Molecular evolution of antibody affinity for sensitive detection of botulinum neurotoxin type A

Botulism is caused by botulinum neurotoxin (BoNT), the most poisonous substance known. Potential use of BoNT as a biothreat agent has made development of sensitive assays for toxin detection and potent antitoxin for treatment of intoxication a high priority. To improve detection and treatment of botulism, molecular evolution and yeast display were used to increase the affinity of two neutralizing single chain Fv (scFv) antibodies binding BoNT serotype A (BoNT/A). Selection of yeast displayed scFv libraries was performed using methods to select for both increased association rate constant (k(on)) and decreased dissociation rate constants (k(off)). A single cycle of error prone mutagenesis increased the affinity of the 3D12 scFv 45-fold from a K(D) of 9.43x10(-10)M to a K(D) of 2.1x10(-11)M. Affinity of the HuC25 scFv was increased 37-fold from 8.44x10(-10)M to 2.26x10(-11)M using libraries constructed by both random and site directed mutagenesis. scFv variable region genes were used to construct IgG for use in detection assays and in vivo neutralization studies. While IgG had the same relative increases in affinity as scFv, (35-fold and 81-fold, respectively, for 3D12 and HuC25) higher solution equilibrium binding constants were observed for the IgG, with the 3D12 K(D) increasing from 6.07x10(-11)M to 1.71x10(-12)M and the HuC25 K(D) increasing from 4.51x10(-11)M to 5.54x10(-13)M. Affinity increased due to both an increase in k(on), as well as slowing of k(off). Higher affinity antibodies had increased sensitivity, allowing detection of BoNT/A at concentrations as low as 1x10(-13)M. The antibodies will also allow testing of the role of affinity in in vivo toxin neutralization and could lead to the generation of more potent antitoxin.     

Kinetics of beta-lactam interactions with penicillin-susceptible and -resistant penicillin-binding protein 2x proteins from Streptococcus pneumoniae. Involvement of acylation and deacylation in beta-lactam resistance.

Kinetic interactions of beta-lactam antibiotics such as penicillin-G and cefotaxime with normal, penicillin-susceptible PBP2x from Streptococcus pneumoniae and a penicillin-resistant PBP2x (PBP2x(R)) from a resistant clinical isolate (CS109) of the bacterium have been extensively characterized using electrospray mass spectrometry coupled with a fast reaction (quench flow) technique. Kinetic evidence for a two-step acylation of PBP2x by penicillin-G has been demonstrated, and the dissociation constant, K(d) of 0.9 mm, and the acylation rate constant, k(2) of 180 s(-1), have been determined for the first time. The millimolar range K(d) implies that the beta-lactam fits to the active site pocket of the penicillin-sensitive PBP rather poorly, whereas the extremely fast k(2) value indicates that this step contributes most of the binding affinity of the beta-lactam. The values of K(d) (4 mm) and k(2) (0.56 s(-1)) were also determined for PBP2x(R). The combined value of k(2)/K(d), known as overall binding efficiency, for PBP2x(R) (137 m(-1) s(-1)) was over 1000-fold slower than that for PBP2x (200,000 m(-1) s(-1)), indicating that a major part is played by the acylation steps in penicillin resistance. Most of the decreased binding efficiency of PBP2x(R) comes from the decreased ( approximately 300-fold) k(2). Kinetic studies of cefotaxime acylation of the two PBP2x proteins confirmed all of the above findings. Deacylation rate constants (k(3)) for the third step of the interactions were determined to be 8 x 10(-6) s(-1) for penicilloyl-PBP2x and 5.7 x 10(-4) s(-1) for penicilloyl-PBP2x(R), corresponding to over 70-fold increase of the deacylation rate for the resistant PBP2x(R). Similarly, over 80-fold enhancement of the deacylation rate was found for cefotaxime-PBP2x(R) complex (k(3) = 3 x 10(-4) s(-1)) as compared with that of cefotaxime-PBP2x complex (3.5 x 10(-6) s(-1)). This is the first time that such a significant increase of k(3) values was found for a beta-lactam-resistant penicillin-binding protein. These data indicate that the deacylation step also plays a role, which is much more important than previously thought, in PBP2x(R) resistance to beta-lactams.     

