Hill Number Research Articles

Compared properties of phosphoenolpyruvate carboxylase from dark- and light-adapted Sorghum leaves: use of a rapid purification technique by immunochromatography

Two C 4-type phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPc) forms of Sorghum leaves, differing by their phosphorylation state (the phosphorylated form being predominant during the day), have been purified from dark- and light-adapted leaves by using an immunoaffinity chromatography column. The phosphorylation state of these purified PEPc forms was assessed by incubation with either the endogenous PEPc protein-serine kinase, or the catalytic subunit of the cAMP-dependent protein kinase and alkaline phosphatase on the dark and light enzyme, respectively. HPLC gel filtration studies showed that both forms are tetrameric at concentrations used throughout the work. Comparison of the kinetic properties at pH 7.0, 7.3 and 8.0, indicated a cooperativity for phosphoenolpyruvate (PEP) at all pHs considered, with a Hill number of 2 for both forms and higher V m(2- to 4-fold) and K m for the light form. Malate inhibition mainly affected the V m of the dark form, whereas it was an allosteric inhibitor for the light enzyme. The results established that phosphorylation is the molecular basis which mediates the functional properties of the enzyme. In a medium mimicking an illuminated mesophyll cytosol (2.5 mM PEP, 5 mM glucose-6-P, 20 mM malate and 10 mM MgCl 2 (pH 7.3), the activity of the enzyme from light-adapted leaves was twice that of the dark one. This increased activity due to phosphorylation may be of importance for the regulation of C 4 photosynthesis.

Inhibition of [ 3H]dopamine uptake by flunarizine

The effect of different calcium channel blockers was studied on basal and cocaine-inhibited [ 3H]dopamine uptake in rat striatal synaptosomes. Isradipine (dihydropyridine calcium antagonist) and diltiazem (L-type calaium antagonist) were devoid of effect on [ 3H]dopamine uptake, while flunarizine (T-type calcium antagonist) inhibited [ 3H]dopamine uptake. Flunarizine inhibition was competitive and the inhibitory curve was biphasic with a Hill coefficient of 2.1. The high Hill number suggested a mechanism of positive cooperativity between two sites. Flunarizine inhibition showed a complex interaction with cocaine inhibition. While flunarizine at low concentrations interacts with a distinct site, at higher concentrations it interacts with the same site as cocaine. The relevance of this finding for the potentiation by flunarizine of cocaine-induced dopamine release in vivo is discussed.

Isolation and Characterization of Dihydrodipicolinate Synthase from Maize

Dihydrodipicolinate synthase (EC 4.2.1.52), the first enzyme specific to lysine biosynthesis in plants, was purified from maize (Zea mays L.) cell suspension cultures and leaves. The subunit molecular weight of maize dihydrodipicolinate synthase was estimated to be 38,000 based on SDS-PAGE. The condensation of l-aspartate semialdehyde and pyruvate by highly purified dihydrodipicolinate synthase exhibited kinetics characteristic of a Ping Pong Bi Bi ordered reaction in which pyruvate binds first to the enzyme. Substrate inhibition evident at higher concentrations of l-aspartate semialdehyde was partially alleviated by increasing concentrations of pyruvate. Pyruvate binding exhibited cooperativity with an apparent number of 2 and 1.86 millimolar concentration required for 50% of maximal activity. The K(m) for aspartate semialdehyde was estimated to be 0.6 millimolar concentration. Lysine was an allosteric cooperative inhibitor of dihydrodipicolinate synthase with an estimated Hill number of 4 and 23 micromolar concentration required for 50% inhibition. The physical and kinetic data are consistent with a homotetramer model for the native enzyme.

