Curves of ligand binding. The use of hyperbolic functions for expressing titration curves

1. The dependences of the concentrations of the non-ligated, uni-ligated and bi-ligated forms of a molecule that binds two molecules of ligand are expressed as functions of the logarithm of free ligand concentration by means of hyperbolic functions. Expressions are also given for the saturation both of an individual site and of the molecule as a whole. This form of expression allows derivation of the following points. 2. The sharpness of bell-shaped curves of concentration of the uni-ligated form is analysed in terms of the heights of their points of inflexion; these can rise to 1/ radical2 of the curve. 3. A single group can exhibit a doubly sigmoid saturation curve if this group and another have comparable affinities for a ligand, and if ligand binding at one of them diminishes the affinity at the other. If the molecular pK values pK(1) and pK(2) for the first and second molecules of ligand are called pK*+/-logm, so that K*(2)=K(1)K(2) and m(2)=K(1)/K(2), then the doubly sigmoid curve can be represented by the sum of two independent one-site saturation curves, in general of unequal height, of pK values pK*+/-log(1/2)[m+ radical(m(2)-4)]. The error in such representation is small either if the mutual interaction between the groups (i.e. m) is large, or if the groups have very similar affinities for the ligand. 4. The sum of two one-site saturation curves, again of pK values of pK*+/-log(1/2)[m+ radical(m(2)-4)] but of equal heights, gives a precise value for the total saturation, provided that the binding of one molecule does not promote the binding of a second, i.e. providing that m>/=2. Hence determinations of saturation cannot distinguish interacting and possibly identical sites from independent and different ones.     

Casein Kinase II-Type Protein Kinase from Pea Cytoplasm and Its Inactivation by Alkaline Phosphatase in Vitro

A casein kinase II-type protein kinase has been purified from the cytosolic fraction of etiolated pea (Pisum sativum L.) plumules to about 90% purity as judged from Coomassie blue stained sodium dodecyl sulfate-polyacrylamide gels. This kinase has a tetrameric [alpha][alpha]'[beta]2 structure with a native molecular mass of 150 kD, and subunit molecular masses of 41 and 40 kD for the two catalytic subunits ([alpha] and [alpha]') and 35 kD for the putative regulatory subunit ([beta]).Casein and phosvitin can be used as artificial substrates for this kinase. Both serine and threonine residues were phosphorylated when mixed casein, [beta]-casein, or phosvitin were used as the substrate, whereas only serine was phosphorylated if [alpha]-casein or histone III-S was the substrate. The kinase activity was stimulated 130% by 0.5 mM spermine (the concentration required for 50% of maximal enzyme activity [A50] = 0.1 mM) and 80% by 2.5 mM spermidine (A50 = 0.4 mM), whereas putrescine and cadaverine had no effect. The kinase was very sensitive to inhibition by heparin (concentration for 50% inhibition [I50] = 0.025 [mu]g/mL). In contrast to most other casein kinase II-type protein kinases, this preparation was inhibited by K+ and Na+, with I50 values of 75 and 65 mM, respectively. Pretreatment of the purified kinase preparation in vitro with alkaline phosphatase caused a 5-fold decrease in its activity. Additionally, this kinase also lost its activity when its [beta] subunit was autophosphorylated in the absence of substrate. These results suggest that the activity of this casein kinase II protein kinase may be regulated by the phosphorylation state of two different sites in its multimeric structure.     

Substrate- and alkali-metal-ion-induced pK shifts in intestinal brush-border sucrase, according to the three-protons model.

To define adequately enzyme activation/inhibition mechanisms as a function of pH, it is necessary to characterize the effector-induced pK shifts on both the free enzyme and on the enzyme-substrate complex. On the basis of our recent three-protons model for sucrase [Vasseur, van Melle, Frangne & Alvarado (1988) Biochem. J. 251, 667-675], we show how the 'fundamental' pK values, deduced from the classical double-logarithmic transformations, are insufficient to generate the required information. This insufficiency derives from the fact that, for sucrase, the acid ionization constant, K1, is a molecular constant that involves complex, V-type plus K-type, activatory and inhibitory kinetic effects. As a consequence, substrate-induced pK shifts cannot be interpreted correctly only by using the fundamental pK approach, because an unequal number of key protons is involved, depending on whether the free enzyme or the enzyme-substrate complex is considered. We demonstrate how this problem can be solved by using the 'theoretical' pK values, derived from the reciprocals of the Michaelis pH functions, i.e. Cha's fractional concentration factors. The procedure we propose, which is general, has the advantage of yielding all the macroscopic pK values for any given model, as calculated from the microscopic pK values. Furthermore, it permits predicting pK shifts as a function of [S] and/or [A] (where S is the substrate and A is the allosteric modifier), an objective that cannot be attained by using the double-logarithmic plot approach. Finally, we describe the relation existing between the fundamental and the theoretical pK values.     