Measurement of the affinity and phosphorylation constants governing irreversible inhibition of cholinesterases by di-isopropyl phosphorofluoridate

1. A procedure is described for determining the affinity constant K(a) and the phosphorylation constant k(p) for the inhibition by di-isopropyl phosphorofluoridate of erythrocyte acetylcholinesterase and serum cholinesterase. The procedure depends on the use of a specially designed reaction vessel with which incubation times as short as 1.2sec. could be obtained at any convenient temperature. 2. The K(a) of acetylcholinesterase decreased from 1.58 (+/-0.22)x10(-3)m at 5 degrees to 1.17 (+/-0.10)x10(-3)m at 25 degrees and the associated change in enthalpy was 2980 cal. 3. The k(p) of acetylcholinesterase increased from 11.9 (+/-0.7)min.(-1) at 5 degrees to 40.7 (+/-1.4)min.(-1) at 25 degrees , indicating an activational energy of 9600 cal. The change in entropy associated with K(a) was 23.5 cal. degree(-1) at 25 degrees . 4. At 5 degrees , the K(a) and k(p) of serum cholinesterase were 9.95 (+/-1.10)x10(-6)m and 11.2 (+/-0.63)min.(-1) respectively. 5. The 150-fold difference in the inhibitory power of di-isopropyl phosphorofluoridate for the two cholinesterases was attributed entirely to differences in affinity.     

Efflux of a suppressive neurotransmitter, GABA, across the blood-brain barrier

In this study, GABA efflux transport from brain to blood was estimated by using the brain efflux index (BEI) method. [3H]GABA microinjected into parietal cortex area 2 (Par2) of the rat brain was eliminated from the brain with an apparent elimination half-life of 16.9 min. The blood-brain barrier (BBB) efflux clearance of [3H]GABA was at least 0.153 mL/min/g brain, which was calculated from the elimination rate constant (7.14 x 10(-2) x min(-1)) and the distribution volume in the brain (2.14 mL/g brain). Direct comparison of the apparent BBB influx clearance [3H]GABA (9.29 microL/min/g brain) and the apparent efflux clearance (153 microL/min/g brain) indicated that the efflux clearance was at least 16-fold greater than the influx clearance. In order to reduce the effect of metabolism in the neuronal cells following intracerebral microinjection, we determined the apparent efflux of [3H]GABA in the presence of nipecotic acid, a GABA transport inhibitor in parenchymal cells, using the BEI method. Under such conditions, the elimination of [3H]GABA across the BBB showed saturation and inhibition by probenecid in the presence of nipecotic acid. Furthermore, the uptake of [3H]GABA by MBEC4 cells was inhibited by GABA, taurine, beta-alanine and nipecotic acid in a concentration-dependent manner. It is likely that GABA inhibits the first step in the abluminal membrane uptake by brain endothelial cells, and that probenecid selectively inhibits the luminal membrane efflux transport process from the brain capillary endothelial cells based on the in vivo and in vitro evidence. The BBB acts as the efflux pump for GABA to reduce the brain interstitial fluid concentration.     

Kinetic evidence for two interconvertible forms of the folate transport protein from Lactobacillus casei.