Kinetic regulation of yeast NAD-specific isocitrate dehydrogenase by citrate

The present results suggest that the enzyme modifier citrate and the substrate isocitrate are bound at different sites on yeast NAD-specific isocitrate dehydrogenase and that citrate diminishes the binding of the positive effector 5'-AMP, thereby causing a decreased rate of enzyme catalysis. This interpretation differs from the earlier proposal that citrate can replace isocitrate at an activator site on the enzyme and can cause inhibition by binding at its catalytic site [Atkinson et al. (1965) J. Biol. Chem. 240, 2682]. The present proposal is supported by the following observations: At constant subsaturating levels of isocitrate, NAD+, and Mg2+ without AMP, up to 10 mM citrate was an activator and not an inhibitor. Citrate decreased velocity for AMP-activated enzyme; however, with increasing citrate the specific activity with AMP asymptotically approached but did not decrease below the level of the enzyme maximally activated by citrate in the absence of AMP. When added singly, AMP decreased S0.5 for isocitrate without changing the Hill number (n), whereas citrate lowered n without changing S0.5 for isocitrate. The difference in action of these modifiers indicated that they were bound at separate sites on the enzyme. The binding of citrate appeared to cause a conformational change in the protein that lowered the enzyme's affinity for AMP. This was consistent with the findings that citrate (or the citrate agonist fluorocitrate) (i) resulted in an increase in S0.5 for isocitrate with the AMP-activated enzyme and (ii) decreased binding of the positive effector analogue TNP-AMP as measured by fluorescence change.(ABSTRACT TRUNCATED AT 250 WORDS)

6,7-Dinitroquinoxaline-2,3-dione and 6-nitro,7-cyanoquinoxaline-2,3-dione antagonize responses mediated by [formula omitted] and NMDA-associated glycine recognition sites in vivo: Measurements of cerebellar cyclic-GMP

Direct intracerebellar administration of quisqualate resulted in marked increases in levels of cGMP in the cerebellum of the mouse, with a Hill number of 2.0. Quinoxalinediones, DNQX (6,7-dinitroquinoxaline-2,3-dione) and CNQX (6-nitro, 7-cyanoquinoxaline-2, 3-dione) attenuated the quisqualate-induced response. 6,7-Dinitroquinoxaline-2,3-dione also attenuated the d-serine-induced increases in levels of cGMP in a competitive manner. Intracerebellar injection of DNQX also antagonized the response to parenterally-administered harmaline. Similar results were also obtained with CNQX. These results indicate that these quinoxalinediones can attenuate the responses, mediated through the NMDA-associated glycine recognition sites, as well as the NMDA receptor complex. However, the glycine antagonist HA-966 (3-amino-1-hydroxypyrrolidone-2), at doses which completely reversed the increases induced by d-serine, failed to alter the response to quisqualate, indicating a lack of effect of glycine antagonists on quisqualate-mediated synaptic events. These results further support the interaction of the quinoxalinediones, DNQX and CNQX, with the NMDA receptor complex as established in receptor binding and electrophysiological studies.

Regulation of platelet protein kinase C by oleic acid. Kinetic analysis of allosteric regulation and effects on autophosphorylation, phorbol ester binding, and susceptibility to inhibition.

The regulation of protein kinase C by oleic acid was studied, and parameters that characterize the activation of protein kinase C by oleic acid and distinguish its effects from those of diacylglycerol (DAG) and phosphatidylserine (PS) were delineated. Activation of protein kinase C by sodium oleate required the presence of calcium and showed mild cooperative behavior (Hill number of 1.25) suggesting that Ca(oleate)2 is the active species. Kinetic analysis of the interaction of sodium oleate with substrates indicated that sodium oleate acted to increase the activity of the enzyme without modulating the KM for either MgATP or histone substrates. In this respect, sodium oleate action resembled that of DAG but not PS. However, multiple parameters distinguished the effects of sodium oleate from those of DAG. Unlike DAG, sodium oleate was unable to inhibit phorbol dibutyrate binding to protein kinase C. Sodium oleate also failed to interact with micelle-bound protein kinase C and preferentially activated "soluble" protein kinase C. The addition of histone caused protein/lipid aggregation in the presence of DAG but not in the presence of oleate. Activation of protein kinase C by sodium oleate or by PS/DAG demonstrated differential susceptibility to the action of inhibitors. Sphingosine and NaCl were more potent in inhibiting activation of protein kinase C by PS/DAG than by sodium oleate. Sodium oleate also expressed PS-like activity in that calcium and oleate acted as cofactors in activation of protein kinase C by DAG. Similar to PS, the ability of oleate to act in synergy with DAG resulted from "competitive" activation with a decrease in KM(app) of protein kinase C for DAG. Finally, sodium oleate was unable to induce autophosphorylation of protein kinase C. These studies demonstrate that oleate activates protein kinase C by a mechanism that is distinct from PS/DAG but partially overlaps the kinetic effects of both PS and DAG. The significance of these studies is discussed in relation to mechanisms of protein kinase C activation and to the possible physiological relevance of activation of protein kinase C by fatty acids.

