Single Dissociation Constant Research Articles

A novel dual-color fluorescent sensor with two pKas for on-site detection of pH in food

Tracking pH fluctuations in food samples is important for ensuring food freshness. Fluorescent probes have been widely applied as promising tools for the on-site detection of pH changes; however, most of them can be applied only at either lower or higher pH ranges because their response structures commonly have a single acid dissociation constant (pKa). To address this problem, we designed a fluorescent sensor, called HMB, containing a methylpiperazine group with two pKa values, which exhibited a unique dual-color response to pH changes over a wide pH range. Furthermore, the HMB-based test strips are easily prepared and used as portable labels for the visual monitoring of food spoilage that results in microbial and anaerobic glycolytic pathways in real food (such as cheese and shrimp). To the best of our knowledge, this is the first fluorescent pH sensor with two pKa values, and we expect that this work will inspire more sensor designs for food quality control.

Detail study on the interaction between perfluorooctanoic acid (PFOA) with human hemoglobin (Hb)

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) are often referred to as legacy perfluoroalkyl substances (PFAS). Human exposure to PFAS leads to severe negative health impacts including cancers, infertility, and dysfunction in the kidneys. Steady-state absorbance, fluorescence, and circular dichroism (CD) methods were used to study the interactions between PFOA and Hb. The results demonstrate the presence of multiple PFOA binding sites on the Hb protein. The detailed analysis of the ferric hemoglobin protein (met Hb) absorbance data as a function of PFOA concentration indicates the presence of at least two binding sites with equilibrium dissociation constants of 0.8 ± (0.2) × 10−6 M and 63 ± (15) × 10−5 M. A competitive binding study with 1,8-ANS showed PFOA can bind to the same binding site as 1,8-ANS on the Hb protein. The titration curve for PFOA binding to Hb in its CO bound form (CO-Hb) yields a single equilibrium dissociation constant of 139 ± (20) × 10−6 M. PFOA binding at low concentrations occurs at the high-affinity sites leading to the destabilization of the protein structure as reflected by changes in the CD spectrum. PFOA interactions with Hb also interfere with the kinetics of CO association to this protein. The rate for CO association to Hb increases at low PFOA concentrations, whereas at elevated PFOA concentrations, the ligand association is biphasic as a new kinetic process with a different rate constant was observed. Overall, this study provides a detailed explanation of PFOA-induced structural and conformational changes to the Hb protein based on the spectroscopy data.

The measurement of binding affinities by NMR chemical shift perturbation

We have carried out chemical shift perturbation titrations on three contrasting proteins. The resulting chemical shifts have been analysed to determine the best way to fit the data, and it is concluded that a simultaneous fitting of all raw shift data to a single dissociation constant is both the most accurate and the most precise method. It is shown that the optimal weighting of 15N chemical shifts to 1H chemical shifts is protein dependent, but is around the consensus value of 0.14. We show that chemical shift changes of individual residues can be fit to give residue-specific affinities. Residues with affinities significantly stronger than average are found in close contact with the ligand and are suggested to form a rigid contact surface, but only when the binding involves little conformational change. This observation may be of value in analysing binding and conformational change.

Interaction between thrombin and oligonucleotide RA36 is a two-stage process

Oligonucleotide RA36 contains two G-quadruplex modules with thrombin binding aptamer sequence GGTTGGTGTGGTTGG. Each of the modules potentially can bind thrombin while differing in functional activity. Despite that, previously published studies report a single dissociation constant for the thrombin:RA36 complex, which value varies widely. Here we address this discrepancy using electrophoretic mobility shift assay. Our results reveal that the interaction between RA36 and thrombin is a two-stage process. The two modules have different affinities for thrombin, which explains the discrepancy in the published data.

Probing Amyloid β and the Antibody Interaction Using Atomic Force Microscopy.

Amyloid β (Aβ) peptide is considered to be the critical causative factor in the pathogenesis of Alzheimer's disease (AD) because the hydrophilic molecules accumulated outside of the neural cells and results in the formation of highly toxicity amyloid plaque. In this study, we probed the interaction between Aβ and the antibody using atomic force microscopy (AFM). We compared two kinds of antibodies which are the antibody for Aβ 1-42 (antibody42) and the antibody for Aβ 1-16 (antibody16). To detect the interaction between Aβ and the antibodies, the single molecular force spectroscopy was carried out using Aβ modified glass substrate and the antibodies modified AFM probes. In the results, the single Aβ-antibody42 dissociation constant was estimated to be 5.2 × 10-3 s-1 and the single Aβ-antibody16 dissociation constant was 2.8×10-2 s-1. The Aβ-antibody42 showed 5.3 times longer bond life time compare with Aβ-antibody16. It suggested that antibody42 is better choice for the Aβ sensor development.