Lactobacillus casei cells contain a folate transport protein which exhibits a high affinity for folate. The dissociation constant (KD) for folate derived from binding parameters at the steady state (at 0 degree C) is 0.4 nM at pH 7.5 and 0.1 nM at pH 6.0. In the present study, folate binding to this protein at pH 7.5 (and 0 degree C) was shown to follow second-order kinetics and to proceed with an association constant (k+1) of 4.9 X 10(7) liter/mol per min. K+1 was not affected by preincubation conditions which alter the energetic state of the cell. Measurements on the extent of binding showed further that (at 0 degree C) essentially all unoccupied folate-binding sites reside at or are readily accessible to the outer surface of the membrane. In contrast, after saturating the binding site with [3H]folate, the first-order rate constant (k-1) for dissociation of the bound substrate (at 0 degree C) was found to vary substantially with the conditions employed. k-1 was 0.028/min in freshly harvested cells, but it increased by 2.8-fold in cells preincubated at 23 degrees C for 60 min and by 5.4-fold in isolated membranes. In addition, the faster rate observed in preincubated cells (k-1 = 0.077/min) returned to a slower rate after brief exposure of the cells to pH 6.0 (k-1 = 0.041/min), glucose (k-1 = 0.050/min), or both (k-1 = 0.012/min). k-1 was twofold lower at pH 6.0 than at pH 7.5 and was less dependent on the preincubation conditions, although it also increased substantially (5.5-fold) when the cells were converted to plasma membranes. The proposed explanation for these results is that folate transport protein of L. casei exists in two forms which can be distinguished by the accessibility of the binding site to the external medium and whose amounts are dependent upon the presence of bound folate, the pH, and the energetic state of the cell. It is suggested that these forms are transport proteins with binding sites oriented towards the inner and outer surfaces of the membrane.     

Characterization of beta-adrenergic receptors by (3H) isoproterenol in adipocytes of humans and rats

The specific binding of (3H) isoproterenol to isolated fat cells of human and rat was characterized. Binding of (3H) isoproterenol to isolated fat cells of rat was saturable with 420 pmol of (3H) isoproterenol bound/100 mg of lipid. Half-maximal saturation occurred at 5 microM providing an estimate of the equilibrium dissociation constant, KD, for the interaction of (3H) isoproterenol with its binding sites. Kinetic analysis of (3H) isoproterenol binding provided a value of 2.01 x 10(4) min-1. M-1 for the forward bimolecular rate constant, k1. Dissociation of (3H) isoproterenol was a first order reaction with a rate constant, k2, of 0.62 x 10(-1) min-1. The ratio k2/k1 = 3.07 microM provides an independent measurement of the KD for the interaction of (3H) isoproterenol with its binding sites which is in agreement with the values obtained by steady state analysis (3 to 5 microM). The apparent equilibrium dissociation constant, KD, for the interaction of (3H) isoproterenol with its receptor in human fat cells obtained by steady state analysis was 1 to 0.9 microM. Scatchard- and Hill-analysis suggest the possibility of different negatively cooperative interactions among the binding sites in human and rat. beta-Adrenergic agonists competed for the binding sites. The order of potency was isoproterenol greater than epinephrine greater than norepinephrine. Compounds such as DOPA, dopamine and (m-Hydroxyphenyl)2-methyl-aminoethanol which are structurally related to catecholamines had little or no affinity for (3H) isoproterenol binding sites.     

Prediction of the pharmacokinetic profiles of doxycycline in humans based on the results of experiments in animals

Applicability of the "pharmacokinetic time" concept in animal scale-up was evaluated by the findings of the pharmacokinetic study of doxycycline after its bolus intravenous administration to rats in doses of 9 and 18 mg/kg and to cats as 1-hour constant rate infusion in doses of 3.8 and 7.6 mg/kg. Analysis of the pharmacokinetic profiles in the plot of the logarithmic ratio of concentration/dose to "pharmacokinetic time" i. e. time related to body weight raised to the power 0.25 showed that the slopes of the curves for rats, cats and humans (the literature data, intravenous bolus administration in a dose of 2.9 mg/kg) were practically similar. However, no complete coincidence of the curves was observed. When expressed in the "pharmacokinetic time" scale the half lives were equal to 4.6-5.4, 3.5-3.7 and 5.2 h.kg-0.25 respectively. The difference was 1.5-fold while with using the chronological time the difference was about 5-fold (3.1 hours in rats and 15.1 hours in humans). Therefore, with using the "pharmacokinetic time" 10-fold species differences in the total clearance (0.55 and 0.056 l.h-1.kg-1 in rats and humans respectively) transformed into 2-fold differences (0.37 and 0.16 l.h-1.kg-0.75 respectively). Prediction of doxycycline half lives in humans by the experimental findings was successful.     