Sodium dependency of GABA uptake into glial cells in bullfrog sympathetic ganglia

The kinetics of sodium dependency of GABA uptake by satellite glial cells was studied in bullfrog sympathetic ganglia. GABA uptake followed simple Michaelis-Menten kinetics at all sodium concentrations tested. Increasing external sodium concentration increased both Km and Vmax for GABA uptake, with an increase in the Vmax/Km ratio. The initial rate of uptake as a function of the sodium concentration exhibited sigmoid shape at 100 microM GABA. Hill number was estimated to be 2.0. Removal of external potassium ion or 10 microM ouabain reduced GABA uptake time-dependently. The effect of ouabain was potentiated by 100 microM veratrine. These results suggest that at least two sodium ions are involved with the transport of one GABA molecule and that sodium concentration gradient across the plasma membrane is the main driving force for the transport of GABA. The essential sodium gradient may be maintained by Na+, K(+)-ATPase acting as an ion pump.

Kinetics and thermodynamics of oxygen and carbon monoxide binding to the T-state hemoglobin of Urechis caupo

The tetrameric hemoglobin from Urechis caupo is nearly ideal for studying ligation to the T-state. Our previous EXAFS study had shown that the Fe is displaced 0.35 A from the mean plane of the porphyrin in the HbCO derivative. We have carried out detailed kinetic studies of oxygen and CO ligation as a function of temperature in order to characterize both the kinetics and thermodynamics of ligation in this hemoglobin. The entropy change associated with ligation essentially corresponds to simple immobilization of the ligand and is virtually the same as that we have determined for leghemoglobin, an extreme R-state-type hemoglobin. The low ligand affinities thus derive from small enthalpies of ligation, which can be correlated with the large out of plane displacement of the Fe. Only oxygen pulse measurements revealed kinetic evidence for cooperative oxygen binding, but a direct measurement of oxygen binding gave a Hill number of 1.3. An allosteric analysis gave L = 2.6 and c = 0.048 (oxygen) and c = 0.77 (CO). The higher affinity state in this weakly cooperative hemoglobin is denoted T*, and it is for this state that thermodynamic quantities have been determined. The small differences between T and T* in CO binding were nevertheless sufficient to allow us to measure by flash photolysis the rate of the T*----T conformational change in terms of an allosteric model. The half-time for this transition was calculated to be 8-14 ms at 20 degrees C.

Sample-size dependence of diversity indices and the determination of sufficient sample size in a high-diversity deep-sea environment

