Deuterium nuclear magnetic resonance study of the interaction of substrates and inhibitors with soybean lipoxygenase

Deuterium NMR spectra for a series of selectively deuterated substrates and inhibitors in the presence of lipoxygenase-1 (EC 1.13.11.12) are presented. Extrapolation of the 2H NMR line widths yielded transverse relaxation rates for the bound inhibitors [2H21]dodecanoic acid (protonated at the 2,2-position), [2,2-2H]dodecanoic acid, and [12,12,12-2H]dodecanoic acid which are 1/T2,bd = 5.0 X 10(3), 1.12 X 10(4), and 1.16 X 10(3) s-1, respectively. The substrates [9,10,12,13-2H]linoleic acid and [11,11-2H]linoleic acid had 1/T2,bd = 8.2 X 10(3) and 7.95 X 10(3) s-1, respectively. Kinetic measurements established Ki = 1.5 X 10(-3) M for dodecanoic acid (lauric acid) inhibition of lipoxygenase when the substrate is linoleic acid (Km = 2.6 X 10(-5) M). Lipoxygenase, with Mr 102,000, is predicted to have a rotational correlation time tau c - 1.2 X 10(-7) s, yielding a 1/T2,bd = 1.56 X 10(4) s-1 for tightly bound ligand. Hence, the correlation times of the selectively deuterated inhibitors indicate internal motions are present in the bound species.     

Occluded calcium sites in soluble sarcoplasmic reticulum Ca2+-ATPase

Rabbit muscle sarcoplasmic reticulum Ca2+-ATPase has been shown to bind gadolinium ion (Gd3+) at two high affinity Ca2+ sites (Stephens, E. M., and Grisham, C. M. (1979) Biochemistry 18, 4876-4885). Gd3+ bound at these sites exhibits an unusually long electron spin relaxation time, consistent with occlusion of these sites and reduced contact with solvent H2O. In this report, the nature of the Gd3+ sites was examined in preparations of the enzyme solubilized with the detergent C12E8. The frequency dependence of water proton relaxation in solutions containing the solubilized Ca2+-ATPase yields dipolar correlation times, tau c, for the 1H-Gd3+ interaction of 1.04 X 10(-9) s for Gd3+ bound at site 1 and 1.98 X 10(-9) s for Gd3+ bound at site 2. The correlation time itself is frequency dependent below 30 MHz, indicating that the correlation time is dominated by the electron spin relaxation time of bound Gd3+. The long values of the correlation time found in the present study are consistent with a poor accessibility of these Gd3+ sites (particularly site 2) to solvent water molecules. Analytical ultracentrifugation and molecular sieve high performance liquid chromatography indicated that the active fraction of the soluble Ca2+-ATPase was monomeric. Thus occlusion of the Ca2+ sites in this enzyme is largely dependent on the tertiary structure of the monomeric ATPase and does not appear to depend on multimeric membrane structures.     

Specific binding of leukotriene B4 to receptors on human polymorphonuclear leukocytes

Leukotriene B4 (5(S),12(R)-di-hydroxy-eicosa-6,14-cis-8,10-trans-tetraenoic acid [LTB4]) is a product of the 5-lipoxygenation of arachidonic acid, which elicits human PMN leukocyte chemotactic responses in vitro that are 50% of the maximal level at concentrations of 3 X 10(-9) M to 10(-8) M and are maximal at 2 X 10(-8) M to 10(-7) M. The specific binding of highly purified [3H]LTB4 to human PMN leukocytes was assessed both by extracting the unbound and weakly bound [3H]LTB4 with acetone at -78 degrees C and by centrifuging the PMN leukocytes through cushions of phthalate oil to separate the unbound from bound [3H]LTB4. The levels of total binding of [3H]LTB4 and of nonspecific binding of [3H]LTB4, in the presence of a 1500-fold molar excess of nonradioactive LTB4, were approximately two times higher with the phthalate oil method. Scatchard plots of the concentration dependence of the specific binding (total - nonspecific binding) of [3H]LTB4 to PMN leukocytes were linear for the acetone extraction and phthalate oil methods and revealed dissociation constants of 10.8 X 10(-9) M and 13.9 X 10(-9) M, respectively, and mean of 2.6 X 10(4) and 4.0 X 10(4) receptors per PMN leukocyte. The 5(S),12(S)-all-trans-di-HETE analog of LTB4 and 5-HETE competitively inhibited by 50% the binding of [3H]LTB4 to PMN leukocytes at respective concentrations that evoked half-maximal chemotactic responses, whereas neither N-formyl-methionyl-leucyl-phenylalanine nor chemotactic fragments of C5 inhibited the binding. Human erythrocytes exhibited no specific binding sites for [3H]LTB4. Human PMN leukocytes possess a subset of receptors for LTB4 that are distinct from those specific for peptide chemotactic factors.     