Pharmacokinetics of pralidoxime chloride in the rat

The pharmacokinetics of pralidoxime chloride (2-PAM) was studied in rats. Different groups of rats were given an intramuscular injection of 2-PAM at one of three doses (20, 40, or 80 mg/kg). This range of doses is used commonly in studies concerned with the efficacy of 2-PAM against poisoning by potent organophosphorus inhibitors of cholinesterase enzyme. Individual, sequential blood samples were collected during the course of the experiment. From these blood samples the plasma concentrations of 2-PAM were determined over time for each animal. Next the relationship of plasma concentration to time was expressed in terms of a standard pharmacokinetic model. Estimates of various pharmacokinetic parameters were calculated using an open, one-compartment model: volume of distribution (Vd), maximal plasma concentration (Cmax), elimination rate constant (k10), absorption rate constant (k01), area under the curve (AUC) and clearance (CL). Of the pharmacokinetic estimates, only Cmax and AUC were found to be statistically significant (p less than 0.0001) when compared across all the doses; these pharmacokinetic estimates were highly correlated with doses with r = 0.998 and r = 0.997, respectively. However, when AUC and Cmax were normalized by dividing through by dose, no significant differences were found in the transformed data. The results of this study in rat indicate that the pharmacokinetics of 2-PAM is linearly related to dose in a range employed in therapeutic studies of 2-PAM.     

Analysis of the binding of the Fab fragment of monoclonal antibody NC10 to influenza virus N9 neuraminidase from tern and whale using the BIAcore biosensor: effect of immobilization level and flow rate on kinetic analysis.

The binding of the Fab fragment of monoclonal antibody NC10 to influenza virus N9 neuraminidase, isolated from tern and whale, was measured using an optical biosensor. Both neuraminidases, homotetramers of 190 kDa, were immobilized to avoid multivalent binding, and the binding of the monovalent NC10 Fab to immobilized neuraminidase was analyzed using the 1:1 Langmuir binding model. A contribution of mass transport to the kinetic constants was demonstrated at higher surface densities and low flow rates, and was minimized at low ligand densities and relatively high flow rates (up to 100 microl/min). Application of a global fitting algorithm to a 1:1 binding model incorporating a correction term for mass transport indicated that mass transport was minimized under appropriate experimental conditions; analysis of binding data with a mass transport component, using this model, yielded kinetic constants similar to those obtained with the 1:1 Langmuir binding model applied to binding data where mass transport had been minimized experimentally. The binding constant for binding of NC10 Fab to N9 neuraminidase from tern influenza virus (K(A) = 6.3 +/- 1.3 x 10(7) M(-1)) was about 15-fold higher than that for the NC10 Fab binding to N9 neuraminidase from whale influenza virus (K(A) = 4.3 +/- 0.7 x 10(6) M(-1)). This difference in binding affinity was mainly attributable to a 12-fold faster dissociation rate constant of the whale neuraminidase-NC10 Fab complex and may be due to either (i) the long-range structural effects caused by mutation of two residues distant from the binding epitope or (ii) differences in carbohydrate residues, attached to Asn(200), which form part of the binding epitope on both neuraminidases to which NC10 Fab binds.     

The Caulobacter crescentus CgtA Protein Displays Unusual Guanine Nucleotide Binding and Exchange Properties

The Caulobacter crescentus CgtA protein is a member of the Obg-GTP1 subfamily of monomeric GTP-binding proteins. In vitro, CgtA specifically bound GTP and GDP but not GMP or ATP. CgtA bound GTP and GDP with moderate affinity at 30 degrees C and displayed equilibrium binding constants of 1.2 and 0.5 microM, respectively, in the presence of Mg(2+). In the absence of Mg(2+), the affinity of CgtA for GTP and GDP was reduced 59- and 6-fold, respectively. N-Methyl-3'-O-anthranoyl (mant)-guanine nucleotide analogs were used to quantify GDP and GTP exchange. Spontaneous dissociation of both GDP and GTP in the presence of 5 to 12 mM Mg(2+) was extremely rapid (k(d) = 1.4 and 1.5 s(-1), respectively), 10(3)- to 10(5)-fold faster than that of the well-characterized eukaryotic Ras-like GTP-binding proteins. The dissociation rate constant of GDP increased sevenfold in the absence of Mg(2+). Finally, there was a low inherent GTPase activity with a single-turnover rate constant of 5.0 x 10(-4) s(-1) corresponding to a half-life of hydrolysis of 23 min. These data clearly demonstrate that the guanine nucleotide binding and exchange properties of CgtA are different from those of the well-characterized Ras-like GTP-binding proteins. Furthermore, these data are consistent with a model whereby the nucleotide occupancy of CgtA is controlled by the intracellular levels of guanine nucleotides.     