Diversity indices, although designed for comparative purposes, often cannot be used as such, due to their sample-size dependence. I t is argued here that this dependence is more pronounced in h~gh divers~ty than in low diversity assemblages and that indices more sensitive to rarer species require larger sample sizes to estimate diversity with reasonable precision than indices which put more weight on commoner species. This was tested for Hill's diversity numbers No to N, (Hill 1973) and some other commonly used diversity indices for a high-diversity nematode assemblage in the Mediterranean deep sea. Although diversity indices were introduced into the ecological literature more than 20 yr ago and have very often been criticized since, their use in applied ecological research, mainly in pollution impact studies, is still very popular ( e . g . Heip et al. 1988a). A fundamental drawback of many diversity indices is their sample-size dependence (Sanders 1968 and references therein), making comparison between studies difficult. Yet, the main purpose of quantifying diversity by a numerical index is to provide means for comparison between different communities. One way of avoiding incomparability of measurements resulting from different-sized samples was provided by the rarefaction method of Sanders (1968). In this method, one calculates the number of species expected from each sample if sampling size is standardized. Hurlbert (1971) showed that the rarefaction method generally overestimates the expected number of species present and h e introduced an exact computational formula for this index: the expected number of species in a sample with size n , drawn from a population of size N which had S species, is given by i' Inter-Research/Printed in F. R. Germany where Ni represents the number of individuals in the i th species in the full sample (Hurlbert 1971). This index was used by Heck et al. (1975) to estimate sufficient sample size for the calculation of the number of species in a sample. However, a mere species count, like ES (n), does not cover all information present in the community as it is not related to the way the individuals are divided among the species. Thus other diversity measures should be considered as well. Sample size dependence of diversity indices. Intuitively, one expects that not all diversity indices are equally influenced by sample size, and that also the type of community (with high or low diversity) plays a role. Let us consider the influence of sample size on Hill's diversity numbers (N,) of various orders (Hill 1973). Hill's diversity number of order a is given by: S Na = I C Pia I(l-al

Purification and Characterization of Rat Liver GTP Cyclohydrolase I: Cooperative Binding of GTP to the Enzyme

Abstract GTP cyclohydrolase I, an enzyme that catalyzes the first step in the biosynthetic pathway of tetrahydrobiopterin, has been purified about 38,000-fold to apparent homogeneity from rat liver extract with a yield of 5%. The molecular weight of the enzyme was estimated to be 300,000 by gel filtration on Ultrogel AcA 34. The purified enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at a position corresponding to a molecular weight of 30,000. N-terminal amino acid sequence analysis gave a single amino acid at every step of the Edman degradation up to residue 10. These results suggest that the enzyme is probably a homopolymer. The enzyme showed positive cooperativity with a Hill coefficient of 2.4 at a substrate (GTP) concentration of 10-50 microM. The Vmax value of the enzyme was 45 nmol/min.mg protein. The GTP concentration producing half-maximal velocity was 30 microM at a KCl concentration of 0.1 M. This value increased as the KCl concentration rose, without any change in Vmax or Hill number. Biosynthesis of tetrahydrobiopterin may be controlled by the intracellular level of GTP.

Autoradiographic mapping of a selective cyclic adenosine monophosphate phosphodiesterase in rat brain with the antidepressant [ 3H]rolipram

Rolipram is a clinically effective antidepressant with selective cAMP phosphodiesterase (PDE) inhibiting properties. (±)-[ 3 H] Rolipram binds with high affinity ( K d = 2.52 ± 0.47 nM) to sections of rat brain (Hill number = 0.90 ± 0.05). Binding is stereospecific. Association of (±)-[ 3 H] rolipram to sections is rapid (47% of specific binding in the first minute, k obs = 0.52 min −1). Dissociation of (±)-[ 3 H] rolipram exhibits non first order kinetics (3 component model: t 1 2 = 2.5 min , 50 min and 6 h, respectively). A number of PDE inhibitors reduce (±)-[ 3 H] rolipram binding to the level of nonspecific binding ((−)-rolipram, IC 50 = 0.9 nM; (±)-rolipram, IC 50 = 1.5 nM; Ro 20–1724, IC 50 = 11 nM; ICI 63.197, IC 50 = 35 nM; medazepam, IC 50 = 240 nM; diazepam, IC 50 = 1200 nM; IBMX, IC 50 = 3800 nM). In vitro autoradiography reveals high binding site densities in the cerebellum, olfactory bulb, lateral septal nucleus, frontal cortex, subiculum and CA1 of hippocampus. Most of the labeled structures are part of the limbic system. In vivo autoradiography of (±)-[ 3 H] rolipram binding shows much more nonspecific binding than in vitro, nevertheless the distribution pattern of (±)-[ 3 H] rolipram binding sites is similar. A comparison of the distribution pattern of (±)-[ 3 H] rolipram binding sites with that of an antidepressant (monoamine oxidase inhibitor, monoamine uptake inhibitor) reveals no overlap. Limited, though significant correlations exist with the distribution of β 2-adrenergic, adenosine 1, and glutamate/quisqualate receptors as well as protein kinase C, but not with β 2-adrenergic receptors and forskolin binding sites.