Soluble complex formation between low-density lipoprotein and glycosaminoglycans. A 2H and 31P-NMR, and quasi-elastic light scattering study

Soluble complex formation between LDL and heparin (HEP) and chondroitin sulfate (CS) has been studied by 2H- and 31P-NMR and light scattering. The 2H-NMR linewidths of [2H]HEP and [2H]C4S increase substantially upon binding to LDL, with the [2H]HEP linewidths broader at low glycosaminoglycan (GAG)/low density lipoprotein (LDL) ratios. Preliminary analysis of the bound C2H3 group correlation times suggests that the observed linewidths are determined by the complex size, and that both [2H]GAGs have similar motions when bound to LDL. The 31P-NMR data demonstrate that large LDL-HEP complexes (diameter approx. 50 nm) are formed only over a narrow range of HEP concentrations, whereas the size of LDL-CS complexes increases continuously over the range of CS concentrations studied, reaching values of 32-35 nm for both C4S and C6S. At the lower protein concentrations studied by light scattering (less than or equal to 1 mg/ml), the same trends are observed, although the mean diameters are less than those estimated by 31P-NMR. Soluble complex formation was unaffected by the presence of 2 mM Ca2+. Dilution studies demonstrate that complex size varies with protein concentration. The binding of GAGs to LDL was also examined by HEP-CS competition studies. HEP has the higher affinity while no differences in binding could be detected between C4S and C6S.     

Unusual mechanism for an aminomutase rearrangement: retention of configuration at the migration termini

The phenylalanine aminomutase from Taxus catalyzes the vicinal exchange of the amino group and the pro-3S hydrogen of (2S)-alpha-phenylalanine to make (3R)-beta-phenylalanine. While the migration of the amino group from C2 of the substrate to C3 of the product is already known to proceed intramolecularly with retention of configuration, the stereochemistry of the hydrogen transfer remained unknown, until now. The chemical shifts of the prochiral hydrogens of authentic (3R)-beta-phenylalanine were established by 1H NMR, and the configuration of each hydrogen was assigned by 2H NMR analysis of a racemic mixture of [2,3-2H2]-(2S,3R)- and (2R,3S)-beta-phenylalanines synthesized via syn addition of deuterium gas with palladium catalyst to stereospecifically reduce the double bond of an N-acetyl enamine. After the aminomutase was incubated with [3,3-2H2]-(2S)-alpha-phenylalanine, the derived deuterium-labeled beta-diastereoisomer product, derivatized as the N-acetyl methyl ester, was analyzed by 2H NMR, which revealed that the mutase shuttles the pro-3S hydrogen to C2 of the beta-isomer product (designated 2S,3R) with retention of configuration. Retention of configuration at both reaction termini is unique among all aminomutase mechanisms examined so far. Furthermore, the dynamics of the Cbeta-H bond of the substrate were measured in a competitive experiment with deuterium-labeled substrate to calculate a primary kinetic isotope effect on Vmax/KM of 2.0 +/- 0.2, indicating that C-H bond cleavage is likely rate limiting. Isotope exchange data indicate that the migratory deuterium of [2H8]-(2S)-alpha-phenylalanine, at saturation, dynamically exchanges up to 75%, with protons from the solvent during the reaction after the first 10% of product is formed. The calculated equilibrium constant of 1.1 indicates that the beta-isomer was slightly favored relative to the alpha-isomer at 30 degrees C.     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affinity (Kd = 10(-9) M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [3H]tamoxifen binding capacity is thermolabile, being most stable at 4 degrees C and rapidly lost at 37 degrees C. More [3H]tamoxifen, however, is specifically bound at incubation temperatures of 25 degrees C and 37 degrees C than at 4 degrees C although prewarming nuclei has no effect, suggesting exchange of [3H]tamoxifen for an unidentified endogeneous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (Kd = 10(-9) M) [3H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78 +/- 0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000 X g, 30 min) contains high-capacity (955 +/- 405 (S.D.) fmol/mg protein), low-affinity (Kd = 10.9 +/- 4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000 X g, 60 min) contains low-capacity (46 +/- 15 (S.D.) fmol/mg protein), high-affinity (Kd = 0.61 +/- 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%-3.2%, and nuclear sites less than 0.5% of total sites.     