Using two-site binding models to analyze microscale thermophoresis data

The emergence of microscale thermophoresis (MST) as a technique for determining the dissociation constants for bimolecular interactions has enabled these quantities to be measured in systems that were previously difficult or impracticable. However, most models for analyses of these data featured the assumption of a simple 1:1 binding interaction. The only model widely used for multiple binding sites was the Hill equation. Here, we describe two new MST analytic models that assume a 1:2 binding scheme: the first features two microscopic binding constants (KD(1) and KD(2)), while the other assumes symmetry in the bivalent molecule, culminating in a model with a single macroscopic dissociation constant (KD,M) and a single factor (α) that accounts for apparent cooperativity in the binding. We also discuss the general applicability of the Hill equation for MST data. The performances of the algorithms on both real and simulated data are assessed, and implementation of the algorithms in the MST analysis program PALMIST is discussed.

Effect of osmolytes on the interaction of flavin adenine dinucleotide with muscle glycogen phosphorylase b

The effect of three osmolytes, trimethylamine N-oxide (TMAO), betaine and proline, on the interaction of muscle glycogen phosphorylase b with allosteric inhibitor FAD has been examined. In the absence of osmolyte, the interaction is described by a single intrinsic dissociation constant (17.8 μM) for two equivalent and independent binding sites on the dimeric enzyme. However, the addition of osmolytes gives rise to sigmoidal dependencies of fractional enzyme-site saturation upon free inhibitor concentration. The source of this cooperativity has been shown by difference sedimentation velocity to be an osmolyte-mediated isomerization of phosphorylase b to a smaller dimeric state with decreased affinity for FAD. These results thus have substantiated a previous inference that the tendency for osmolyte-enhanced self-association of dimeric glycogen phosphorylase b in the presence of AMP was being countered by the corresponding effect of molecular crowding on an isomerization of dimer to a smaller, nonassociating state.

Dynorphin B is an agonist of nuclear opioid receptors coupling nuclear protein kinase C activation to the transcription of cardiogenic genes in GTR1 embryonic stem cells.

The cardiac differentiation of embryonic stem (ES) cells was found to involve prodynorphin gene and dynorphin B expression and was associated with the interaction of secreted dynorphin B with cell surface opioid receptors coupled with protein kinase C (PKC) signaling and complex subcellular redistribution patterning of selected PKC isozymes. Here, confocal microscopy revealed the presence of immunoreactive dynorphin B-like material in GTR1 ES cells, suggesting that dynorphin peptides may also act intracellularly. Opioid binding sites were identified in ES cell nuclei, with a single dissociation constant in the low nanomolar range. A significant increase in Bmax for a kappa opioid receptor ligand was observed in nuclei isolated from ES-derived cardiomyocytes compared with nuclei from undifferentiated cells. Direct exposure of nuclei isolated from undifferentiated ES cells to dynorphin B or U-50,488H, a synthetic kappa opioid receptor agonist, time- and dose-dependently activated the transcription of GATA-4 and Nkx-2.5, 2 cardiac lineage-promoting genes. Nuclear exposure to dynorphin B also enhanced the rate of prodynorphin gene transcription. These responses were abolished in a stereospecific fashion by the incubation of isolated nuclei with selective opioid receptor antagonists. Nuclei isolated from undifferentiated cells were able to phosphorylate the acrylodan-labeled MARCKS peptide, a high-affinity fluorescent PKC substrate. Exposure of isolated nuclei to dynorphin B induced a remarkable increase in nuclear PKC activity, which was suppressed by opioid receptor antagonists. Nuclear treatment with PKC inhibitors abolished the capability of dynorphin B to prime the transcription of cardiogenic genes.

Equilibrium and stop-flow kinetic studies of fluorescently labeled DNA substrates with DNA repair proteins XPA and replication protein A.