Substance P and antagonists. Surface activity and molecular shapes.

The molecular properties of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met amide) and three of its antagonists were derived by measuring the Gibbs adsorption isotherm, providing information on the surface activity, the molecular shape, and the pK values of the different molecules. The following three antagonists were investigated: [D-Arg1,D-Pro2,D-Trp7,9,Leu11]SP, ANT I; [D-Arg1,D-Trp7,9,Leu11]SP, ANT II and [D-Pro2,D-Trp7,9]SP, ANT III. SP is only moderately surface active. The amino acid substitutions lead, however, to an increased surface activity of the antagonists. From the concentration dependence of the surface activity it was possible to quantify the packing characteristics of the individual neuropeptides. SP shows cross-sectional areas of 300 +/- 5 A2 to 240 +/- 5 A2 (pH 5 to 8, 154 mM NaCl) at concentrations below 10(-5) M, i.e., in the physiological concentration range, indicating a folded SP conformation. Upon increasing the packing density to concentrations larger than 10(-5) M the surface area was only half as large (148 +/- 5 A2 to 124 +/- 3 A2) suggesting now a relatively extended conformation of the SP molecule with its long molecular axis perpendicular to the air/water interface. In contrast, the three antagonists were characterized by surface areas of 147 +/- 3 A2 to 126 +/- 3 A2 which were almost independent of concentration. The antagonists thus adopt a relatively extended conformation in the whole concentration range measured. This is further supported by computer modelling which shows that the antagonists are motionally restricted and can adopt neither a bent nor a alpha-helical conformation. The surface activity of the neuropeptides was dependent on the pH of the solution. At low peptide concentrations (about 10(-6) M) it was possible to resolve and determine the pK values of all individual charged amino acid side chains. The pK values observed for the neuropeptides were about two pK units lower than those of the free amino acids in solution. The pK shifts of the neuropeptides at the air/water interface are explained in terms of the Gouy-Chapman theory. SP and its antagonists bind to lipid bilayers in the order of their surface activity. While the binding of SP is mainly due to electrostatic interactions, hydrophobic peptide-lipid interactions contribute to the binding of the antagonists.     

Effect of the alkaline cations on the stability of the model polynucleotide poly(dG-dC)·poly(dG-dC)

When the model polynucleotide poly(dG-dC)?poly(dG-dC) [polyGC] is titrated with a strong acid (HCl) in unbuffered aqueous solutions containing the chlorides of the alkali metals in the concentration range 0.010?M-0.600?M, two transitions in the absorbance vs. pH plots are evidenced, characterized by the constants pK(a(?)) and pK(a(?)). The limiting values at infinite saline concentrations of these two constants, namely pK(∞)(a(?)) and pK(∞)(a(?)) obtained making use of the "one site saturation constant" equation or, in turn, of the double logarithmic plot: pK(a) vs. log([salt]?1), exhibit a clear dependence on the nature of the cations. The effects of the different alkali cations on the pK(∞)(a) values follow the Hofmeister series. In fact, the pK(∞)(a(?)) and the pK(∞)(a(?)) values are smaller for Li+ and Na+ than for Rb+ and Cs+, with K+ at the border between the two, showing that the transitions require higher concentrations of protons to occur in the presence of high concentrations of the cosmotropic ions.     

Pharmacological evaluation of selected arylpiperazines with atypical antipsychotic potential

Six active compounds, among previously synthesized and screened arylpiperazines, were selected and evaluated for the binding affinity to rat dopamine, serotonin and alpha(1) receptors. Two compounds with benztriazole group had a 5-HT(2A)/D(2) binding ratio characteristic for atypical neuroleptics (>1, pK(i) values). Compound 2, 5-[2-[4-(2,3-dimethyl-phenyl)-piperazin-1-yl]ethyl]1H-benzotriazole, expressed clozapine-like in vitro binding profile at D(2), 5-HT(2A) and alpha 1 receptors and a higher affinity for 5-HT(1A) receptors than clozapine. Also, it exhibited the noncataleptic behavioural pattern of atypical antipsychotics and antagonized d-amphetamine-induced hyperlocomotion in rats.     

