Low Affinity Form Research Articles

Physical mechanism for regulation of phosphoenolpyruvate-dependent glucose transport activity in Escherichia coli.

The activity of the phosphoenolpyruvate-dependent glucose phosphotransferase system (PTS) in Escherichia coli is coupled to the oxidation-reduction potential. It is inhibited when the redox potential is increased above -300 mV either via substrate oxidation or via direct addition of oxidizing agents. Depending on the point of addition, dithiothreitol either blocks or reverses these effects. Inhibition occurs at the level of sugar binding to EII. A sulfhydryl group associated with EII activity undergoes reversible oxidation to, presumably, a disulfide, resulting in the conversion of EII from a reduced, high-affinity form to an oxidized, low-affinity form which has a 10(2)-10(3) times lower affinity for the sugar. An identical change in affinity occurs as the result of the generation of a delta mu H+ during the oxidation of reduced N-methylphenazonium methosulfate or nicotinamide adenine dinucleotide. In this case, uncouplers and ionophores reverse the change. A mechanism is proposed in which the electrical potential difference across the membrane regulates the glucose PTS by shifting the midpoint potential of the EII-associated redox transition to more negative values. As a result, EII is converted to the oxidized, low-affinity state in the presence of a delta mu H+.

In vivo regulation of muscarinic acetylcholine receptor number and function in embryonic chick heart.

We have used the chick embryonic heart to study the regulation of the muscarinic acetylcholine receptor in vivo. Sustained activation of the muscarinic receptor in vivo with the cholinergic agonist carbachol decreases muscarinic receptor number as much as 87% as measured by the specific binding of the potent muscarinic receptor in antagonist [3H]quinuclidinyl benzilate. The decrease in receptor number is both dose and time dependent. After carbachol-induced down regulation, the receptor number recovers to control levels when further receptor activation is blocked by the muscarinic receptor antagonist atropine. When receptor number is decreased 50% in vivo, isolated atria require a 12-fold greater concentration of carbachol than controls to arrest spontaneous beating in an organ bath. Analysis of the binding of carbachol to the muscarinic receptor indicates that this shift in the dose response of atria is accompanied by a change in the relative fraction of the high and low affinity forms of the muscarinic receptor with no change in their respective affinities for carbachol. In addition, this analysis suggests that the low agonist affinity form of the cardiac muscarinic receptor is the physiologically active form.

Disulfide reduction converts the insulin receptor of human placenta to a low affinity form.

Treatment of human placenta membranes with dithiothrietol (DTT) followed by N-ethylmaleimide results in a 60% reduction in insulin binding. Treatment with N-ethylmaleimide alone has little effect. The decrease in insulin binding that results from DTT treatment is due to a decrease in affinity for insulin, with little change in total receptor number. DTT has similar effects on receptor solubilized from placenta membranes with Triton X-100, indicating that its effects are not attributable to changes in the arrangement of receptors in the membrane. In contrast to placenta membranes, treatment of liver membranes with DTT does not decrease insulin binding. These results suggest that reduction of a critical disulfide bond in insulin receptors from human placenta converts the receptor to a low affinity form.

Differential regulation of the α2-adrenergic receptor by Na+ and guanine nucleotides

Many hormones interact with receptors which stimulate the enzyme adenylate cyclase. Less well characterized ar those receptors which mediate an inhibition of adenylate cyclase activity. However, guanine nucleotides are clearly important in the regulation of both stimulatory and inhibitory receptors. Monovalent cations, notably Na+, regulate many inhibitory receptor systems but apparently not stimulatory receptors. We investigate here the effects of Na+ and guanine nucleotides on the adenylate cyclase-coupled inhibitory alpha 2-adrenergic receptor of the rabbit platelet. Computer modelling of adrenaline competition curves with 3H-dihydroergocryptine (3H-DHE) indicates that adrenaline induces two distinct affinity states of the alpha 2 receptor--one of higher (alpha 2H) and the other of lower (alpha 2L) affinity. Guanyl-5'-yl-imidodiphosphate (Gpp(NH)p) seems to reduce adrenaline affinity to converting the high-affinity state into the low-affinity form of the receptor. In contrast, Na+ reduces adrenaline affinity at both the high- and low-affinity states of the alpha 2 receptor while preserving receptor heterogeneity. Thus, guanine nucleotides and Na+ differ in the manner by which each reduces agonist affinity for the alpha 2-adrenergic receptor.