Interactions of Lycopodium alkaloids with acetylcholinesterase investigated by 1H NMR relaxation rate

In order to further understand the interaction processes between the Lycopodium alkaloids and acetylcholinesterase, the binding properties of N-acetyl huperzine A (1), huperzine B (2) and huperzine F (3) with Torpediniforms Nacline acetylcholinesterase (TnAchE) were investigated by 1H NMR methods. The nonselective, selective and double-selective spin-lattice relaxation rates were acquired in the absence and presence of TnAchE at a ratio of [ligand]/[protein]=1:0.005. The selective relaxation rates show protons of 1-3 have dipole-dipole interaction with protons of TnAchE at the binding interface. The molecular rotational correlation time of bound ligands was calculated by double-selective relaxation rate at 298 K, which showed that 1-3 had high affinity with the protein. The results indicate that investigation of 1H NMR relaxation data is a useful method to locate the new Lycopodium alkaloids as AchE inhibitors.     

Nuclear magnetic relaxation studies of cytidine and its complexing dimethyl sulfoxide solution.

13C spin-lattice relazation times (T1 values) in cytidine were determined experimentally to investigate molecular motions of both metal-free and ion-complexed cytidines in dimethylsulfoxide solutions. It was found that the correlation times of the protonated carbons were equal within experimental error, and this equality of correlation times of different sites of the molecule suggests strongly isotropic random motion of the molecule. Correlation times for internal motion of the amino group obtained from the observed T1 of the amino protons are 4.6 -10(-11) S, 2.0 - 10(-9) S and 1.1 - 10(-10) S for the metal-free cytidine and the cytidine complexed with either CaCl2 or ZnCl2, respectively. An experimental value of T1 of the H6 proton of the cytidine base agrees very well with the value estimated from a conformation determined by the nuclear "Overhauser" effect. Spin-lattice relaxation time measurements of the 7Li nucleus in the LiCl/cytidine system strongly suggested that the 7Li cation is directly coordinated with cytidine.     

A detailed analysis of the motions of cholesterol in biological membranes by 2H-NMR relaxation

Spin-lattice relaxation, T1z, measurements of [2,2,3,4,4,6-2H6]cholesterol in model membranes of DMPC were performed as a function of temperature, Larmor frequency and position of labelling in the fused ring system. The results are interpreted according to a hierarchy of motions, such that motion i of correlation time tau i reduces the residual ordering set, characterizing motions i-1, i-2, etc..., by the amount Si = d(2)00(beta i), where beta i is the angle between the axes of motional averaging of motions i and i-1, respectively and d(2)00 is the Wigner rotation matrix element. The appearance of minima in the temperature dependence of T1z for cholesterol, at 46.1 MHz and 30.7 MHz, and the scaling of these T1z (min) according to the orientation of each individual C-2H bond with respect to the axis of motional averaging of cholesterol, allows assignment of the sterol axial rotation to the second fastest motion, characterized by a correlation time of 3.2 X 10(-9) s at 25 degrees C and an activation energy of 32 +/- 5 kJ X mole-1. The fastest motion of cholesterol in DMPC could be a very rapid libration, 'wobbling', which does not contribute significantly to the T1z relaxation of cholesterol at physiological temperatures and Larmor frequencies smaller than 50 MHz, but does reduce the ordering of the cholesterol molecule in DMPC from S0 = 1 to S1 = 0.8, at 25 degrees C.     