Role of ions and membrane potential in uptake of serotonin into plasma membrane vesicles from mouse brain

Plasma membrane vesicle preparations from mouse cerebral cortex actively accumulated [ 3H]serotonin upon the imposition of a K + gradient (in > out), a Na + gradient (out > in), and the presence of external Cl −. Maximal stimulation of uptake by internal K + occurred at 15 mM and half-maximal stimulation at 2 mM. Internal K + did not enhance uptake merely via generation of a membrane potential because simultaneous parallel increases in internal and external K + concentration also stimulated uptake. External Cl − increased serotonin uptake with a K m of 18 mM and a Hill number of 1.0, suggesting a requirement for one chloride ion for transport. Uptake could not be driven by internal H + instead of K +. Estimation of the membrane potential by the distribution of triphenylmethylphosphonium ion showed a modest effect of valinomycin (1–20 μM) in increasing the potential from −19 to −31 mV accompanied by an increase in serotonin uptake. Proton ionophores prevented this effect of valinomycin and, by themselves, reduced the potential to −6 mV, but did not affect serotonin transport. A model is proposed for serotonin transport in brain plasma membrane vesicles that is similar to the model for porcine blood platelet vesicles as far as electroneutrality and stimulation by K + Na +, and Cl − are concerned, but that is different in substitution of internal H + for K +.

Thermodynamics of Agonist and Antagonist Interaction with the Strychnine‐Sensitive Glycine Receptor

The thermodynamic parameters associated with the interactions of agonists and antagonists with glycine receptors in rat spinal cord membranes were determined. The binding of the antagonist [3H]strychnine and the inhibition of strychnine binding by 11 different glycinergic ligands were examined at temperatures between 0.5 and 37 degrees C. The density of receptors was not affected by the temperature at which the incubation was performed, but the ability of glycine receptor agonists and antagonists to compete with [3H]strychnine binding varied markedly. The affinity of the receptor for the antagonists strychnine, 2-aminostrychnine, RU-5135, 5,6,7,8-tetrahydro-4H-isoxazolo[5,4-c]azepin-3-ol, and the ligands bicuculline, norharmane, and PK-8165 decreased at higher temperatures. The binding of these ligands was enthalpy-driven. In contrast, the affinity of the agonists glycine, beta-alanine, and taurine and of the antihelmintic ivermectin increased at higher temperatures, and their binding was characterized by substantial increases in entropy. In addition, temperature affected the allosteric interaction between the glycine and strychnine sites of the receptor, as indicated by changes in the Hill number of the competition curves for glycine. Our results clearly indicate that the binding of agonists and antagonists to the glycine receptor is differentially affected by temperature, probably as a consequence of the different changes induced in the receptor conformation.

N‐[3H]Butyl‐β‐Carboline‐3‐Carboxylate, a Putative Endogenous Ligand, Binds Preferentially to Subtype 1 of Central Benzodiazepine Receptors