Synthesis and acid ionization constants of cyclic cystine peptides H-Cys-(Gly)n-Cys-OH (n = 0-4)

Cyclic peptide disulfides of the general formula H-Cys-(Gly)n-Cys-OH (n = 0-4) were synthesized from the corresponding peptide derivatives [Boc-Cys(Trt)(Gly)-n-Cys(Trt)-OBut] by oxidation with iodine in methanol and by subsequent removal of the terminal groups with trifluoroacetic acid. Acid ionization constants of the obtained peptides were determined by potentiometric titration in aqueous KCl (0.1 mol/L) medium. All compounds have two dissociable hydrogens, corresponding to carboxyl (pK1 = 2.35-2.84) and to terminal amino group (pK2 = 5.61-6.93); pK1 values show first an upward and then a downward trend with the increase in ring size; the opposite is true for pK2 values. These trends could be tentatively attributed to the intramolecular salt bridge (-COO- ----NH+3-) formation.     

Replacement of lysine 269 by arginine in Escherichia coli tryptophan indole-lyase affects the formation and breakdown of quinonoid complexes

Lysine 269 in Escherichia coli tryptophan indole-lyase (tryptophanase) has been changed to arginine by site-directed mutagenesis. The resultant K269R mutant enzyme exhibits kcat values about 10% those of the wild-type enzyme with S-(o-nitrophenyl)-L-cysteine, L-tryptophan, and S-benzyl-L-cysteine, while kcat/Km values are reduced to 2% or less. The pH profile of kcat/Km for S-benzyl-L-cysteine for the mutant enzyme exhibits two pK alpha values which are too close to separate, with an average value of 7.6, while the wild-type enzyme exhibits pK alpha values of 6.0 and 7.8. The pK alpha for the interconversion of the 335 and 412 nm forms of the K269R enzyme is 8.3, while the wild-type enzyme exhibits a pK alpha of 7.4. Steady-state kinetic isotope effects on the reaction of [alpha-2H]S-benzyl-L-cysteine with the K269R mutant enzyme (Dkcat = 2.0; D(kcat/Km) = 3.9) are larger than those of the wild-type enzyme (Dkcat = 1.4; D(kcat/Km) = 2.9). Rapid scanning stopped-flow kinetic studies demonstrate that the K269R mutant enzyme does not accumulate quinonoid intermediates with L-alanine, L-tryptophan, or S-methyl-L-cysteine, but does form quinonoid absorption peaks in complexes with S-benzyl-L-cysteine and oxidolyl-L-alanine, whereas wild-type enzyme forms prominent quinonoid bands with all these amino acids. Single wavelength stopped-flow kinetic studies demonstrate that the alpha-deprotonation of S-benzyl-L-cysteine is 6-fold slower in the K269R mutant enzyme, while the intrinsic deuterium kinetic isotope effect is less for the K269R enzyme (Dk = 4.2) than for the wild-type (Dk = 7.9). The decay of the K269R quinonoid intermediate in the presence of benzimidazole is 7.1-fold slower than that of the wild-type enzyme. These results demonstrate that Lys-269 plays a significant role in the conformational changes or electrostatic effects obligatory to the formation and decomposition of the quinonoid intermediate, although it is not an essential basic residue.     

Contraction-induced increases in Na+-K+-ATPase mRNA levels in human skeletal muscle are not amplified by activation of additional muscle mass

