Bell-shaped Dependence Research Articles

Calcium-dependent changes of paired-pulse modulation at single GABAergic synapses

Whole-cell patch-clamp and local electrical stimulation were used for measuring of monosynaptic GABAergic currents from rat hippocampal neurons in culture. Under control conditions (normal extracellular calcium, 2 mM) paired-pulse depression with 150 ms interpulse interval was observed. The mean current amplitude for both 1st and 2nd IPSCs displayed bell-shaped dependency from the stimulus amplitude. When extracellular Ca was either decreased to 0.5 mM or increased to 5 mM both mean amplitude of IPSCs and mean release probability decreased/increased correspondingly yet their dependences from the stimulus strength remained bell-shaped. Mean paired-pulse ratio was also affected—we observed facilitation of second IPSC in low-calcium whereas the latter was depressed in high-calcium solution. Our results suggest that calcium concentrations not only regulate the strength of paired-pulse plasticity but also can invert its direction.

Sugar-binding Properties of VIP36, an Intracellular Animal Lectin Operating as a Cargo Receptor

The vesicular integral protein of 36 kDa (VIP36) is an intracellular animal lectin that acts as a putative cargo receptor, which recycles between the Golgi and the endoplasmic reticulum. Although it is known that VIP36 interacts with glycoproteins carrying high mannose-type oligosaccharides, detailed analyses of the sugar-binding specificity that discriminates isomeric oligosaccharide structures have not yet been performed. In the present study, we have analyzed, using the frontal affinity chromatography (FAC) method, the sugar-binding properties of a recombinant carbohydrate recognition domain of VIP36 (VIP36-CRD). For this purpose, a pyridylaminated sugar library, consisting of 21 kinds of oligosaccharides, including isomeric structures, was prepared and subjected to FAC analyses. The FAC data have shown that glucosylation and trimming of the D1 mannosyl branch interfere with the binding of VIP36-CRD. VIP36-CRD exhibits a bell-shaped pH dependence of sugar binding with an optimal pH value of approximately 6.5. By inspection of the specificity and optimal pH value of the sugar binding of VIP36 and its subcellular localization, together with the organellar pH, we suggest that VIP36 binds glycoproteins that retain the intact D1 mannosyl branch in the cis-Golgi network and recycles to the endoplasmic reticulum where, due to higher pH, it releases its cargos, thereby contributing to the quality control of glycoproteins.

Inhibition of phospholipase C activity in Drosophila photoreceptors by 1,2-bis(2-aminophenoxy)ethane N, N, N′, N′-tetraacetic acid (BAPTA) and di-bromo BAPTA

In vivo light-induced and basal hydrolysis of phosphatidyl inositol 4,5-bisphosphate (PIP 2) by phospholipase C (PLC) were monitored in Drosophila photoreceptors using genetically targeted PIP 2-sensitive ion channels (Kir2.1) as electrophysiological biosensors for PIP 2. In cells loaded via patch pipettes with varying concentrations of Ca 2+ buffered by 4 mM free BAPTA, light-induced PLC activity, showed an apparent bell-shaped dependence on free Ca 2+ (maximum at “100 nM”, ∼10-fold inhibition at <10 nM or ∼1 μM). However, experiments where the total BAPTA concentration was varied whilst free [Ca 2+] was maintained constant indicated that inhibition of PLC at higher (>100 nM) nominal Ca 2+ concentrations was independent of Ca 2+ and due to inhibition by BAPTA itself (IC 50 ∼ 8 mM). Di-bromo BAPTA (DBB) was yet more potent at inhibiting PLC activity (IC 50 ∼ 1 mM). Both BAPTA and DBB also appeared to induce a modest, but less severe inhibition of basal PLC activity. By contrast, EGTA, failed to inhibit PLC activity when pre-loaded with Ca 2+, but like BAPTA, inhibited both basal and light-induced PLC activity when introduced without Ca 2+. The results indicate that both BAPTA and DBB inhibit PLC activity independently of their role as Ca 2+ chelators, whilst non-physiologically low (<100 nM) levels of Ca 2+ suppress both basal and light-induced PLC activity.

