Tight binding of bulky fluorescent derivatives of adenosine to the low affinity E2ATP site leads to inhibition of Na+/K+-ATPase. Analysis of structural requirements of fluorescent ATP derivatives with a Koshland-Némethy-Filmer model of two interacting ATP sites

A Koshland-Némethy-Filmer model of two cooperating ATP sites has previously been shown to explain the kinetics of inhibition of Na+/K+-ATPase (EC 3.6.1.37) by dansylated ATP (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321). The present work demonstrates that this model adequately describes all types of interactions and kinetics of a number of ATP analogs that differ in their cooperativity of the high and low affinity ATP binding sites of the enzyme. 2',3'-O(2,4,6-trinitrophenyl)ATP binds in a negative cooperative way to the E1ATP site (Kd = 0.7 microM) and to the E2ATP site (Kd = 210 microM), but 3'(2')-O-methylanthraniloyl-ATP in a positive cooperative way with a lower affinity to the E1ATP binding site (Kd = 200 microM) than to the E2ATP binding site (Kd = 80 microM). 3'(2')-O(5-Fluor-2,4-dinitrophenyl)-ATP, however, binds in a noncooperative way, with equal affinities to both ATP binding sites (Kd = 10 microM). In a research for the structural parameters determining ATP site specificity and cooperativity, we became aware that structural flexibility of ribose is necessary for catalysis. Moreover, puckering of the ring atoms in the ribose is essential for the interaction between ATP sites in Na+/K+-ATPase. A number of derivatives of 2'(3')-O-adenosine with bulky fluorescent substitutes bind with high affinity to the E2ATP site and inhibit Na+/K+-ATPase activity. Evidently, an increased number of interactions of such a bulky adenosine with the enzyme protein tightens binding to the E2ATP site.     

The binding of receptor-recognized alpha2-macroglobulin to the low density lipoprotein receptor-related protein and the alpha2M signaling receptor is decoupled by oxidation

Receptor-recognized forms of alpha2-macroglobulin (alpha2M*) bind to two classes of cellular receptors, a high affinity site comprising approximately 1500 sites/cell and a lower affinity site comprising about 60,000 sites/cell. The latter class has been identified as the so-called low density lipoprotein receptor-related protein (LRP). Ligation of receptors distinct from LRP activates cell signaling pathways. Strong circumstantial evidence suggests that the high affinity binding sites are responsible for cell signaling induced by alpha2M*. Using sodium hypochlorite, a powerful oxidant produced by the H2O2-myeloperoxidase-Cl- system, we now demonstrate that binding to the high affinity sites correlates directly with activation of the signaling cascade. Oxidation of alpha2M* using 200 microM hypochlorite completely abolishes its binding to LRP without affecting its ability to activate the macrophage signaling cascade. Scatchard analysis shows binding to a single class of high affinity sites (Kd - 71 +/- 12 pM). Surprisingly, oxidation of native alpha2-macroglobulin (alpha2M) with 125 microM hypochlorite results in the exposure of its receptor-binding site to LRP, but the ligand is unable to induce cell signaling. Scatchard analysis shows binding to a single class of lower affinity sites (Kd - 0.7 +/- 0.15 nM). Oxidation of a cloned and expressed carboxyl-terminal 20-kDa fragment of alpha2M (RBF), which is capable of binding to both LRP and the signaling receptor, results in no significant change in its binding Kd, supporting our earlier finding that the oxidation-sensitive site is predominantly outside of RBF. Attempts to understand the mechanism responsible for the selective exposure of LRP-binding sites in oxidized native alpha2M suggest that partial protein unfolding may be the most likely mechanism. These studies provide strong evidence that the high affinity sites (Kd - 71 pM) are the alpha2M* signaling receptor.     