Binding of (6R,S)-methyltetrahydrofolate to methyltransferase from Clostridium thermoaceticum: role of protonation of methyltetrahydrofolate in the mechanism of methyl transfer

The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoacetium catalyzes transfer of the N5-methyl group of (6S)-methyltetrahydrofolate (CH3-H4folate) to the cob(I)amide center of a corrinoid/iron-sulfur protein (CFeSP), forming H4folate and methylcob(III)amide. We have investigated binding of 13C-enriched (6R,S)-CH3-H4folate and (6R)-CH3-H4folate to MeTr by 13C NMR, equilibrium dialysis, fluorescence quenching, and proton uptake experiments. The results described here and in the accompanying paper [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 5728-5735] constitute the first evidence for protonation of the pterin ring of CH3-H4folate. The pH dependence of the chemical shift in the 13C NMR spectrum for the N5-methyl resonance indicates that MeTr decreases the acidity of the N5 tertiary amine of CH3-H4folate by 1 pK unit in both water and deuterium oxide. Binding of (6R,S)-CH3H4folate is accompanied by the uptake of one proton. These results are consistent with a mechanism of activation of CH3-H4folate by protonation to make the methyl group more electrophilic and the product H4folate a better leaving group toward nucleophilic attack by cob(I)amide. When MeTr is present in excess over (6R,S)-13CH3-H4folate, the 13C NMR signal is split into two broad signals that reflect the bound states of the two diastereomers. This unexpected ability of MeTr to bind both isomers was confirmed by the observation of MeTr-bound (6R)-13CH3-H4folate by NMR and by the measurement of similar dissociation constants for (6R)- and (6S)-CH3-H4folate diastereomers by fluorescence quenching experiments. The transversal relaxation time (T2) of 13CH3-H4folate bound to MeTr is pH independent between pH 5.50 and 7.0, indicating that neither changes in the protonation state of bound CH3-H4folate nor the previously observed pH-dependent MeTr conformational change contribute to broadening of the 13C resonance signal. The dissociation constant for (6R,S)-CH3-H4folate is also pH independent, indicating that the role of the pH-dependent conformational change is to stabilize the transition state for methyl transfer, and not to favor the binding of CH3-H4folate.     

Enzyme-inhibitor complexes of lysozyme with glucosamine inhibitors. A molecular dynamics study through 2H-NMR

2H relaxation measurements coupled with multiple specific 2H labeling have provided insight into the molecular dynamics of N-acetyl-D-glucosamine (GlcNAc) inhibitors bound to lysozyme. Deuteron T1 and T2 data for the bound state of methyl alpha- and -beta-GlcNAc 2H-labeled in the glycosidic methyl and C2 positions have been derived from measurements at different enzyme/inhibitor ratios. Rotational correlation times calculated therefrom for the labeled sites indicate, in both cases, tight binding for the sugar ring (tau(b) = 3.0 x 10(-9) s) accompanied by fast internal rotation, about one axis, of the glycosidic methyl groups (tau(r) = 5.5-7.6 x 10(-11) s). The small but consistent difference in the rates of internal rotation for the alpha- and beta-anomeric inhibitors may be indicative of different solution structures of the enzyme-inhibitor complexes.     

Wheat germ agglutinin dimers bind sialyloligosaccharides at four sites in solution: proton nuclear magnetic resonance temperature studies at 360 MHz