The present study tested the hypothesis that exercise with a large compared with a small active muscle mass results in a higher contraction-induced increase in Na(+)-K(+)-ATPase mRNA expression due to greater hormonal responses. Furthermore, the relative abundance of Na(+)-K(+)-ATPase subunit alpha(1), alpha(2), alpha(3), alpha(4), beta(1), beta(2), and beta(3) mRNA in human skeletal muscle was investigated. On two occasions, eight subjects performed one-legged knee extension exercise (L) or combined one-legged knee extension and bilateral arm cranking (AL) for 5.00, 4.25, 3.50, 2.75, and 2.00 min separated by 3 min of rest. Leg exercise power output was the same in AL and L, but heart rate at the end of each exercise interval was higher in AL compared with L. One minute after exercise, arm venous blood lactate was higher in AL than in L. A higher level of blood epinephrine and norepinephrine was evident 3 min after exercise in AL compared with L. Nevertheless, none of the exercise-induced increases in alpha(1), alpha(2), beta(1), and beta(3) mRNA expression levels were higher in AL compared with L. The most abundant Na(+)-K(+)-ATPase subunit at the mRNA level was beta(1), which was expressed 3.4 times than alpha(2). Expression of alpha(1), beta(2), and beta(3) was less than 5% of the alpha(2) expression, and no reliable detection of alpha(3) and alpha(4) was possible. In conclusion, activation of additional muscle mass does not result in a higher exercise-induced increase in Na(+)-K(+)-ATPase subunit-specific mRNA.     

A possibility of a protein-bound water molecule as the ionizable group responsible for pKe at the alkaline side in human matrix metalloproteinase 7 activity

Human matrix metalloproteinase 7 (MMP-7) activity exhibits broad bell-shaped pH profile with the acidic and alkaline pK(a) (pK(e1) and pK(e2)) values of about 4 and 10. The ionizable group for pK(e2) was assigned to Lys or Arg by thermodynamic analysis; however, no such residues are present in the active site. Hence, based on the crystal structure, we hypothesized that a water molecule bound to the main-chain nitrogen of Ala162 (W1) or the main-chain carbonyl oxygen of Pro217 (W2) is a candidate for the ionizable group for pK(e2) [Takeharu, H. et al. (2011) Biochim. Biophys. Acta 1814, 1940-1946]. In this study, we inspected this hypothesis. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2), all 19 variants, in which one of all Lys and Arg residues was replaced by Ala, retained activity, indicating that neither Lys nor Arg is the ionizable group. pK(e2) values of A162S, A162V and A162G were 9.6 ± 0.1, 9.5 ± 0.1 and 10.4 ± 0.2, respectively, different from that of wild-type MMP-7 (WT) (9.9 ± 0.1) by 0.3-0.5 pH unit, and those of P217S, P217V and P217G were 10.1 ± 0.1, 9.8 ± 0.1 and 9.7 ± 0.1, respectively, different from that of WT by 0.1-0.2 pH unit. These results suggest a possibility of W1 or W2 as the ionizable group for pK(e2).     

Ligand modulation of [35S]GTPgammaS binding at human alpha(2A), alpha(2B) and alpha(2C) adrenoceptors

Affinities and efficacies of chemically diverse ligands--some of them used as clinical agents--were examined, employing [3H]RX821,002 and [35S]GTPgammaS binding assays, respectively, at human (h) cloned, halpha(2A), halpha(2B) and halpha(2C) adrenoceptors (AR) expressed in Chinese hamster ovary (CHO) cells. As compared to noradrenaline (NA, efficacy defined as 100%), the majority of the 13 agonists tested generally behaved as partial agonists. Amongst 18 antagonists, pK(B) and pK(i) values, which were highly correlated for each alpha(2)-AR subtype, failed to reveal any strikingly selective agents. Inverse agonist properties were not detected for any antagonist, consistent with a lack of constitutive activity suggested by the monophasic inhibition of [35S]GTPgammaS binding by GTPgammaS. These data should facilitate interpretation of experimental and clinical actions of adrenergic agonists. Moreover, they emphasize the continuing need for alpha(2)-AR subtype-selective antagonists in order to define further the roles and therapeutic relevance of halpha(2A)-, halpha(2B)-, and halpha(2C)-AR.     

Determination of dissociation constants of (4RS)-[4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oic acid] in water and biological fluids by means of nuclear magnetic resonance spectroscopy and the DISCO software package

The pK(A) values of (4RS)-[4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oic acid] (BOPTA), a polyprotic molecule whose gadolinium complex is an important magnetic resonance imaging contrast agent for clinical use, have been determined in water, in physiologic solution (PS), in serum (S), and in cerebrospinal fluid (CSF), by means of 13C nuclear magnetic resonance spectroscopy data processed by a dedicated software package called DISCO. The aim of this study was to supply the BOPTA pK(A) values in media very similar to the in vivo environment and, consequently, to get a picture of the in vivo behavior of its Gd complex, whose thermodynamic stability is directly linked to the pK(A) values. The pK(A) values appeared to be almost equal both in D(2)O and in PS, while pK(1) and pK(5) values in CSF differ a little. In S, only pK(2) and pK(3) were calculated due to the narrow pH range used for data collection. However, these pK(A) values were found equal to those in the other media. These results represent the first direct spectroscopic evidence of a substantial invariability of BOPTA behavior in different media and they justify the extrapolation to biological fluids of the data obtained in water. The values also confirmed the high-quality performance of DISCO in calculating pK(A) values of polyprotic molecules in complex media.     