Nucleotide excision repair (NER) is a crucial pathway in the maintenance of genome stability requiring at least two dozen proteins. XPA and RPA have essential roles in the damage recognition step of NER. To better understand the mechanism of their interactions with DNA, we utilized equilibrium and stop-flow kinetic approaches with fluorescently labeled oligonucleotides. Fluorescein is a bona fide NER lesion because a circular plasmid with a single defined fluorescein was repaired by efficient extracts from Xenopus oocyte nuclei. Single-stranded and double-stranded oligonucleotides 5'-labeled with fluorescein were used in the subsequent studies. Oligonucleotide fluorescence was quenched upon specific binding to full-length recombinant Xenopus XPA (xXPA) and/or human RPA. The binding was highly sensitive to the buffer conditions. Analysis of equilibrium binding data with ds DNA and xXPA revealed a single dissociation constant (K(d)) of 24.4 nM. Stopped-flow kinetic experiments were described by a first-order on-rate constant k(on) of 9.03 x 10(8) M(-1) s(-1) and k(off) of 26.1 s(-1). From the ratio of off-rate to on-rate, a calculated K(d) of 28.9 nM was obtained, revealing that the kinetic and equilibrium studies were consistent. The affinity of xXPA for ds undamaged DNA determined in our spectrofluorometry experiments was up to 3 orders of magnitude higher than previously reported values using different substrates, conditions, and assays [gel-shifts (EMSA), filter-binding, anisotropy, and surface plasmon resonance]. The same substrate DNA containing a 4-bp mismatch in the middle yielded a K(d) five times higher (158 nM), indicating weaker binding by xXPA. The differences in K(d) values for these two substrates were mainly attributable to the k(on), rather than k(off) rates. Fluorescence intensity changes upon interaction of xXPA with ss 50-mer were too low to calculate an accurate K(d). Although recombinant human RPA binding to the ds 50-mer was very weak (K(d) > 1 mM), stop-flow and equilibrium measurements to ss oligonucleotide yielded K(d) values of 96 and 20.3 nM, respectively, which correlated with previously reported values using gel mobility shift assays and a similarly sized poly-dT. Equilibrium and stop-flow measurements to the cognate and mismatched ds oligonucleotides using both xXPA and hRPA yielded a 2- to 3-fold increase in the K(d).

Characterization of Proexosite I on Prothrombin

Activation of prothrombin by factor Xa is accompanied by expression of regulatory exosites I and II on the blood coagulation proteinase, thrombin. Quantitative affinity chromatography and equilibrium binding studies with a fluorescein-labeled derivative of the exosite I-specific peptide ligand, hirudin(54-65) ([5F]Hir(54-65) (SO(3)(-)), were employed to identify and characterize this site on human and bovine prothrombin and its expression on thrombin. [5F]Hir(54-65)(SO(3)(-)) showed distinctive fluorescence excitation spectral differences in complexes with prothrombin and thrombin and bound to human prothrombin and thrombin with dissociation constants of 3.2 +/- 0.3 micrometer and 25 +/- 2 nm, respectively, demonstrating a 130-fold increase in affinity for the active proteinase. The bovine proteins similarly showed a 150-fold higher affinity of [5F]Hir(54-65)(SO(3)(-)) for thrombin compared with prothrombin, despite a 2-5-fold lower affinity of the peptides for the bovine proteins. Unlabeled, Tyr(63)-sulfated and nonsulfated hirudin peptides bound competitively with [5F]Hir(54-65)(SO(3)(-)) to human and bovine prothrombin and thrombin, exhibiting similar, 40-70-fold higher affinities for the proteinases, although nonsulfated Hir(54-65) bound with 7-17-fold lower affinity than the sulfated analog. These studies characterize proexosite I for the first time as a specific binding site for hirudin peptides on both human and bovine prothrombin that is present in a conformationally distinct, low affinity state and is activated with a approximately 100-fold increase in affinity when thrombin is formed.

Laminin inhibition of beta-amyloid protein (Abeta) fibrillogenesis and identification of an Abeta binding site localized to the globular domain repeats on the laminin a chain.