Equilibrium binding studies have been performed over a range of temperatures from 25.4 to 47.3 degrees C between wheat germ agglutinin isolectin I (WGA I) and the alpha 2-3 isomer of (N-acetylneuraminyl)lactose (NeuNAc alpha 2-3Gal beta 1-4G1c). Proton nuclear magnetic resonance spectroscopy at 360 MHz has been used to monitor titrations in this system under conditions where the fraction of total ligand which is bound is small, yet the fractional occupation of sites covers a wide range. Several of the ligand resonances, including the N-acetyl methyl and the axial and equatorial hydrogens at carbon 3 of the NeuNAc residue, are shifted and broadened in the presence of WGA due to chemical exchange between the free and bound environments. The lifetime broadening of the N-acetyl resonance at room temperature of a series of related sialyloligosaccharides has been previously used by us to measure binding affinities to two WGA isolectins [Kronis, K.A., & Carver, J.P. (1982) Biochemistry 21, 3050-3057]. In this paper we report the temperature dependence of the apparent bound shifts and the apparent bound line widths of the N-acetyl, H3a, and H3e peaks. The true bound shifts for the three resonances have been obtained from these data by using the equations derived by Swift and Connick [Swift, T.J., & Connick, R.E. (1962) J. Chem. Phys. 37, 307-320]. The total bound shifts, per monomer, were found to be -1.98, -4.0, and -0.8 ppm for the N-acetyl, the H3a, and the H3e resonances, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation-specific monoclonal antibodies to the calcium-induced structure of protein C

Monoclonal antibodies to various domains of human protein C were characterized, and the cross-reactivity of these antibodies with other vitamin K-dependent proteins was explored. Three antibodies, JTC-1, -2, and -3 reacted with protein C only in the presence of Ca2+ and were shown to bind to the light chain of protein C. It is suggested that these antibodies recognize a gamma-carboxyglutamic acid domain-related conformational change induced by metal ions, evidenced by the fact that half-maximal binding was observed at calcium concentration of 0.5, 0.6, and 0.7 mM, respectively, by the fact that these antibodies, even in the presence of Ca2+, do not react with gamma-carboxyglutamic acid domainless protein C, and by the fact that Zn2+ and Tb3+ support binding in essentially the same way. Each cell line was stabilized by recloning five times. In addition each antibody had a single isoelectric point and was of the IgG1 kappa class. The interaction of antibodies JTC-1, -2; and -3 with protein C-Ca2+ was characterized by a single class of binding sites with Kd of 3.98 X 10(-9) M, 4.01 X 10(-9) M, and 6.76 X 10(-9) M, respectively. However, antibodies JTC-1, -2, and -3 bound to prothrombin-Ca2+ with Kd of 7.81 X 10(-9) M, 2.0 X 10(-7) M, and higher than 1.0 X 10(-5) M, respectively. In addition they had weak affinity for factor X in the presence of Ca2+. The results indicate that the antibodies JTC-1, -2, and -3 are conformation-specific monoclonal antibodies directed against an at least partially common metal ion-induced three-dimensional structure in protein C, prothrombin, and factor X.     

Interaction of (3H) bongkrekic acid with the mitochondrial adenine nucleotide translocator.

Chemical labeling by 3H and biosynthetic labeling by 14C of bongkrekic acid (BA) are described. In the rat liver cell, mitochondria are the only subcellular particles to bind [3H]BA with high affinity. The high affinity sites for BA in mitochondria are located in the inner membrane. High affinity binding sites for BA are only displayed at pH below 7; they amount to 0.15-0.20 nmol/mg of protein in rat liver mitochondria and to 1.1-1.3 nmol/mg of protein in rat heart mitochondria. These values are similar to those found for the high affinity atractyloside binding sites and for the carboxyatractyloside binding sites. The kinetic parameters for BA binding to rat heart mitochondria at 20 degrees C are Kd = 10-40 X 10(-9) M, k+1 = 0.7 X 10(5) M-1 s-1, k-1 = 1.4 X 10(-3) M s-1. Binding assays carried out with rat heart mitochondria, under equilibrium conditions, showed that the amount of BA bound to high affinity sites increases with temperature and reaches the maximum value of 1.1-1.3 nmol/mg of protein at 32-35 degrees C. At lower temperatures, and under equilibrium conditions, a significant fraction of high affinity sites remains masked and is not titrated by BA; these masked BA sites are revealed by addition of micromolar concentrations of ADP or by energization of the mitochondria. Carboxyatractyloside added to rat heart mitochondria preloaded with [3H]BA is able to displace part of the bound [3H]BA. Displacement of the bound BA is enhanced by simultaneous additions of carboxyatractyloside plus ADP, or by energization of the mitochondria. The synergistic effect of carboxyatractyloside and ADP on displacement of bound [3H]BA is also observed in isolated inner membrane vesicles from rat liver mitochondria. When BA is preincubated with rat heart mitochondria before addition of [14C]ADP for assay of ADP transport, the inhibition of ADP transport is a mixed-type inhibition. When BA is preincubated with the mitochondria together with a very small concentration of ADP (less than 0.5 muM), the inhibition of [14C]ADP transport is markedly increased (up to ten times) and it becomes typically uncompetitive, which suggests the formation of a ternary complex, carrier-ADP-BA. The transition from a mixed-type inhibition, with high Ki value, to an uncompetitive type of inhibition, with low Ki value, upon addition of ADP, is explained by an ADP-induced conformational change of the ADP translocator.     

