Apparent Affinity Constant Research Articles

Specificity and Stoichiometry of the ArabidopsisH+/Amino Acid Transporter AAP5

The H+-dependent AAP5 amino acid transporter from Arabidopsis thaliana was expressed in Xenopus oocytes, and we used radiotracer flux and electrophysiology methods to investigate its substrate specificity and stoichiometry. Inward currents of up to 9 microA were induced by a broad spectrum of amino acids, including anionic, cationic, and neutral amino acids. The apparent affinity of AAP5 for amino acids was influenced by the position of side chain branches, bulky ring structures, and charged groups. The maximal current was dependent on amino acid charge, but was relatively independent of amino acid structure. A detailed kinetic analysis of AAP5 using lysine, alanine, glutamate, and histidine revealed H+-dependent differences in the apparent affinity constants for each substrate. The differences were correlated to the effect of H+ concentration on the net charge of each amino acid and suggested that AAP5 transports only the neutral species of histidine and glutamate. Stoichiometry experiments, whereby the uptake of 3H-labeled amino acid and net inward charge were simultaneously measured in voltage-clamped oocytes, showed that the charge:amino acid stoichiometry was 2:1 for lysine and 1:1 for alanine, glutamate, and histidine. The results confirm that histidine is transported in its neutral form and show that the positive charge on lysine contributes to the magnitude of its inward current. Thus, the transport stoichiometry of AAP5 is 1 H+:1 amino acid irrespective of the net charge on the transported substrate. Structural features of amino acid molecules that are involved in substrate recognition by AAP5 are discussed.

Functional epitope mapping of insulin-like growth factor I (IGF-I) by anti-IGF-I monoclonal antibodies.

Based on a collection of monoclonal antibodies (mAb) against insulin-like growth factor I (IGF-I), we have defined the IGF-I epitopes involved in the interaction with IGF-binding proteins (IGFBP) and IGF-I receptors. We have also characterized the ability of these antibodies to block IGF-I-induced survival of the IL-3-dependent Ba/F3 cell line. More than 140 hybridomas secreting IGF-I-specific mAb were characterized, of which 28 were studied in detail. They display apparent affinity constants ranging from less than 10(6) to 10(10) M-1 and varying crossreactivity with IGF-II, including 2 mAb with higher affinity for IGF-II than for IGF-I. None crossreact with insulin or any other growth factor tested. Using both enzyme immunoassays and real-time biospecific interaction analysis, we have identified 8 epitopic clusters related to the primary structure of IGF-I, according to mAb reactivity to synthetic peptides, proteolytic fragments of IGF-I, and various IGF-I mutants. The mAb panel also was used to map the IGF domains implicated in the interaction with IGFBP and IGF-I receptors. An IGF-I domain has been identified that remains exposed after IGF-I binding to IGFBP-1 or to IGFBP-3, which is recognized by 6 different mAb. The mAb in this group also bind IGF-I, when complexed to the type-1 IGF receptor on the murine pro-B cell line Ba/F3, and BALB/c 3T3 fibroblasts overexpressing the human receptor. Finally, IGF-I-promoted survival can be blocked with mAb specific for target epitopes, and their potential use in tumor cell growth control is discussed.

Functional Epitope Mapping of Insulin-Like Growth Factor I (IGF-I) by Anti-IGF-I Monoclonal Antibodies

Based on a collection of monoclonal antibodies (mAb) against insulin-like growth factor I (IGF-I), we have defined the IGF-I epitopes involved in the interaction with IGF-binding proteins (IGFBP) and IGF-I receptors. We have also characterized the ability of these antibodies to block IGF-I-induced survival of the IL-3-dependent Ba/F3 cell line. More than 140 hybridomas secreting IGF-I-specific mAb were characterized, of which 28 were studied in detail. They display apparent affinity constants ranging from less than 106 to 1010m−1 and varying crossreactivity with IGF-II, including 2 mAb with higher affinity for IGF-II than for IGF-I. None crossreact with insulin or any other growth factor tested. Using both enzyme immunoassays and real-time biospecific interaction analysis, we have identified 8 epitopic clusters related to the primary structure of IGF-I, according to mAb reactivity to synthetic peptides, proteolytic fragments of IGF-I, and various IGF-I mutants. The mAb panel also was used to map the IGF domains implicated in the interaction with IGFBP and IGF-I receptors. An IGF-I domain has been identified that remains exposed after IGF-I binding to IGFBP-1 or to IGFBP-3, which is recognized by 6 different mAb. The mAb in this group also bind IGF-I, when complexed to the type-1 IGF receptor on the murine pro-B cell line Ba/F3, and BALB/c 3T3 fibroblasts overexpressing the human receptor. Finally, IGF-I-promoted survival can be blocked with mAb specific for target epitopes, and their potential use in tumor cell growth control is discussed.