beta-Amyloid protein (Abeta) is a major component of neuritic plaques and cerebrovascular amyloid deposits in the brains of patients with Alzheimer's disease (AD). Inhibitors of Abeta fibrillogenesis are currently sought as potential future therapeutics for AD and related disorders. In the present study, the basement membrane protein laminin was found to bind Abeta 1-40 with a single dissociation constant, K(d) = 2.7 x 10(-9) M, and serve as a potent inhibitor of Abeta fibril formation. 25 microM of Abeta 1-40 was incubated at 37 degrees C for 1 week in the presence of 100 nM of laminin or other basement membrane components, including perlecan, type IV collagen, and fibronectin to determine their effects on Abeta fibril formation as evaluated by thioflavin T fluorometry. Of all the basement membrane components tested, laminin demonstrated the greatest inhibitory effect on Abeta-amyloid fibril formation, causing a ninefold inhibition at 1 and 3 days and a 21-fold inhibition at 1 week. The inhibitory effects of laminin on Abeta fibrillogenesis occurred in a dose-dependent manner and were still effective at lower concentrations. The inhibitory effects of laminin on Abeta 1-40 fibril formation was confirmed by negative stain electron microscopy, whereby laminin caused an almost complete inhibition of Abeta fibril formation and assembly by 3 days, resulting in the appearance of primarily amorphous nonfibrillar material. Laminin also caused partial disassembly of preformed Abeta-amyloid fibrils following 4 days of coincubation. Laminin was not effective as an inhibitor of islet amyloid polypeptide fibril formation, suggesting that laminin's amyloid inhibitory effects were Abeta-specific. To identify a potential Abeta-binding site(s) on laminin, laminin was first digested with V8, trypsin, or elastase. An Abeta-binding elastase digestion product of approximately 120-130 kDa was found. In addition, a approximately 55 kDa fragment derived from V8 and elastase-digested laminin interacted with biotinylated Abeta 1-40. Amino acid sequencing of the approximately 55 kDa fragment identified a conformationally dependent Abeta-binding site within laminin localized to the globular repeats on the laminin A chain. These studies demonstrate that laminin not only binds Abeta with relatively high affinity but is a potent inhibitor of Abeta-amyloid fibril formation. In addition, further identification of an Abeta-binding domain within the globular repeats on the laminin A chain may lead to the design of new therapeutics for the inhibition of Abeta fibrillogenesis.

Sulfate content and specific glycosaminoglycan backbone of perlecan are critical for perlecan's enhancement of islet amyloid polypeptide (amylin) fibril formation.

Islet amyloidosis is characterized by the deposition and accumulation of amylin in pancreatic beta-cells and is observed in 90% of patients with type 2 diabetes. Previous studies have also revealed the presence of the specific heparan sulfate proteoglycan, perlecan, colocalized to islet amyloid deposits, similar to perlecan's known involvement with other amyloid proteins. In the present study, perlecan purified from the Engelbreth-Holm-Swarm (EHS) tumor was used to define perlecan's interactions with amylin (i.e., islet amyloid polypeptide) and its effects on amylin fibril formation. Using a solid phase-binding immunoassay, human amylin, but not rat amylin, bound immobilized EHS perlecan with a single dissociation constant (Kd) = 2.75 x 10(-6) mol/l. The binding of human amylin to perlecan was similarly observed using perlecan heparan sulfate glycosaminoglycans (GAGs), and was completely abolished by 10 micromol/l heparin. Using thioflavin T fluorometry, Congo red staining, and electron microscopy methodology, intact perlecan was found to enhance amylin fibril formation in a dosage-dependent manner, with the majority of these effects attributed to the heparan sulfate GAG chains of perlecan. Other sulfated GAGs and related macromolecules were also effective in the enhancement of amylin fibril formation in the order of heparin > heparan sulfate > chondroitin-4-sulfate = dermatan sulfate = dextran sulfate > pentosan polysulfate, implicating the importance of the specific GAG/carbohydrate backbone. The sulfate content of heparin/heparan sulfate was also important for the enhancement of amylin fibril formation in the order of heparin > N-desulfated N-acetylated heparin > completely desulfated N-sulfated heparin > completely desulfated N-acetylated heparin. These studies suggest that the enhancement effects of perlecan on amylin fibril formation are mediated primarily by both specific GAG chain backbone and GAG sulfate content, and implicate perlecan as an important macromolecule that is likely involved in the pathogenesis of islet amyloidosis.

