady-state concentrations of GTP and GDP complexes that may represent active and inactive states. The results are discussed in terms of the likely role of p21ras in transmembrane signalling.     

The binding of nucleotides and metal ions to elongation factor Tu from Bacillus stearothermophilus as studied by equilibrium dialysis

EF-Tu from B. stearothermophilus binds divalent metal ions even in the absence of guanine nucleotides. The association constants necessary for characterizing the multiple equilibria between EF-Tu, GDP and the divalent ions magnesium and manganese were determined by equilibrium dialysis. The constants are 4.6 X 10(4) M-1 and 5.4 X 10(5) M-1 for the binding of Mg2 and 1.0 X 10(5) M-1 and 1.1 X 10(6) M-1 for the binding of Mn2 to EF-Tu and EF-Tu . GDP, respectively. In the absence of divalent ions EF-Tu binds GMP, GDP and GTP with association constants of 3 x 10(3) M-1, 1.7 x 10(7) M-1 and 1.3 x 10(6) M-1, respectively. The binding of GDP in the presence of metal ions is an order of magnitude stronger than in the absence of metal ions.     

Electron-paramagnetic-resonance studies of manganese(II) complexes with elongation factor Tu from Bacillus stearothermophilus. Observation of a GTP hydrolysis intermediate state complex

Changes in the coordination of Mn2+ to nucleotide, water and protein at the active site of elongation factor Tu (EF-Tu) have been studied by electron paramagnetic resonance (EPR) spectroscopy. From the time dependence of the Mn2+ spectrum after addition of GTP to EF-Tu X Mn, it was apparent that three complexes with different EPR linewidths could be detected. Using additional information from the kinetics of 32Pi production and release from EF-Tu X Mn X [gamma-32P]GTP these were identified as EF-Tu X Mn X GTP (linewidth 4.2 mT), EF-Tu X Mn X GDP X Pi (1.20 mT) and EF-Tu X Mn X GDP (1.29 mT). The linewidth for EF-Tu X Mn was 1.51 mT. The rate constant for GTP cleavage on EF-Tu was 0.01 min-1 at 24 C, for Pi release from the EF-Tu X GDP X Pi complex 0.0033 min-1. The corresponding rate constants in the presence of Mg2+ were 0.003 min-1 and 0.0065 min-1. The rate constant for reversal of the cleavage step was found to be much smaller than that for the rate of Pi release (and consequently much smaller than in the forward direction), as shown by 31P-NMR experiments on the incorporation of 18O into Pi from GTP hydrolyzed in the presence of H2 18O. EPR experiments using specifically 17O-labelled GTPs demonstrated an interaction of Mn2+ with the beta-phosphate in both the EF-Tu X GDP X Pi and EF-Tu X GDP complexes. Inorganic phosphate in the EF-Tu X GDP X Pi complex was found not to interact with the metal ion. From EPR experiments in H2 17O, it was concluded that the most probable number of water molecules in the different complexes was 4 (EF-Tu X Mn), 5 (EF-Tu X Mn X GDP X Pi) and 3 (EF-Tu X Mn X GDP), with 2, 0 and 2 metal-protein interactions respectively.     

The effect of divalent metal cations on the inhibition of human coagulation factor Xa by plasma proteinase inhibitors