Mechanism of malic enzyme from pigeon liver. Magnetic resonance and kinetic studies of the role of Mn2+

As determined by EPR, malic enzyme from pigeon liver binds Mn2+ with a half-site stoichiometry of two tight binding sites (KD=6 to 10 mum) per enzyme tetramer and at two to four weak binding sites (KD=0.43 to 1.34 mM). The activation of malic enzyme by Mn2+ at high levels of L-malate shows biphasic kinetics yielding two activator constants for Mn2+. The dissociation constants of Mn2+ for both classes of sites are of the same order as the kinetically determined activator constants of Mn2+, indicating active site binding at both classes of binding sites. The binding of Mn2+ to the tight sites enhances the paramagnetic effect of Mn2+ on 1/T1 of water protons by a factor (epsilon) of 17, while binding at the weak sites yields a smaller epsilon of 11. The coenzymes TPN and TPNH have no effects on epsilon, while the carboxylic acid substrates L-malate and pyruvate and the inhibitors D-malate and oxalate significantly decrease epsilon. TPNH causes a 38-fold tightening of binding of the substrate L-malate to the enzyme-Mn2+ complex, consistent with the previously described highly ordered kinetic scheme, but only a 2-fold tightening of binding of the competitive inhibitor D-malate. The dissociation constant of L-malate from the quaternary E-Mn2+-TPNH-L-malate complex (32 muM) agrees with the Km of L-malate (25 muM), indicating active site binding. The dissociation constants of pyruvate from the ternary E-Mn2+-pyruvate complex (12 mM) and from the quaternary E-Mn2+-TPN-pyruvate complex (20 mM) are similar to the Km of pyruvate (5 mM), also indicating active site binding and a less highly ordered kinetic scheme for the reactions of pyruvate than for those of L-malate. Analysis of the frequency dependence of 1/T1 of water protons indicates that two fast exchanging water ligands remain coordinated to Mn2+ in the binary E-Mn2+ complex. The binding of the substrates L-malate and pyruvate and of the transition state analog oxalate to the E-Mn2+ complex decrease the number of fast exchanging water ligands on Mn2+ by approximately 1, but the binding of D-malate has no significant effect on this parameter, indicating the occlusion or replacement of a water ligand of the enzyme-bound Mn2+ by a properly oriented substituent on C-2 of the substrate. Occlusion rather than replacement of a water ligand by pyruvate is established by studies of 1/T1 of 13COO- and 13CO-enriched pyruvate which indicate second sphere Mn2+ to pyruvate distances of 4.6 A (COO-) and 4.8 A (CO) in the ternary enzyme-Mn2+-pyruvate complex. Formation of the quaternary complex with TPN increases these distances by 0.8 A, indicating the participation of a second sphere enzyme-Mn2+-(H2O)-pyruvate complex in catalysis. Thus, malic enzyme, like five other enzymes which utilize metals to polarize carbonyl groups, forms a second sphere complex with its substrate.     

Binding of the aflatoxin-glutathione conjugate to mouse glutathione S-transferase A3-3 is saturated at only one ligand per dimer

The binding of two different reaction products (p-nitrobenzyl glutathione and the aflatoxin-glutathione conjugate) to mouse glutathione S-transferase A3-3 (mGSTA3-3) has been measured using equilibrium dialysis and a direct fluorescence quenching technique. As expected, p-nitrobenzyl glutathione was found to bind with a stoichiometry of 2.24 +/- 0.17 mol/mol of dimeric enzyme. However, the much larger aflatoxin-glutathione conjugate, 8, 9-dihydro-8-(S-glutathionyl)-9-hydroxyl-aflatoxin B1 (AFB-GSH), was found to bind with a stoichiometry of 1.12 +/- 0.08 mol/mol of dimeric enzyme. p-Nitrobenzyl glutathione bound mGSTA3-3 with a dissociation constant (Kd) of 59 +/- 17 microM while the aflatoxin-glutathione conjugate bound the enzyme with a Kd of 0.86 +/- 0.19 microM. Glutathione competitively inhibited binding of AFB-GSH to mGSTA3-3 with a Ki of 1.5 mM, suggesting that AFB-GSH was binding to the enzyme active site. Although AFB-GSH bound to mGSTA3-3 with a stoichiometry of 1 mol/mol of dimeric enzyme, AFB-GSH completely inhibited activity toward 1-chloro-2, 4-dinitrobenzene, indicating that AFB-GSH binding to one active site alters affinity for 1-chloro-2,4-dinitrobenzene in the active site of the other subunit. To our knowledge, this is the first report of a glutathione S-transferase reaction product which binds to the enzyme with a stoichiometry of 1 mol/mol of dimer.     