Synergistic interactions between Ca2+ entries through L‐type Ca2+ channels and Na+–Ca2+ exchanger in normal and failing rat heart

We used confocal Ca2+ imaging and the patch-clamp technique to investigate the interplay between Ca2+ entries through L-type Ca2+ channels (LCCs) and reverse-mode Na+-Ca2+ exchange (NCX) in activating Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) in cardiac myocytes from normal and failing rat hearts. In normal myocytes exposed to N(6),2'-O-dibutyryl adenosine-3',5'-cyclic monophosphate (db-cAMP, membrane-permeable form of cAMP), the bell-shaped voltage dependence of cytosolic Ca2+ transients was dramatically broadened due to activation of SR Ca2+ release at high membrane potentials (30-120 mV). This broadening of Ca2+-transient voltage dependence could be prevented by KB-R7943, an inhibitor of the reverse-mode NCX. Trans-sarcolemmal Ca2+ entries were measured fluorometrically in myocytes during depolarizing steps to high membrane potentials. The total Ca2+ entry (deltaF(Tot)) was separated into two Ca2+ entry components, LCC-mediated (deltaF(LCC)) and NCX-mediated (deltaF(NCX)), by exposing the cells to the specific inhibitors of LCCs and reverse-mode NCX, nifedipine and KB-R7943, respectively. In the absence of protein kinase A (PKA) stimulation the amplitude of the Ca2+-inflow signal (deltaF(Tot)) corresponded to the arithmetic sum of the amplitudes of the KB-R7943- and nifedipine-resistant components (deltaF(Tot)=deltaF(LCC)+deltaF(NCX)). PKA activation resulted in significant increases in deltaF(Tot) and deltaF(LCC). Paradoxically, deltaF(Tot) became approximately threefold larger than the sum of the deltaF(NCX) and deltaF(LCC) components. In myocytes from failing hearts, stimulation of PKA failed to induce a shift in Ca2+ release voltage dependence toward more positive membrane potentials. Although the total and NCX-mediated Ca2+ entries were increased again, deltaF(Tot) did not significantly exceed the sum of deltaF(LCC) and deltaF(NCX). We conclude that the LCC and NCX Ca2+-entry pathways interact synergistically to trigger SR Ca2+ release on depolarization to positive membrane potentials in PKA-stimulated cardiac muscle. In heart failure, this new form of Ca2+ release is diminished and may potentially account for the compromised contractile performance and reduced functional reserve in failing hearts.

NO signalling decodes frequency of neuronal activity and generates synapse‐specific plasticity in mouse cerebellum

Nitric oxide (NO) is an intercellular messenger regulating neuronal functions. To visualize NO signalling in the brain, we generated a novel fluorescent NO indicator, which consists of the heme-binding region (HBR) of soluble guanylyl cyclase and the green fluorescent protein. The indicator (HBR-GFP) was expressed in the Purkinje cells of the mouse cerebellum and we imaged NO signals in acute cerebellar slices upon parallel fibre (PF) activation with a train of burst stimulations (BS, each BS consisting of five pulses at 50 Hz). Our results showed that the intensity of synaptic NO signal decays steeply with the distance from the synaptic input near PF-Purkinje cell synapses and generates synapse-specific long-term potentiation (LTP). Furthermore, the NO release level has a bell-shaped dependence on the frequency of PF activity. At an optimal frequency (1 Hz), but not at a low frequency (0.25 Hz) of a train of 60 BS, NO release as well as LTP was induced. However, both NO release and LTP were significantly reduced at higher frequencies (2-4 Hz) of BS train due to cannabinoid receptor-mediated retrograde inhibition of NO generation at the PF terminals. These results suggest that synaptic NO signalling decodes the frequency of neuronal activity to mediate synaptic plasticity at the PF-Purkinje cell synapse.