The inactivation of human coagulation factor Xa by the plasma proteinase inhibitors alpha 1-antitrypsin, antithrombin III and alpha 2-macroglobulin in purified systems was found to be accelerated by the divalent cations Ca2+, Mn2+ and Mg2+. The rate constant for the inhibition of factor Xa by antithrombin III rose from 2.62 X 10(4) M-1 X min-1 in the absence of divalent cations to a maximum of 6.40 X 10(4) M-1 X min-1 at 5 mM Ca2+, 8.10 X 10(4) M-1 X min-1 at 5 mM Mn2+, with a slight decrease in rate at higher cation concentrations. Mg2+ caused a gradual rise in rate constant to 5.65 X 10(4) M-1 X min-1 at 20 mM. The rate constant for the inhibition of factor Xa by alpha 1-antitrypsin in the absence of divalent cations was 5.80 X 10(3) M-1 X min-1. Ca2+ increased the rate to 1.50 X 10(4) M-1 X min-1 at 5 mM and Mn2+ to 2.40 X 10(4) M-1 X min-1 at 6 mM. The rate constant for these cations again decreased at higher concentrations. Mg2+ caused a gradual rise in rate constant to 1.08 X 10(4) M-1 X min-1 at 10 mM. The rate constant for the factor Xa-alpha 2-macroglobulin reaction was raised from 6.70 X 10(3) M-1 X min-1 in the absence of divalent cations to a maximum of 4.15 X 10(4) M-1 X min-1 at 4 mM Ca2+, with a decrease to 3.05 X 10(4) M-1 at 10 mM. These increases in reaction rate were correlated to the binding of divalent cations to factor Xa by studying changes in the intrinsic fluorescence and dimerization of factor Xa. The changes in fluorescence suggested a conformational change in factor Xa which may be responsible for the increased rate of reaction, whilst the decrease in rate constant at higher concentrations of Ca2+ and Mn2+ may be due to factor Xa dimerization.     

Nitrogenase of Klebsiella pneumoniae. Kinetic studies on the Fe protein involving reduction by sodium dithionite, the binding of MgADP and a conformation change that alters the reactivity of the 4Fe-4S centre.

The kinetics of reduction of indigocarmine-dye-oxidized Fe protein of nitrogenase from Klebsiella pneumoniae (Kp2ox) by sodium dithionite in the presence and absence of MgADP were studied by stopped-flow spectrophotometry at 23 degrees C and at pH 7.4. Highly co-operative binding of 2MgADP (composite K greater than 4 X 10(10) M-2) to Kp2ox induced a rapid conformation change which caused the redox-active 4Fe-4S centre to be reduced by SO2-.(formed by the predissociation of dithionite ion) with k = 3 X 10(6) M-1.s-1. This rate constant is at least 30 times lower than that for the reduction of free Kp2ox (k greater than 10(8) M-1.s-1). Two mechanisms have been considered and limits obtained for the rate constants for MgADP binding/dissociation and a protein conformation change. Both mechanisms give rate constants (e.g. MgADP binding 3 X 10(5) less than k less than 3 X 10(6) M-1.s-1 and protein conformation change 6 X 10(2) less than k less than 6 X 10(3) s-1) that are similar to those reported for creatine kinase (EC 2.7.3.2). The kinetics also show that in the catalytic cycle of nitrogenase with sodium dithionite as reductant replacement of 2MgADP by 2MgATP occurs on reduced and not oxidized Kp2. Although the Kp2ox was reduced stoichiometrically by SO2-. and bound two equivalents of MgADP with complete conversion into the less-reactive conformation, it was only 45% active with respect to its ability to effect MgATP-dependent electron transfer to the MoFe protein.     

Phalloidin enhances actin assembly by preventing monomer dissociation

Incubation of the isolated acrosomal bundles of Limulus sperm with skeletal muscle actin results in assembly of actin onto both ends of the bundles. These cross-linked bundles of actin filaments taper, thus allowing one to distinguish directly the preferred end for actin assembly from the nonpreferred end; the preferred end is thinner. Incubation with actin in the presence of equimolar phalloidin in 100 mM KCl, 1 mM MgCl2 and 0.5 mM ATP at pH 7.5 resulted in a slightly smaller association rate constant at the preferred end than in the absence of the drug (3.36 +/- 0.14 X 10(6) M-1 s-1 vs. 2.63 +/- 0.22 X 10(6) M-1 s- 1, control vs. experimental). In the presence of phalloidin, the dissociation rate constant at the preferred end was reduced from 0.317 +/- 0.097 s-1 to essentially zero. Consequently, the critical concentration at the preferred end dropped from 0.10 microM to zero in the presence of the drug. There was no detectable change in the rate constant of association at the nonpreferred end in the presence of phalloidin (0.256 +/- 0.015 X 10(6) M-1 s-1 vs. 0.256 +/- 0.043 X 10(6) M-1 s-1, control vs. experimental); however, the dissociation rate constant was reduced from 0.269 +/- 0.043 s-1 to essentially zero. Thus, the critical concentration at the nonpreferred end changed from 1.02 microM to zero in the presence of phalloidin. Dilution-induced depolymerization at both the preferred and nonpreferred ends was prevented in the presence of phalloidin. Thus, phalloidin enhances actin assembly by lowering the critical concentration at both ends of actin filaments, a consequence of reducing the dissociation rate constants at each end.     