Ligand Binding Specificities of the Two Mannose 6-Phosphate Receptors

Two mannose 6-phosphate (Man-6-P) receptors (MPRs) direct the vesicular transport of newly synthesized lysosomal enzymes that contain Man-6-P from the Golgi to a prelysosomal compartment. In order to understand the respective roles of the Mr = 46,000 cation-dependent (CD-) MPR and the Mr = 300,000 cation-independent (CI-) MPR in lysosomal targeting, an assay has been developed that simultaneously measures the relative affinity of each MPR for multiple ligands. Glycoproteins containing Man-6-P were affinity-purified from the metabolically labeled secretions of mutant mouse fibroblasts lacking both MPRs. They were incubated with purified MPRs, and the resulting receptor-ligand complexes were immunoprecipitated by anti-MPR monoclonal antibodies coupled to agarose beads. Ligands were eluted with Man-6-P and then quantified following SDS-polyacrylamide gel electrophoresis. Saturating concentrations of CI-MPR resulted in the complete recovery of each Man-6-P glycoprotein in receptor-ligand complexes. Apparent affinity constants ranged between 1 and 5 nM for the individual species. Ligands precipitated by the CD-MPR appeared identical to those bound by the CI-MPR, with apparent affinity constants ranging between 7 and 28 nM. The binding affinities of the two receptors for different ligands were not correlated, indicating that the two MPRs preferentially recognize different subsets of lysosomal enzymes. In addition, saturating levels of CD-MPR resulted in the precipitation of only 50% of the total input ligands, suggesting that the CD-MPR binds a subpopulation of the Man-6-P glycoproteins bound by the CI-MPR. These results provide a biochemical mechanism, which, in part, may explain the interaction of the two MPRs with overlapping yet distinct subsets of ligands in vivo.

Biochemical Characterization of Recombinant Fusions of Lipopolysaccharide Binding Protein and Bactericidal/Permeability-increasing Protein: IMPLICATIONS IN BIOLOGICAL ACTIVITY

The physiological response to endotoxin (lipopolysaccharide (LPS)) can be regulated by two closely related LPS-binding proteins, LPS-binding protein (LBP), which potentiates LPS' inflammatory activity via interaction with the monocytic antigen CD14, and bactericidal/permeability-increasing protein (BPI), which neutralizes LPS. Both proteins bind LPS with high affinity sites in their N-terminal domains, whereas interaction between LBP and CD14 is dependent upon the LBP C-terminal domain. We have created fusions of the N- and C-terminal domains from each protein and compared the functional activities and pharmacokinetics of these fusions, the individual N-terminal domains, and the parent proteins. The N-terminal domains of BPI and LBP bound lipid A with their characteristic apparent affinity constants, regardless of the C-terminal fusion partner. In addition, the C-terminal domain of LBP allowed transfer of LPS to CD14 in conjunction with either N-terminal LPS binding domain. Proteins containing a BPI N-terminal domain had greater heparin binding capacities in vitro and were cleared more rapidly from the plasma of whole animals. Taken together, these data better define how closely related proteins such as BPI and LBP can have opposing effects on the body's response to LPS.