Electrostatic effects in highly charged proteins: salt sensitivity of pKa values of histidines in staphylococcal nuclease

The pK(a) values of most histidines in small peptides and in myoglobin increase on average by 0.30 unit between 0.02 and 1.5 M NaCl [Kao et al. (2000) Biophys. J. 79, 1637]. The DeltapK(a) values reflect primarily the ionic strength dependence of the solvation energy; screening of Coulombic interactions contributes only in a minor way. This implies that Coulombic interactions are weak, or that attractive and repulsive contributions to the pK(a) values are balanced. To distinguish experimentally between these two possibilities, and to further characterize the magnitude and salt sensitivity of surface electrostatic interactions in proteins, the salt dependence of pK(a) values of histidines in staphylococcal nuclease was measured by (1)H NMR spectroscopy. Three of the four histidines titrated with significantly depressed pK(a) values, and the salt sensitivity of all histidine pK(a) values was substantial. In three cases, the pK(a) values increased by a full unit between 0.01 and 1.5 M KCl. Anion-specific effects were found; the pK(a) values measured under equivalent ionic strengths in SCN(-) and SO(4)(2-) were higher than in Cl(-); the order of the sensitivity of pK(a) values to anions was SCN(-) > Cl(-) > SO(4)(2-). Structure-based pK(a) calculations with continuum methods were performed to interpret the measured effects structurally and to test their ability to capture the experimental behavior. Calculations in which the protein interior was treated empirically with a dielectric constant of 20 reproduced the pK(a) values and their dependence on the concentration of Cl(-). According to the calculations, the pK(a) values are depressed because of unfavorable self-energies and repulsive Coulombic interactions. Their striking salt sensitivity reflects screening of weak, repulsive, Coulombic interactions among charges separated by more than 10 A. Long-range Coulombic interactions on the surfaces of proteins are weak, but they can add up to produce substantial electrostatic effects when positive and negative charges are not balanced.     

31P nuclear magnetic resonance study of phosphoribosyldiphosphate and its interaction with magnesium ions

31P NMR has been used to study phosphoribosyldiphosphate (P-Rib-PP) over a wide range of pH values, both in the absence and presence of MgCl2. In the absence of MgCl2, the chemical shift variations of the three 31P nuclei in the molecule, over the pH range 4 to 9, were found to be largest for the terminal 1-diphosphate (1P beta) oxyanion and the 5-phosphate (5P) moiety. Apparent pK alpha values of approximately 6.1 and 6.3 were estimated for protonation of the 1P beta and 5P groups, respectively. Variations in the apparent pK alpha values associated with 1P beta and 5P oxyanions in the presence of various concentrations of MgCl2 were consistent with P-Rib-PP having two independent metal ion binding sites with different affinities for Mg2+ ions. The binding of Mg2+ reduced the apparent pK alpha of the 1P beta moiety by approximately 1.6 units and the apparent pK alpha of the 5P group by approximately 0.7 unit. This behavior is analogous to the situation reported for the terminal phosphooxyanion of ADP and observed for the phosphate group of ribose 5-phosphate, respectively. In the presence of an equimolar concentration of added MgCl2, the 1P alpha and 1P beta resonances of P-Rib-PP were shifted downfield and the 31P-31P coupling constant was decreased. Changes in both these parameters were very similar to those reported for the MgADP- complex. The observed chemical shifts and spin-spin coupling constants suggest that the diphosphate and monophosphate moieties of P-Rib-PP act as independent binding sites for Mg2+ in a manner similar to the phosphooxyanion groups of ADP and ribose 5-phosphate, respectively.     