Kinetics and thermodynamics of the interaction of elongation factor Tu with elongation factor Ts, guanine nucleotides, and aminoacyl-tRNA

The exchange of elongation factor Tu (EF-Tu)-bound GTP in the presence and absence of elongation factor Ts (EF-Ts) was monitored by equilibrium exchange kinetic procedures. The kinetics of the exchange reaction were found to be consistent with the formation of a ternary complex EF-Tu X GTP X EF-Ts. The equilibrium association constants of EF-Ts to the EF-Tu X GTP complex and of GTP to EF-Tu X EF-Ts were calculated to be 7 X 10(7) and 2 X 10(6) M-1, respectively. The dissociation rate constant of GTP from the ternary complex was found to be 13 s-1. This is 500 times larger than the GTP dissociation rate constant from the EF-Tu X GTP complex (2.5 X 10(-2) s-1). A procedure based on the observation that EF-Tu X GTP protects the aminoacyl-tRNA molecule from phosphodiesterase I-catalyzed hydrolysis was used to study the interactions of EF-Tu X GTP with Val-tRNAVal and Phe-tRNAPhe. Binding constants of Phe-tRNAPhe and Val-tRNAVal to EF-Tu X GTP of 4.8 X 10(7) and 1.2 X 10(7)M-1, respectively, were obtained. The exchange of bound GDP with GTP in solution in the presence of EF-Ts was also examined. The kinetics of the reaction were found to be consistent with a rapid equilibrium mechanism. It was observed that the exchange of bound GDP with free GTP in the presence of a large excess of the latter was accelerated by the addition of aminoacyl-tRNA. On the basis of these observations, a complete mechanism to explain the interactions among EF-Tu, EF-Ts, guanine nucleotides, and aminoacyl-tRNA has been developed.     

Oxalyl hydroxamates as reaction-intermediate analogues for ketol-acid reductoisomerase

N-Hydroxy-N-isopropyloxamate (IpOHA) is an exceptionally potent inhibitor of the Escherichia coli ketol-acid reductoisomerase. In the presence of Mg2+ or Mn2+, IpOHA inhibits the enzyme in a time-dependent manner, forming a nearly irreversible complex. Nucleotide, which is essential for catalysis, greatly enhances the binding of IpOHA by the reductoisomerase, with NADPH (normally present during the enzyme's rearrangement step, i.e., conversion of a beta-keto acid into an alpha-keto acid, in either the forward or reverse physiological reactions) being more effective than NADP. In the presence of Mg2+ and NADPH, IpOHA appears to bind to the enzyme in a two-step mechanism, with an initial inhibition constant of 160 nM and a maximum rate of formation of the tight, slowly reversible complex of 0.57 min-1 (values that give an association rate of IpOHA, at low concentration, of 5.9 X 10(4) M-1 s-1). The rate of exchange of [14C]IpOHA from an enzyme-[14C]IpOHA-Mg2(+)-NADPH complex with exogenous, unlabeled IpOHA has a half-time of 6 days (150 h). This dissociation rate (1.3 X 10(-6) s-1) and the association rate determined by inactivation kinetics define an overall dissociation constant of 22 pM. By contrast, in the presence of Mn2+ and NADPH, the corresponding association and dissociation rates for IpOHA are 8.2 X 10(4) M-1 s-1 and 3.2 X 10(-6) s-1 (half-time = 2.5 days), respectively, which define an overall dissociation constant of 38 pM. In the presence of NADP or in the absence of nucleotide (both in the presence of Mg2+), the enzyme-IpOHA complex is far more labile, with dissociation half-times of 28 and 2 h, respectively. In the absence of Mg2+ or Mn2+, IpOHA does not exhibit time-dependent inhibition of the reductoisomerase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mg2+ dependence of guanine nucleotide binding to tubulin