Biophysical Characteristics of the Pig Kidney Na+/Glucose Cotransporter SGLT2 Reveal a Common Mechanism for SGLT1 and SGLT2

The Na+-dependent, low affinity glucose transporter SGLT2 cloned from pig kidney is 76% identical (at the amino acid level) to its high affinity homologue SGLT1. Using two-microelectrode voltage clamp, we have characterized the presteady-state and steady-state kinetics of SGLT2 expressed in Xenopus oocytes. The kinetic properties of the steady-state sugar-evoked currents as a function of external Na+ and alpha-methyl-D-glucopyranoside (alphaMG) concentrations were consistent with an ordered, simultaneous transport model in which Na+ binds first. Na+ binding was voltage-dependent and saturated with hyperpolarizing voltages. Phlorizin was a potent inhibitor of the sugar-evoked currents (KiPz approximately 10 microM) and blocked an inward Na+ current in the absence of sugar. SGLT2 exhibited Na+-dependent presteady-state currents with time constants 3-7 ms. Charge movements were described by Boltzmann relations with apparent valence approximately 1 and maximal charge transfer approximately 11 nC, and were reduced by the addition of sugar or phlorizin. The differences between SGLT1 and SGLT2 were that (i) the apparent affinity constant (K0.5) for alphaMG (approximately 3 mM) was an order of magnitude higher for SGLT2; (ii) SGLT2 excluded galactose, suggesting discrete sugar binding; (iii) K0.5 for Na+ was lower in SGLT2; and (iv) the Hill coefficient for Na+ was 1 for SGLT2 but 2 for SGLT1. Simulations of the six-state kinetic model previously proposed for SGLT1 indicated that many of the kinetic properties observed in SGLT2 are expected by simply reducing the Na+/glucose coupling from 2 to 1.

Variations in the ensemble of potassium currents underlying resonance in turtle hair cells.

1. Potassium currents were characterized in turtle cochlear hair cells by whole-cell voltage clamp during superfusion with the potassium channel antagonists, tetraethylammonium (TEA) and 4-aminopyridine (4-AP). The estimated resonant frequency, f0, was inferred from tau, the time constant of deactivation of outward current upon repolarization to -50 mV, according to the empirical relation, f0 = k1 tau-1/2 + k2. 2. Dose-response relations for TEA and 4-AP were obtained by exposing single cells to ten concentrations exponentially distributed over four orders of magnitude. Potassium current in cells tuned to low frequencies was carried by a single class of channels with an apparent affinity constant, K1, for TEA of 35.9 mM. Half-blocking concentrations of 4-AP were correlated with the time constant of deactivation and varied between 26.2 and 102 microM. In cells tuned to higher frequencies, K+ current was carried by a single class of channels with high affinity for TEA (K1 = 0.215 mM) and low affinity for 4-AP (K1 = 12.3 mM). This pharmacological profile suggests that K+ current in low frequency cells is purely voltage gated and in high frequency cells, it is gated by both Ca2+ and voltage. 3. For each current type, the voltage dependence of activation was determined from tail current amplitude at -50 mV. The purely voltage-gated current, IK(V), was found to increase e-fold in 4.0 +/- 0.3 mV (n = 3) in low frequency cells exposed to TEA (25 mM). The Ca(2+)- and voltage-gated current, IK(Ca), was more steeply voltage dependent, increasing e-fold in 1.9 mV (n = 2) in high frequency cells exposed to 4-AP (0.8 mM). 4. IK(V) was found to inactivate slowly during prolonged voltage steps (approximately 10 s). Steady-state inactivation increased with depolarization from -70 mV and was incomplete such that on average IK(v) did not fall below approximately 0.39 of its maximum value. 5. Superfusion of 4-AP (0.8 mM) reversibly depolarized a low frequency cell and eliminated steady voltage oscillations, while TEA (6 mM) had no effect. In a high frequency cell, voltage oscillations were abolished by TEA, but not by 4-AP. 6. The differential pharmacology of IK(V) and IK(Ca) was used to measure their contribution to K+ current in cells tuned to different frequencies. Both currents exhibited a frequency-dependent increase in maximum conductance. IK(V) accounted for nearly all K+ current in cells tuned to less than 60 Hz, while IK(Ca) was the dominant current in higher frequency cells. 7. Mapping resonant frequency onto epithelial position suggests an exponential relation between K+ current size and position. IK(V) appeared to be limited to the apical or low frequency portion of the basilar papilla and coincided with maximal expression of a K(+)-selective inward rectifier, IK(IR). This finding is consistent with the notion that low frequency resonance is produced by interaction of IK(V) and IK(IR) with the voltage-gated Ca2+ current, ICa, and the cell's capacitance. The ionic events underlying higher frequency resonance are dominated by the action of IK(Ca) and ICa and include a contribution from IK(IR).