High affinity binding of human interleukin 4 to cell lines

Purified human recombinant interleukin 4 (IL-4) was radio iodinated to high specific radioactivity without loss of biological activity. 125I-IL-4 bound specifically to the Burkitt lymphoma Jijoye cells and other cell lines. Jijoye cells showed a high affinity for 125I-IL-4 (Kd approximately equal to 7 10(-11) M) and displayed 1200-1400 specific receptors per cell at 4 degrees C or 37 degrees C. The equilibrium dissociation constant (Kd) corresponds to the IL-4 concentration which induces 50% maximal expression of the low affinity IgE receptor (Fc epsilon RL/CD23) on Jijoye cells. At 4 degrees C the rate constant of association K1 is 1.7 x 10(6) M-1 s-1 and the rate contant of dissociation k -1 is 1.3 x 10(-4) s-1 (t 1/2 = 91 min.) No human recombinant lymphokines other than IL-4 were able to compete for the binding of 125I-IL-4 to its receptor.     

Binding characteristics of [3H]17beta-estradiol in the hypothalamus of juvenile rainbow trout, Oncorhynchus mykiss

Gonadal steroids in the salmonid brain, acting through cellular receptors, may be responsible for the modulation of neuronal activity and organization of reproductive behaviors. We report our findings on the use of [3H]17beta-estradiol (E2) to identify intracellular estrogen receptors (ERs) in the hypothalamus of juvenile rainbow trout, Oncorhynchus mykiss. Specific binding (B(SP)) of [3H]E2 was tissue dependent between 0.5 and 2.25 hypothalamus equivalents for cytosol and nuclear extract preparations, respectively. B(SP) in cytosol fractions increased with time and reached maximum levels (4.18 nM) at 2.5 h incubation; by contrast, B(SP) in nuclear extract increased with time to achieve maximum levels (3.9 nM) by 2 h incubation. The association rate constants (k(+1)) for cytosol and nuclear extract preparations were 1.10 +/- 0.02 x 10(8) M(-1) min(-1) and 1.27 +/- 0.04 x 10(8) M(-1) min(-1), respectively. Equilibrium bound B(SP) dissociated from cytosol preparations with a half life (t1-2) of 42 min and a dissociation rate constant (k(-1)) of 1.01 +/- 0.03 min(-1). B(SP) dissociated from nuclear extract preparations with a t1-2 = 45 min and k(-1)= 0.92 +/- 0.01 min(-1) x B(SP) was saturable in both extract preparations with a calculated equilibrium dissociation constant (Kd) of 1.46 +/- 0.1 nM (cytosol) and 2.37 +/- 0.2 nM (nuclear), and a maximum number of binding sites (B(MAX)) of 50.85 +/- 3.2 fmol mg(-1) protein and 61.74 +/- 2.65 fmol mg(-1) protein, respectively. In both preparations, B(SP) was differentially displaced by structurally similar compounds with a rank order of potency of E2 > estrone > estriol > 17alpha-ethynyl estradiol > testosterone > progesterone = tamoxifen > cortisol > dexamethasone > > beta-sitosterol. These properties of specifically bound [3H]E2 suggest the presence of an ER in the hypothalamus of juvenile rainbow trout comparable with ERs identified in salmonid liver.     