Preparation of 2-thioltryptophan-glucagon and (tryptophan-S-glucagon)2. Differences in binding to the glucagon receptor in the hepatic adenylate cyclase system.

The synthesis of 2-thioltryptophan-glucagon is described. Oxidation of this compound gives the dimer (Trp-S-glucagon)2. Both monomer and dimer are equi-potent on a molar basis with native glucagon as activators of adenylate cyclase in hepatic plasma membranes. However, from the ability to compete with 125I-glucagon for binding at the glucagon receptor, the dimer has one-fourth the binding affinity for the receptor as does native glucagon, 2-thiol-Trp-glucagon, and Trp-(2,4-dinitrophenylsulfenyl)-glucagon which have equal affinities for the receptor. Addition of GTP, which converts the receptor from a tight-binding to a lower affinity form and which activates adenylate cyclase in the presence of glucagon, allows (Trp-S-glucagon)2 to bind equally with native glucagon and the other thiol derivatives of the hormone. This effect of GTP on the binding of the glucagon dimer and the uses of 2-thiol-Trp-glucagon in the semisynthesis of new glucagon derivatives are discussed.

Enhanced excitability of the uterus of the pregnant rabbit by imidazole stimulation of cyclic AMP phosphodiesterase.

Imidazole, at concentrations between 10(-3) and 10(-2) M, exerts a profound stimulatory effect on rabbit uterine strips obtained during pregnancy and studied isometrically in vitro. The action is not duplicated by N-alkylimidazoles which have greater potency as inhibitors of thromboxane synthetase but the effect of imidazole was antagonized by isoproterenol or theophylline. Biochemical analysis indicated that imidazole at concentrations greater than 5 x 10(-4) M stimulated both high and low affinity forms of cyclic AMP phosphodiesterase. The uterus of pregnant rabbits is profoundly refractory to any kind of pharmacological stimulation and the effects of imidazole, acting to stimulate phosphodiesterase, suggest that the integrity of the adenyl cyclase-cyclic AMP-protein kinase system is a necessary requirement for this organ to remain quiescent during pregnancy.

Conservation of the kinetic and allosteric properties of the acetylcholine receptor in its Na cholate soluble 9 S form : Effect of lipids

The rapid kinetics of interaction of the fluorescent cholinergic agonist Dns-C6-Cho with the acetylcholine receptor protein (AChR) were followed by stopped-flow measurements. Dissolution of AChR rich membrane fragments from Torpedomarmorata by Na cholate and subsequent dilution in the absence of lipids resulted in the stabilization of the AChR in a state which exhibited a low affinity for Dns-C6-Cho and could no longer be interconverted to the high affinity state in the presence of agonist. On the other hand, when the concentration of lipids was kept elevated in the presence of Na cholate, a soluble, 9S, low affinity form of the AChR was obtained which showed most of the characteristic properties of the membrane-bound AChR : in particular, the slow interconversion to the high affinity state and the effect of non-competitive blockers on this transition. After elimination of the detergent, this form lead systematically to successful reconstitution.

Differential effects of ethanol and other inducers of drug metabolism on the two forms of hamster liver microsomal aniline hydroxylase

The aniline hydroxylase activity of microsomes isolated from hamster liver can be differentiated kinetically into high affinity (low K m , form I) and low affinity (high K m , form II) forms. Microsomes isolated from uninduced animals contain slightly more form I activity. The activity of the low affinity form (form II) is preferentially enhanced by Aroclor or 3-methylcholanthrene treatment, while phenobarbital treatment increases the activity of both forms. Chronic ethanol consumption results in enhancement of only the high affinity form (form I).

Electron paramagnetic resonance study of nitrosyl haemoglobin M Iwate

The nitrosyl derivative of haemoglobin M Iwate, a haemoglobin variant which has functional beta chains and non-functional alpha chains, is known to exist only in a low affinity form. It shows a nine-line superhyperfine (SHF) pattern in the low temperature electron paramegnetic resonance (e.p.r.) spectrum, even in the presence of inositol hexaphosphate. This provides further support for the suggestion (Chevion, M., Stern, A., Peisach, J., Blumberg, W. E. and Simon, S. Biochemistry 1978 bd17, 1745) that the beta chains of tetrameric, fully ligated, nitrosyl derivatives of all haemoglobins invariably exhibit an e.p.r. spectrum with a nine-line SHF pattern regardless of the affinity state of the molecule. On the other hand, nitrosyl ferrous alpha chains within the haemoglobin tetramer can exhibit an e.p.r. spectrum with either a nine-line or a three-line SHF pattern which is dependent upon the affinity state of the molecule.