A semiconserved residue inhibits complex formation by stabilizing interactions in the free state of a theophylline-binding RNA

The theophylline-binding RNA aptamer contains a 15 nucleotide motif that is required for high-affinity ligand binding. One residue within this RNA motif is only semiconserved and can be an A or C. This residue, C27, was disordered in the previously determined three-dimensional structure of the complex, suggesting that it is dynamic in solution. 13C Relaxation measurements are reported here, demonstrating that C27 is highly dynamic in the otherwise well-ordered RNA-theophylline complex. A synthetic complex with an abasic residue at position 27 was found to exhibit wild-type binding affinity (Kd approximately 0.2 microM), indicating that the base of residue 27 is not directly involved with theophylline binding. Surprisingly, the U27 and G27 RNAs were found to bind theophylline with low affinity (Kd values > 4 microM). NMR spectroscopy on the U27 RNA revealed the presence of an A7-U27 base pair in the free RNA that prevents formation of a critical base-platform structural motif and therefore blocks theophylline binding. Similarly, a protonated A7H+-C27 base pair forms in the absence of theophylline at low pH, which explains the unusual pH dependence of theophylline binding of the C27 RNA aptamer. Thus the weak binding for various nucleotides at position 27 arises not from unfavorable interactions in the RNA-theophylline complex but instead from stable interactions in the free state of the RNA that inhibit theophylline binding.     

Guinea worm disease--a chance for successful eradication in the Volta Region, Ghana

Parsley cells recognize the fungal phytopathogen Phytophthora sojae through a plasma membrane receptor. A 13 amino acid oligopeptide fragment (Pep-13) of a 42 kDa fungal cell wall glycoprotein was shown to bind to the receptor and stimulate a complex defense response in cultured parsley cells. The Pep-13 binding site solubilized from parsley microsomal membranes by non-ionic detergents exhibited the same ligand affinity and ligand specificity as the membrane-bound receptor. Chemical crosslinking and photoaffinity labeling assays with [125I]Pep-13 revealed that a monomeric 100 kDa integral plasma membrane protein is sufficient for ligand binding and may thus constitute the ligand binding domain of the receptor. Ligand affinity chromatography of solubilized microsomal membrane protein on immobilized Pep-13 yielded a 5000-fold enrichment of specific receptor activity.     

Transient-state kinetic analysis of the oxidative decarboxylation of D-malate catalyzed by tartrate dehydrogenase

The oxidative decarboxylation of D-malate catalyzed by tartrate dehydrogenase has been analyzed by transient-state kinetic methods and kinetic isotope effect measurements. The reaction time courses show a burst of NADH formation prior to the attainment of the steady-state velocity. The binding of the inhibitor tartronate to the enzyme was examined by monitoring the quenching of the protein's intrinsic fluorescence; the tartronate concentration dependence of the observed rate constant for association was hyperbolic, supporting a two-step model for inhibitor binding. Analysis of the time courses for D-malate oxidation yielded values for many of the microscopic rate constants governing the reaction. The range of possible solutions for the microscopic rate constants was constrained by comparison of the time course for oxidation of unlabeled malate with that of deuterated malate; this analysis relied on the determination of the intrinsic isotope effect on hydride transfer via measurement of D(V/K), T(V/K), and the oxaloacetate partition ratio. The results of the transient-state kinetic analyses suggest that the rate of D-malate oxidation is largely limited by the rate of decarboxylation of the intermediate oxaloacetate which occurs at 11 s-1. Hydride transfer from D-malate to NAD+ occurs with a rate constant of 300 s-1, and (D)k for this step is 5.5. The agreement between experimentally measured steady-state kinetic parameters and kinetic isotope effects and their values calculated from the microscopic rate constants derived from the transient-state kinetic analyses was quite good.     