The relationship between the concentration of Mg2+ and the binding of GDP and GTP to tubulin dimers was investigated by measuring the displacement of the nucleotide bound at the exchangeable site (E-site) by radiolabeled GDP and GTP. A wide range of concentrations of GTP, GDP, and Mg2+ was explored. In the near absence of Mg2+, the affinity of tubulin for GDP was found to be much greater than its affinity for GTP. In the presence of 1.0 mM Mg2+, however, its affinity for GDP was slightly less than for GTP. The results could be quantitatively described in terms of a small number of reversible equilibria. Equilibrium constants, pertaining to measurements at 0 degrees C, in 0.1 M piperazine-N,N'-bis(2-ethanesulfonic acid), 0.2 mM dithioerythritol, 2 mM EGTA, pH 6.9, were obtained by nonlinear least squares fitting of the data. When the association constant of tubulin for GDP uncomplexed with Mg2+ was taken to be 1.6 X 10(7) M-1, that for uncomplexed GTP was found to be no larger than 1.4 x 10(4) M-1, at least 1100-fold smaller. The association constant of tubulin for the GDP.Mg2+ complex was found to be 2.5-2.7 x 10(7) M-1, while that for the GTP.Mg2+ complex is 6.4-9.0 x 10(7) M-1.     

Kinetic studies of calcium binding to parvalbumins from bullfrog skeletal muscle

In addition to steady-state properties of calcium binding to parvalbumins, kinetic studies are required for adequate evaluation of the physiological roles of parvalbumins. By using a dual-wavelength spectrophotometer equipped with a stopped-flow accessory, the transient kinetics of calcium binding to parvalbumins (PA-1 and 2) from bullfrog skeletal muscle was examined at 20 degrees C in medium containing 20 mM MOPS-KOH, pH 6.80, 0.13 mM tetramethylmurexide, 25 microM CaCl2, metal-deprived PA-1 or PA-2, various concentrations of Mg2+, and KCl to adjust the ionic strength of the medium to 0.106. The results can be explained in terms of the following rate constants under the conditions mentioned above when a second-order kinetic scheme is assumed. For PA-1, the association and apparent dissociation rate constants for Ca2+ are 1.5 X 10(7) M-1 X s-1 and 1.5 s-1, respectively, or more. The rate constants for Mg2+ are 7,500 M-1 X s-1 and 5-6 s-1, respectively. For PA-2, the rate constants for Ca2+ are 7 X 10(6) M-1 X s-1 and 1.16 s-1, respectively, and those for Mg2+ are 3,500 M-1 X s-1 and 3.5-4 s-1, respectively. Increased affinities for Ca2+ and Mg2+ at 10 degrees C are largely due to decreased apparent dissociation rate constants for these divalent cations, because no significant change in the association rate constants was found.     

Functional properties of human skeletal muscle acetylcholine receptors expressed by the TE671 cell line

Functional properties of acetylcholine receptors from intact TE671 human medulloblastoma cells were examined using tracer ion flux, ligand competition against 125I-labeled alpha-bungarotoxin binding, and single channel recording measurements. 125I-Labeled alpha-bungarotoxin binds to surface receptors with the forward rate constant 1.8 X 10(5) M-1 s-1 and dissociates with the rate constant 4.6 X 10(-5) s-1, at 21 degrees C; the apparent dissociation constant is 2.6 X 10(-10) M. alpha-Bungarotoxin binds to at least two sites/receptor, but blocks agonist-induced 22Na+ uptake when bound to only one site. The reversible antagonists, dimethyl-d-tubocurarine and gallamine, occupy two sites which exhibit nearly equivalent affinities, but block agonist-induced uptake by occupying only one site. Strong agonists activate rapid sodium uptake with relatively low affinity, but desensitize with a much higher affinity; among agonists, the ratio of low to high affinity dissociation constants ranges from 1600 to 4000. By using the estimated dissociation constants, the allosteric model of Monod, Wyman, and Changeux (MWC) can be fitted to the concentration dependencies of both steady-state agonist occupancy and desensitization. The fitting analysis discloses an allosteric constant of 3 X 10(-5), which is the ratio of activatable to desensitized receptors in the absence of agonist. The rate of recovery from desensitization can exceed the rate of onset of desensitization elicited by low concentrations of agonist, further supporting the general MWC framework. Single channel recordings show that the channel opening probability is greater than 0.7 at high agonist concentrations. Favorable channel opening is shown to only slightly oppose strong desensitization.     