Reconstitution of local Ca2+ signaling between cardiac L-type Ca2+ channels and ryanodine receptors: insights into regulation by FKBP12.6

Ca+-induced Ca2+ release (CICR) in the heart involves local Ca2+ signaling between sarcolemmal L-type Ca2+ channels (dihydropyridine receptors, DHPRs) and type 2 ryanodine receptors (RyR2s) in the sarcoplasmic reticulum (SR). We reconstituted cardiac-like CICR by expressing a cardiac dihydropyridine-insensitive (T1066Y/Q1070M) alpha1-subunit (alpha1CYM) and RyR2 in myotubes derived from RyR1-knockout (dyspedic) mice. Myotubes expressing alpha1CYM and RyR2 were vesiculated and exhibited spontaneous Ca2+ oscillations that resulted in chaotic and uncontrolled contractions. Coexpression of FKBP12.6 (but not FKBP12.0) with alpha1CYM and RyR2 eliminated vesiculations and reduced the percentage of myotubes exhibiting uncontrolled global Ca2+ oscillations (63% and 13% of cells exhibited oscillations in the absence and presence of FKBP12.6, respectively). alpha1CYM/RyR2/FKBP12.6-expressing myotubes exhibited robust and rapid electrically evoked Ca2+ transients that required extracellular Ca2+. Depolarization-induced Ca2+ release in alpha1CYM/RyR2/FKBP12.6-expressing myotubes exhibited a bell-shaped voltage dependence that was fourfold larger than that of myotubes expressing alpha1CYM alone (maximal fluorescence change was 2.10 +/- 0.39 and 0.54 +/- 0.07, respectively), despite similar Ca2+ current densities. In addition, the gain of CICR in alpha1CYM/RyR2/FKBP12.6-expressing myotubes exhibited a nonlinear voltage dependence, being considerably larger at threshold potentials. We used this molecular model of local alpha1C-RyR2 signaling to assess the ability of FKBP12.6 to inhibit spontaneous Ca2+ release via a phosphomimetic mutation in RyR2 (S2808D). Electrically evoked Ca2+ release and the incidence of spontaneous Ca2+ oscillations did not differ in wild-type RyR2- and S2808D-expressing myotubes over a wide range of FKBP12.6 expression. Thus a negative charge at S2808 does not alter in situ regulation of RyR2 by FKBP12.6.

Noise-flatness-induced hypersensitive transport in tilted periodic sawtooth potentials

The noise-flatness-induced hypersensitive transport of overdamped Brownian particles in a tilted periodic sawtooth potential driven by a multiplicative nonequilibrium three-level noise and additive white noise is considered. The study is a follow-up of one of our earlier papers on trichotomous-noise-induced hypersensitive transport [Mankin et al., Phys. Rev. E 68 (2003) 011105]. In the current paper the exact formula for the stationary current is presented. All the physical results discussed are computed by means of the exact formula. It is demonstrated that (i) at low temperatures the enhancement of current is hypersensitive to the applied small static tilting force, (ii) the stationary macroscopic current has a bell-shaped dependence on the parameters, correlation time and flatness, of multiplicative noise which occurs even in the case of large (compared to the ultrasmall DC signal) additive thermal noise, and (iii) at low temperatures and small values of the switching rate, the differential mobility as a function of the tilting force has local minima in a finite range of the force. Implications of the case of multiplicative trichotomous noise are compared with the results of a deterministic square-wave stimulus.

The Pore, not Cytoplasmic Domains, Underlies Inactivation in a Prokaryotic Sodium Channel

Kinetics and voltage dependence of inactivation of a prokaryotic voltage-gated sodium channel (NaChBac) were investigated in an effort to understand its molecular mechanism. NaChBac inactivation kinetics show strong, bell-shaped voltage dependence with characteristic time constants ranging from ∼50 ms at depolarized voltages to a maximum of ∼100 s at the inactivation midpoint. Activation and inactivation parameters for four different covalently linked tandem dimer or tandem tetramer constructs were indistinguishable from those of the wild-type channel. Point mutations in the outer part of the pore revealed an important influence of the S195 residue on the process of inactivation. For two mutants (S195D and S195E), the maximal and minimal rates of inactivation observed were increased by ∼2.5-fold, and the midpoint of the steady-state inactivation curve was shifted ∼20 mV in the hyperpolarizing direction, compared to the wild-type channel. Our data suggest that pore vestibule structure is an important determinant of NaChBac inactivation, whereas the inactivation mechanism is independent of the number of free cytoplasmic N- and C-termini in the functional channel. In these respects, NaChBac inactivation resembles C-type or slow inactivation modes observed in other voltage-gated K and Na channels.