Imaging of the Buffering Effect of Insulin Antibodies in the Autoimmune Hypoglycemic Syndrome1

Insulin autoimmune hypoglycemia is characterized by recurrent hypoglycemia and high levels of immunoreactive insulin in the presence of insulin autoantibodies. The mechanisms inducing hypoglycemia are largely unknown. An [123I]insulin scintigraphic scanning was performed to directly demonstrate the effect of antibodies on insulin biodistribution in one patient with this syndrome both before and after treatment. The patient had insulin autoantibodies IgG3 lambda, which had a single site dissociation constant (Kd = 10(-7) mol/L, by Scatchard analysis), a very fast dissociation rate of immune complexes, and a very rapid association of [125I]insulin. Insulin receptors on red blood cells were down-regulated. The [123I]insulin scintigraphic study imaged the buffering effect of antibodies on insulin bioavailability. [123I]Insulin was not removed from the blood, and no liver or kidney uptake of the hormone occurred. The frequency and severity of hypoglycemic episodes required treatment. Insulin antibody levels decreased and [123I]insulin biodistribution improved after treatment with plasmapheresis and prednisone. Improved hormone bioavailability was further evidenced by the reduction in the hypoglycemic delay after i.v. insulin from 90 min before any treatment to 60 min after plasmapheresis and 30 min after steroid administration. Glucose tolerance was normal after treatment. Plasmapheresis followed by steroid treatment can lower the insulin antibody concentration, abolish severe hypoglycemia, and improve insulin biodistribution and glucose tolerance in insulin autoimmune hypoglycemia.

Coupling of the human Y2 receptor for neuropeptide Y and peptide YY to guanine nucleotide inhibitory proteins in permeabilized SMS-KAN cells.

Using guanine nucleotides, pertussis toxin, and specific antisera against the COOH-terminals of the alpha-subunits of Gi1/2, Gi3, and G(o), the binding and biological response of the Y2 receptor (Y2R) for peptide YY (PYY) was probed in SMS-KAN neuroblastoma cells. The specific binding of radiolabeled PYY exhibited a single apparent dissociation constant, KD = 76 pM for intact cells and KD = 906 pM for permeabilized cells. However, other data suggested existence of multiple receptor affinity states. A shift in KD and a decrease in apparent number of binding sites (Bmax) was observed in permeabilized cells when incubated with guanine nucleotides. By contrast, in membrane preparations guanine nucleotides induced only a decrease in Bmax. In intact cells, agonist exposure inhibited the intracellular accumulation of forskolin-stimulated cyclic AMP by 80% (IC50 = 420 nM) compared with 94% inhibition (IC50 = 380 nM) in permeabilized cells. In permeabilized cells, preincubation with antisera against alpha i1/2 and alpha i3 blocked the functional response of PYY, with anti-alpha i3 being the most potent; whereas anti-alpha o failed to affect the cyclic AMP levels. These results suggest that permeabilized SMS-KAN cells serve as a good model system for analysis of Y2R binding kinetics and functional response and that the Y2R interacts directly with several different GiS (but not G(o)).

Yeast TATA binding protein interaction with DNA: fluorescence determination of oligomeric state, equilibrium binding, on-rate, and dissociation kinetics.

A combination of steady-state, stopped-flow, and time-resolved fluorescence of intrinsic tryptophan and extrinsically labeled fluorescent DNA is utilized to examine the interaction of yeast TATA binding protein (TBP) with DNA. TBP is composed of two structural domains, the carboxy domain (residues 61-240), which is responsible for DNA binding and initiation of basal level transcription, and an amino terminal domain (residues 1-60), whose function is currently unknown. The steady-state fluorescence emission spectrum of the single tryptophan in the amino terminal domain of TBP undergoes a huge (30-40 nm) red-shift upon interaction with stoichiometric amounts of TATA box containing DNA. From time-resolved tryptophan fluorescence anisotropy studies, we demonstrate that, in the absence of DNA, the protein exists as a multimer in solution and it contains (at least) two primary conformations, one with the amino terminus associated tightly with the protein(s) in a hydrophobic environment and one with the amino terminus decoupled away from the rest of the protein and solvent-exposed. Upon binding DNA, the protein dissociates into a monomeric complex, upon which only the solvent-exposed amino terminus conformation remains. Kinetic and equilibrium binding studies were performed on TATA box containing DNA which was extrinsically labeled with a fluorescent probe Rhodamine-X at the 5'-end. This "fluorescent" DNA allowed for the collection of quantitative spectroscopic binding, kinetic on-rate, and kinetic off-rate data at physiological concentrations. Global analysis of equilibrium binding studies performed from 500 pM to 50 nM DNA reveals a single dissociation constant (Kd) of approximately 5 nM. Global analysis of stopped-flow anisotropy on-rate experiments, with millisecond timing resolution and TBP concentrations ranging from 20 to 600 nM (20 nM DNA), can be perfectly described by a single second-order rate constant of 1.66 x 10(5) M(-1) s(-1). These measurements represent the very first stopped-flow anisotropy study of a protein/DNA interaction. Stopped-flow anisotropy off-rate experiments reveal a single exponential k(off) of 4.3 x 10(-2) min-1 (1/k(off) = 23 min) From the ratio of on-rate to off-rate, a predicted Kd of 4.3 nM is obtained, revealing that the kinetic and equilibrium studies are internally consistent. Deletion of the amino terminal domain of TBP decreases the k(on) of TBP approximately 45-fold and eliminates classic second-order behavior.