Biostructural chemistry of magnesium ion: characterization of the weak binding sites on tRNA(Phe)(yeast). Implications for conformational change and activity

The thermodynamics and kinetics of magnesium binding to tRNA(Phe)(yeast) have been studied directly by 25Mg NMR. In 0.17 M Na+(aq), tRNA(Phe) exists in its native conformation and the number of strong binding sites (Ka greater than or equal to 10(4)) was estimated to be 3-4 by titration experiments, in agreement with X-ray structural data for crystalline tRNA(Phe) (Jack et al., 1977). The set of weakly bound ions were in slow exchange and 25Mg NMR resonances were in the near-extreme-narrowing limit. The line shapes of the exchange-broadened magnesium resonance were indistinguishable from Lorentzian form. The number of weak magnesium binding sites was determined to be 50 +/- 8 in the native conformation and a total line-shape analysis of the exchange-broadened 25 Mg2+ NMR resonance gave an association constant Ka of (2.2 +/- 0.2) X 10(2) M-1, a quadrupolar coupling constant (chi B) of 0.84 MHz, an activation free energy (delta G*) of 12.8 +/- 0.2 kcal mol-1, and an off-rate (koff) of (2.5 +/- 0.4) X 10(3) s-1. In the absence of background Na+(aq), up to 12 +/- 2 magnesium ions bind cooperatively, and 73 +/- 10 additional weak binding sites were determined. The binding parameters in the nonnative conformation were Ka = (2.5 +/- 0.2) X 10(2) M-1, chi B = 0.64 MHz, delta G* = 13.1 +/- 0.2 kcal mol-1, and koff = (1.6 +/- 0.4) X 10(3) s-1. In comparison to Mg2+ binding to proteins (chi B typically ca. 1.1-1.6 MHz) the lower chi B values suggest a higher degree of symmetry for the ligand environment of Mg2+ bound to tRNA. A small number of specific weakly bound Mg2+ appear to be important for the change from a nonnative to a native conformation. Implications for interactions with the ribosome are discussed.     

Parameters controlling the kinetics of ferric and ferrous hemeproteins reduction by hydrated electrons

To clarify the processes of hemeproteins reduction, three classes of these proteins (ferric, ferrous and desFe) were reduced by hydrated electrons generated by pulse radiolysis. Spectral and kinetic investigations were made on alpha hemoglobin chain and myoglobin. Human alpha chain has been chosen to avoid all ferric contaminations and horse ferric myoglobin to eliminate all ferrous protein fractions. We have successively studied the influences of: the iron presence, its oxidation state (II and III), the protein charge and the iron-ligand nature (H2O, OH-, N3- and CN-). For alpha human hemoglobin chain without metallic ion or with ferrous iron, the reduction rates are the same: 1.1 +/- 0.2.10(10) M-1.s-1. In the case of horse ferric myoglobin, the reduction rates depend principally on the protein charge (from pH 6.3 to pH 9.5, the reduction rate of Mb(FeIII)N3- decreases from 2.5 +/- 0.5.10(10) M-1.s-1 to 1.2 +/- 0.2.10(10) M-1.s-1) and are also modulated by the equilibrium constant of the hemeprotein-ligand association (1.2 +/- 0.2.10(10) M-1.s-1 for Mb(FeIII)N3- and 0.8 +/- 0.2.10(10) M-1.s-1 for Mb(FeIII)CN-, at pH 9.8).     

Binding of guanine nucleotides and Mg2+ to tubulin with a nucleotide-depleted exchangeable site