Characterization of the ethenoadenosine diphosphate binding site of myosin subfragment 1. Energetics of the equilibrium between two states of nucleotide.S1 and vanadate-induced global conformation changes detected by energy transfer

The fluorescence decay of 1,N6-ethenoadenosine diphosphate (epsilon ADP) bound to myosin subfragment 1 (S1) was studied as a function of temperature. The decay was biexponential, and the two lifetimes were quenched relative to the single lifetime of free epsilon ADP. The temperature dependence of the fractional intensities of the decay components showed two states of the S1.epsilon ADP complex. At pH 7.5 in 30 mM TES, 60 mM KCl, and 3 mM MgCl2, the equilibrium constant for the conversion of the low-temperature state (S1L.epsilon ADP) to the high-temperature state (S1H.epsilon ADP) was 40 at physiological temperatures, and delta H degrees = 13 kcal.mol-1 and delta S degrees = 49 cal.deg-1.mol-1. At 10 degrees C the equilibrium constant of S1 for epsilon ADP was 5, indicating that S1H.epsilon ADP was the dominant state, and that for the vanadate complex epsilon ADP.Vi was 0.7, suggesting that in S1.epsilon ADP.Vi the dominant state of the S1-nucleotide complex was converted from S1H.epsilon ADP to S1L.epsilon ADP. The single rotational correlation time of bound epsilon ADP at 10 degrees C decreased from 107 ns in S1.epsilon ADP to 74 ns in S1+.epsilon ADP.Vi. Conversion of the binary complex to the ternary vanadate complex resulted in a 3-A decrease in the energy transfer distance between bound epsilon ADP and N-[4-(dimethylamino)-3,5-dinitrophenyl]maleimide attached to SH1 and a decrease of the average distance between bound epsilon ADP and bound Co2+ from 12.6 to 8.3 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibodies directed against gonococcal protein I vary in bactericidal activity

Monoclonal antibodies (Mab) with specificity for protein I (PI) from Neisseria gonorrhoeae (GC) were examined for bactericidal activity. Mab 4G5 (gamma 3), ID3 (gamma 2a), and 1G6 (gamma 2a) bound to surface-exposed epitopes on PI of GC strain R11 (IA serotype) as assessed by co-agglutination and 125I protein A uptake. Mab 2H1 (gamma 3) that were directed against IB serotype strains and Mab 2E9 (gamma 2a) were negative in co-agglutination and protein A uptake assays and served as controls for some experiments. Only 4G5 and 1D3 were bactericidal for R11 when presensitized organisms were incubated in 10% absorbed, pooled normal human serum (PNHS) or 10% hypogammaglobulinemic serum (H gamma S) despite binding of nearly equivalent numbers of 4G5, 1D3, and 1G6 to R11 during presensitization, as assessed by 125I-protein A uptake. These Mab activated complement to a similar extent on GC R11, leading to deposition of 56.4 X 10(3), 61.9 X 1093), and 47.1 X 10(3) molecules of C3/organism during incubation in 10% C8-deficient serum. Deposition occurred almost exclusively via the classical complement pathway. Measurement of complement component C9 binding to R11 during incubation in H gamma S showed 35,700 molecules of C9/organism with 4G5, 32,600 C9/organism with 1D3, and surprisingly, 29,600 C9/organism with 1G6. Eight thousand four hundred molecules of C9/organism bound to 2E9-coated organisms, 6000 C9/organism to 2H1-coated bacteria, and 3600 C9/organism to nonpresensitized organisms. The C5b-9 complex deposited by 4G5 had a different sedimentation profile by sucrose density gradient analysis from the C5b-9 complex deposited by 1G6, consistent with a different molecular configuration of the bound complex. Mab 1G6 and 1D3, but not 2E9 or 2H1, were able to compete with 125I-4G5 for binding to GC R11. A Mab (2E6) directed against protein III of GC competed weakly with 125I-4G5 for binding to GC R11. Mab 1G6, but not 1D3, blocked 4G5-dependent killing in a dose-related fashion. Both 4G5 and IG6 reacted weakly with native PI of GC R11 by immunoblotting, but neither Mab recognized the 34,800 m.w. fragment of PI generated by trypsin and chymotrypsin treatment of outer membranes. In contrast, 2E9 reacted strongly by immunoblot with both native and cleaved PI of GC R11, suggesting binding to buried determinants of PI. These experiments show that Mab directed against identical or closely associated, surface-exposed epitopes on gonococcal PI differ markedly in bactericidal activity, despite leading to deposition of nearly equivalent numbers of C3 and C9 molecules per organism.     