Studies on cobalt-reconstituted trout hemoglobins

1. Introduction Thermodynamic and kinetic studies of the oxygen and carbonmonoxide binding to trout Hb IV, the main hemoglobin component of trout’s blood, have recently contributed to the understanding of the molecular mechanism and the physiological role of the Root effect, i.e., the drop in oxygen affinity and cooperativity observed at low pH. This effect has been interpreted in the framework of a classical two states model [ 13, as a stabilization by protons or organic phosphate of the low affinity form of the ligand-bound derivative [2]. On the other hand, in the last few years recon- stitution of myoglobin and hemoglobin containing cobalt in the place of iron have been successfully employed to elucidate structure-function relation- ships in hemeproteins [3,4]. The reaction of these cobalt-reconstituted hemeproteins with oxygen has been shown to be cooperative and reversible. More- over, the paramagnetic character of the Co(I1) allowed a detailed EPR study of the deoxy- and oxy-state of the reconstituted proteins [4]. The availability of globins from Hb I and Hb IV from trout blood [5] and the purpose of gaining new relevant information on the effect of protons on the liganded and the un~ganded derivative of these proteins, prompted to prepare cobalt containing trout hemoglobins and to study their functional and EPR properties. 2. Materials and methods Cobalt-meso-Hb IV and cobaltlproto- and meso-

Multiple forms of 2-deoxy-d-glucoside-2-sulphamate sulphohydrolase from human placenta

2-Deoxyglucoside-2-sulphamate sulphohydrolase was purified about 10 000-fold from the soluble extract of human placenta by using as substrate [N-sulpho-35S]heparin. Differently charged enzyme forms were observed on chromatography on DEAE-cellulose, all of which had an apparent mol.wt. of 110 000 as determined by gel filtration. By using immobilized heparan sulphate as affinity matrix the sulphamate sulphohydrolase could be separated into two forms, a minor one with low and a major one with high affinity for the adsorbent. When tested with [N-sulpho-35S]heparan sulphate the low-affinity form had a Km of 0.2 mM, and the high-affinity form a Km of 0.03 mM. Both forms exhibited the same Km of 10 microM towards [N-sulpho-35S]heparin and were equally well adsorbed to immobilized heparin. The two forms could be distinguished by their pH-optima and by the influence of KCl on heparan sulphate sulphohydrolase activity.

Conformational influences on brain tryptophan hydroxylase by submicromolar calcium: opposite effects of equimolar lithium.

Tryptophan hydroxylase from rat midbrain, EGTA-pretreated and dialyzed, manifested allosteric properties with respect to its substrate tryptophan, cofactor tetrahydrobiopterin, and the calcium ion. Kinetic studies suggest two preferred enzyme conformations in the presence of low concentrations of the cosubstrates: a higher affinity form manifesting hyperbolic substrate kinetics, induced by submicromolar (0.4--0.8 microM) calcium in vitro and cocaine in vivo, and a lower affinity form exaggerating cooperativity with respect to substrate, induced by submicromolar (0.4 to 0.8 microM) lithium in vitro and lithium in vivo. Lithium's effect on serotonin biosynthesis may be due to its antagonism of the positive effector influence of calcium on tryptophan hydroxylase, either as a negative effector or by blocking the calcium site.

Patterns of apparent co-operativity in a simple random non-equilibrium enzyme–substrate–modifier mechanism. Comparison with equilibrium allosteric models

It has often been claimed that random non-equilibrium mechanisms can result in apparent homotropic and heterotropic effects in steady-state kinetics of the kind more usually attributed to intersubunit allosteric interactions. However, it has never been shown whether any simple random mechanism could in fact give patterns of apparent interaction similar to those predicted by the well-known allosteric models. The patterns of apparent substrate co-operativity and affinity given by the steady-state of a standard simple random substrate-modifier mechanism in which catalytic velocity is proportional to substrate binding have been analysed mathematically and numerically. All patterns possible with this model are described. Some of them rather resemble those possible with standard allosteric models, in that there is a high-affinity and a low-affinity form at zero and infinite modifier concentrations (or vice versa) which show Michaelian behaviour, apparent co-operativity passing through a maximum or minimum at intermediate affinities. Unlike the allosteric models the family of curves is in principle not symmetrical. The random model can also give behaviour not possible with the standard allosteric models, such as higher substrate affinity at intermediate modifier concentrations than at either zero or infinite modifier, with concomitant negative apparent substrate co-operativity, or a single change of sign of apparent substrate co-operativity. The analysis uses recently discovered simplified forms of steady-state equations for random models.