Thermal annealing of MgO substrate increases 1/ f noise in YBaCuO x at 300 K and reduces the sheet resistance in the superconducting thin film

In this work, we characterize YBaCuO high T c superconducting thin films deposited on (0 0 1) MgO substrates and compare their noise properties. The films were sputtered under the same conditions on substrates that were annealed at different temperatures prior to deposition. The noise measurements were performed at 300 K. The films exhibit 1/ f noise due to the conductivity fluctuations and the results are compared with Hooge’s empirical relation. The normalized noise spectral density ( S v/ V 2), measured at 300 K as a function of the substrate annealing temperature T a, displays a bell-shaped dependence with a maximum at an anneal temperature T a = 1050 °C. Two models based on the empirical relation are proposed to explain the observed results: a slight reduction in sample resistance goes hand in hand with a strong increase in 1/ f noise after annealing of the substrate.

Activation of Isolated NADH:Ubiquinone Reductase I (Complex I) from Escherichia coli by Detergent and Phospholipids. Recovery of Ubiquinone Reductase Activity and Changes in EPR Signals of Iron−Sulfur Clusters

NADH:ubiquinone oxidoreductase (NDH-1 or complex I) from Escherichia coli was purified using a combination of anion exchange chromatography and centrifugation in sucrose density gradient. The dependence of enzyme activity on detergent and phospholipids was studied. Artificial hexaammineruthenium reductase activity was not affected by dodecyl maltoside (DDM) and asolectin. Ubiquinone reductase activity had a bell-shape dependence on DDM concentration; 7-10-fold activation could be achieved. Treatment with asolectin subsequently yields additional 2-fold activation with a corresponding increase in the apparent V(max) and without significant changes in apparent K(m). Comparative EPR studies of complex I reduced with NADH, "as prepared" and "activated by asolectin" showed an increase in the signals derived mainly from two [4Fe-4S] clusters in the activated enzyme. One of these signals could be simulated with an axial spectrum with g values of g(xyz)= 1.895, 1.904, 2.05, which corresponds to the parameters reported for the N2 cluster. This data indicates conformational rearrangements of catalytic importance in complex I upon binding of phospholipids.

Electrical Coupling in Sustentacular Cells of the Mouse Olfactory Epithelium

Sustentacular cells (SCs) line the apical surface of the olfactory epithelium (OE) and provide trophic, metabolic, and mechanical support for olfactory receptor neurons. Morphological studies have suggested that SCs possess gap junctions, although physiological evidence for gap junctional communication in mammalian SCs is lacking. In the present study we investigated whether coupling exists between SCs situated in tissue slices of OE from neonatal (P0-P4) mice. Using whole cell and cell-attached patch recordings from SCs, we demonstrate that SCs are electrically coupled by junctional resistances on the order of 300 M(omega). Under whole cell recording conditions, Alexa 488 added to the pipette solution failed to reveal dye coupling between SCs. Electrical coupling was deduced from the biexponential decay of capacitive currents recorded from SCs and from the bell-shaped voltage dependency of a P2Y-receptor-activated current, both of which were abolished by 18beta-glycyrrhetinic acid (20-50 microM), a blocker of gap junctions. These data provide strong evidence for functional coupling between SCs, the physiological importance of which is discussed.

The effect of cholesterol on the solubilization of phosphatidylcholine bilayers by the non-ionic surfactant Triton X-100