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.     

Kinetic study of interaction between [14C]amphotericin B derivatives and human erythrocytes: relationship between binding and induced K+ leak

The relationship between polyene antibiotic binding to red cells and their membrane permeabilization was studied using two 14C-labelled amphotericin B (AmB) derivatives: N-fructosyl AmB and N-acetyl methyl ester AmB. The binding kinetics of both derivatives were determined on whole red cells and ghosts. The resulting experimental points were well fitted by monoexponential functions, and the characteristic t1/2 for both derivatives were calculated from these functions. At 2 X 10(-5) M, the half time t1/2 for N-acetyl methyl ester AmB (30.2 min) which forms aqueous aggregates was longer than the t1/2 for the more soluble species N-fructosyl AmB (4.5 min). At lower concentrations (10(-7) M), the t1/2 for N-acetyl methyl ester AmB (6.3 min) in a more solubilized form was close to that of N-fructosyl AmB (7.9 min). These results suggest that only solubilized species bound to red cell membranes and that disaggregation of aggregates is the limiting step in the binding process. The permeabilization of red cell membranes by N-fructosyl AmB, measured as intracellular K+ leak, was not instantaneous and at 10 degrees C external K+ was only detected 20 min after antibiotic addition. In contrast, binding occurs without lag time. Consequently, different mecanisms underlie binding and K+ permeability inducement. Absorption spectroscopy data showed that bound antibiotic is located in the hydrophobic membrane interior and that this penetration of the membrane by AmB derivatives occurs without lag time. Consequently, the lag time occurring for K+ permeability inducement would be due to some steps subsequent to binding and probably located in the hydrophobic membrane interior. This statement is further supported by the observation that the lag time is sensitive to changes in membrane fluidity as shown here by the break between 20 and 30 degrees C in the slope of the Arrhenius plot for the lag time, coinciding with the phase transition in red cell membranes.     

Heavy metal binding to heparin disaccharides. II. First evidence for zinc chelation.