Duration-dependent changes in calcium responsiveness in the alloxan-diabetic rat intestine

Disturbances of the gastrointestinal tract that are common in diabetes mellitus seem to be related to intestinal motility. In experimental models of diabetes, decreased calcium sensitivity has been demonstrated in various smooth muscles including those in the gastrointestinal tract. The main purpose of the present study was to examine further the calcium sensitivity in diabetic rat intestine and to understand if changes in the calcium sensitivity occur at an earlier stage of the disease. For this purpose, the effects of potassium and calcium were evaluated on nondepolarized and depolarized duodenum from rats with alloxan diabetes for 1 and 8 weeks and their age-matched controls. To evaluate the calcium sensitivity in rat duodenum, apparent affinity constants (pD 2 values) and intrinsic activities (α E values) were calculated for every experimental conditions examined in this study. Both values (pD 2 and α E) for the effects of potassium and calcium on the nondepolarized and depolarized duodenum, respectively, were not changed in 1-week diabetic rats. In contrast, intrinsic activities for the effects of potassium and calcium were found to be significantly decreased ( p < 0.001) in the nondepolarized and depolarized duodenum from rats with alloxan diabetes for 8 weeks, whereas apparent affinity constants were not altered in this case. Taking into consideration all these experimental findings, the decreased calcium sensitivity in gastrointestinal tract seems to be closely related to decreased calmodulin levels and may occur at a later stage of diabetes as a linkage to long-term gastrointestinal complications.

NMR investigation of pefioxacin/cation/DNA interactions. Mg 2+ and Ca 2+ binding

Pefloxacin at low concentration (10 −4 mol l −1 and high [cation] 0/[quinolone] 0 ratios forms 1:1 and 1:2 (drug:cation) complexes with magnesium or calcium cation, at pH 7.4. The binding sites are, first, the carbonyl and carboxyl groups, then, the N-4′ piperazinyl atom, as shown by the investigation of the pefloxacin ethyl ester/magnesium interaction. As expected, the apparent affinity constants are larger for magnesium than for calcium. When the pefloxacin concentration increases to 10 −3 mol −1, the affinity of this drug for magnesium becomes much larger. That is due to the formation of a 2:2 complex, what enhances the stacking of this fluoroquinolone.

Relative location of epitopes involved in synergistic antibody binding using human chorionic gonadotropin as a model.

We systematically screened a large panel of well-characterized monoclonal antibodies (mAb) directed towards various epitopes on human chorionic gonadotropin (hCG) for synergistic binding of 125I-hCG when they were adsorbed to a solid phase. The epitope locations involved in synergy were then related to the crystal structure of hCG and discussed in accordance with available data on the hCG epitopes. Enhanced binding of hCG was specific for certain pairs of mAb and was reflected in a 3-50-fold increased apparent functional affinity constant for hCG. Surface plasmon resonance revealed that when the mAb were captured by a polyclonal anti-IgG1 coupled to the Biacore chip, the off rates for hCG were significantly slower with synergistic mAb combinations than for the corresponding single mAb or nonsynergistic pairs of mAb, whereas the on rates did not differ appreciably. Each of the two antibodies involved in synergistic binding of hCG (more than 3-fold compared to additive binding of the two mAb) always belonged to a different epitope cluster in a separate antigenic domain on hCG. Synergistic epitope combinations on holo-hCG were located in similar structural planes. Combinations of mAb directed towards the epitope clusters alpha 2/beta 3/5, alpha 2/hCG beta CTP (C-terminal peptide) and beta 3/5/hCG beta CTP showed the strongest enhancement, with binding more than 10-fold greater than the sum of 125I-hCG bound to the individual mAb, followed by pairs of mAb directed towards the epitope groups beta 1/beta 3/5, c 1/2/beta 3/5, beta 1/alpha 2, and alpha 2/alpha 3/5 (3-9-fold). The greater frequency of synergy obtained with the linear epitopes of the hCG beta CTP can be ascribed to their greater molecular flexibility relative to the constrained discontinuous epitopes on hCG alpha and core-hCG beta (residues 1-112). In general, these studies provide a method for rapid screening of synergistic antibody pairs which also helps to identify non-overlapping epitopes that are accessible in similar structural planes. In turn, this facilitates the design of high-affinity bispecific antibodies targetted to a single antigen molecule.