Development of muscarinic cholinergic receptors in inbred strains of mice: Identification of receptor heterogeneity and relation to audiogenic seizure susceptibility

The concentrations and biochemical properties of muscarinic acetylcholine receptors in the brains of two highly inbred strains of mice, DBA/2J and C57BL/6J, have been studied using [ 3H]quinuclidinyl benzilate (QNB), a potent and specific receptor antagonist. As is the case with rat brain, murine muscarinic receptors exist in at least two forms, which differ in their affinities for receptor agonists but which have the same high affinity for receptor antagonists. Carbamylcholine binding to mouse neural membranes can be resolved into two components with K Ds of 5.2 × 10 −7 and 7.9 × 10 −5 M. There is a regional heterogeneity of brain receptors with respect to their distribution between these high and low agonist affinity forms. Brain stem and hypothalamus receptors display binding properties that would be expected if over 60% of their receptors were in the high affinity state, while only 30–40% of cortex, striatum and thalamus receptors appear to be in the high affinity form. Hippocampal receptors display the least amount of high agonist affinity character. Saturation curves and Scatchard plots of QNB binding at 2, 14, 21 and 42 days postnatal age in both strains indicate no differences or changes in the affinity or nature of the binding with age. Significant increases in QNB binding per mg membrane protein were observed between 14 and 42 days in the cortex, hippocampus, striatum, thalamus and hypothalamus, but not in the midbrain-pons-medulla region. In the hippocampus the DBA mice had significantly more QNB bindin. In the hypothalamus decreases with age in total binding were noted in DBA, while slight increases were noted in C57. Compared to C57, hippocampal receptors in DBA displayed lower agonist affinity at 14 and 21 days, a trait which was not apparent when DBA had outgrown their audiogenic seizure sensitivity at 42 days. The differences in receptor density and agonist state distribution between the two strains may be related to audiogenic seizure sensitivity.

The reversible binding of oxygen to sulfhemoglobin.

The O2 binding properties of sulfhemoglobin were studied. The oxygen tension required for half-saturation of sulfhemoglobin is more than 2 orders of magnitude higher than that for hemoglobin A. The binding of O2 exhibits an alkaline Bohr effect larger than that observed for hemoglobin, yet the Hill number is unity. From the Bohr titration curve, 0.68 proton is released during O2 binding at 0 degrees C. Sulfhemoglobin prepared from carboxypeptidase A-treated hemoglobin has an affinity for O2 which is about the same as that of sulfhemoglobin at the theoretical limit of the Bohr titration curve. Like its carboxypeptidase A-treated hemoglobin precursor, this sulfhemoglobin does not bind O2 cooperatively. Thus, sulfhemoglobin appears to be in a high affinity form at alkaline pH and a low affinity form at acid pH, similar to hemoglobin A. These results demonstrate that the magnitude of the Hill number is not always an indicator of the interaction between oxygen binding and other functions in a hemoglobin.