Interactions between lonidamine and beta- or hydroxypropyl-beta-cyclodextrin

Equilibrium dialysis and Scatchard plots were used to establish that human and rabbit paraoxonases both have two calcium binding sites. Independent-site and stepwise constant analyses were used to calculate a higher affinity site (Kd1) of 3.6 +/- 0.9 x 10(-7) M for human A paraoxonase, and 1.4 +/- 0.5 x 10(-8) M for rabbit paraoxonase, and a lower affinity site (Kd2) of 6.6 +/- 1.2 x 10(-6) M for human A paraoxonase, and 5.3 +/- 0.94 x 10(-6) M for rabbit paraoxonase. In both species, the higher affinity sites were found to be essential to maintain hydrolytic activity; complete removal of calcium led to irreversible inactivation. The lower affinity sites were required for catalytic activity, and their binding of calcium was reversible. Experimentally estimated values of Kd2 based on the concentration of calcium required to obtain half the maximum enzymatic activity were 3 microM for human A and B paraoxonases, and also in the order of 3 microM for rabbit paraoxonase, using three different substrates. Calcium was the only metal found that protects against denaturation and also confers hydrolytic activity with these two mammalian paraoxonases.     

High affinity binding of latent matrix metalloproteinase-9 to the alpha2(IV) chain of collagen IV

Association of matrix metalloproteinases (MMPs) with the cell surface and with areas of cell-matrix contacts is critical for extracellular matrix degradation. Previously, we showed the surface association of pro-MMP-9 in human breast epithelial MCF10A cells. Here, we have characterized the binding parameters of pro-MMP-9 and show that the enzyme binds with high affinity (Kd approximately 22 nM) to MCF10A cells and other cell lines. Binding of pro-MMP-9 to MCF10A cells does not result in zymogen activation and is not followed by ligand internalization, even after complex formation with tissue inhibitor of metalloproteinase-1 (TIMP-1). A 190-kDa cell surface protein was identified by ligand blot analysis and affinity purification with immobilized pro-MMP-9. Microsequencing and immunoblot analysis revealed that the 190-kDa protein is the alpha2(IV) chain of collagen IV. Specific pro-MMP-9 surface binding was competed with purified alpha2(IV) and was significantly reduced after treatment of the cells with active MMP-9 before the binding assay since alpha2(IV) is hydrolyzed by MMP-9. A pro-MMP-9.TIMP-1 complex and MMP-9 bind to alpha2(IV), suggesting that neither the C-terminal nor the N-terminal domain of the enzyme is directly involved in alpha2(IV) binding. The closely related pro-MMP-2 exhibits a weaker affinity for alpha2(IV) compared with that of pro-MMP-9, suggesting that sites other than the gelatin-binding domain may be involved in the binding of alpha2(IV) to pro-MMP-9. Although pro-MMP-9 forms a complex with alpha2(IV), the proenzyme does not bind to triple-helical collagen IV. These studies suggest a unique interaction between pro-MMP-9 and alpha2(IV) that may play a role in targeting the zymogen to cell-matrix contacts and in the degradation of the collagen IV network.     

Differential effects of CD28 costimulation on HIV production by CD4+ cells

S 5627 is a synthetic analogue of chlorogenic acid. S 5627 is a potent linear competitive inhibitor of glucose 6-phosphate (Glc-6-P) hydrolysis by intact microsomes (Ki = 41 nM) but is without effect on the enzyme in detergent- or NH4OH-disrupted microsomes. 3H-S 5627 was synthesized and used as a ligand in binding studies directed at characterizing T1, the Glc-6-P transporter. Binding was evaluated using Ca2+-aggregated microsomes, which can be sedimented at low g forces. Aside from a modest reduction in K values for both substrate and S 5627, Ca2+ aggregation had no effect on glucose-6-phosphatase (Glc-6-Pase). Scatchard plots of binding data are readily fit to a simple "two-site" model, with Kd = 21 nM for the high affinity site and Kd = 2 microM for the low affinity site. Binding to the high affinity site was competitively blocked by Glc-6-P (Ki = 9 microM), whereas binding was unaffected by mannose-6-phosphate, Pi, and PPi and only modestly depressed by 2-deoxy-D-glucose 6-phosphate, a poor substrate for Glc-6-Pase in intact microsomes. Thus the high affinity 3H-S 5627 binding site fits the criteria for T1. Permeabilization of the membrane with 0.3% (3-[(chloramidopropyl)-dimethylammonio]-1-propanesulfonate) activated Glc-6-Pase and broadened its substrate specificity, but it did not significantly alter the binding of 3H-S 5627 to the high affinity sites or the ability of Glc-6-P to block binding. These data demonstrate unequivocally that two independent Glc-6-P binding sites are involved in the hydrolysis of Glc-6-P by intact microsomes. The present findings are the strongest and most direct evidence to date against the notion that the substrate specificity and the intrinsic activity of Glc-6-Pase in native membranes are determined by specific conformational constraints imposed on the enzyme protein. These data constitute compelling evidence for the role of T1 in Glc-6-Pase activity.     