Cobalt and the iron acquisition pathway: competition towards interaction with receptor 1

During iron acquisition by the cell, complete homodimeric transferrin receptor 1 in an unknown state (R1) binds iron-loaded human serum apotransferrin in an unknown state (T) and allows its internalization in the cytoplasm. T also forms complexes with metals other than iron. Are these metals incorporated by the iron acquisition pathway and how can other proteins interact with R1? We report here a four-step mechanism for cobalt(III) transfer from CoNtaCO(3)(2-) to T and analyze the interaction of cobalt-loaded transferrin with R1. The first step in cobalt uptake by T is a fast transfer of Co(3+) and CO(3)(2-) from CoNtaCO(3)(2-) to the metal-binding site in the C-lobe of T: direct rate constant, k(1)=(1.1+/-0.1) x 10(6) M(-1) s(-1); reverse rate constant, k(-1)=(1.9+/-0.6) x 10(6) M(-1) s(-1); and equilibrium constant, K=1.7+/-0.7. This step is followed by a proton-assisted conformational change of the C-lobe: direct rate constant, k(2)=(3+/-0.3) x 10(6) M(-1) s(-1); reverse rate constant, k(-2)=(1.6+/-0.3) x 10(-2) s(-1); and equilibrium constant, K(2a)=5.3+/-1.5 nM. The two final steps are slow changes in the conformation of the protein (0.5 h and 72 h), which allow it to achieve its final thermodynamic state and also to acquire second cobalt. The cobalt-saturated transferrin in an unknown state (TCo(2)) interacts with R1 in two different steps. The first is an ultra-fast interaction of the C-lobe of TCo(2) with the helical domain of R1: direct rate constant, k(3)=(4.4+/-0.6)x10(10) M(-1) s(-1); reverse rate constant, k(-3)=(3.6+/-0.6) x 10(4) s(-1); and dissociation constant, K(1d)=0.82+/-0.25 muM. The second is a very slow interaction of the N-lobe of TCo(2) with the protease-like domain of R1. This increases the stability of the protein-protein adduct by 30-fold with an average overall dissociation constant K(d)=25+/-10 nM. The main trigger in the R1-mediated iron acquisition is the ultra-fast interaction of the metal-loaded C-lobe of T with R1. This step is much faster than endocytosis, which in turn is much faster than the interaction of the N-lobe of T with the protease-like domain. This can explain why other metal-loaded transferrins or a protein such as HFE-with a lower affinity for R1 than iron-saturated transferrin but with, however, similar or higher affinities for the helical domain than the C-lobe-competes with iron-saturated transferrin in an unknown state towards interaction with R1.     

Inhibition of fly head acetylcholinesterase by bis-[(m-hydroxyphenyl)trimethylammonium iodide] esters of polymethylenedicarbamic acids

A series of bis-[(m-hydroxyphenyl)trimethylammonium iodide] esters of polymethylenedicarbamic acids and a number of (m-hydroxyphenyl)trimethylammonium iodide esters of straight-chain N-alkylcarbamic acids have been examined as inhibitors of acetylcholinesterase from fly head. Evidence is presented suggesting that inhibition of acetylcholinesterase by the bis-carbamates is due to carbamoylation of the enzyme, as is generally thought to be the case with esters of N-alkylcarbamic acids. Inhibition is irreversible. The (m-hydroxyphenyl)trimethylammonium iodide ester of N-hexylcarbamic acid also inhibits fly head acetylcholinesterase irreversibly. There is therefore no need to implicate a second functional group in bis-carbamate esters to explain the irreversible inhibition of the enzyme. An unusual feature of the inhibition is that inhibition lines do not pass through 100% enzyme activity at t=0, except for rather low concentrations of inhibitor (<10mum for the octamethylene compound). Also, inhibition lines tend towards a maximum slope as inhibitor concentration is increased. The first observation indicates complex-formation, even in the presence of high concentrations of substrate, and by using measurements of inhibition at relatively high inhibitor concentrations, affinity constants K'(a) have been calculated. K'(a) varies from 0.1mum for the dodecamethylene compound to 10mum for the tetramethylene compound, in the presence of 3.75mm-acetylthiocholine, indicating high affinity for the enzyme. The second observation shows that, owing to this high affinity, the enzyme becomes saturated with inhibitor under the experimental conditions employed, and from the limiting slope values of the carbamoylation rate constant (k(2)) have been calculated. k(2) varies from 0.15min(-1) for the tetramethylene compound to 1min(-1) for the decamethylene compound. Variations of potency in this series are therefore mainly due to changes in affinity (100-fold) rather than in carbamoylation rate (sevenfold). The observation that large molecules may acylate the enzyme raises certain problems, which are discussed.     