Identification of the Fy6 epitope recognized by two monoclonal antibodies in the N-terminal extracellular portion of the Duffy antigen receptor for chemokines

The epitope Fy6 recognized by two monoclonal antibodies (i3A and BG6), which inhibit binding of chemokines to the Duffy antigen, was characterized by means of peptides synthesized on pins (Epitope Scanning Kit) and deletion mutagenesis. Both antibodies showed very similar specificities. They recognized a linear epitope, the essential portion of which was the heptapeptide Gln-Leu-Asp-Phe-Glu-Asp-Val comprising amino acid residues 21–27, located between two glycosylation sites of the Duffy protein. All the amino acid residues of the epitope, except Glu, were essential for antibody binding, since they could not be replaced by any other amino acid residues or by only one or two. The Glu residue could be replaced by most other amino acid residues, and its replacement by 10 amino acid residues gave a distinct increase in the antibody binding. The results were in full agreement with the finding that the mutant of the Duffy antigen, lacking amino acid residues 23–25 (-Asp-Phe-Glu-), did not bind the i3A antibody, but bound the anti-Fy3 monoclonal antibody similarly to the wild type of the Duffy antigen. The apparent affinity constants of both anti-Fy6 antibodies were determined by surface plasmon resonance, using immunopurified Duffy protein as a ligand.

Influence of oestrogens on muscarinic receptor density and contractile response in the guinea-pig uterus.

The objective of this study was to investigate the contractile response in vitro of uterine and myometrial strips from control and oestrogen-treated guinea-pigs to carbachol. In addition, the muscarinic receptor parameters in subcellular preparations from the myometrium of the same animals were evaluated. Strips of guinea-pig uterus contract in response to carbachol in a concentration dependent manner and the effect of carbachol is independent of the presence of an intact endometrium. However, treatment with oestrogen reduces the response to carbachol, when expressed with respect to the effect of KCl. Measurement of the specific binding of [3H]quinuclidinyl benzylate ([3H]QNB) to myometrial membranes from control and oestradiol-treated immature guinea-pigs showed that oestradiol significantly decreased the number of specific binding sites of [3H]QNB, but did not change the apparent affinity of specific [3H]QNB binding. Moreover, the apparent affinity constants for the different antagonists showed a close similarity to those obtained at muscarinic receptors present in the myometrium from control and oestradiol-treated guinea-pigs.

Effect of Sodium on the Energetics of Thrombin – Thrombomodulin Interaction and its Relevance for Protein C Hydrolysis

Measurements of the apparent affinity constant for thrombomodulin (TM) binding to human α-thrombin as a function of both Na +and temperature at constant ionic strength (0.15 M) showed that TM affinity increases in the presence of Na +and vice versa. Moreover, this experimental strategy allowed us to accurately split the free energy of sodium binding into its entropic and enthalpic components for both the TM-free and TM-bound enzyme. Namely, at 25°C, the value of Δ Gof sodium binding was found equal to −2.4 kcal/mol in the absence of TM and −3.6 kcal/mol for the thrombin-TM complex. The enthalpic contribution to the free energy of sodium binding is equal to −27 kcal/mol and −21 kcal/mol in the TM-free and TM-bound thrombin forms, respectively. Finally, the entropy change for sodium binding was also affected by TM, being equal to −83 cal/(mol deg) and −58 cal/(mol deg) in TM-free and TM-bound thrombin species, respectively. Moreover, the thermodynamic parameters for TM binding to Na +-free thrombin species were solved. TM binding is characterized by an enthalpy and entropy change equal to −10 kcal/mol and 2 cal/(mol deg), respectively, for Na +-free thrombin. It is well known that Na +binding to thrombin causes conformational transitions and functional activation of the enzyme molecule. The finding that binding of thrombomodulin enhances thrombin affinity for sodium and vice versaraises the question as to whether the change of Na +ligation induced by TM binding could contribute to the change in thrombin specificity for the hydrolysis of Protein C. Therefore, the effect of sodium binding to thrombin on the hydrolysis of human Protein C was extensively investigated. At both 25 and 37°C the value of k cat/ K mfor Protein C hydrolysis by thrombin in the absence of TM was found to be enhanced by Na +over a concentration ranging from 0 to 150 mM. Application of thermodynamic principles demonstrated that the Na +-thrombomodulin linkage contributes, under physiological conditions of sodium activity and temperature, to reduce significantly the tran- sition-state stabilization free energy for Protein C hydrolysis.