Simultaneous binding of basic peptides at intracellular sites on a large conductance Ca2+-activated K+ channel. Equilibrium and kinetic basis of negatively coupled ligand interactions

The homologous Kunitz inhibitor proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin I (DTX-I), interact with large conductance Ca2+-activated K+ channels (maxi-KCa) by binding to an intracellular site outside of the pore to produce discrete substate events. In contrast, certain homologues of the Shaker ball peptide produce discrete blocking events by binding within the ion conduction pathway. In this study, we investigated ligand interactions of these positively charged peptide molecules by analysis of single maxi-KCa channels in planar bilayers recorded in the presence of DTX-I and BPTI, or DTX-I and a high-affinity homologue of ball peptide. Both DTX-I (Kd, 16.5 nM) and BPTI (Kd, 1, 490 nM) exhibit one-site binding kinetics when studied alone; however, records in the presence of DTX-I plus BPTI demonstrate simultaneous binding of these two molecules. The affinity of BPTI (net charge, +6) decreases by 11.7-fold (Kd, 17,500 nM) when DTX-I (net charge, +10) is bound and, conversely, the affinity of DTX-I decreases by 10.8-fold (Kd, 178 nM) when BPTI is bound. The ball peptide homologue (BP; net charge, +6) exhibits high blocking affinity (Kd, 7.2 nM) at a single site when studied alone, but has 8.0-fold lower affinity (Kd, 57 nM) for blocking the DTX-occupied channel. The affinity of DTX-I likewise decreases by 8.4-fold (Kd, 139 nM) when BP is bound. These results identify two types of negatively coupled ligand-ligand interactions at distinct sites on the intracellular surface of maxi-KCa channels. Such antagonistic ligand interactions explain how the binding of BPTI or DTX-I to four potentially available sites on a tetrameric channel protein can exhibit apparent one-site kinetics. We hypothesize that negatively coupled binding equilibria and asymmetric changes in transition state energies for the interaction between DTX-I and BP originate from repulsive electrostatic interactions between positively charged peptide ligands on the channel surface. In contrast, there is no detectable binding interaction between DTX-I on the inside and tetraethylammonium or charybdotoxin on the outside of the maxi-KCa channel.     

Highly potent, selective, and low cost bis-tetrahydroaminacrine inhibitors of acetylcholinesterase. Steps toward novel drugs for treating Alzheimer's disease

We report highly potent, selective, and low cost bifunctional acetylcholinesterase (AChE) inhibitors developed by our two-step prototype optimization strategy utilizing computer modeling of ligand docking with target proteins: 1) identify low affinity sites normally missed by x-ray crystallography; and 2) design bifunctional analogs capable of simultaneous binding at the computer-determined low affinity site and the x-ray-identified high affinity site. Applying this strategy to 9-amino-1,2,3,4-tetrahydroacridine (THA), a drug for Alzheimer's disease, we obtained alkylene linked bis-THA analogs. These analogs were up to 10,000-fold more selective and 1,000-fold more potent than THA in inhibiting rat AChE and yet required one simple reaction to synthesize. Additionally, alkylene linked benzyl-THA analogs were developed to examine the specificity of the docking-derived low affinity THA peripheral site in AChE. The present work and our previous computational studies strongly suggest that a low affinity THA peripheral site exists in AChE. This peripheral site provides a structural basis for design of improved cholinesterase ligands for treating Alzheimer's disease and for other health-related purposes.     