Kinetic scheme for intermolecular RNA cleavage by a ribozyme derived from hepatitis delta virus RNA

A minimal kinetic mechanism for a trans-acting ribozyme derived from the HDV antigenomic RNA self-cleaving element was established from steady-state, pre-steady-state, single-turnover, and binding kinetics. Rate constants for individual steps, including substrate binding and dissociation, cleavage, and product release and binding, were measured at 37 degrees C at pH 8.0 in 10 mM Mg(2+) using oligonucleotides as either substrates, noncleavable analogues or 3' product mimics. A substrate containing a normal 3',5'-linkage was cleaved with a first-order rate constant (k(2)) of 0.91 min(-)(1). The association rate constant for the substrate to the ribozyme (2.1 x 10(7) M(-)(1) min(-)(1)) was at the lower range of the expected value for RNA duplex formation, and the substrate dissociated with a rate constant (1.4 min(-)(1)) slightly faster than that for cleavage. Thus the binary complex was not at equilibrium with free enzyme and substrate prior to the cleavage step. Following cleavage, product release was kinetically ordered in that the 5' product was released rapidly (>12 min(-)(1)) relative to the 3' product (6.0 x 10(-)(3) min(-)(1)). Rapid 5' product release and lack of a demonstrable binding site for the 5' product could contribute to the difficulty in establishing the ribozyme-catalyzed reverse reaction (ligation). Slow release of the 3' product was consistent with the extremely low turnover under steady-state conditions as 3' product dissociation was rate-limiting. The equilibrium dissociation constant for the substrate was 24-fold higher than that of the 3' cleavage product. A substrate with a 2',5'-linkage at the cleavage site was cleaved with a rate constant (k(2)) of 1.1 x 10(-)(2) min(-)(1). Thus, whereas cleavage of a 3',5'-linkage followed a Briggs-Haldane mechanism, 2', 5' cleavage followed a Michaelis-Menten mechanism.     

Vasodilator effects of L-arginine are stereospecific and augmented by insulin in humans

The amino acid l-arginine, the precursor of nitric oxide (NO) synthesis, induces vasodilation in vivo, but the mechanism behind this effect is unclear. There is, however, some evidence to assume that the l-arginine membrane transport capacity is dependent on insulin plasma levels. We hypothesized that vasodilator effects of l-arginine may be dependent on insulin plasma levels. Accordingly, we performed two randomized, double-blind crossover studies in healthy male subjects. In protocol 1 (n = 15), subjects received an infusion of insulin (6 mU x kg(-1) x min(-1) for 120 min) or placebo and, during the last 30 min, l-arginine or d-arginine (1 g/min for 30 min) x In protocol 2 (n = 8), subjects received l-arginine in stepwise increasing doses in the presence (1.5 mU x kg(-1) x min(-1)) or absence of insulin. Renal plasma flow and glomerular filtration rate were assessed by the para-aminohippurate and inulin plasma clearance methods, respectively. Pulsatile choroidal blood flow was assessed with laser interferometric measurement of fundus pulsation, and mean flow velocity in the ophthalmic artery was measured with Doppler sonography. l-arginine, but not d-arginine, significantly increased renal and ocular hemodynamic parameters. Coinfusion of l-arginine with insulin caused a dose-dependent leftward shift of the vasodilator effect of l-arginine. This stereospecific renal and ocular vasodilator potency of l-arginine is enhanced by insulin, which may result from facilitated l-arginine membrane transport, enhanced intracellular NO formation, or increased NO bioavailability.     