Identification of a Mg(2+)- and guanyl nucleotide-dependent glucagon receptor cycle by use of permeabilized canine hepatocytes.

We have investigated (by use of intact and saponinpermeabilized canine hepatocytes) the roles of Mg2+ and guanyl nucleotides in regulating glucagon-receptor interactions. In contrast to intact cells, saponinpermeabilized hepatocytes bind [[125I]iodo-Tyr10]glucagon according to a single first-order process and exhibit a single apparent dissociation constant for glucagon binding during steady-state incubations. Further analysis of the permeabilized cell system demonstrated (a) the temperature-sensitive action of Mg2+ to enhance the extent and affinity of glucagon-receptor interactions at steady-state, (b) the conversion of Mg(2+)-independent hormone-receptor complexes to Mg(2+)-dependent complexes, (c) the effect of guanyl nucleotides to inhibit specifically the Mg(2+)-dependent component of glucagon-receptor interactions, (d) the more rapid association of glucagon with receptor during cell incubations occurring in the presence of guanyl nucleotides or in the absence of Mg2+, and (e) the ability of guanyl nucleotides to induce both high and low affinity states of glucagon-receptor interactions. Additional experiments identified an effect of cell incubations in the presence of glucagon to limit the subsequent binding of hormone, the ability of GDP, GTP, or guanosine-5'-3-O-(thio)triphosphate (GTP gamma S) to dissociate previously bound glucagon, and a specific requirement for GDP to re-activate the glucagon receptor for additional cycles of hormone binding. A model is presented in which (a) glucagon binds to receptor in a Mg(2+)-independent fashion, (b) glucagon-receptor complexes are converted to a Mg(2+)-dependent state, (c) guanyl nucleotide exchange initiates both an alteration in glucagon-receptor affinity and the subsequent dissociation of hormone, and (d) in the context of the intact cell, G protein-mediated hydrolysis of GTP to GDP is required to reinitialize the system.

Bradykinin binding to B2 kinin receptors and stimulation of phosphoinositide turnover and arachidonic acid release in primary cultures of cells from late pregnant rat myometrium.

Primary cultures of cells from late pregnant rat myometrium contain B2 kinin receptors through which bradykinin (BK) stimulates inositol phosphate (InsP) formation and arachidonic acid (20:4) release. Equilibrium binding at 4 degrees C revealed that [3H]BK identified a maximal number of cell surface B2 kinin receptor binding sites on rat myometrial cells of 308 +/- 78 fmol/10(6) cells with apparently a single equilibrium dissociation constant of 1.8 +/- 0.2 nM. At 37 degrees C, [3H]BK binding was associated with a time-dependent decrease in the reversibility of the binding. This decrease was due in part to formation of slowly dissociating cell surface receptor [3H]BK binding and in part to internalization of the receptor-bound [3H]BK. Exposure of labeled cells to BK resulted in dose-dependent increases in [3H]InsP3, [3H]InsP2 ([3H]Ins(1,4)P2), and [3H]InsP1 ([3H]Ins(1)P1) formation and [3H]20:4 release. Pretreatment with 100 ng/mL pertussis toxin did not perturb BK stimulation of [3H]InsP formation but partially (approximately 30%) inhibited BK stimulation of [3H]20:4 release. BK stimulation of [3H]20:4 release was directly proportional to the number of receptor sites occupied by BK. In contrast, stimulation of [3H]InsP formation required a threshold level of receptor occupancy, which decreased as a function of time of BK exposure. These results show that BK interacts with B2 kinin receptors on rat myometrial cells with apparently a single affinity through which BK stimulates [3H]InsP formation and [3H]20:4 release. BK stimulation of [3H]InsP formation requires a threshold BK concentration, which decreases with time, and we suggest that the decrease is due to a time-dependent formation of a BK receptor binding state from which BK slowly dissociates.