Enzymic analysis of the structure of oxidized potato starches

The binding of TNP-ATP (2' or 3'-O-(2,4,6-trinitrophenyl)-ATP) to cytochrome c oxidase (COX) from bovine heart and liver and to the two-subunit COX of Paracoccus denitrificans was measured by its change of fluorescence. Three binding sites, two with high (dissociation constant Kd = 0.2 microM) and one with lower affinity (Kd = 0.9 microM), were found at COX from bovine heart and liver, while the Paracoccus enzyme showed only one binding site (Kd = 3.6 microM). The binding of [35S]ATP alpha S was measured by equilibrium dialysis and revealed seven binding sites at the heart enzyme (Kd = 7.5 microM) and six at the liver enzyme (Kd = 12 microM). The Paracoccus enzyme had only one binding site (Kd = 16 microM). The effect of variable intraliposomal ATP/ADP ratios, but at constant total concentration of [ATP + ADP] = 5 mM, on the H+/e- stoichiometry of reconstituted COX from bovine heart and liver were studied. Above 98% ATP the H+/e- stoichiometry of the heart enzyme decreased to about half of the value measured at 100% ATP. In contrast, the H+/e-stoichiometry of the liver enzyme was not influenced by the ATP/ADP ratio. It is suggested that high intramitochondrial ATP/ADP ratios, corresponding to low cellular work load, will decrease the efficiency of energy transduction and result in elevated thermogenesis for the maintenance of body temperature.     

Multiple alpha 2 adrenergic receptor subtypes. I. Comparison of [3H]RX821002-labeled rat R alpha-2A adrenergic receptors in cerebral cortex to human H alpha2A adrenergic receptor and other populations of alpha-2 adrenergic subtypes

In the present study, we examined the binding of the alpha-2 adrenergic receptor (AR) antagonist [3H]-(2-(2-methoxy-1,4-benzodioxan- 2yl)-2-imidazoline ([3H]RX821002) to alpha-2 AR in rat cerebral cortex (CC) and compared the properties of these sites to those of rat alpha-2A (R alpha-2A) AR in submaxillary gland (SMG), human alpha-2A (H alpha-2A) AR in human platelets and alpha-2B AR in neonatal rat lung. In the presence of guanidinium phosphate, [3H]RX821002 bound with high affinity to a large and homogeneous population of sites in CC (Kd = 0.30 +/- 0.03 nM and Bmax = 271 +/- 7 fmol/mg of protein), SMG (Kd = 0.7 and Bmax = 274), human platelets (Kd = 0.6 nM and Bmx = 189) and neonatal rat lung (kd = 0.9 and Bmax = 161). A total of 34 chemically diverse AR ligands monophasically inhibited the binding of [3H]RX821002 from each site with, for the CC, the most potent ligand being atipamezole (Ki = 0.2 nM). For all ligands, and at each site, Hill coefficients did not differ significantly from unity. Although the profiles of inhibition of [3H]RX821002 were virtually identical in rat CC and SMG, these populations revealed several marked differences to human platelets; the alkaloids, rauwolscine and yohimbine, as well as the benzodioxane, [2-(2,6- dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxane] (WB 4101), displayed about 10-fold lower affinity for R alpha-2A as compared to H alpha-2A sites, whereas the benzopyrrolidines, fluparoxan and des-fluorofluparoxan, showed about 10-fold greater affinity for R alpha-2A sites. Further, whereas the calculation of potency ratios for selected pairs of ligands, as well as of correlation coefficients, revealed virtual identity between R alpha-2A AR in CC and SMG, these analyses revealed that each of these populations of R alpha-2A AR clearly differed to H alpha-2A AR in human platelets. In addition, both R alpha-2A AR in rat CC and SMG as well as H alpha-2A AR in human platelets markedly differed to alpha-2B AR in neonatal rat lung; thus, they showed 20-fold higher affinity for [2-(2H-(1-methyl-1,3-dihydroisoindole)methyl)-4,5- dihydroimidazoline] (BRL 44408), oxymetazoline, guanfacine and guanabenz yet 10- to 100-fold lower affinity for [2-(2-4-o- methoxyphenyl)piperazine-1-yl)-ethyl)-4,4-dimethyl-1,3-(2H,4H)- isoquinolinedione] (ARC 239) prazosin, chlorpromazine and corynanthine. Similar differences in R alpha-2A and H alpha-2A sites to alpha-2C sites were apparent upon analysis of literature data.(ABSTRACT TRUNCATED AT 400 WORDS)     