Photoaffinity labeling of the ouabain binding site in Na, K-ATPase in developing brine shrimp

Analysis of purified Na,K-ATPase from brine shrimp nauplii by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals two large (alpha) subunits [G.L. Peterson, R.D. Ewing, S.R. Hootman, and F.P. Conte (1978) J. Biol. Chem. 253:4762]. The band with lower mobility in a neutral or alkaline gel is designated alpha 1 and the band with higher mobility alpha 2. Ouabain prevents dephosphorylation of both alpha 1 and alpha 2 as documented by gel analysis, but a higher concentration of ouabain is required to prevent dephosphorylation of alpha 2. The photoaffinity label, [3H]4'(2-ethyldiazomalonyl) digitoxigenin monodigitoxiside, specifically labels alpha in a ouabain-protectable manner without labeling other contaminating proteins in the preparation. Greater than 93% of the total ouabain-protectable labeling of the alpha subunits is associated with alpha 1. The photoaffinity label, [3H]4"' (2-ethyldiazomalonyl) digitoxin, specifically labels alpha 1 and beta in a ouabain-protectable manner without labeling other contaminating proteins. These data show that in the brine shrimp the third digitoxose residue of digitoxin binds in a region in which the alpha 1 and beta chains are in close proximity. Less than 5% of the specific ouabain-protectable labeling of total alpha is associated with alpha 2. These studies indicate that cardioactive steroids have higher affinity for the alpha 1 subunit.     

Dissociation of the contractile and hypertrophic effects of vasoconstrictor prostanoids in vascular smooth muscle.

To more clearly define the physiologic roles of thromboxane (TX)A2 and primary prostaglandins (PG) in vascular tissue we examined vascular contractility, cell signaling, and growth responses. The growth-promoting effects of (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619; TXA2 agonist), PGF2 alpha, and PGE2 consisted of protein synthesis and proto-oncogene expression, but not DNA synthesis or cell proliferation. U46619 contracted rat aortas and increased cultured rat aortic vascular smooth muscle cell intracellular free calcium concentration [Ca2+]i, [3H]inositol monophosphate (IP) accumulation, myosin light chain phosphorylation, and protein synthesis ([3H]leucine incorporation) with EC50 values ranging from 10 to 50 nM. Each of these responses was inhibitable with the TXA2 receptor antagonist [1S]1 alpha,2 beta(5Z),3 beta,4 alpha-7-(3-[2- [(phenylamino)carbonyl]hydrazino]methyl)-7-oxabicyclo[2.2.1]hept-2- yl-5-heptenoic acid (SQ29548). In contrast, PGF2 alpha increased [Ca2+]i, [3H]IP, and protein synthesis with EC50 values of 30-230 nM but contracted rat aortas with an EC50 of 4800 nM. PGE2 increased [Ca2+]i, [3H]IP accumulation, protein synthesis, and contracted rat aortas with EC50 values of 2.5-3.5 microM. TXA2 receptor blockade prevented PGF2 alpha- and PGE2-induced aortic contraction and cell myosin light chain phosphorylation, but not cell signaling or protein synthesis. Binding studies to vascular smooth muscle TXA2 receptors using 1S-[1 alpha,2 beta(5Z),3 alpha(1E,3S),4 alpha]-7-(3-[3-hydroxy-4-(p- [125I]iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-hepte noic acid ([125I]BOP) showed U46619, SQ29548, PGF2 alpha, and PGE2 competition for TXA2 receptor binding at concentrations similar to their EC50 values for aortic contraction, while binding competition with [3H]PGF2 alpha and [3H]PGE2 demonstrated the specificity of [125I]BOP and SQ29548 for TXA2 receptors. The results suggest that 1) PGF2 alpha- and E2-stimulated vessel contraction is due to cross-agonism at vascular TXA2 receptors; 2) PGF2 alpha stimulates TXA2 receptor-independent vascular smooth muscle protein synthesis at nanomolar concentrations, consistent with an interaction at its primary receptor; and 3) TXA2 is a potent stimulus for vascular smooth muscle contraction and protein synthesis. We suggest that the main physiologic effect of PGF2 alpha may be as a stimulus for vascular smooth muscle cell hypertrophy, not as a contractile agonist.     

Heterogeneity of pig kidney Na,K-ATPase as indicated by ADP- and ouabain-binding stoichiometry.