Mechanisms of the Human Intestinal H+-coupled Oligopeptide Transporter hPEPT1

The hPEPT1 cDNA cloned from human intestine (Liang, R., Fei, Y.-J., Prasad, P. D., Ramamoorthy, S., Han, H., Yang-Feng, T. L., Hediger, M. A., Ganapathy, V., and Leibach, F. H. (1995) J. Biol. Chem. 270, 6456-6463) encodes a H+/oligopeptide cotransporter. Using two-microelectrode voltage-clamp in Xenopus oocytes expressing hPEPT1, we have investigated the transport mechanisms of hPEPT1 with regard to voltage dependence, steady-state kinetics, and transient charge movements. The currents evoked by 20 mM glycyl-sarcosine (Gly-Sar) at pH 5.0 were dependent upon membrane potential (Vm) between -150 mV and +50 mV. Gly-Sar-evoked currents increased hyperbolically with increasing extracellular [H+], with Hill coefficient approximately 1, and the apparent affinity constant (K0.5H) for H+ was in the range of 0.05 1 microM. K0.5 for Gly-Sar (K0.5GS) was dependent upon Vm and pH; at -50 mV, K0.5H was minimal (approximately 0.7 mM) at pH 6.0. Following step-changes in Vm, in the absence of Gly-Sar, hPEPT1 exhibited H+-dependent transient currents with characteristics similar to those of Na+-coupled transporters. These charge movements (which relaxed with time constants of 2-10 ms) were fitted to Boltzmann relations with maximal charge (Qmax) of up to 12 nC; the apparent valence was determined to be approximately 1. Qmax is an index of the level of transporter expression which for hPEPT1 was in the order of 1011/oocyte. In general our data are consistent with an ordered, simultaneous transport model for hPEPT1 in which H+ binds first.

Understanding the molecular mechanism of dominant negative action of mutant thyroid hormone beta 1-receptors: the important role of the wild-type/mutant receptor heterodimer.

The clinical manifestations of patients with resistance to thyroid hormone result from inhibition of the functions of wild-type thyroid hormone receptors (wTRs) by the dominant negative effect of mutant TR beta 1 receptors (mTR beta 1). One of the proposed mechanisms by which mTR beta 1 exerts its dominant negative action is via formation of the putative inactive wTR beta 1/mTR beta 1 heterodimer. However, the nature of the wTR beta 1/mTR beta 1 heterodimer is poorly understood. The present study characterizes the wTR beta 1/mTR beta 1 heterodimer by electrophoretic mobility shift assay. The mutant TR beta 1 used was PV, which contains a frame shift mutation in the C-terminal part of TR beta 1 and has less than 1% of the T3 binding affinity of the wTR beta 1. Because of the difficulty in resolving wTR beta 1 and mutant PV dimers, we used a truncated wTR beta 1 in which the A/B domain was deleted (delta TR beta 1) to demonstrate the formation of the heterodimer on thyroid hormone response elements (TREs) in which the half-site binding motifs are oriented in an inverted repeat (F2), a direct repeat separated by four nucleotides (DR4), or an inverted repeat (Pal). Deletion of the A/B domain had no effect on the binding of T3 and TREs to wTR beta 1. In the presence of equal amounts of delta TR beta 1 and PV, three types of molecular complexes. delta TR beta 1 homodimer, delta TR beta 1/PV heterodimer, and PV homodimer bound to each TRE in a ratio of approximately 1:2:1. The identities of these complexes were confirmed by their ability to be supershifted by anti-TR beta 1 and/or anti-PV antibodies. delta TR beta 1/PV heterodimer formation varied with different TREs. The ratio of apparent affinity constant (Ka) in the binding of delta TR beta 1/PV to TREs was F2:DR4:Pal = approximately 6:2:1. The effect of T3 on delta TR beta 1/PV heterodimer formation was TRE dependent. No T3-induced dissociation was observed for the delta TR beta 1/PV heterodimer when bound to F2 and Pal. In contrast, the delta TR beta 1/PV heterodimer bound to DR4 was dissociated by T3 with an ED50 of 3.9 +/- 0.9 nM. The T3-induced dissociation of delta TR beta 1 homodimer bound to F2, DR4, and Pal had ED50 values of 4.1 +/- 1.2, 1.3 +/- 0.3, and more than 100 nM, respectively. By transfection assays, the dominant negative action of PV was found to be TRE dependent with the rank order of F2 >> Pal > ME (a DR4-like TRE in the rat malic enzyme gene). Taken together, these results indicate a strong correlation between wTR beta 1/mTR beta 1 heterodimer formation and the dominant negative potency of PV. These results suggest that the wTR beta 1/mTR beta 1 heterodimer could play an important role in the dominant negative action of mTR beta 1.