Methodologic standards for diagnostic test research in pulmonary medicine

The presence of functional nongenomic progesterone (P) receptors in human spermatozoa has been investigated by equilibrium binding studies in intact spermatozoa, ligand blot and Western blot analysis of sperm lysates, as well as determination of the effects of the steroid on sperm intracellular Ca2+ concentrations. Binding experiments were performed using progesterone-11alpha-glucuronide-[125I]iodotyramine as tracer. Computer analysis of competition curves using different steroids as competitors indicated the presence of two distinct binding sites for P. The high affinity site (Kd in the nanomolar range) appears to be specific for P, whereas the low affinity one (Kd in the micromolar range) binds with equal affinity 11beta-hydroxyprogesterone (11betaOHP) and 17alpha-hydroxyprogesterone (17alphaOHP). A significant correlation exists among affinity constants (as determined by binding studies) and EC50 values for the effects of P, 11betaOHP, and 17alphaOHP on intracellular Ca2+ in fura-2-loaded spermatozoa, strongly indicating the involvement of P-binding sites in the biological effect of the steroid. In particular, dose-response curves for P were biphasic, with an EC50 in the nanomolar range and another in the micromolar range. Conversely, curves for 11betaOHP and 17alphaOHP were monophasic, with an EC50 just in the micromolar range. Ligand blot analysis of sperm total lysates performed with peroxidase-conjugated P revealed the presence of two binding proteins of 54 and 57 kDa that were specific for P. Indeed, peroxidase-conjugated P binding was blocked by the simultaneous presence of the unconjugated steroid. Using alpha c262 antibody, which is directed against the P-binding domain of genomic receptor, we detected two proteins of similar molecular mass (54 and 57 kDa), whereas using antibodies directed against the DNA-binding and N-terminal domains of the genomic P receptors, the two proteins were not detected. In addition, p54 and p57 appear to be mostly localized in sperm membranes and virtually absent in the cytoplasm. The involvement of these proteins in the biological effects of P is indicated by the strong inhibitory effect of alpha c262 on P-induced acrosome reaction of capacitated human spermatozoa.     

2B4, the natural killer and T cell immunoglobulin superfamily surface protein, is a ligand for CD48

2B4 is a cell surface glycoprotein related to CD2 and implicated in the regulation of natural killer and T lymphocyte function. A recombinant protein containing the extracellular region of mouse (m)2B4 attached to avidin-coated fluorescent beads bound to rodent cells, and binding was completely blocked by CD48 monoclonal antibodies (mAbs). Using surface plasmon resonance, we showed that purified soluble mCD48 bound m2B4 with a six- to ninefold higher affinity (Kd approximately 16 microM at 37 degreesC) than its other ligand, CD2. Human CD48 bound human 2B4 with a similar affinity (Kd approximately 8 microM). The finding of an additional ligand for CD48 provides an explanation for distinct functional effects observed on perturbing CD2 and CD48 with mAbs or by genetic manipulation.     