Synthetic n-butyl beta-carboline-3-carboxylate, an endogenous central benzodiazepine receptor inhibitor found in brain, was tritium-labeled from the butenyl ester. Binding of this [3H]beta-carboline was concentrated particularly in the synaptosomal membrane fraction of the cerebral cortex; this fraction showed a single type of high-affinity site (KD = 2.7 +/- 0.1 nM) with a Bmax of 1.16 +/- 0.08 pmol/mg of protein. The number of sites labeled was about half of that obtained with [3H]flunitrazepam binding (Bmax = 2.36 +/- 0.06 pmol/mg of protein). On the other hand, in the cerebellum, both ligands bound to practically the same number of sites. When [3H]flunitrazepam binding was done in the presence of 10(-11)-10(-5) M butyl beta-carboline, the differences between the two brain regions were more apparent. In cerebellar membranes the data fitted a straight line in the Eadie-Hofstee plot; this finding and a Hill number near unity suggest a single type of binding site. In the cortical membranes the data of binding fitted a concave curve, and the Hill number was 0.6. These are characteristics of two types of binding sites with different affinities (KD1 = 0.6-1.5 nM and KD2 = 12-18 nM). The differentiation of a high- and low-affinity site in the cerebral cortex was corroborated by experiments in which [3H]butyl beta-carboline binding was displaced by the triazolopyridazine CL 218,872. These results demonstrate that in the cerebral cortex there are two subtypes of sites (1 and 2) of central benzodiazepine receptors and that CL 218,872 binds preferentially to subtype 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Une méthode rapide et simple pour l'estimation de la position de la courbe de dissociation de l'oxyhémoglobine

The methods currently available for assessing the oxyhaemoglobin dissociation curve parameters are expensive, lengthy, require a large volume of blood, and the results obtained are modified by anaesthetic gases. The equipment required for the method described includes : a microtonometre, microcuvettes, 3 gas bottles containing different oxygen, carbon dioxide and nitrogen mixtures (4.5 %, 5.6 %, 89.9 % ; 3.5 %, 5.6 %, 90.9 % ; 2.5 %, 5.6 %, 91.9 % respectively), a microxymetre, and a micropHmetre. The samples in the microcuvettes are incubated at 37 °C in a gas flow of 45 ml · min −1 from the bottles. SO 2 is then read using the microxymetre. P 50, i.e. PO 2 at 50 % saturation, is calculated, as well as Hill's number « n(the angle of the oxyhaemoglobin dissociation curve). The PO 2 required for Hill's formula is also calculated from the atmospheric pressure, the water vapour partial pressure, and the oxygen fraction in the gas bottle used. Normal values obtained by this method were P 50 : 26.5 ± 1.5 mmHg ; Hill's number : 2.65 ± 0.35. The method presented here, requiring only 300 μl of blood, is cheap, reliable, not affected by anaesthetic drugs, and quick.

Octopamine-binding sites in the brain of Locusta migratoria

Binding sites for octopamine in the brain of Locusta migratoria were characterized using [ 3H]octopamine as radioligand. Equilibrium binding of [ 3H]octopamine to a membrane fraction from brains was achieved after a 60-min incubation period at 26°C. Binding of the radioligand was a function of tissue concentration, with a linear correlation over the range of 0.25–1 brain equivalents. Incubation of aliquots of a membrane fraction from brains with increasing concentrations of [ 3H]octopamine indicated saturable binding at radioligand concentrations of 100 nM and above. Scatchard analysis of the binding data suggested the presence of a single binding site with the equilibrium dissociation constant ( K d) being 3±0.5 × 10 −8 M, and the capacity of binding sites ( B max) being 0.2±0.1 pmol/mg protein. The Hill number was nearly equal to one, which indicates that one molecule of octopamine binds to one octopamine-binding site. A Scatchard analysis of the displacement data using dl-octopamine, also suggested the presence of a single binding site. The rank order of potency of different octopamine agonists and antagonists in competing for binding, based on IC 50 values was: naphazoline > clonidine > metoclopramide > chlorpromazine, which indicates that the binding site for [ 3H]octopamine is an octopamine 2 receptor. Since naphazoline > tolazoline, it is likely that the binding site for [ 3H]cotopamine is an octopamine 2A receptor.