A novel antioxidant mechanism of ebselen involving ebselen diselenide,a substrate of mammalian thioredoxin and thioredoxin reductase

The antioxidant mechanism of ebselen involves recently discovered reductions by mammalian thioredoxin reductase (TrxR) and thioredoxin (Trx) forming ebselen selenol. Here we describe a previously unknown reaction; ebselen reacts with its selenol forming an ebselen diselenide with a rate constant of 372 m(-1)s(-1). The diselenide also was a substrate of TrxR forming the selenol with K(m) of 40 microm and k(cat) of 79 min(-1) (k(cat)/K(m) of 3.3 x 10(4) m(-1)s(-1)). Trx increased the reduction because of its fast reaction with diselenide (rate constant 1.7 x 10(3) m(-1)s(-1)). Diselenide stimulated the H2O2 reductase activity of TrxR, even more efficiently with Trx present. Because the mechanism of ebselen as an antioxidant has been assumed to involve glutathione peroxidase-like activity, we compared the H2O2 reductase activity of ebselen with the GSH and Trx systems. TrxR at 50 nm, far below the estimated physiological level, gave 8-fold higher activity compared with 1 mm GSH; addition of 5 microm Trx increased this difference to 13-fold. The rate constant of ebselen selenol reacting with H2O2 was estimated to be faster than 350 m(-1)s(-1). We propose novel mechanisms for ebselen antioxidant action involving ebselen selenol and diselenide formation, with the thioredoxin system rather than glutathione as the predominant effector and target.     

Characterization of azadirachtin binding to Sf9 nuclei in vitro

[22,23-(3)H(2)]dihydroazadirachtin was incorporated by Sf9 cells in culture and was bound specifically to the nuclear fraction. The observed association constant of the binding of the radioligand to a purified nuclear fraction was determined to be 0.037 +/- 0.008 min(-1) using a one-phase exponential association equation, and binding appeared to be to a single population of sites. The binding was essentially irreversible, and the dissociation constant was estimated to be 0.00065 +/- 0.00013 min(-1). An association rate constant of 7.3 x 10(6) M(-1) min(-1) was calculated from these data. Binding was saturable, and the receptor number and affinity were determined as B(max) = 23.87 +/- 1.15 pmol/mg protein, K(d) = 18.1 +/- 2.1 nM. The order of potency of semisynthetic azadirachtin analogues for competition for the binding site was as follows (IC(50) in parentheses): azadirachtin (1.55 x 10(-8) M) > dihydroazadirachtin (3.16 x 10(-8) M) > dansyl dihydroazadirachtin (7.40 x 10(-8) M) > DNP-azadirachtin (7.50 x 10(-8) M) > biotin dihydroazadirachtin (1.27 x 10(-7) M) > 11-methoxy 22,23-dihydroazadirachtin (6.67 x 10(-7) M). [Originally published in Volume 34, Archives of Insect Biochemistry and Physiology, 34:461-473 (1997).] Copyright 1997 Wiley-Liss, Inc.     

Solubilization and partial characterization of the sex hormone-binding globulin receptor from human prostate

The sex hormone-binding globulin (SHBG) receptor was solubilized from the membranes of human prostate glands with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid). The binding activity of the soluble receptor was measured by allowing it to bind to 125I-SHBG and precipitating the complex with polyethylene glycol-8000. The binding activity was stable for at least 4 months at -20 degrees C and had a half-life of 23 days at 4 degrees C. Like the membrane-bound receptor, Scatchard analysis revealed two sets of binding sites for the soluble one. At equilibrium (24 h), the high affinity site had an association constant (KA) of 6.8 x 10(8) M-1 and a binding capacity of 1.4 pmol/mg protein, whereas the low affinity site had a KA of 4.7 x 10(6) M-1 and a binding capacity of 43 pmol/mg protein. At 37 degrees C, the association rate constant (k1) was 8.37 x 10(5) M-1 min-1 and the dissociation rate constant (k2) was 3.43 x 10(-4) min-1. The soluble receptor was retarded on Sepharose CL-6B and had an apparent Mr = 167,000.