Association and direct activation of signal transducer and activator of transcription1alpha by platelet-derived growth factor receptor

The binding parameters of [3H]nociceptin were examined in membrane preparations of rat heart and compared with those of [3H]dynorphin A-(1-13) ([3H]Dyn A-(1-13)). Scatchard analysis of [3H]nociceptin binding revealed the presence of two distinct sites: a high affinity (Kd: 583 nM) low capacity (Bmax: 132 pmol/mg protein) site and a low affinity (Kd: 10,316 nM) high capacity (1552 pmol/mg protein) site. Dyn A and related peptides were potent competitors of the binding to the high affinity site with the following rank order of potency: alpha-neo-endorphin > Dyn A-(2-13) = Dyn A-(3-13) > Dyn A-(5-13) > Dyn A-(1-13) > Dyn A > Dyn B > Dyn A-(6-10) > Dyn A-(1-8). Nociceptin was 6.7 times less potent than Dyn A with a Ki of 4.8 microM as compared with 0.72 microM for Dyn A. The order of potency of the various peptides in inhibiting [3H]nociceptin binding correlated well (r = 0.93) with their ability to complete with the binding of [3H]Dyn A-(1-13) (Dumont and Lemaire, 1993). In addition, the high affinity [3H]nociceptin and non-opioid [3H]Dyn A-(1-13) sites were both sensitive to NaCl (120 mM) and the phospholipase C (PLC) inhibitors, U-73122 and neomycin (100 microM). The binding activities were less affected by the weak PLC inhibitor, U-73343, and no effect was observed with the non-hydrolysable GTP analogs. Gpp(NH)p and GTP-gamma-S. Nociceptin (1-50 microM) was also shown to inhibit the uptake of [3H]noradrenaline ([3H]NA) by cardiac synaptosomal preparations. In spontaneously hypertensive rats (SHR), the potency of nociceptin in inhibiting [3H]NA uptake was increased by 1.6-fold as compared with Wistar Kyoto (WKY) control rats and such effect was accompanied by comparable increased levels of cardiac ORL1 mRNA and [3H]nociceptin high affinity sites. These changes correlated well with the previously observed increased levels of non-opioid cardiac [3H]Dyn A-(1-13) sites in SHR (1.3 times as compared with WKY) and increased potency of Dyn A-(1-13) in inhibiting [3H]NA uptake by cardiac synaptosomes in SHR (2.2-fold as compared with WKY) (Dumont and Lemaire, 1995). The results demonstrate that in rat heart the characteristics of the high affinity, low capacity [3H]nociceptin binding site are similar to those of the non-opioid Dyn binding site. The stimulation of this site by nociceptin, Dyn A or related peptides is more likely to produce a modulation of PLC activity and [3H]NA uptake and may participate to the pathophysiology of hypertension.     

Enzymatic characterization of immunopurified prohormone convertase 2: potent inhibition by a 7B2 peptide fragment

Prohormone convertase (PCs) are thought to mediate the controlled proteolysis of prohormones and neuropeptide precursors. While recombinant PC1 and furin are currently available, thus far it has not been possible to produce recombinant PC2. We have used conditioned medium obtained from the mouse insulinoma cell line beta TC3 to generate a working preparation of enzymatically active PC2 through immunopurification. Immunopurified PC2 cleaved the fluorogenic substrate Cbz-Arg-Ser-Lys-Arg-AMC in a time- and calcium-dependent manner. It was half-maximally stimulated at 75 microM Ca2+, had an optimum pH of 5, and exhibited PCMS and EDTA sensitivity similar to that reported for furin and PC1. The tight-binding inhibitor 27 kDa 7B2 was used to calculate the Kd for this inhibitor and the active enzyme concentration. The Kd was 7.3 +/- 1.7 nM, and the turnover rate of PC2 was 5.2 molecules substrate per enzyme molecule per minute. The specific activity was 4.9 nmol/micrograms/h (assuming a molecular mass for PC2 of 64 kDa). The enzyme preparation was able to cleave recombinant proenkephalin at at least four of the expected paired basic sites in the absence, but not in the presence, of 27 kDa 7B2. Since 21 kDa 7B2 is functionally inactive as a proteinase inhibitor, we examined the inhibitory activity of the carboxy-terminal portion of 27 kDa 7B2 (7B2 CT-peptide). Synthetic peptides were used to demonstrate that the 7B2 CT-peptide (a) represents a potent inhibitor of PC2 (Ki = 57 nM), (b) can block the conversion of proPC2 to PC2, and (c) can block the PC2-mediated conversion of proenkephalin to smaller peptide fragments.(ABSTRACT TRUNCATED AT 250 WORDS)