Anomeric Specificity and Kinetics of Glucokinase: Theoretical Unsuitability of the Hill Equation

The kinetics of the low-Km hexokinase isoenzymes, which obey the Michaelis-Menten equation, can be established from the Km (Michaelis constant) and Vmax (maximal velocity) values for either equilibrated D-glucose or its alpha- and beta-anomers. In the case of the high-Km glucokinase isoenzyme, however, the sigmoidal substrate dependency and the competition between the two anomers of D-glucose do not allow, theoretically, to assign any meaningful value to either the Km, Vmax or n (Hill number) constants for equilibrated D-glucose. Thus, with equilibrated D-glucose, the concentration dependency fails to display a rectilinear relationship in the Hill plot. These observations illustrate the shortcomings of current biochemical studies in which the anomeric heterogeneity of D-glucose is ignored.

Taurine uptake by glial cells in the bullfrog sympathetic ganglia

Taurine uptake was studied in the bullfrog sympathetic ganglia. High- and low-affinity components were detected after subtraction of nonsaturable influx from total uptake in a concentration range from 34 nM to 8mM. Taurine uptake was strictly sodium dependent and the sodium dependency curve was sigmoidal, with a Hill number of 1.6, indicating that at least two sodium ions are required for the transport of one taurine molecule. External sodium ion affected both K m and V max for taurine uptake. Taurine uptake was inhibited by ouabain, but not by tetrodotoxin. These results suggest that sodium concentration gradient across plasma membrane may be the main driving force for taurine uptake. Electron and light microscopic autoradiography showed that glial cells were heavily labeled by [ 3H]taurine while ganglion cells were slightly labeled. The present data suggest that glial uptake may contribute to terminating the effect of taurine in the bullfrog sympathetic ganglia.

Site-directed alteration of Glu197 and Glu66 in a pyruvoyl-dependent histidine decarboxylase.

Site-directed mutagenesis has been used to explore the role of two carboxylates in the active site of histidine decarboxylase from Lactobacillus 30a. The most striking observation is that conversion of Glu197 to either Gln or Asp causes a major decrease in catalytic rate while enhancing substrate binding. This is consistent with models based on X-ray diffraction results which suggest that the acid may protonate a reaction intermediate during catalysis. The Asp197 protein undergoes a suicide reaction with substrate, apparently triggered by inappropriate protonation of the intermediate. This leads to decarboxylation-dependent transamination which converts the pyruvoyl cofactor to an alanine, inactivating the enzyme. Conversion of Glu66 to Gln affects parameters of kinetic cooperativity. The mutation fixes the Hill number at approximately 1.5, midway between the pH-dependent values of the wild-type enzyme.

Effects of arachidonic acid and the other long-chain fatty acids on the membrane currents in the squid giant axon.

The effects of arachidonic acid and some other long-chain fatty acids on the ionic currents of the voltage-clamped squid giant axon were investigated using intracellular application of the test substances. The effects of these acids, which are usually insoluble in solution, were examined by using alpha-cyclodextrin as a solvent, alpha-cyclodextrin itself had no effect on the excitable membrane. Arachidonic acid mainly suppresses the Na current but has little effect on the K current. These effects are completely reversed after washing with control solution. The concentration required to suppress the peak inward current by 50% (ED50) was 0.18 mM, which was 10 times larger than that of medium-chain fatty acids like 2-decenoic acid. The Hill number was 1.5 for arachidonic acid, which is almost the same value as for medium-chain fatty acids. This means that the mechanisms of the inhibition are similar in both long- and medium-chain fatty acids. When the long-chain fatty acids were compared, the efficacy of suppression of Na current was about the same value for arachidonic acid, docosatetraenoic acid and docosahexaenoic acid. The suppression effects of linoleic acid and linolenic acid on Na currents were one-third of that of arachidonic acid. Oleic acid had a small suppression effect and stearic acid had almost no effect on the Na current. The currents were fitted to equations similar to those proposed by Hodgkin and Huxley (Hodgkin, A.L., Huxley, A.F. (1952) J. Physiol (London) 117:500-544) and the change in the parameters of these equations in the presence of fatty acids were calculated.(ABSTRACT TRUNCATED AT 250 WORDS)