Binding of GTP and GDP to tubulin in the presence or absence of Mg2+ was measured following depletion of the exchangeable site--(E-site) nucleotide. The E-site nucleotide was displaced with a large molar excess of the nonhydrolyzable GTP analogue, GMPPCP, followed by the removal of the analogue. Using a micropartition assay, the equilibrium constant measured in 0.1 M 1.4-piperazinediethanesulfonic acid (Pipes), pH 6.9, 1 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, 1 mM dithiothreitol, and 1 mM MgSO4 at 4 degrees C was 9.1 x 10(6) M-1 for GTP and 4.4 x 10(6) M-1 for GDP. Removal of Mg2+ reduced the binding affinity of GTP by 160-fold while the affinity of GDP remained essentially unchanged. Similar values were obtained if 0.1 M Tris, pH 7.0, was used instead of Pipes. Binding of Mg2+ to tubulin containing GTP, GDP, or no nucleotide at the E-site was also examined by the micropartition method. Tubulin-GTP contained one high affinity Mg2+ site (K alpha = 1.2 x 10(6) M-1) in addition to the one occupied by Mg2+ as tubulin is isolated, while only weak Mg2+ binding to tubulin-GDP and to tubulin with a vacant E-site (K alpha = 10(3) M-1) was observed. It is suggested that Mg2+ binds to the beta and gamma phosphates of GTP, and only to the beta phosphate of GDP, as shown for the H. ras p21 protein.     

Kinetic studies of three different molecular forms of urokinase for the activation of native human plasminogen

The kinetic parameters of three different molecular forms of urokinase (UK) for the activation of native Glu-plasminogen were compared. The apparent Michaelis constant (Km. app.) of each UK was almost of the same order of magnitude (31-38 microM), but the catalytic constants (kc) were observed to be different: UKh (high molecular weight form, molecular weight 53,000), 2.4 +/- 0.2 s-1; UK+ (low molecular weight form, molecular weight 33,000), 0.83 +/- o.10 s-1, and UKl (trypsin-digested form, molecular weight 36,000), 0.91 +/- 0.18 s-1. The overall second order rate constant, kc/Km calculated for UKh was 7.7 X 10(4) M-1 s-1, higher than for UKl (2.2 X 10(4) M-1 s-1) or UKt (2.4 X 10(4) M-1 s-1), indicating the possibility of a much higher degree of enzymatic specificity and efficiency.     

Reactions of prostaglandin H synthase in the presence of the stabilizing agents diethyldithiocarbamate and glycerol

Both cyclooxygenase and peroxidase reactions of prostaglandin H synthase were studied in the presence and absence of diethyldithiocarbamate and glycerol at 4 degrees C in phosphate buffer (pH 8.0). Diethyldithiocarbamate reacts with the high oxidation state intermediates of prostaglandin H synthase; it protects the enzyme from bleaching and loss of activity by its ability to act as a reducing agent. For the reaction of diethyldithiocarbamate with compound I, the second-order rate constant k2,app, was found to fall within the range of 5.8 x 10(6) +/- 0.4 x 10(6) M-1.s-1 less than k2,app less than 1.8 x 10(7) +/- 0.1 x 10(7) M-1.s-1. The reaction of diethyldithiocarbamate with compound II showed saturation behavior suggesting enzyme-substrate complex formation, with kcat = 22 +/- 3 s-1, Km = 67 +/- 10 microM, and the second-order rate constant k3,app = 2.0 x 10(5) +/- 0.2 x 10(5) M-1.s-1. In the presence of both diethyldithiocarbamate and 30% glycerol, the parameters for compound II are kcat = 8.8 +/- 0.5 s-1, Km = 49 +/- 7 microM, and k3,app = 1.03 x 10(5) +/- 0.07 x 10(5) M-1.s-1. The spontaneous decay rate constants of compounds I and II (in the absence of diethyldithiocarbamate) are 83 +/- 5 and 0.52 +/- 0.05 s-1, respectively, in the absence of glycerol; in the presence of 30% glycerol they are 78 +/- 5 and 0.33 +/- 0.02 s-1, respectively. Neither cyclooxygenase activity nor the rate constant for compound I formation using 5-phenyl-4-pentenyl-1-hydroperoxide is altered by the presence of diethyldithiocarbamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rate constants and equilibrium constants for binding of the gelsolin-actin complex to the barbed ends of actin filaments in the presence and absence of calcium