Most of the studies on the solubilization of model membranes by Triton X-100 (TR) involve one lipid. The aim of the present study was to evaluate the effect of the addition of cholesterol on the solubilization of bilayers made of palmitoyloleoylphosphatidylcholine (POPC) or dipalmitoylphosphatidylcholine (DPPC). Detailed investigation of the kinetics of solubilization of the cholesterol-containing bilayers by TR at different temperatures reveals that: (i) At 4 °C, solubilization of both systems is relatively slow. Hence, in order to prevent misleading conclusions from turbidity measurements it is important to monitor the solubilization after steady-state values of optical density (OD) are reached. (ii) Studies of the temperature-induced changes of the aggregates present in mixtures of TR, POPC and cholesterol indicate that the state of aggregation at all temperatures (including 4 °C) represents equilibrium. By contrast, for DPPC/cholesterol/TR mixtures “kinetic traps” may occur not only at 4 °C but at higher temperatures as well (e.g. 37 °C). (iii) The presence of cholesterol in POPC bilayers makes the bilayers more resistant to solubilization at low temperatures, especially at 4 °C. As a consequence, the temperature dependence of the TR concentration required for complete solubilization ( D t sol ) is no longer a monotonically increasing function (as for POPC bilayers) but a bell-shaped function, with a minimum at about 25 °C. Inclusion of cholesterol in DPPC bilayers makes the bilayers more resistant to solubilization at all temperatures except 4 °C. In this system, we observe a bell-shaped dependence of D t sol on temperature, with a minimum at 37 °C. (iv) Both the rate of vesicle size growth and the rate of the solubilization of POPC vesicles are not affected by the inclusion of cholesterol in the bilayers. Similarly, cholesterol did not affect significantly the rate of size growth of DPPC bilayers at all temperatures, but reduced the rate of solubilization at 4 °C.

Modal Gating of Human CaV2.1 (P/Q-type) Calcium Channels

The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing these calcium channels. Human CaV2.1 channels showed a complex modal gating, which is described in this and the preceding paper (Luvisetto, S., T. Fellin, M. Spagnolo, B. Hivert, P.F. Brust, M.M. Harpold, K.A. Stauderman, M.E. Williams, and D. Pietrobon. 2004. J. Gen. Physiol. 124:445–461). Here, we report the characterization of the so-called b gating mode. A CaV2.1 channel in the b gating mode shows a bell-shaped voltage dependence of the open probability, and a characteristic low open probability at high positive voltages, that decreases with increasing voltage, as a consequence of both shorter mean open time and longer mean closed time. Reversible transitions of single human CaV2.1 channels between the b gating mode and the mode of gating in which the channel shows the usual voltage dependence of the open probability (nb gating mode) were much more frequent (time scale of seconds) than those between the slow and fast gating modes (time scale of minutes; Luvisetto et al., 2004), and occurred independently of whether the channel was in the fast or slow mode. We show that the b gating mode produces reversible uncoupling of inactivation in human CaV2.1 channels. In fact, a CaV2.1 channel in the b gating mode does not inactivate during long pulses at high positive voltages, where the same channel in both fast-nb and slow-nb gating modes inactivates relatively rapidly. Moreover, a CaV2.1 channel in the b gating mode shows a larger availability to open than in the nb gating modes. Regulation of the complex modal gating of human CaV2.1 channels could be a potent and versatile mechanism for the modulation of synaptic strength and plasticity as well as of neuronal excitability and other postsynaptic Ca2+-dependent processes.

Salt-Induced Multilayer Growth: Correlation with Phase Separation in Solution

We report salt-induced phase separation of polyelectrolyte complexes in solution correlates with deposition of polymer multilayers at a surface. Multilayer growth was followed in situ using FTIR-ATR (Fourier transform infrared spectroscopy by attenuated total reflection). The polymer system was quaternized poly(4-vinylpyridine) (QPVP) and poly(methacrylic acid) (PMAA), with various ratios of PMAA to QPVP chain lengths. In solution, phase diagrams were produced for QPVP/PMAA mixtures containing excess of PMAA or QPVP, in both cases showing salt-induced phase separation. These phase diagrams controlled deposition of PMAA or QPVP chains at the surface and explained a bell-shaped dependence of the multilayer thickness on the salt concentration. The order of chain removal (occurring at the step of QPVP or PMAA deposition) could also be predicted from the phase state of QPVP/PMAA mixtures in solution. Finally, we show that, by using short equilibration times with solution, robust multilayers can be produced at surfaces even in conditions when multilayer deposition is aggravated by desorption of polymer chains.