Basis for the anomalous effect of competitive inhibitors on the kinetics of hydrolysis of short-chain phosphatidylcholines by phospholipase A2.

The effect of four specific competitive inhibitors on the kinetics of hydrolysis of short-chain diacyl-sn-glycero-3-phosphocholines below their critical micelle concentrations was examined. The kinetics of hydrolysis of short-chain substrates dispersed as solitary monomers were generally consistent with the classical Michaelis-Menten formalism; i.e., hydrolysis began without any latency period, the steady-state rate was observed at higher substrate concentrations, the steady-state initial rate showed a linear dependence on the enzyme concentration, and the hyperbolic dependence of the initial rate on the substrate concentration could be described in terms of KM and Vmax parameters. The competitive nature of the inhibitors used in this study has been established by a variety of techniques, and the equilibrium dissociation constants for the inhibitors bound to the enzyme were measured by the protection method [Jain et al. (1991) Biochemistry 30, 7306-7317]. The kinetics of hydrolysis in the presence of competitive inhibitors could be described by a single dissociation constant. However, the value of the dissociation constant obtained under the kinetic conditions was comparable to that obtained by the protection method for the inhibitor-enzyme complex bound to a neutral diluent, rather than to the value of the dissociation constant obtained with solitary monomeric inhibitors and the enzyme in the aqueous phase. Spectroscopic methods showed that the effectively lower dissociation constant of an inhibitor bound to PLA2 at the interface is due to the stabilization of the enzyme-inhibitor complex by interaction with other amphiphiles present in the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of Zn2+ with the bovine‐heart mitochondrial bc1 complex

A study is presented of the effect of Zn2+ on the enzymatic properties of the bovine-heart cytochrome-bc1 complex. Micromolar concentrations of Zn2+ reversibly inhibit the cytochrome-c reductase activity of either the cholate-solubilized or liposome-reconstituted complex. Kinetic analysis of the redox reactions of the cytochromes indicate that Zn2+ affects the activity of the complex at the quinol oxidation site. The following have been determined: (a) Zn2+ inhibits the pre-steady-state reduction of cytochrome c1 by duroquinol either in the absence or in the presence of antimycin, (b) it does not inhibit the reduction of b cytochromes in the absence of antimycin or in the presence of myxothiazol, (c) it inhibits cytochrome-b reduction in the presence of antimycin. Furthermore Zn2+ inhibits the antimycin-promoted oxidant-induced extrareduction of b cytochromes. Addition of Zn2+ to reduced bc1 complex causes a red shift in the absorption spectrum of cytochrome b566 and a substantial decrease in the signal intensity of the EPR spectrum of the Fe-S protein. This is interpreted as an interaction of Zn2+ with the 2Fe-2S-cluster region of the Fe-S protein, thus giving rise to inhibition of the reductase activity and of the antimycin-insensitive reduction route of b cytochromes. A Scatchard-plot of 65Zn2+ binding to the native isolated complex gave a straight line from which a value of three binding sites and a single dissociation constant of 3 x 10(-6) M can be calculated, which is practically equal to the concentration causing 50% inhibition of electron flow.

Heparin-modulated Binding of Pancreatic Lipase and Uptake of Hydrolyzed Triglycerides in the Intestine

Utilizing small intestine membranes that contain heparin (50 micrograms/mg protein), binding of triglyceride lipase (homogeneous 52 kDa, specific activity, 70 nmol/mg.h) to membranes was shown to be concentration dependent and saturable, and it was characterized by a single dissociation constant (KD = 86 +/- 16 nM) with a maximal binding capacity of 54 +/- 8 pmol/mg of vesicle protein. Specific binding was decreased in a concentration-dependent manner by the addition of exogenous heparin, and binding was virtually eliminated (less than 6% control values) by pretreatment of membranes with bacterial heparinase. Cultured intestinal epithelial cells (CaCo-2), shown to possess membrane-associated heparin, also bound pancreatic triglyceride lipase in a specific and saturable manner, with KD = 77 +/- 12 nM and Bmax = 13.7 +/- 6 pmol/10(6) cells. Soluble heparin not only decreased binding, but it also diminished the enzyme-mediated cellular uptake of [14C]oleate from [14C]triolein by over 75%. Therefore, intestinal heparin, a component of the brush border membrane, localizes pancreatic triglyceride lipase in a receptor-like manner to the plasma membrane to promote the subsequent absorption of fatty acids derived from hydrolyzed triglycerides.