Understanding the molecular mechanism of dominant negative action of mutant thyroid hormone beta 1-receptors: the important role of the wild- type/mutant receptor heterodimer

The clinical manifestations of patients with resistance to thyroid hormone result from inhibition of the functions of wild-type thyroid hormone receptors (wTRs) by the dominant negative effect of mutant TR beta 1 receptors (mTR beta 1). One of the proposed mechanisms by which mTR beta 1 exerts its dominant negative action is via formation of the putative inactive wTR beta 1/mTR beta 1 heterodimer. However, the nature of the wTR beta 1/mTR beta 1 heterodimer is poorly understood. The present study characterizes the wTR beta 1/mTR beta 1 heterodimer by electrophoretic mobility shift assay. The mutant TR beta 1 used was PV, which contains a frame shift mutation in the C-terminal part of TR beta 1 and has less than 1% of the T3 binding affinity of the wTR beta 1. Because of the difficulty in resolving wTR beta 1 and mutant PV dimers, we used a truncated wTR beta 1 in which the A/B domain was deleted (delta TR beta 1) to demonstrate the formation of the heterodimer on thyroid hormone response elements (TREs) in which the half-site binding motifs are oriented in an inverted repeat (F2), a direct repeat separated by four nucleotides (DR4), or an inverted repeat (Pal). Deletion of the A/B domain had no effect on the binding of T3 and TREs to wTR beta 1. In the presence of equal amounts of delta TR beta 1 and PV, three types of molecular complexes. delta TR beta 1 homodimer, delta TR beta 1/PV heterodimer, and PV homodimer bound to each TRE in a ratio of approximately 1:2:1. The identities of these complexes were confirmed by their ability to be supershifted by anti-TR beta 1 and/or anti-PV antibodies. delta TR beta 1/PV heterodimer formation varied with different TREs. The ratio of apparent affinity constant (Ka) in the binding of delta TR beta 1/PV to TREs was F2:DR4:Pal = approximately 6:2:1. The effect of T3 on delta TR beta 1/PV heterodimer formation was TRE dependent. No T3-induced dissociation was observed for the delta TR beta 1/PV heterodimer when bound to F2 and Pal. In contrast, the delta TR beta 1/PV heterodimer bound to DR4 was dissociated by T3 with an ED50 of 3.9 +/- 0.9 nM. The T3-induced dissociation of delta TR beta 1 homodimer bound to F2, DR4, and Pal had ED50 values of 4.1 +/- 1.2, 1.3 +/- 0.3, and more than 100 nM, respectively. By transfection assays, the dominant negative action of PV was found to be TRE dependent with the rank order of F2 >> Pal > ME (a DR4-like TRE in the rat malic enzyme gene). Taken together, these results indicate a strong correlation between wTR beta 1/mTR beta 1 heterodimer formation and the dominant negative potency of PV. These results suggest that the wTR beta 1/mTR beta 1 heterodimer could play an important role in the dominant negative action of mTR beta 1.

Batch growth and transport kinetics of utilization of mixtures of sucrose and maltose by Saccharomyces cerevisiae