Copper-dependent protein–protein interactions studied by yeast two-hybrid analysis

An important step in copper homeostasis is delivery of copper to a specific P-type ATPase in the Golgi apparatus (Ccc2 in yeast, ATP7A and ATP7B in humans) by a small copper chaperone protein (Atx1 in yeast, ATOX1 in humans). Atx1 and ATOX1 both contain an MXCXXC motif that is also present in Ccc2 (two motifs) and ATP7A/B (six motifs). Protein–protein interactions probably require coordination of one Cu(I) by cysteines from both MXCXXC motifs. We applied yeast two-hybrid analysis to screen systematically all possible interactions between MXCXXC-containing domains in these proteins. We demonstrate that ATOX1 and Atx1 preferentially interact with domains 2 and 4 of ATP7B and that Atx1 interacts with both Ccc2 domains. All combinations show a remarkable bell-shaped dependency on copper concentration that is maximal just below normal copper levels. Our results suggest that yeast two-hybrid analysis can be used to study the intracellular copper status of a cell.

Regulation of GABA release by depolarisation-evoked Ca2+ transients at a single hippocampal terminal.

We correlated dynamic changes in free cytosolic [Ca2+] ([Ca2+]i) within single presynaptic terminals of cultured hippocampal neurones with the postsynaptic GABA-mediated currents. The local changes in [Ca2+]i and evoked inhibitory postsynaptic currents (eIPSCs) were recorded simultaneously using Fura-2 fluorescence and whole-cell patch-clamp respectively. The Ca2+ signals and eIPSCs were evoked by direct extracellular electrical stimulation of a single presynaptic terminal by short depolarising pulses. The presynaptic Ca2+ transient was graded by varying the amplitude of extracellular stimulating pulses. The probability of the release event, P, estimated for each stimulation strength, reached a maximum (P=1) when the Ca2+ signal became maximal and remained at this level at higher stimulation strength, despite the subsequent decrease in the amplitude of the Ca2+ transient. A gradual, linear increase in stimulation amplitude (Vstim) resulted in a bell-shaped dependence of the averaged amplitudes of Ca2+ signals and corresponding averaged amplitudes of eIPSCs. Analysis of the eIPSC demonstrated that the decrease in both the mean eIPSC amplitude and the mean quantal content of release resulted from a reduction in the probability of multivesicular release, i.e. in the disappearance of failures and in the decrease of individual eIPSC amplitude. The Ca2+ signals of similar amplitude resulted in both random and determinate (non-random) neurotransmitter release. We conclude that depolarisation-induced elevation of [Ca2+]i within the terminal is necessary but not sufficient for activation of vesicular release of neurotransmitter.

Calcium Dependence of Polycystin-2 Channel Activity Is Modulated by Phosphorylation at Ser812

Polycystin-2 (PC-2) is a non-selective cation channel that, when mutated, results in autosomal dominant polycystic kidney disease. In an effort to understand the regulation of this channel, we investigated the role of protein phosphorylation in PC-2 function. We demonstrated the direct incorporation of phosphate into PC-2 in cells and tissues and found that this constitutive phosphorylation occurs at Ser(812), a putative casein kinase II (CK2) substrate domain. Ser(812) can be phosphorylated by CK2 in vitro and substitution S812A results in failure to incorporate phosphate in cultured epithelial cells. Non-phosphorylated forms of PC-2 traffic normally in the endoplasmic reticulum and cilial compartments and retain homo- and hetero-multimerization interactions with PC-2 and polycystin-1, respectively. Single-channel studies of PC-2, S812A, and a substitution mutant, T721A, not related to phosphorylation show that PC-2 and S812A function as divalent cation channels with similar current amplitudes across a range of holding potentials; the T721A channel is not functional. Channel open probabilities for PC-2 and S812A show a bell-shaped dependence on cytoplasmic Ca(2+) but there is a shift in this Ca(2+) dependence such that S812A is 10-fold less sensitive to Ca(2+) activation/inactivation than the wild type PC-2 channel. In vivo analysis of PC-2-dependent enhanced intracellular Ca(2+) transients found that S812A resulted in enhanced transient duration and relative amplitude intermediate between control cells and those overexpressing wild type PC-2. Phosphorylation at Ser(812) modulates PC-2 channel activity and factors regulating this phosphorylation are likely to play a role in the pathogenesis of polycystic kidney disease.