A centrifugation method has been used for determination of [14C]ADP and [3H]ouabain binding to Na,K-ATPase from pig kidney with high specific activity. In the presence of K+, the fit of the [14C]ADP binding data to a two-site model gives a component with high affinity which accounts for 12 +/- 2% of the total sites. The figure is significantly different from 50%, i.e., two components of equal size cannot be assumed. This contrasts with a ratio between the sites of 1:1 obtained by the rate dialysis technique. The discrepancy may be due to the fact that the centrifugation method enables bound ADP to be determined at lower concentrations of free ligand. [3H]Ouabain binding in the absence of Na+ is compatible with a straight line in a Scatchard plot if the isotope is purified shortly before use. An unspecific binding of ouabain can be neglected if the concentration of free ouabain is not too high. In the presence of Na+, the isotherms become upward concave. An analysis of the binding data gives a 19:81% division, although equilibrium is not quite attained. This is a maximum value because the lack in equilibrium will be most pronounced at the small values of free ouabain. Thus the ADP-binding studies are supported. The finding here is in some agreement with the semiquantitative immunoassay showing that pig kidney enzyme contains the isoenzymes alpha 1, alpha 2 and alpha 3 in a proportion of 84:12:4, respectively. Determination of ADP- and ouabain-binding site stoichiometry favours a theory with one substrate site per (alpha beta)2.     

Noradrenaline Release from Cultured Mouse Postganglionic Sympathetic Neurons

The possible existence of alpha2-autoreceptors, P2-autoreceptors, and adenosine A1- or A2A-receptors was studied in cultured thoracolumbar postganglionic sympathetic neurons from mice. The cells were preincubated with [3H]noradrenaline and then superfused. The selective alpha2-adrenoceptor agonist UK 14,304 reduced the electrically evoked overflow of tritium. When the cultures were stimulated by trains of increasing pulse number, ranging from a single pulse to 72 pulses at 3 Hz, the concentration-inhibition curve of UK 14,304 was shifted progressively to the right and the maximal inhibition obtainable became progressively smaller. Six alpha-adrenoceptor antagonists shifted the concentration-inhibition curve of UK 14,304 in a parallel manner to the right. Neither ATP (3-300 microM), adenosine (0.01-100 microM), the selective A1-receptor agonist cyclopentyladenosine (1-1,000 nM), nor the selective A2A-receptor agonist CGS-21680 (1-10,000 nM) changed the basal or the electrically evoked overflow of tritium. It is concluded that the cultured neurons possess presynaptic, release-inhibiting alpha2-autoreceptors. As in intact tissues, the effectiveness of presynaptic alpha2-adrenergic inhibition depends on the "strength" of the releasing stimulus. The pK(D) values of the six antagonists against UK 14,304 indicate that the autoreceptors belong to the pharmacological alpha2D and hence the genetic alpha(2A/D) subtype of alpha2-adrenoceptor. Neither P2-autoreceptors nor receptors for adenosine, the degradation product of ATP, were detected.     

pH dependence of kinetic parameters of pepsin, rhizopuspepsin, and their active-site hydrogen bond mutants.

The pH dependence of the kinetic parameters of pepsin, rhizopuspepsin, and their active-site hydrogen bond mutants has been determined. These data have permitted the calculation of two active-site ionization constants in the free enzymes (pKe1 and pK32) and in the enzyme-substrate complexes (pKes1 and pKes2). The pKe1 of rhizopuspepsin (2.8) is near that of a normal carboxyl group and near the pKe1 of human immunodeficiency virus type 1 (HIV-1) protease (3.32) (Ido, E., Han, H. P., Kezdy, F. J., and Tang, J. (1991) J. Biol. Chem. 266, 24359-24366). The pKe1 of pepsin (1.57) is thus abnormally low. The pKe2 of rhizopuspepsin (4.44) is lower than that of pepsin (5.02) and HIV protease (6.80). The binding of substrate to rhizopuspepsin causes the lowering of pKes1 to 1.8 and the elevating of pKes2 to above 6. The pK alpha shifts due to substrate binding are much less pronounced in pepsin. Thus, the two enzyme-substrate complexes have similar pK alpha values. For both pepsin and rhizopuspepsin, the removal of hydrogen bonds to the active-site carboxyls by mutagenesis results in negligible changes in the four pK alpha values. The major alteration caused by these mutations is the decrease in kcat values, while there is little change in Km. These observations suggest that these hydrogen bonds to the active-site aspartyls contribute little to the pH-activity relationships of the aspartic proteases. The role of the active-site hydrogen bonds may well be to preserve the conformational rigidity of the catalytic apparatus.