Sucrose and maltose are commonly used sugars in brewing, baking and/or ethanol production industries. Since both sugars have been shown to be transported directly into the yeast cells of Saccharomyces cerevisiae (for maltose, it is the only mode of utilization), the way the two transport systems interact when the two disaccharides are present in mixture was investigated using both fermentation and radiometric studies. Two strains of S. cerevisiae were used in this study: 248 UNSW 703100 (laboratory strain) and A11 (industrial strain). The yeast cells were adapted on the disaccharides for up to 14 d before fermentation or radiometric studies were carried out. In batch culture studies, the adapted cells on either maltose for 2 d or sucrose for 14 d were grown on mixtures of sucrose and maltose containing different proportions of the two disaccharides. In both strains, the growth on the mixture was diauxic with sucrose and its hydrolysis products being utilized first. In radiometric studies, the affinity constant for maltose in maltose-adapted cells of strain 248 UNSW 703100 increased from 4.3 mM when the cells were incubated with [U- 14C] maltose to 19.3 mM when incubated with equimolar mixture of [U- 14C] maltose and unlabelled sucrose. The apparent affinity constant for sucrose transport in sucrose-adapted cells increased from 6.4 mM when the cells were incubated with [U- 14C] sucrose to 18.9 mM when incubated with equimolar mixture of [U- 14C] sucrose and unlabelled maltose.

Cloning and expression of a murine Fab fragment recognizing a defined linear epitope of Chironomus thummi thummi major allergen Chi t 1-9.

We have cloned and expressed in bacteria the genes coding for the Fab fragment of the monoclonal antibody (MoAb) M3. M3 is a murine IgG1 antibody reactive with the major allergen Chi t 1-9 of Chironomus thummi thummi. The major allergen Chi t 1-9 is known to be an aggressive inhalant allergen and causes type-I allergy. The immunoglobulin (Ig) fragment genes were cloned as a synthetic dicistronic operon. In this operon each gene is preceded by a bacterial signal sequence to direct the recombinant protein to the periplasmic space of the bacteria. The cloned genes were expressed in Escherichia coli using the strong T7-RNA-polymerase-based system. Sequence analysis revealed that the light and heavy chains of MoAb M3 belong to the V kappa II and V kappa IIC group of the Ig family, respectively. Genes of the V kappa II and V kappa IIC group are known to be used in response to haptens. The apparent affinity constants of the parent antibody M3 and the recombinant Fab fragment are nearly equivalent.

Unusual affinity of cyanide for ferrous and ferric Scapharca inaequivalvis homodimeric hemoglobin. Equilibria and kinetics of the reaction.

The homodimeric hemoglobin from the mollusk Scapharca inaequivalvis (HbI) yields very stable ferrous and ferric cyanide adducts. The stability of the ferrous complex is particularly unusual such that it enabled determination of the spectroscopic properties of the complex and the characterization of the cyanide binding reaction to deoxygenated HbI at equilibrium and kinetically. The absorption spectrum of the ferrous cyanide complex is typical of a low-spin derivative; in the near-infrared region, it displays two bands at 695 and 840 nm attributable to charge transfer transitions. At pH 9.2, cyanide binds to deoxy HbI with no cooperativity and an apparent affinity constant of 17 M-1, which is about 10-fold higher than that for deoxy horse heart myoglobin. The rate of cyanide dissociation from both the ferrous and the ferric HbI adducts is slow relative to those of the other hemoproteins investigated to date and provides the major contribution to the unusual affinity for the ligand. The rate of cyanide binding to the ferric protein, in which the pentacoordinate derivative is the dominant species, is about 100-fold faster relative to that of the ferrous protein. In structural terms, the high affinity for cyanide of Scapharca hemoglobin has been ascribed to the decreased overall polarity of the heme pocket which is related to the localization of the heme groups at the subunit interface.

Development of monoclonal antibodies for the selective isolation of human plasma apolipoprotein A-containing particles.

Monoclonal antibodies (MAbs) to human plasma apolipoprotein A-I (apo A-I), denoted FF9 and 6B9, and apolipoprotein A-II (apo A-II), 3F5, were developed to be used in an immunoaffinity chromatography procedure to isolate lipoprotein particles Lp A-I and Lp A-I:A-II. MAb FF9 and MAb 6B9 reacted with apo A-I and high-density lipoprotein (HDL) while MAb 3F5 was directed to apo A-II and HDL. The apparent affinity constant (Kapp) for apo A-I of the MAb FF9 was higher (2 x 10(7) M-1) than that of 6B9 (5 x 10(6) M-1). MAb 3F5 recognized the apo A-II with a Kapp value of 1 x 10(9) M-1. The isolated lipoparticles Lp A-I and Lp A-I:A-II will be used to standardize an immunoassay for the measurement of these apo A-I-containing lipoprotein particles in human plasma.