Oxygen binding to cobalt porphyrins

The thermodynamic constants of oxygen binding to cobalt picket fence porphyrin complexes, meso-tetra(a,o,a,o- o-pivalamidophenyl)porphyrinatocobalt(II)- 1 -methylimidazole and 1,2-dimethylimidazole, are reported. In contrast to pre- viously studied cobalt porphyrins, these complexes bind oxygen with the same affinity as cobalt substituted myoglobin and he- moglobin. Solvation effects are discussed as the source of this difference. The use of sterically hindered axial bases as models of T state hemoglobin is discussed. In studies of myoglobin (Mb) and hemoglobin (Hb), the replacement of the neutral iron porphyrin prosthetic group with different metalloporphyrins has proved to be a useful tech- nique.2 Artificial hemoglobins containing ~inc,~.~ manga- ne~e,~-* ~opper,~ and nickel9 have been reconstituted, and their properties compared with those of the native iron proteins. These artificial systems, however, are incapable of reversible oxygenation. In contrast, cobalt substituted hemoglobin and myoglobin (CoHb and CoMb)l0 are functional,I1-l3 although their oxygen affinities are 10-100 times less than those of native Hb and Mb. CoHb exhibits cooperativity in oxygen binding, though to a lesser degree than Hb. The extent of this cooperativity is conveniently expressed as AG;, , the free energy difference between the intrinsic binding of the first and the fourth 02 to Hb.13b,c For CoHb, AG:l is roughly one-third that of Hb under comparable condition^.'^^ Because of the different stereo- chemical and electronic factors involved in binding oxygen to a cobalt porphyrin, the observation of cooperativity in CoHb has been variously used either to que~tion~9~~J~ or s~pport'~~,'~ the elegant proposal of Perutz concerning the molecular mechanism of cooperativity in natural Hb.16 At the heart of this proposal is the assumption, based on earlier ideas of Hoard and Williams,18 that the high-spin iron in the unli- gated, low 02 affinity form of Hb (T state) lies out of the porphyrin plane, and that on binding 02, the iron becomes low spin and moves into the plane. The resulting motion of the proximal imidazole (0.6 A) then causes conformational changes in the protein which produce a higher 02 affinity quaternary form of the protein (R state). In the deoxy form of coboglobin, cobalt is low spin rather than high spin and the best estimates from simple cobalt model systems indicate that the proximal imidazole in CoHb moves -0.4 A upon oxygen- ation'4,'9,20 as opposed to the 0.6 8, for Hb. The resulting motion of the proximal histidine upon binding 02 will therefore only be two-thirds as great. This seems to be qualitatively consistent with the lowered AGil of CoHb.

Stopped flow kinetics of carbamylcholine binding to membrane bound acetylcholine receptor

Conformational changes upon binding of carbamylcholine to acetylcholine receptor-enriched membrane fragments have been observed by stopped-flow methods using the fluorescent probe ethidium bromide. A model consistent with both equilibrium and kinetic experiments is proposed in which the receptor binds two molecules of carbamylcholine with high affinity in a non-cooperative manner followed by binding of a third and possibly a fourth molecule with increasingly lower affinity. The receptor ligand precomplexes isomerize to different non-interconvertible complexes depending on the number of ligands bound. This kinetic model fits the data for carbamylcholine interactions with receptor prepared initially either in a low or high affinity form for ligands.

Cooperativity in O2 binding to iron porphyrins.

The solid-gas O2 binding equilibrium has been studied for for ferrous "picket fence" porphyrinates with sterically hindered axial imidazoles. Such systems show significant cooperativity in their binding of O2: at low O2 pressures a low O2 affinity form exists, and at high O2 pressures a higher O2 affinity form develops. Direct analogies are drawn to the cooperativity shown in O2 binding by hemoglobin. These model systems mimic hemoglobin quantitatively.

Physical and kinetic distinction of two ornithine decarboxylase forms in Physarum

Two forms of ornithine decarboxylase ( l-ornithine carboxy-lyase, EC 4.1.1.17) can be isolated from crude plasmodial homogenates of Physarum polycephalum. Both forms catalyze the stoichiometric production of putrescine and CO 2 from ornithine, yet they are distinguished by (a) a large difference in their affinity for coenzyme (apparent K m values of 0.13 and 33 μM); (b) a differential stability to extended dialysis of crude homogenates at 4°C; and (c) the tendency of the low affinity form to polymerize when suspended in low ionic strength borate and phosphate buffers. These forms appear to be alternate states of a basic catalytic subunit in that (a) they both demonstrate monomer and dimer molecular forms of 80 000 and 160 000 daltons, respectively, depending on the buffer content; (b) they coelute from DEAE-cellulose ion-exchange columns; and (c) they vary in activity in approximately equivalent yet opposite directions in response to factors which alter this organism's growth or metabolism. These data suggest that ornithine decarboxylase activity may be modulated by the control of the transition of this enzyme between the active and the relatively less active form.

Ligand interactions with the acetylcholine receptor from Torpedo californica. Extensions of the allosteric model for cooperativity to half-of-site activity

The solubilized acetylcholine receptor from Torpedo californica showed positive cooperativity in acetylcholine binding with a dissociation constant of 1.2 X 10(-8) M. Blockade of acetylcholine binding by nicotine was competitive; blockade by d-tubocurarine appeared to result from an allosteric interaction that altered half of the acetylcholine binding sites to a lower affinity form; decamethonium blockade displayed properties of competitive and allosteric inhibition suggesting less specificity for decamethonium binding than seen with either nicotine or d-tubocurarine. The d-tubocurarine inhibition data were evaluated by several possible models involving either differential competitive inhibition or allosteric inhibiton. The data were best described by the allosteric model.