The equilibrium constant for binding of the gelsolin-actin complex to the barbed ends of actin filaments was measured by the depolymerizing effect of the gelsolin-actin complex on actin filaments. When the gelsolin-actin complex blocks monomer consumption at the lengthening barbed ends of treadmilling actin filaments, monomers continue to be produced at the shortening pointed ends until a new steady state is reached in which monomer production at the pointed ends is balanced by monomer consumption at the uncapped barbed ends. By using this effect the equilibrium constant for binding was determined to be about 1.5 X 10(10) M-1 in excess EGTA over total calcium (experimental conditions: 1 mM MgCl2, 100 mM KCl, pH 7.5, 37 degrees C). In the presence of Ca2+ the equilibrium constant was found to be in the range of or above 10(11) M-1. The rate constant of binding of the gelsolin-actin complex to the barbed ends was measured by inhibition of elongation of actin filaments. Nucleation of new filaments by the gelsolin-actin complex towards the pointed ends was prevented by keeping the monomer concentration below the critical monomer concentration of the pointed ends where the barbed ends of treadmilling actin filaments elongate and the pointed ends shorten. The gelsolin-actin complex was found to bind fourfold faster to the barbed ends in the presence of Ca2+ (10 X 10(6) M-1 s-1) than in excess EGTA (2.5 X 10(6) M-1 s-1). Dissociation of the gelsolin-actin complex from the barbed ends can be calculated to be rather slow. In excess EGTA the rate constant of dissociation is about 1.7 X 10(-4) s-1. In the presence of Ca2+ this dissociation rate constant is in the range of or below 10(-4) s-1.     

The oxidation of Pseudomonas cytochrome c-551 oxidase by potassium ferricyanide.

Stopped-flow kinetics were made of the reaction between ascorbate-reduced Pseudomonas cytochrome oxidase and potassium ferricyanide under both N2 and CO atmospheres. Under N2 three kinetic processes were observed, two being dependent on ferricyanide concentration, with second-order rate constants of 9.6 X 10(4)M-1.s-1 and 1.5 X 10(4)M-1.s-1, whereas the other was concentration-independent, with a first-order rate constant of 0.17 +/- 0.03s-1. Measurements of their kinetic difference spectra have allowed the fastest and second-fastest phases of the reaction to be assigned to direct bimolecular reactions of ferricyanide with the haem c and haem d, moieties of the enzyme respectively. Under CO, the second-order rate constant for the reaction of the haem c was, at 1.3 X 10(5)M-1.s-1, slightly enhanced over the rate in a N2 atmosphere, but the reaction velocity of the haem d1 component was greatly decreased, being apparently limited to that of the rates of CO dissociation from the molecule (0.15s-1 and 0.03s-1). The results are compared with those obtained during a previous study of the reaction of reduced Pseudomonas cytochrome oxidase with oxidized azurin.     

Kinetics of cyanide binding to Chromatium vinosum ferricytochrome c'

The kinetics of the reversible binding of cyanide by the ferric cytochrome c' from Chromatium vinosum have been studied over the pH range 6.9-9.6. The reaction is extremely slow at neutral pH compared to the reactions of other high-spin ferric heme proteins with cyanide. The observed bimolecular rate constant at pH 7.0 is 2.25 X 10(-3) M-1 s-1, which is approximately 10(7)-fold slower than that for peroxidases, approximately 10(5)-fold slower than those for hemoglobin and myoglobin, and approximately 10(2)-fold to approximately 10(3)-fold slower than that recently reported for the Glycera dibranchiata hemoglobin, which has anomalously slow cyanide rate constants of 4.91 X 10(-1), 3.02 X 10(-1), and 1.82 M-1 s-1 for components II, III, and IV, respectively [Mintorovitch, J., & Satterlee, J. D. (1988) Biochemistry 27, 8045-8050; Mintorovitch, J., Van Pelt, D., & Satterlee, J. D. (1989) Biochemistry 28, 6099-6104]. The unusual ligand binding property of this cytochrome c' is proposed to be associated with a severely hindered heme coordination site. Cyanide binding is also characterized by a nonlinear cyanide concentration dependence of the observed rate constant at higher pH values, which is interpreted as involving a change in the rate-determining step associated with the formation of an intermediate complex between the cytochrome c' and cyanide prior to coordination. The pH dependence of both the binding constant for the formation of the intermediate complex and the association rate constant for the subsequent coordination to the heme can be attributed to the ionization of HCN, where cyanide ion binding is the predominant process.(ABSTRACT TRUNCATED AT 250 WORDS)