The Preferred Pathway of Glycosaminoglycan-accelerated Inactivation of Thrombin by Heparin Cofactor II

Thrombin (T) inactivation by the serpin, heparin cofactor II (HCII), is accelerated by the glycosaminoglycans (GAGs) dermatan sulfate (DS) and heparin (H). Equilibrium binding and thrombin inactivation kinetics at pH 7.8 and ionic strength (I) 0.125 m demonstrated that DS and heparin bound much tighter to thrombin (K(T(DS)) 1-5.8 microm; K(T(H)) 0.02-0.2 microm) than to HCII (K(HCII(DS)) 236-291 microm; K(HCII(H)) 25-35 microm), favoring formation of T.GAG over HCII.GAG complexes as intermediates for T.GAG.HCII complex assembly. At [GAG] << K(HCII(GAG)) the GAG and HCII concentration dependences of the first-order inactivation rate constants (k(app)) were hyperbolic, reflecting saturation of T.GAG complex and formation of the T.GAG.HCII complex from T.GAG and free HCII, respectively. At [GAG] >> K(HCII(GAG)), HCII.GAG complex formation caused a decrease in k(app). The bell-shaped logarithmic GAG dependences fit an obligatory template mechanism in which free HCII binds GAG in the T.GAG complex. DS and heparin bound fluorescently labeled meizothrombin(des-fragment 1) (MzT(-F1)) with K(MzT(-F1)(GAG)) 10 and 20 microm, respectively, demonstrating a binding site outside of exosite II. Exosite II ligands did not attenuate the DS-accelerated thrombin inactivation markedly, but DS displaced thrombin from heparin-Sepharose, suggesting that DS and heparin share a restricted binding site in or nearby exosite II, in addition to binding outside exosite II. Both T.DS and MzT(-F1).DS interactions were saturable at DS concentrations substantially below K(HCII(DS)), consistent with DS bridging T.DS and free HCII. The results suggest that GAG template action facilitates ternary complex formation and accommodates HCII binding to GAG and thrombin exosite I in the ternary complex.

Electrochemical preparation of conductive poly( N-alkylaniline)s with long N-alkyl chains using appropriate dopant anions and organic solvents

To prepare conductive polymers more soluble in organic solvents and more stable to over-oxidation than polyaniline, some N-alkylanilines were electropolymerized. Several polymerizing solutions with different kinds of anions (SO 4 2− and ClO 4 −) and organic solvents (acetonitrile and dimethyl sulfoxide) were employed. The anions and organic solvents were selected on the basis of the lipophilicity relationship among the poly( N-alkylaniline)s, the dopant anions and the organic solvents. The bell-shaped dependence of the conductivity on the N-alkyl chain length of the poly( N-alkylaniline)s was first observed for the poly( N-alkylaniline)s prepared from acetonitrile solution, and the highest conductivity of 1.0×10 −3 S cm −1 was obtained for SO 4 2−doped poly( N- n-heptylaniline). The dependence of the conductivity on the alkyl chain length of poly( N-alkylaniline)s was explained by the lipophilic affinity of the organic solvents for the poly( N-alkylaniline)s.

Application of a TCA-precipitation method for the determination of 1-alkyl-sn-glycero-3-phosphate:Acetyl-CoA acetyltransferase in human renal tissue

The activity of 1-alkyl-sn-glycero-3-phosphate:Acetyl-CoA acetyltransferase, which catalyses the first step of the de novo biosynthesis of PAF, was determined and characterised in cortical and medullary human renal tissues. A novel thin-layer chromatographic system as well as a trichloroacetic acid precipitation method, were utilised in order to determine the enzyme’s activity. The acetyltransferase activity was associated with the membranous fractions of the renal tissue, it showed an optimum pH of 8.4 and it had a bell-shaped dependence on BSA concentration. One or more disulphide bonds were necessary for the action of acetyltransferase while the enzyme seemed to be independent from divalent cations. Two assay products were extracted from the incubation mixture namely alkylacetylphosphatidic acid, produced by the acetylating action of the acetyltransferase on alkyllyso-phosphatidic acid and alkylacetyl-glycerol, which is produced by the action of a phosphohydrolase on alkylacetylphosphatidic acid. The presence of NaF in the assay mixture resulted to a decreased degradation of alkylacetylphosphatidic acid, as well as to an increased overall product formation. Cortical and medullary acetyltransferases share similar biochemical properties and there is no statistical difference between the two activities.