To map out the heavy metal binding sites of iduronic acid containing oligosaccharides isolated from human kidneys, we studied Zn(II) binding by nuclear magnetic resonance (NMR) and molecular modeling to two disaccharides isolated after nitrous acid depolymerization of heparin and two synthetic disaccharides representative of the heparin structure, namely, IdopA2S (alpha 1,4)AnManOH, 1 alpha, IdopA2S (alpha 1,4)AnManOH6S, 1b, IdopA2S-(alpha 1,4)GlcNS alpha Me, 2a, and IdopA2S (alpha 1,4)GlcNS6S alpha Me, 2b (see previous article in this series). A conformational analysis of the metal free and metal bound solutions was made by comparing calculated [(NOE)]s, [T1]s, and [J]s to experimental values. The 1C4, 4C1, and 2S0 conformations of the L-idopyranosiduronate ring and the 4E and 4T3 of the anhydro-D-mannitol ring are evaluated as are rotations about the C5-C6 hydroxymethylene of the AnManOH(6S) or GlcNS (6S) residues. The NOE between IdopA2S H1 and H3 and the known NOE between H2 and H5, as well as the T1 of IdopA2S H3, are introduced as NMR observables sensitive to the IdopA2S ring conformation. Similarly, a NOE between IdopA2S H5 and AnManOH(6S) or GlcNS(6S) H3 was observed that directly restricts the allowed interglycosidic conformational space. For all disaccharides, the Zn(II) bound spectral data are consistent with models in which these motions are partially "frozen" such that the 1C4 conformation of the IdopA2S is stabilized along with the 4T3 conformation of the AnManOH(6S) ring. The interglycosidic conformation is also stabilized in one of two minima. Electrostatic potential energy calculations gave the best overall agreement with experiment and suggest metal binding conformations with the carboxylate and ring oxygen of the IdopA2S residues (1C4 conformation) and either O3 of the GlcNS(6S) residues or the sulfate oxygens of the 6-sulphate for 2b providing additional chelating sites. These chelation models concur with the observation of marked 13C and 1H NMR chemical shifts for the IdopA2S resonances and of GlcNS H3 for 2 alpha and GlcNS6S C6 for 2b. This study of model compounds implicates the IdopA2S(alpha 1,4)GlcNS6S group as part of the heavy metal binding site in biologically important acidic oligosaccharides such as heparin.     

Mechanism of adenylate kinase. Is there a relationship between local substrate dynamics, local binding energy, and the catalytic mechanism?

Adenylyl (beta,gamma-methylene)diphosphonic acid (AMPPCP) labeled with deuterium at the adenine ring ([8-2H]AMPPCP) and at the beta,gamma-methylene group (AMPPCD2P), as well as adenosine 5'-monophosphate labeled at the adenine ring ([8-2H]AMP), was synthesized and used for deuterium nuclear magnetic resonance (NMR) determination of effective correlation times (tau c) of the free nucleotide and the complexes with adenylate kinase (AK). Extensive and rigorous control experiments and theoretical analysis were performed to justify the validity of the experimental approaches, particularly the fast exchange condition, and the reliability of the tau c values obtained. For the free nucleotide, the results suggest that the phosphonate group of free AMPPCP possesses appreciable local mobility relative to the adenine ring and that complexation with Mg2+ greatly reduced such a local mobility. For the complexes with AK, effective tau c values of 7, 15, 28, 28, and 27 ns were obtained for AMPPCD2P, MgAMPPCD2P, [8-2H]AMPPCP, Mg[8-2H]AMPPCP, and [8-2H]AMP, respectively. These results suggest that the adenine ring of substrates is rigidly bound in all cases, that the phosphonate chain of AMPPCP possesses considerable local mobility, and that Mg2+ reduces such local mobility but does not totally immobilize it. The local dynamics of the analogues bound to AK was correlated with local binding energies for the binding of MgAMPPCP and MgATP to AK estimated from the binding studies by proton NMR and other techniques, in conjunction with the binding theory of Jencks [Jencks, W. P. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4046-4050]. The results suggest that no general correlation exists between the local rigidity of portions of a bound substrate and the corresponding (ground state) local binding energy contributed by these portions. In particular, the adenosine moiety contributes little to the binding energy despite the fact that the adenine ring is rigidly bound; the triphosphate (PPPi) moiety behaves oppositely; Mg2+ immobilizes the triphosphate chain but does not enhance binding. Finally, isomers of the substitution-inert beta,gamma-bidentate Cr(III) complexes of adenosine 5'-triphosphate (CrATP) were used to probe two unresolved catalytic problems implicitly related to the local mobility of the phosphonate chain of AMPPCP in the AK-MgAMPPCP complex. The first problem concerns the result of electron paramagnetic resonance (EPR) studies that (Rp)- but not (Sp)-[beta-17O]ATP caused a line broadening in the Mn(II) EPR spectrum of the AK-MnATP complex [Kalbitzer, H. R., Marquetant, R., Connolly, B. A., & Goody, R. S. (1983) Eur. J. Biochem. 133, 221-227].(ABSTRACT TRUNCATED AT 400 WORDS)